Polypeptides binding to neonatal FC receptor

Abstract

The present technology relates to polypeptides that bind to neonatal Fc receptors. More specifically, the present technology provides polypeptides that bind to neonatal Fc receptors and comprise (i) at least one domain comprising a serum albumin protein and / or at least one domain that specifically binds to a serum albumin protein and (ii) an Fc domain of immunoglobulin G (IgG).

Description

1. Technical Field

[0001] The present technology relates to polypeptides that bind to the neonatal Fc receptor. More specifically, the present technology provides polypeptides that bind to the neonatal Fc receptor and comprise (i) at least one domain comprising a serum albumin protein and / or at least one domain that specifically binds to a serum albumin protein and (ii) an Fc domain of immunoglobulin G (IgG).

[0002] The technology further relates to constructs, compounds, molecules or chemical entities comprising at least one of these polypeptides.

[0003] In addition, the present technology further relates to methods for producing such polypeptides and the use of such polypeptides for various applications, including but not limited to extending the in vivo half-life of therapeutic compounds and / or other groups or parts and / or reducing their clearance rate in vivo and / or preventing and / or treating diseases and / or disorders, such as but not limited to proliferative diseases, inflammatory diseases, infectious diseases or autoimmune diseases. 2. Technical Background

[0004] Peptides and proteins are two classes of molecules that are attractive for potential therapeutic applications. However, a bottleneck in their development into clinically and commercially relevant drugs is their short half-life in vivo, typically only minutes to hours.

[0005] The half-life of peptides and proteins in human serum is determined by several factors, including size, charge, susceptibility to proteolysis, their biological properties, the turnover rate of the proteins to which they bind, etc. Those with a molecular weight less than about 70 kDa are eliminated primarily by renal filtration and generally have very short serum half-lives. Larger proteins may persist in the circulation for several days.

[0006] Albumin and IgG are the two most abundant soluble proteins present in the blood circulation. They are exceptions to most proteins in the circulation because they share the remarkable characteristic of having an extended serum half-life of about 19 to 21 days in humans. The key factor regulating the plasma half-life of IgG and albumin is a cellular receptor called the neonatal Fc receptor (FcRn). FcRn is a heterodimer composed of N-glycosylated transmembrane MHC class I heavy chains that are non-covalently bound to soluble b2-microglobulin. Both IgG and albumin are ligands that bind to different epitopes of FcRn. Their interaction with FcRn is strictly pH-dependent, with strong binding occurring at acidic pH < 6.5 and not binding at neutral physiological pH. Generally, cellular models of half-life regulation rely on the uptake of IgG or albumin, possibly through liquid phase pinocytosis, and then binding to the FcRn in the acidified endosome where the receptor mainly resides. The FcRn-IgG or FcRn-albumin complex is then derouted for lysosomal degradation and recycled to the cell surface where exposure to the near-neutral pH of blood results in the release of IgG or albumin into the extracellular environment.

[0007] The most currently explored strategy for extending the half-life of peptide- and protein-based therapeutics is based on the above-mentioned FcRn-mediated recycling mechanism by attaching the therapeutically active compound directly or indirectly to albumin or the Fc domain.

[0008] However, extending the in vivo half-life of therapeutic peptides and small proteins to or exceeding that of albumin or full-length antibodies has not been achieved so far.

[0009] Most protein-Fc or peptide-Fc fusion proteins produced so far have a half-life value of only 4 to 21 days in the human body. These low values ​​may be due to the lower affinity of FcRn compared with the structure of conventional antibodies. When compared with complete IgG (which has a half-life of approximately 3 weeks), Fc fusion proteins exhibit a shorter half-life. The factors affecting this are complex and include generally lower affinity to FcRn, lower stability, clearance pathways of effector molecules, and the lack of Fab domains.

[0010] Similarly, even though serum albumin has a half-life of approximately 19 days in humans, albiglutide (GLP-1-HSA fusion protein) (United States) or (EU), also known as Albugon) has a half-life of only about 5 days. So far, other fusion partners tested in the clinic, such as CTP or ELP, have not performed better, with the fusion proteins having half-life values ​​of 2.5 or 4-5 days, respectively.

[0011] For most of the above proteins, the optimal dosing schedule is expected to be once a week, with some potentially requiring twice-weekly dosing. While this is far better than the natural peptide or protein alone, it is still far more frequent than most therapeutic antibodies. Currently, almost all protein-based drug formulations available on the market are administered intravenously or subcutaneously at highly frequent dosing intervals, ultimately leading to complications related to dose fluctuations and greatly reducing patient compliance.

[0012] Therefore, there is a need to extend the serum persistence of peptide- and protein-based therapeutics and / or any other groups or moieties, thereby leading to even more uniform serum concentrations of the drug / moiety, lower doses without compromising efficacy, and less frequent dosing. This may well translate into less toxicity and side effects and improved compliance. 3. Summary of the Invention

[0013] The present inventors have identified polypeptides that specifically bind and / or are otherwise directed to FcRn, said polypeptides comprising (i) at least one domain comprising a serum albumin protein and / or at least one domain that specifically binds a serum albumin protein and (ii) an Fc domain of immunoglobulin G (IgG).

[0014] Compared to known half-life extension peptides and proteins (including full-length immunoglobulins) as described in the prior art, the FcRn binding polypeptides provided by the technology of the present invention have the advantages of showing a significant increase in serum half-life in vivo and a reduction in clearance rate. Therefore, the polypeptides with extended in vivo persistence in the blood circulation according to the technology of the present invention can be used in a variety of applications, including but not limited to extending the in vivo half-life of (existing or future) therapeutic compounds and / or reducing their clearance rate. The benefits of extending the half-life of therapeutic molecules will be apparent to those skilled in the art. Such benefits include lower dosage and / or frequency of administration, which reduces the risk of adverse events in subjects and reduces costs. Therefore, therapeutic agents with extended half-life have substantial added value in terms of pharmaceutical significance.

[0015] In one aspect, the present technology provides polypeptides (such as FcRn targeting polypeptides) comprising (i) at least one domain comprising a serum albumin protein and / or at least one domain that specifically binds a serum albumin protein and (ii) an Fc domain of immunoglobulin G (IgG) or a fragment thereof.

[0016] In certain embodiments, the present technology provides polypeptides (such as FcRn targeting polypeptides) comprising (i) at least one domain comprising a serum albumin protein and (ii) an Fc domain of immunoglobulin G (IgG) or a fragment thereof.

[0017] In certain embodiments, the present technology provides polypeptides (such as FcRn targeting polypeptides) comprising (i) at least one domain that specifically binds to a serum albumin protein and (ii) an Fc domain of immunoglobulin G (IgG) or a fragment thereof.

[0018] In certain embodiments, the present technology provides polypeptides (such as FcRn targeting polypeptides) comprising (i) at least one domain comprising a serum albumin protein and at least one domain that specifically binds to a serum albumin protein and (ii) an Fc domain of immunoglobulin G (IgG) or a fragment thereof.

[0019] In certain embodiments, the at least one domain comprising a serum albumin protein is a portion, fragment, derivative or variant of a serum albumin protein.

[0020] In certain embodiments, the at least one serum albumin protein-containing domain is human serum albumin or a portion, fragment, derivative or variant of human serum albumin.

[0021] In another specific embodiment, the at least one domain that specifically binds to the serum albumin protein specifically binds to an amino acid residue on the serum albumin protein that is not involved in the binding of the serum albumin protein to FcRn.

[0022] In further specific embodiments, said at least one domain that specifically binds to a serum albumin protein specifically binds to domain II of human serum albumin.

[0023] In certain embodiments, the at least one domain that specifically binds to a serum albumin protein is a peptide or protein comprising between 5 and 500 amino acids.

[0024] In yet other specific embodiments, the at least one domain that specifically binds to a serum albumin protein is selected from (affibrioles), scFv, Fab, designed ankyrin repeat proteins Albumin binding domain (ABD), (also known as affitin) and immunoglobulin variable domain sequence (ISVD).

[0025] In certain additional specific embodiments, the at least one domain that specifically binds to a serum albumin protein is at least one ISVD.

[0026] In further specific embodiments, the at least one domain that specifically binds to a serum albumin protein is at least one ISVD that specifically binds to domain II of serum albumin, such as domain II of human serum albumin.

[0027] In certain additional specific embodiments, the present technology provides FcRn targeting polypeptides characterized in that at least one domain that specifically binds to a serum albumin protein is at least one ISVD that specifically binds to human serum albumin, wherein the ISVD is a (single) domain antibody, VHH, VHH, humanized VHH or camelized VH.

[0028] In certain additional specific embodiments, the at least one domain that specifically binds to a serum albumin protein is at least one ISVD that specifically binds to human serum albumin, said ISVD consisting essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0029] a. CDR1 comprises the amino acid sequence of SEQ ID NO: 1 according to Kabat numbering or has 3, 2 or 1 amino acid differences from SEQ ID NO: 1;

[0030] b. CDR2 comprises the amino acid sequence of SEQ ID NO: 2 according to Kabat numbering or has 3, 2 or 1 amino acid differences from SEQ ID NO: 2; and

[0031] c. CDR3 comprises the amino acid sequence of SEQ ID NO: 3 according to Kabat numbering or has 3, 2 or 1 amino acid differences from SEQ ID NO: 3.

[0032] In certain additional specific embodiments, the at least one domain that specifically binds to a serum albumin protein is at least one ISVD that specifically binds to human serum albumin, said ISVD consisting essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0033] a. CDR1 comprises the amino acid sequence of SEQ ID NO: 4 according to AbM numbering or has 3, 2 or 1 amino acid differences from SEQ ID NO: 4;

[0034] b. CDR2 comprises the amino acid sequence of SEQ ID NO: 5 according to AbM numbering or has 3, 2 or 1 amino acid differences from SEQ ID NO: 5; and

[0035] c. CDR3 comprises the amino acid sequence of SEQ ID NO: 6 according to AbM numbering or has 3, 2 or 1 amino acid differences from SEQ ID NO: 6.

[0036] In certain additional specific embodiments, the at least one domain that specifically binds to a serum albumin protein is at least one ISVD that specifically binds to a human serum albumin protein and has:

[0037] a. a degree of sequence identity with any one of SEQ ID NOs: 7 to 21 or 61-69 (wherein any CDRs and any C-terminal extensions that may be present are not taken into account when determining the degree of sequence identity) of at least 85%, preferably at least 90%, more preferably at least 95%; and / or

[0038] b. has no more than 7, preferably no more than 5 (such as only 3, 2 or 1) "amino acid differences" (as defined herein, and not taking into account CDRs and any C-terminal extension that may be present) with the sequence of SEQ ID NOs: 7 to 21 or 61-69.

[0039] In further specific embodiments, the at least one domain that specifically binds to serum albumin is at least one ISVD that specifically binds to human serum albumin and has a sequence selected from the group consisting of SEQ ID NOs: 7 to 21 and 61 to 69.

[0040] In yet other specific embodiments, the at least one peptide or protein that specifically binds to serum albumin is at least one ISVD that specifically binds to serum albumin with a dissociation constant (K D ) in 10 -6 M and 10 -11 M or less (as determined using Proteon, Kinexa, BLI or SPR).

[0041] In certain embodiments, the FcRn binding polypeptide further comprises at least one IgG Fc domain selected from the group consisting of an Fc domain of immunoglobulin G type 1 (IgG1), an Fc domain of immunoglobulin G type 2 (IgG2), an Fc domain of immunoglobulin G type 3 (IgG3), and an Fc domain of immunoglobulin G type 4 (IgG4), preferably IgG1 or IgG4, even more preferably IgG4.

[0042] In further specific embodiments, the at least one IgG Fc domain is a native (ie, wild-type) Fc domain of IgG, or a portion or fragment thereof.

[0043] In other further embodiments, the at least one IgG Fc domain is a variant Fc domain of IgG, or a portion or fragment thereof.

[0044] In certain embodiments, at least one IgG Fc domain binds to FcRn at a pH between about 5.0 and 6.8, preferably at a pH of about 6.0, with a K D In another specific embodiment, at least one IgG Fc domain binds to FcRn at a pH between about 5.0 and 6.8, preferably at a pH of about 6.0, with a K of less than about 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100 nM. D In yet other specific embodiments, at least one IgG Fc domain binds to FcRn at a pH between about 5.0 and 6.8, preferably at a pH of about 6.0, with a K of less than about 75 nM, such as less than 50 nM, 25 nM, 20 nM, 17 nM, 15 nM, 10 nM, 5 nM, 2.5 nM, 1 nM (at a pH between 5.0 and 6.8). In yet other specific embodiments, at least one IgG Fc domain binds to FcRn at a pH between about 5.0 and 6.8, preferably at a pH of about 6.0, with a K of less than about 75 nM, such as less than 50 nM, 25 nM, 20 nM, 17 nM, 15 nM, 10 nM, 5 nM, 2.5 nM, 1 nM (at a pH between 5. D Values ​​are between about 250 nM and 1 nM, such as between 100 nM and 1 nM, preferably between 75 nM and 1 nM, such as between 50 nM and 1 nM, most preferably between 25 nM and 1 nM, such as about 20 nM, such as about 17 nM.

[0045] In certain embodiments, polypeptides according to the present technology are such that their serum half-life in humans (expressed as t 1 / 2 β) is more than 6 hours, preferably more than 12 hours, more preferably more than 24 hours, even more preferably more than 72 hours; for example, about one week, two weeks and up to the half-life of serum albumin in humans (estimated to be about 19 days), and even up to three weeks, four weeks, one month, two to three months and more.

[0046] In certain embodiments, the polypeptides according to the present technology are such that they have a serum half-life in humans of at least 5%, such as at least 10%, at least 25%, at least 50%, at least 100%, up to 200%, 300%, 400% and 500% or more of the half-life of serum albumin in humans.

[0047] In certain embodiments, the at least one ISVD contains one or more mutations that reduce binding to a pre-existing antibody compared to any one of the sequences of SEQ ID NOs: 7 to 21 or 61-69.

[0048] In certain embodiments, at least one ISVD is a VHH and contains one or more humanizing substitutions compared to any one of the sequences of SEQ ID NOs: 7 to 21 or 61-69.

[0049] In certain embodiments, the present technology provides an FcRn-binding polypeptide as described herein, characterized in that the polypeptide further comprises a therapeutic moiety.

[0050] In certain embodiments, the present technology provides an FcRn binding polypeptide as described herein, characterized in that the polypeptide further comprises a therapeutic moiety comprising an ISVD, such as a (single) domain antibody, VHH, VHH, humanized VHH or camelized VH.

[0051] In another aspect, the present technology provides nucleic acids or nucleic acid sequences encoding polypeptides according to the present technology.

[0052] In another aspect, the present technology provides a vector comprising a nucleic acid or a nucleic acid sequence according to the present technology.

[0053] In yet another aspect, the present technology provides a host cell or a (non-human) host organism transformed or transfected with a nucleic acid or nucleic acid sequence according to the present technology or with a vector according to the present technology.

[0054] In another aspect, the present invention provides a method or process for producing a polypeptide according to the present invention, the method comprising at least the following steps:

[0055] a. expressing the nucleic acid sequence (encoding a polypeptide according to the present technology) in a suitable host cell or (non-human) host organism or in another suitable expression system, optionally followed by:

[0056] b. Isolation and / or purification of polypeptides according to the technology of the present invention.

[0057] In yet another aspect, the present technology provides pharmaceutical compositions comprising a polypeptide according to the present technology or a polypeptide produced by a method according to the present technology.

[0058] In another aspect, the present technology provides polypeptides of the present invention or polypeptides produced according to the methods of the present technology for use in treating a subject in need thereof.

[0059] In another aspect, the present technology provides a method for delivering a prophylactic or therapeutic polypeptide to a specific location, tissue or cell type in the body, comprising the step of administering to a subject a polypeptide of the present technology or a polypeptide produced by a method according to the present technology.

[0060] In another aspect, the present technology provides polypeptides of the present technology or polypeptides produced according to the methods of the present technology for use in delivering prophylactic or therapeutic polypeptides to specific locations, tissues or cell types in the body.

[0061] In yet another aspect, the present technology provides polypeptides of the present technology, or polypeptides produced according to methods of the present technology, for use in therapy.

[0062] On the other hand, the present invention provides polypeptides of the present invention or polypeptides produced according to the methods of the present invention, which are used to prevent, treat or improve diseases selected from proliferative diseases, inflammatory diseases, infectious diseases and autoimmune diseases.

[0063] On the other hand, the present technology provides a method for preventing, treating or ameliorating a disease selected from proliferative diseases, inflammatory diseases, infectious diseases and autoimmune diseases, which method comprises at least the step of administering a polypeptide of the present technology or a polypeptide produced by the method of the present technology to a subject in need.

[0064] In another aspect, the present technology provides a kit comprising a polypeptide of the present technology, a nucleic acid or nucleic acid sequence of the present technology, a vector of the present technology, or a host cell of the present technology.

[0065] Unless otherwise indicated or defined, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is made, for example, to the standard handbook mentioned in paragraph a) on page 46 of WO 08 / 020079.

[0066] It must be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes one or more of such different agents, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that can modify or substitute the methods described herein.

[0067] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the technology of the present invention described herein using only routine experimentation. Such equivalents are intended to be encompassed by the technology of the present invention.

[0068] The term "and / or" wherever used herein includes the meanings of "and," "or," and "all or any other combinations of the elements connected by the term."

[0069] The term "about" used in the context of the parameters or parameter ranges provided herein should have the following meanings. Unless otherwise indicated, when the term "about" is applied to a specific value or range, the value or range is interpreted as being as accurate as the method used to measure it. If the error range is not specified in the application, the last decimal place of the numerical value represents its accuracy. Where no other error ranges are given, the maximum range is determined by applying the rounding convention to the last decimal place, for example, for a pH value of about pH 2.7, the error range is 2.65-2.74. However, for the following parameters, a specific range should be used: the temperature indicated in ° C and without decimal places should have an error range of ± 1 ° C (for example, a temperature value of about 50 ° C represents 50 ° C ± 1 ° C); the time indicated in hours should have an error range of 0.1 hours, regardless of the decimal place (for example, a time value of about 1.0 hours represents 1.0 hours ± 0.1 hours; a time value of about 0.5 hours represents 0.5 hours ± 0.1 hours).

[0070] In this application, any parameter indicated with the term "about" is also considered to be disclosed without the term "about." In other words, embodiments that use the term "about" to refer to a parameter value should also describe the embodiment itself that relates to the numerical value of the parameter. For example, an embodiment that specifies a pH value of "about pH 2.7" should also disclose the embodiment itself that specifies a pH value of "pH 2.7"; an embodiment that specifies a pH range of "between about pH 2.7 and about pH 2.1" should also describe an embodiment that specifies a pH range of "between pH 2.7 and pH 2.1," etc.

[0071] Throughout this specification and claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term "comprises" may be replaced by the term "contains" or "includes" or sometimes when used herein, by the term "having". 4. Brief Description of the Figures

[0072] Figure 1 . Schematic diagram of the structural forms of polypeptides TP003, TP006, TP008, TP009, TP016, and TP019 according to specific embodiments of the present technology.

[0073] TP006, TP009, TP016, and TP019 are fusion polypeptides according to specific embodiments of the present technology comprising an IgG4 FALA Fc domain (as described herein and produced using the knob-in-mold technique) linked to (i) a Fab domain that specifically binds serum albumin; VHH (grey hatched ovals) and / or (ii) do not bind serum albumin or any other envisioned target VHH (black oval). The Fc domain and The VHH sequences are fused to the N-terminus and / or C-terminus of the Fc chain via a linker (as described in detail herein), ie via an IgG1 hinge, e.g. SEQ ID NO: 38, and / or a GS linker, see e.g. Table A-2.

[0074] P003 and TP008 were prepared as control fusion polypeptides containing the same components of the corresponding test constructs (i.e., TP009, TP016, and TP006 and TP019, respectively), except for the serum albumin binding VHHs were substituted to not bind serum albumin or any other envisioned target VHH (black oval). As a second control, a full-length monoclonal antibody (TP013) was generated that contained the same IgG4 FALA Fc framework containing the knob-to-hole mutation.

[0075] TP016 and TP019 contain additional amino acid variations in the Fc backbone sequence (i.e., I253A, H310A, H435A) compared to the native Fc IgG4 domain. These Fc sequence variants were made to test constructs that showed no binding to FcRn and will be further referred to herein as non-binding Fc variants.

[0076] Figure 2 Mean (+ / -SD, n=2) serum concentration-time curves of polypeptides TP006 and TP009 in competition with IgG (hIgG) in female Tg32 mice following intravenous bolus administration at 5-8 mg / kg compared to control Fc-ISVD construct (TP003) or monoclonal antibody (TP013). To simulate relevant competition with hIgG, Tg32 mice were preloaded with 250 mg / kg of 4 doses of TP006 and TP009 intravenously. Injections were given once a week, with the first administration taking place 2 days prior to the start of the PK study. The black triangles in the X-axis indicate the time points of Privigen injections.

[0077] Figure 3. Schematic diagram of the structural forms of polypeptides TP108, TP111, TP117, TP118, TP121 and TP123 according to specific embodiments of the present technology.

[0078] Figure 4 Mean (+ / - SD, n=3) plasma concentration-time profiles of polypeptides TP108 and TP111 following intravenous bolus administration at 5 mg / kg in female Tg32 mice.

[0079] Figure 5 Mean (+ / - SD, n=3) plasma concentration-time profiles of polypeptides TP117, TP118, TP121 and TP123 following intravenous bolus administration at 5 mg / kg in female Tg32 mice.

[0080] Figure 6 . Schematic diagram of the structural forms of polypeptides TPP-66143, TPP-66145, TPP-66146, TPP-66147, TPP-66148, TPP-66149, TPP-66144, TPP-66150, TPP-66151, TPP-66152, TPP-66176, TPP-66177, TPP-66153, TPP-66154, TPP-66175 and TPP-66174 according to specific embodiments of the present technology.

[0081] Figure 7 Mean (+ / - SD, n=3) plasma concentration-time profiles of ALB23002 ISVD-Fc polypeptides TPP-66144, TPP-66147 (with 9 and 35 GS linkers, respectively) compared to control ISVD-Fc polypeptide TPP-66143 following intravenous bolus administration at 5 mg / kg in female Tg32 mice.

[0082] Figure 8 Mean (+ / - SD, n=3) plasma concentration-time profiles of albumin binding domain-Fc polypeptides TPP-66150, TPP-66151, and TPP-66152 compared to ALB23002 ISVD-Fc polypeptide TPP-66147 and control ISVD-Fc polypeptide TPP-66143 following 5 mg / kg intravenous bolus administration in female Tg32 mice.

[0083] Figure 9Mean (+ / - SD, n=3) plasma concentration-time profiles of different albumin-binding ISVD-Fc polypeptides TPP-66145, TPP-66146, TPP-66147, TPP-66148, TPP-66149 compared to the control ISVD-Fc polypeptide TPP-66143 following intravenous bolus administration at 5 mg / kg in female Tg32 mice.

[0084] Figure 10 Mean (+ / - SD, n=3) plasma concentration-time profiles of human albumin-Fc polypeptide TPP-66153 and human albumin (QMP)-Fc polypeptide TPP-66154 compared to ALB23002 ISVD-Fc polypeptide TPP-66147 and control ISVD-Fc polypeptide TPP-66143 following 5 mg / kg intravenous bolus administration in female Tg32 mice.

[0085] Figure 11 Mean (+ / - SD, n=3) plasma concentration-time profiles of ALB23002 ISVD-Fc(YTE) polypeptide TPP-66174 and ALB23002 ISVD-Fc polypeptide TPP-66147 compared to control ISVD-Fc(YTE) polypeptide TPP-66175 and control ISVD-Fc polypeptide TPP-66143 following intravenous bolus administration at 5 mg / kg in female Tg32 mice.

[0086] Figure 12 Mean (+ / - SD, n=3) plasma concentration-time profiles of the symmetric ISVD-Fc polypeptide TPP-66176 and the symmetric ALB23002 ISVD-Fc polypeptide TPP-66177 compared to the ALB23002 ISVD-Fc polypeptide TPP-66147 and the control ISVD-Fc polypeptide TPP-66143 following intravenous bolus administration at 5 mg / kg in female Tg32 mice. 5. Specific implementation plan

[0087] The inventors of the present application have developed a novel polypeptide that binds to FcRn, comprising (i) at least one domain comprising a serum albumin protein and / or at least one domain that specifically binds to a serum albumin protein and (ii) an Fc domain of immunoglobulin G (IgG) or a fragment thereof, preferably an FcRn-binding fragment thereof.

[0088] In further specific embodiments, the polypeptides as disclosed herein, in addition to a domain comprising a serum albumin protein and / or a domain that specifically binds to a serum albumin protein as described in detail below, further comprise an IgG Fc region that may or may not specifically bind to human FcRn (SEQ ID NO: 24) or a (polymorphic) variant or isotype thereof, as also described in detail below.

[0089] Isoforms are alternative protein sequences that can be produced from the same gene by a single biological event or by a combination of biological events, such as alternative promoter usage, alternative splicing, alternative initiation, and ribosomal frameshifting, all of which are known in the art.

[0090] Amino acid residues will be referred to herein interchangeably according to the standard three-letter or one-letter amino acid code as set forth in Table B-1 below.

[0091] Table B-1: Common amino acids

[0092] 1-letter code 3-letter code Amino acid name A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu glutamate F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val thiazolyl amino acid W Trp Tryptophan X Xaa not specified Y Tyr Tyrosine

[0093] When an amino acid residue is indicated as "X" or "Xaa", this 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 a CDR, where one (or more) of the amino acid residues is indicated by "X", the description may further specify which amino acid residue(s) is (can be) present at that particular position in the CDR.

[0094] The amino acids are those L-amino acids commonly found in naturally occurring proteins and are listed in Table B-1. Amino acid sequences containing D-amino acids are not intended to be encompassed by this definition. Any amino acid sequence containing post-translationally modified amino acids can be described as the originally translated amino acid sequence using the symbols and modification positions shown in Table B-1; for example, hydroxylation or glycosylation, but these modifications should not be explicitly shown in the amino acid sequence. Any peptide or protein that can be represented as a sequence modification, such as linkage, cross-linking, end caps, non-peptidyl bonds, etc., is encompassed by this definition. The terms "protein," "peptide," "protein / peptide," and "polypeptide" are used interchangeably throughout this disclosure and, for the purposes of this disclosure, each have the same meaning. Each term refers to an organic compound composed of a linear chain of two or more amino acids. The compound can 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; or even three hundred or more amino acids. Those skilled in the art will understand that polypeptides generally contain fewer amino acids than proteins, although there is no art-recognized dividing line between the number of amino acids for polypeptides and proteins; polypeptides can be made by chemical synthesis or recombinant methods; and proteins are generally made in vitro or in vivo by recombinant methods known in the art.

[0095] When a nucleotide sequence or amino acid sequence is referred to as "comprising" or "consisting essentially of" another nucleotide sequence or amino acid sequence, respectively, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first-mentioned nucleotide sequence or amino acid sequence, respectively, but more generally this generally means that the first-mentioned nucleotide sequence or amino acid sequence contains within its sequence a stretch of nucleotides or amino acid residues, respectively, having the same nucleotide sequence or amino acid sequence as the latter sequence, regardless of how the first-mentioned sequence was actually generated or obtained (e.g., it may be generated or obtained by any suitable method described herein). By way of non-limiting example, when an ISVD is referred to as comprising a CDR sequence, this may mean that the CDR sequence has been incorporated into the ISVD, but more generally this generally means that the ISVD contains within its sequence a stretch of amino acid residues having 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 later amino acid sequence has a specific biological or structural function, it preferably has substantially the same, similar or equivalent biological or structural function as the first-mentioned amino acid sequence (in other words, the first-mentioned amino acid sequence preferably enables the later sequence to perform substantially the same, similar or equivalent biological or structural function). For example, when an ISVD is referred to as comprising a CDR sequence or a framework sequence, respectively, the CDR sequence and the framework are preferably capable of serving as CDR sequences or framework sequences, respectively, in the ISVD. Furthermore, when a nucleotide sequence is referred to as comprising another nucleotide sequence, the first-mentioned nucleotide sequence is preferably such that, when expressed as an expression product (e.g., a polypeptide), the amino acid sequence encoded by the later nucleotide sequence forms part of the expression product (in other words, the later nucleotide sequence is in the same reading frame as the first-mentioned larger nucleotide sequence).

[0096] As used herein, the term "domain" generally refers to the globular region of a protein. For example, the term "domain" can refer to the globular region of an antibody and particularly the globular region of a heavy chain antibody, or the term "domain" can refer to a polypeptide that is essentially composed of the globular region of a polypeptide. In specific embodiments, the domain in the context of the present disclosure is essentially composed of a serum albumin protein or a fragment, variant or derivative thereof. In other specific embodiments, the domain in the context of the present disclosure will comprise the globular region of an antibody and will comprise, for example, peptide loops (e.g., 3 or 4 peptide loops) that are stabilized as sheets or by disulfide bonds. In specific embodiments, the domain in the context of the present disclosure will essentially consist of the constant region of an antibody (such as the Fc domain of an antibody).

[0097] In the context of the present technology, "binding" to a target molecule has the same ordinary meaning in the art as understood in the context of a protein and its corresponding ligand or an antibody and its corresponding antigen. In certain specific embodiments of the present application, "binding" refers to a direct and specific interaction between two binding partners or molecules (such as, for example, a protein and its ligand or an antibody and its antigen). In certain other specific embodiments, "binding" refers to an indirect interaction between two binding partners or molecules (such as, for example, when a first binding partner and a second binding partner directly and specifically bind to the same target protein to indirectly connect or interact indirectly with each other via the target protein).

[0098] The term "antigenic determinant" refers to an epitope on an antigen that is recognized by an antigen binding molecule (such as an ISVD or a polypeptide comprising an ISVD), more particularly recognized by the antigen binding site of the molecule. The terms "antigenic determinant" and "epitope" can also be used interchangeably herein. An antigen binding molecule (such as an antibody, ISVD, a polypeptide of the present technology, or generally an antigen binding protein or polypeptide or fragment thereof) that can (specifically) bind to a specific antigenic determinant, epitope, antigen or protein (or at least a portion, fragment or epitope thereof), has affinity and / or specificity for a specific antigenic determinant, epitope, antigen or protein (or at least a portion, fragment or epitope thereof) is said to be "for" or "directed against" the antigenic determinant, epitope, antigen or protein.

[0099] 5.1 Immunoglobulin Single Variable Domains

[0100] The term "immunoglobulin single variable domain" (ISVD) is used interchangeably with "single variable domain" to define an immunoglobulin molecule in which the antigen binding site is present on and formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (such as Fab, Fab', F(ab')2, scFv, di-scFv), in which two immunoglobulin domains, in particular 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 will contribute to the antigen binding site, i.e., a total of 6 CDRs will participate in the formation of the antigen binding site.

[0101] In view of the above definition, antigen binding domains of conventional 4-chain antibodies (such as IgG, IgM, IgA, IgD or IgE molecules; known in the art) or Fab fragments, F(ab')2 fragments, Fv fragments (such as disulfide-linked Fv or scFv fragments) or diabodies (all known in the art) derived from such conventional 4-chain antibodies will generally not be regarded as immunoglobulin single variable domains because, in these cases, binding to the corresponding epitope of the antigen generally does not occur through one (single) immunoglobulin domain, but rather through a pair of (associated) immunoglobulin domains (such as light and heavy chain variable domains) that bind together to the epitope of the corresponding antigen, i.e., through a VH-VL pair of immunoglobulin domains.

[0102] In contrast, immunoglobulin single variable domains are able to specifically bind to an antigenic epitope without pairing with another immunoglobulin variable domain.The binding site of an immunoglobulin single variable domain is formed by a single VH, single VHH or single VL domain.

[0103] Therefore, the single variable domain can be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit essentially consisting 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).

[0104] The immunoglobulin single variable domain (ISVD) can be, for example, a heavy chain ISVD, such as VH, VHH, including camelized VH or humanized VHH. Preferably, it is VHH, including camelized VH or humanized VHH. The heavy chain ISVD can be derived from a conventional four-chain antibody or a heavy chain antibody.

[0105] For example, the immunoglobulin single variable domain can be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or a dAb (or an amino acid sequence suitable for use as a dAb), or molecules (as defined herein, and including but not limited to VHH); other single variable domains, or any suitable fragment of either thereof.

[0106] In particular, the immunoglobulin single variable domain may be Immunoglobulin single variable domain (such as VHH, including humanized VHH or camelized VH) or a suitable fragment thereof. [Note: is a registered trademark of Ablynx NV]

[0107] "VHH domains" (also referred to as VHH, VHH antibody fragments, and VHH antibodies) were originally described as "heavy chain antibodies" (i.e., "antibodies without light chains"; Hamers-Casterman et al. Nature 363:446-448, 1993) antigen-binding immunoglobulin variable domains. The term "VHH domain" has been selected to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For further description of VHH, reference is made to Muyldermans' review article (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0108] Typically, the production of immunoglobulins involves immunization of experimental animals, fusion of immunoglobulin-producing cells to produce hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be produced by screening natural or synthetic libraries (e.g., by phage display).

[0109] Immunoglobulin sequences (such as The generation of VHHs has been extensively described in various publications, of which WO 94 / 04678, Hamers-Casterman et al. 1993, and Muyldermans et al. 2001 (reviewed in Molecular Biotechnology 74: 277-302, 2001) can serve as examples. In these methods, camelids are immunized with a target antigen to induce an immune response against the target antigen. The library of VHHs obtained from the immunization is further screened for VHHs that bind to the target antigen.

[0110] In these cases, the generation of antibodies requires purified antigen for immunization and / or screening. The antigen can be purified from a natural source or during recombinant production.

[0111] Immunization and / or screening of immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0112] The technology of the present invention can use immunoglobulin sequences from different sources, including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences. The technology also includes fully human, humanized, or chimeric sequences. For example, the technology of the present invention comprises camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, for example, camelized dAbs as described by Ward et al. (see, for example, WO 94 / 04678 and Riechmann, Febs Lett., 339:285-290, 1994 and Prot. Eng., 9:531-537, 1996). In addition, the present technology also uses fused immunoglobulin sequences, for example, to form multivalent and / or multispecific constructs (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, see also Conrath et al., J. Biol. Chem., Vol. 276, 10.7346-7350, 2001, and for example WO 96 / 34103 and WO 99 / 23221), as well as immunoglobulin sequences that contain tags or other functional parts (e.g., toxins, labels, radiochemicals, etc.), which can be derived from the immunoglobulin sequences of the present technology.

[0113] "Humanized VHHs" comprise an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but which has been "humanized", i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (and in particular the framework sequence) with one or more amino acid residues present at one or more corresponding positions in the VH domain of a conventional 4-chain antibody from a human (e.g., as indicated above). This can be done in a manner known per se, as will be clear to the skilled person, e.g., based on the further description herein and the prior art (e.g., WO 2008 / 020079). Furthermore, it should be noted that such humanized VHHs can be obtained in any suitable manner known per se, and are therefore not strictly limited to polypeptides that have been obtained using a polypeptide comprising a naturally occurring VHH domain as starting material.

[0114] "Camelized VH" comprises an amino acid sequence corresponding to the amino acid sequence of a naturally occurring VH domain but which has been "camelized", i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody with one or more amino acid residues present at one or more corresponding positions in the VHH domain of a heavy chain antibody. This can be done in a manner known per se, which will be clear to the skilled person, for example based on the further description herein and the prior art (e.g., WO 2008 / 020079). As defined herein, such "camelized" substitutions are preferably inserted at amino acid positions that form and / or occur at the VH-VL junction and / or at so-called camelid hallmark residues, as defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996), supra). Preferably, the VH sequence used as the starting material or starting point for generating or designing a camelized VH is preferably a VH sequence from a mammal, more preferably a human VH sequence, such as a VH3 sequence. However, it should be noted that such camelized VH may be obtained in any suitable manner known per se and is therefore not strictly limited to polypeptides that have been obtained using a polypeptide comprising a naturally occurring VH domain as starting material.

[0115] The preferred structure of an immunoglobulin single variable domain sequence can be considered to be composed of four framework regions ("FR"), which are referred to in the art and herein as "Framework Region 1" ("FR1"); "Framework Region 2" ("FR2"); "Framework Region 3" ("FR3"); and "Framework Region 4" ("FR4"), respectively; said framework regions are interrupted by three complementarity determining regions ("CDRs"), which are referred to in the art and herein as "Complementarity Determining Region 1" ("CDR1"); "Complementarity Determining Region 2" ("CDR2"); and "Complementarity Determining Region 3" ("CDR3").

[0116] As further described in paragraph q) on pages 58 and 59 of WO 08 / 020079 (incorporated herein by reference), the amino acid residues of the immunoglobulin single variable domains may be numbered according to the universal numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as adapted for use in Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2):185-195, see e.g., reference 1 to this publication). Figure 2) in the article from camelids. It should be noted that, as is well known in the art for VH and VHH 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 Kabat numbering (that is, one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in VH and VHH domains is typically between 110 and 120, often in the range of between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0117] In this application, unless otherwise indicated, CDR sequences are determined according to the AbM numbering described in Kontermann and Dübel (2010 edition, Antibody Engineering, Volume 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pages 33-51). According to this method, FR1 comprises the amino acid residues at positions 1-25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the amino acids at positions 36-49, CDR2 comprises the amino acid residues at positions 50-58, FR3 comprises the amino acid residues at positions 59-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113.

[0118] The determination of CDR regions can also be carried out according to different methods. In the determination according to Kabat's CDR, the FR1 of the immunoglobulin single variable domain comprises the amino acid residues at positions 1-30, the CDR1 of the immunoglobulin single variable domain comprises the amino acid residues at positions 31-35, the FR2 of the immunoglobulin single variable domain comprises the amino acid residues at positions 36-49, the CDR2 of the immunoglobulin single variable domain comprises the amino acid residues at positions 50-65, the FR3 of the immunoglobulin single variable domain comprises the amino acid residues at positions 66-94, the CDR3 of the immunoglobulin single variable domain comprises the amino acid residues at positions 95-102, and the FR4 of the immunoglobulin single variable domain comprises the amino acid residues at positions 103-113.

[0119] In such immunoglobulin sequences, the framework sequence may be any suitable framework sequence, and examples of suitable framework sequences will be clear to the skilled person, e.g. based on standard manuals and the further disclosure and prior art mentioned herein.

[0120] The framework sequence is preferably an immunoglobulin framework sequence (a suitable combination thereof), or a framework sequence derived from an immunoglobulin framework sequence (e.g., by humanization or camelization). For example, the framework sequence may be a framework sequence derived from a light chain variable domain (e.g., a VL sequence) and / or a heavy chain variable domain (e.g., a VH sequence or a VHH sequence). In a particularly preferred aspect, the framework sequence is a framework sequence derived from a VHH sequence (wherein the framework sequence may optionally be partially or fully humanized), or a conventional VH sequence that has been camelized (as defined herein).

[0121] In particular, the framework sequences present in the ISVD sequences used in the technology of the present invention may contain one or more Hallmark residues (as defined herein) such that the ISVD sequence is Molecules, such as VHHs, include humanized VHHs or camelized VHs. Some preferred, but non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

[0122] Likewise, as generally described herein for immunoglobulin sequences, suitable fragments (or combinations of fragments) of any of the foregoing may also be used, such as fragments containing one or more CDR sequences, suitably flanked by and / or connected via one or more framework sequences (e.g., in the same order as these CDR and framework sequences might appear in the full-size immunoglobulin sequence from which the fragment is derived).

[0123] However, it should be noted that the present technology is not limited with respect to the source of the ISVD sequence (or the nucleotide sequence used to express it), nor is it limited with respect to the manner in which the ISVD sequence or nucleotide sequence is generated or obtained (or has been generated or obtained). Thus, the ISVD sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In specific but non-limiting aspects, 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, and in particular partially or fully humanized VHH sequences), "camelized" (as defined herein) immunoglobulin sequences, and immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veiling, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping heavy primers, and similar techniques for engineering immunoglobulin sequences well known to those skilled in the art, or any suitable combination of any of the foregoing.

[0124] Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence and may, for example, be a sequence isolated by PCR from a suitable naturally occurring sample (e.g. DNA or RNA isolated from a cell), a nucleotide sequence that has been isolated from a library (and in particular an expression library), a nucleotide sequence that has been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence that has been prepared by PCR using overlapping heavy primers or a nucleotide sequence that has been prepared using DNA synthesis techniques known per se.

[0125] As mentioned above, an ISVD can be VHH or its suitable fragment. For the general description of ISVD, reference is made to the further description below, as well as the prior art cited herein. However, in this regard, it should be noted that this description and the prior art primarily describe ISVDs of the so-called "VH3 class" (i.e., ISVDs with a high degree of sequence homology to human germline sequences of the VH3 class, such as DP-47, DP-51, or DP-29). However, it should be noted that the technology of the present invention can generally use any type of ISVD in its broadest sense and, for example, also use ISVDs belonging to the so-called "VH4 class" (i.e., ISVDs with a high degree of sequence homology to human germline sequences of the VH4 class, such as DP-78), as described, for example, in WO 2007 / 118670.

[0126] Typically, an ISVD (particularly a VHH sequence, including (partially) humanized VHH sequences and camelized VH sequences) can be characterized by the presence of one or more "Hallmark residues" (as described herein) in one or more framework sequences (also as further described herein). Thus, in general, an ISVD can be defined as an immunoglobulin sequence having the following (general) structure:

[0127] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0128] wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein one or more of the Hallmark residues are as further defined herein.

[0129] In particular, the ISVD may be an immunoglobulin sequence having the (general) structure

[0130] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0131] wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein the framework sequences are as further defined herein.

[0132] More specifically, the ISVD may be an immunoglobulin sequence having the (general) structure

[0133] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0134] wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein:

[0135] According to Kabat numbering, 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 hallmark residues mentioned in the following Table A-0.

[0136] Table A-0: Hallmark residues in ISVD

[0137]

[0138]

[0139] 5.2 Specificity

[0140] The terms "specificity," "specifically binds," or "specific binding" refer to the number of different target molecules (such as antigens) from the same organism to which a particular binding unit (such as an ISVD) can bind with sufficiently high affinity (see below). "Specificity," "specifically binds," or "specific binding" are used interchangeably herein with "selectivity," "selectively binds," or "selectively binds." A binding unit, such as an ISVD, preferably binds specifically to its designated target. The specificity / selectivity of a binding unit can be determined based on affinity. Affinity indicates the strength or stability of a molecular interaction. Affinity is typically measured by K. D Or dissociation constant, expressed in mol / L (or M). Affinity can also be expressed as the association constant K A , which is equal to 1 / K D And (mol / L) -1 (or M -1 Affinity is a measure of the strength of binding between a moiety and a binding site on a target molecule: K D The lower the value, the stronger the binding strength between the target molecule and the targeting moiety. Typically, the binding units (such as ISVDs) used in the present technology will be 10 -5 to 10-12 mol / L or less, 10 -6 to 10 -12 mol / L or less, and preferably 10 -7 to 10 -12 mol / L or less, and more preferably 10 -8 to 10 -12 The dissociation constant (K) in mol / L D )(i.e., with 10 5 to 10 12 L / mol or higher, 10 6 to 10 12 L / mol or more, and preferably 10 7 to 10 12 L / mol or more, and more preferably 10 8 to 10 12 Association constant (K A )) binds to its target. It is generally believed that any -4 mol / L K D A K value of 0.104 (or any K value less than 104 liters / mol) indicates nonspecific binding. A K value for a biological interaction such as binding of an immunoglobulin sequence to an antigen is considered specific. D Typically in 10 -5 mol / L (10000nM or 10μM) to 10 -12 Thus, specific / selective binding may mean that, using the same measurement method such as SPR, the binding unit (or polypeptide comprising the same) is in the range of 10 -5 to 10 -12 mol / L or lower K D The value binds to FcRn and is greater than 10 -4 mol / L K D Thus, the ISVD preferably exhibits at least half the binding affinity for human FcRn, more preferably at least the same binding affinity, as an ISVD consisting of the amino acids of SEQ ID NO: 14 or 15, wherein the binding affinity is measured using the same method, such as SPR. Specific binding to a target from a certain species does not exclude that the binding unit can also specifically bind to a similar target from a different species.

[0141] In one embodiment, the polypeptides of the present technology bind to HSA, and their K D The value is about 10 -5 to 10 -12 mol / L or less, such as about 10 -6 to 10 -10Mole / L, such as about 10 -7 to 10 -10 mol / L, or about 10 -7 to 10 -9 Mole / L, such as about 10 -8 to 10 -9 Moles / liter, for example, as determined by SPR.

[0142] In another embodiment, the polypeptides of the present technology bind to FcRn at pH 6.0 in the absence of HSA with a K D The value is about 10 -5 to 10 -12 mol / L or less, such as about 10 -6 to 10 -10 Mole / L, such as about 10 -7 to 10 -10 mol / L, or about 10 -7 to 10 -9 mol / L, or about 10 -6 to 10 -8 mol / L, or about 10 -6 to 10 -9 Moles / liter, for example, as determined by SPR.

[0143] For example, specific binding to human FcRn or human serum albumin does not exclude that the binding domain (or a polypeptide comprising the binding domain) can also specifically bind to FcRn or serum albumin from cynomolgus monkey.

[0144] The specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and their different variants known per se in the art; as well as other techniques mentioned herein. As will be clear to the skilled person, the dissociation constant may be an actual or apparent dissociation constant. Methods for determining the dissociation constant will be clear to the skilled person and, for example, include the techniques mentioned below. In this regard, it will also be clear that it may not be possible to measure dissociation constants greater than 10 -4 mol / L or 10 -3 mol / L (e.g., 10 -2 Optionally, as will also be clear to the skilled person, the (actual or apparent) dissociation constant may be based on the (actual or apparent) association constant (K A ) Using the relationship [KD = 1 / K A] to calculate. The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technology (see, for example, Ober et al., 2001, Intern. Immunology 13:301551-1559). As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in protein concentration in a biosensor matrix, in which one molecule is immobilized on a biosensor chip and another molecule passes over the immobilized molecule under flow conditions, thereby generating a k on 、k off The measured value, and thus the K D (or K A ) value. For example, this can be done using the well-known The system was performed using a BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, New Jersey. For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51: 19-26), Jonsson et al. (1991 Biotechniques 11: 620-627), Jonsson et al. (1995, J. Mol. Recognit. 8: 125-131) and Johannson et al. (1991, Anal. Biochem. 198: 268-277). Another well-known biosensor technology for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., Abdiche et al., 2008, Anal. Biochem. 377: 209-217).

[0145] As used herein, the term "biolayer interferometry" or "BLI" refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam). Changes in the number of molecules bound to the biosensor tip result in a shift in the interference pattern, reported as a wavelength shift (nm), which is a direct measure of the number of molecules bound to the biosensor tip surface. Because the interactions can be measured in real time, association and dissociation rates as well as affinities can be determined. For example, BLI can be performed using the well-known System (ForteBio, a division of Pall Life Sciences, Menlo Park, USA). Alternatively, the Kinetic Exclusion Assay (KinExA) can be used (see, for example, Drake et al., 2004, Anal. Biochem., 328: 35-43). Affinity was measured using a PCR platform (Sapidyne Instruments Inc, Boise, USA).

[0146] As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. The equilibrium solution of the antibody / antigen complex is passed through a column with beads pre-coated with the antigen (or antibody), thereby allowing free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is accomplished with a fluorescently labeled protein that binds to the antibody (or antigen). Immunoassay systems provide a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74).

[0147] In certain embodiments, the polypeptides of the present technology (such as FcRn targeting polypeptides) comprise at least one domain that specifically binds to a serum albumin protein with an affinity (K A ) in 10 6 M -1 with 10 11 M -1 between.

[0148] In certain embodiments, the polypeptide (such as an FcRn targeting polypeptide) comprises at least one domain that specifically binds to the serum albumin protein with a dissociation constant (K D ) in 10 -6 M and 10 -11 M or less. Preferably, the K D is measured by Kinexa, BLI or SPR, eg, as determined by SPR.

[0149] In certain embodiments, a polypeptide (such as an FcRn targeting polypeptide) comprises at least one domain that specifically binds to a serum albumin protein with an association rate constant (k on ) is selected from at least about 10 2 M -1 s -1 , at least about 10 3 M -1 s -1 , at least about 10 4M -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 as measured by surface plasmon resonance or BLI.

[0150] In certain embodiments, a polypeptide (such as an FcRn targeting polypeptide) comprises at least one domain that specifically binds to a serum albumin protein with a dissociation rate constant (k off ) is selected from up to about 10 -1 s -1 , up to about 10 -2 s -1 , up to about 10 -3 s -1 , up to about 10 -4 s -1 , up to about 10 -5 s -1 and at most about 10 -6 s -1 , preferably as measured by surface plasmon resonance or BLI.

[0151] In certain embodiments, the polypeptides of the present technology comprising at least one serum albumin binding domain are such that they are cross-reactive between human serum albumin and serum albumins from at least one, preferably at least two, more preferably at least three, and up to substantially all of the following mammalian species: mouse, dog, rat, rabbit, guinea pig, pig, sheep, cow, and cynomolgus monkey.

[0152] When an ISVD is said to exhibit "(improved) cross-reactivity for binding to human serum albumin and non-human primate serum albumin" compared to another ISVD, it is meant that for said ISVD, the binding activity (such as expressed as K) to human serum albumin and to non-human primate serum albumin is D or k off ) is lower than the same ratio calculated for other ISVDs in the same assay. The good cross-reactivity for binding to human serum albumin and non-human primate serum albumin allows the toxicity of serum albumin binding polypeptides according to the present technology to be evaluated in preclinical studies in non-human primates.

[0153] For the purposes of comparing two or more immunoglobulin single variable domains or other amino acid sequences (e.g., such as the polypeptides of the present technology, etc.), the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence (also referred to herein as "amino acid identity") can be calculated or determined as described in paragraph f) of 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 amino acid residues at the corresponding positions 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 as a difference at a single amino acid residue (position), i.e., as an "amino acid difference" as defined herein; alternatively, the degree of sequence identity between two amino acid sequences can be calculated using a known computer algorithm for sequence alignment using standard settings (e.g., NCBI Blast v2.0). Some other techniques, computer algorithms and setups for determining the degree of sequence identity are described in, for example, WO 04 / 037999, EP 0967284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2 357 768-A.

[0154] 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 greatest number of amino acid residues is used as the "first" amino acid sequence, and the other amino acid sequence is used as the "second" amino acid sequence.

[0155] In addition, when determining the degree of sequence identity between two immunoglobulin single variable domains, those skilled in the art may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue of similar chemical structure and has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example from WO 04 / 037999, GB-A-3 357 768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300; and (preferred) types and / or combinations of such substitutions can be selected based on the relevant teachings of WO 04 / 037999 and WO 98 / 49185 and the additional references cited therein. Examples of conservative substitutions are further described below.

[0156] Any amino acid substitutions applied to the polypeptides described herein may also be based on an analysis of the frequency of amino acid variations between homologous proteins from different species, as developed by Schulz et al., 1978 (Principles of Protein Structure, Springer-Verlag), an analysis of structure-forming potential, as developed by Chou and Fasman 1975 (Biochemistry 13:211) and 1978 (Adv. Enzymol. 47:45-149), and an analysis of hydrophobicity patterns in proteins, as developed by 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. 15:321-353), all of which are incorporated herein by reference in their entirety. Information on the primary, secondary, and tertiary structures of ISVDs is provided in the specification and the general background art cited above. Furthermore, for this purpose, the crystal structures of VHH domains from llamas are provided, for example, by Desmyter et al. 1996 (Nature Structural Biology, 3:803), Spinelli et al. 1996 (Natural Structural Biology, 3:752-757), and Decannire et al. 1999 (Structure, 7:361). Further information on some of the amino acid residues that form the VH / VL interface in conventional VH domains and potential camelization substitutions at these positions can be found in the prior art cited above. Immunoglobulin single variable domains and nucleic acid sequences are said to be "identical" if they share 100% sequence identity (as defined herein) over their entire length.

[0157] 5.3 First Domain: (i) A domain comprising a serum albumin protein and / or a domain that has high affinity / specific binding to a serum albumin protein (such as a serum albumin binding ISVD)

[0158] Human serum albumin (HSA) and IgG are two of the most abundant soluble proteins present in the blood circulation and are exceptions to most proteins in the circulation in that they share the remarkable property of having an extended serum half-life of approximately 19 to 21 days in humans.

[0159] HSA is the most abundant plasma protein in the blood and is a carrier protein involved in many processes for maintaining homeostasis in the body, i.e., maintaining tumescent pressure (oncotic pressure). Albumin is widely used as a drug delivery vehicle due to its high serum concentration, long half-life, non-toxicity and low immunogenicity, as well as its uptake in benign and tissue and its ability to combine with various 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). HSA has been well characterized as a polypeptide of 585 amino acids, the sequence of which can be found in, for example, Peters, T., Jr. (1996) " All about Albumin: Biochemistry, Genetics and Medical Applications ", page 10, Academic Press, Inc., Orlando (ISBN 0-12-552110-3).

[0160] The half-life of this extension of serum albumin is mainly due to its being protected from intracellular lysosomal degradation by being combined with neonatal Fc receptor (FcRn). FcRn is a heterodimer composed of N-glycosylated transmembrane MHC class I heavy chains that are non-covalently bound to soluble b2-microglobulin. Both IgG and albumin are ligands that bind to different epitopes of FcRn. Generally, the FcRn recycling mechanism is strictly dependent on pH, and is conducive to being combined with FcRn at low pH (e.g., acidic endosomal pH, which is typically lower than 6.5) after the acidification of the endosomal compartment. When albumin / IgG is combined with FcRn, it escapes degradation in the lysosome. Upon returning to the cell surface, at extracellular physiological pH (typically around pH 7.4), binding is weakened, leading to the release of albumin / IgG into the bloodstream (see, e.g., 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). Thus, HSA has the characteristic of binding to its receptor FcRn, in which it binds at pH 6.0 but not at pH 7.4.

[0161] A natural variant with a lower plasma half-life has been identified (Peach, RJ and Brennan, SO (1991) Biochim Biophys Acta. 1097: 49-54) with the substitution D494N. This substitution creates an N-glycosylation site in this variant that is not present in wild-type albumin. Whether glycosylation or the amino acid change is responsible for the change in plasma half-life is unknown.

[0162] Otagiri et al. (2009), Biol. Pharm, Bull. 32 (4), 527-534 disclose 77 known albumin variants. Of these, 25 are found in domain III. Natural variants lacking the last 175 amino acids at the carboxyl terminus have been shown to have reduced half-lives (Andersen et al. (2010), Clinical Biochemistry 43, 367-372). Iwao et al. (2007) studied the half-lives of naturally occurring human albumin variants using a mouse model and found that K541E and K560E had reduced half-lives, E501K and E570K had increased half-lives, and K573E had little effect on half-life (Iwao et al. (2007) BBA Proteins and Proteomics 1774, 1582-1590).

[0163] The natural variant E505K is disclosed by Galliano et al. (1993) Biochim. Biophys. Acta 1225, 27-32. The natural variant K536E is disclosed by Minchiotti et al. (1990). The natural variant K574N is disclosed by Minchiotti et al. (1987) Biochim. Biophys. Acta 916, 411-418. The natural variant D550G is disclosed by Takahashi et al. (1987) Proc. Natl. Acad. Sci. USA 84, 4413-4417. The natural variant D550A is disclosed by Carlson et al. (1992) Proc. Natl. Acad. Sci. USA 89, 8225-8229.

[0164] In particular, albumin is increasingly being used to improve the pharmacokinetics of short-lived small molecule drugs that can be bound to albumin, as well as bioactive therapeutic peptides and proteins, by genetically fusing such molecules to 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).

[0165] For example, immunoglobulin variable domain sequences (ISVDs) that can bind to serum albumin have been developed, and their coupling to therapeutic compounds, moieties, and entities to extend serum half-life (as defined in these applications) is described in, for example, WO 2004 / 041865, WO 2006 / 122787, WO 2012 / 175400, WO 2015 / 173325, and PCT / EP2016 / 077973. For example, WO 2006 / 122787 discloses a humanized serum albumin-binding ISVD designated Alb-8 as SEQ ID NO: 62 (see SEQ ID NO: 5 herein). WO 2012 / 175400 discloses a humanized serum albumin-binding ISVD designated Alb-23D as SEQ ID NO: 6. Some other references disclosing ISVDs against serum albumin include WO 2003 / 035694, WO 2004 / 003019, EP 2 139 918, WO 2011 / 006915 and WO 2014 / 111550. Preferred examples of albumin binding ISVDs comprise or consist of a polypeptide as defined in SEQ ID NOs: 7-21 or 64-69.

[0166] Other albumin binding proteins (ABPs) such as albumin binding DARPins (designed ankyrin repeat proteins) or Affitins (also known as Nanofitins) 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 serum albumin-binding domains”, Protein Eng Des Sel, 2017, 30(9): 583-591). Preferred examples of the albumin binding portion of the non-ISVD comprise or consist of a polypeptide as defined in SEQ ID NO: 102-104.

[0167] In one embodiment, the polypeptide of the present invention comprises at least one domain comprising a serum albumin protein. In a specific embodiment, the serum albumin protein is human serum albumin (such as "human serum albumin (1)" as defined in SEQ ID NO: 22, "human serum albumin (2) (HSA (25-609))" as defined in SEQ ID NO: 23, or HAS (25-609) (E529Q, T551M, K597P) (HSA (QMP)) as defined in SEQ ID NO: 110, or a polymorphic variant or isoform thereof. Preferably, the serum albumin protein contained in the polypeptide of the present invention is human serum albumin, and more preferably is or comprises a protein sequence as defined in SEQ ID NO: 23 or 110.

[0168] Thus, in certain embodiments, the polypeptides as disclosed herein comprise at least one domain comprising a serum albumin protein and an IgG Fc domain or a fragment thereof. In certain embodiments, the serum albumin protein is human serum albumin (e.g., AAA98797 as defined in SEQ ID NO: 22 or P02768-1 as defined in SEQ ID NO: 23, or HSA-QMP as defined in SEQ ID NO: 110, preferably as defined in SEQ ID NO: 23 or 110) or a polymorphic variant or isoform thereof.

[0169] In certain embodiments, the polypeptides of the present technology comprise at least one serum albumin protein or a fragment or variant thereof, such as, but not limited to, the albumins, fragments and variants disclosed in WO 2011 / 124718, WO 2011 / 051489, WO 2013 / 075066, WO 2013 / 135896 and WO 2014 / 072481.

[0170] According to certain embodiments of the present technology, polypeptides comprising at least one serum albumin protein and at least one Fc domain are generated and tested for their beneficial PK properties.

[0171] In the context of the present technology, the term "serum albumin protein" refers to serum albumin (such as human serum albumin or its derivatives, variants or fragments). Preferably, the serum albumin protein comprises or consists of a polypeptide as defined in SEQ ID NO: 22, 23 or 110.

[0172] The size of the albumin derivative, variant or fragment may vary depending on the size of the fragment, the number of domains, the size of the non-albumin portion of the polypeptide, etc. However, it is preferred that the size of the albumin derivative, variant or fragment is in the range of 40-80 kDa, preferably in the range of 50-70 kDa, more preferably in the range of 55-65 kDa, and most preferably about 60 kDa.

[0173] Human serum albumin is a preferred serum albumin protein according to the present technology and is a protein consisting of about 585 amino acid residues and has a molecular weight of about 67 kDa (e.g., SEQ ID NO: 22 or SEQ ID NO: 23, or SEQ ID NO: 110, preferably SEQ ID NO: 23 or 110, even more preferably SEQ ID NO: 23). The skilled artisan will understand that there may be natural alleles that have substantially the same properties as human serum albumin but have one or more (several) amino acid changes compared to, for example, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 110, and the inventors also contemplate the use of such natural alleles as serum albumin proteins according to the present technology.

[0174] According to the present technology, the term "(serum) albumin derivative" means a non-natural engineered molecule comprising or consisting of one or more portions of one or more domains of a specified serum albumin protein. Serum albumin derivatives can be engineered to increase or decrease FcRn binding. For example, a serum albumin derivative can be HSA (25-609) (E529Q, T551M, K597P) (SEQ ID NO: 110, for increasing FcRn binding). In preferred embodiments, the polypeptides of the present technology comprise at least one domain comprising or consisting of a serum albumin derivative, such as a serum albumin derivative that exhibits increased or decreased FcRn binding compared to a wild-type serum albumin derivative, preferably HSA (25-609) (E529Q, T551M, K597P) as defined in SEQ ID NO: 110.

[0175] 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 modification of the polypeptide, for example, PEGylation and / or conjugation of a desired moiety (such as a therapeutic moiety) to a thiol group, such as provided by an unpaired cysteine. The terms "derivative" and "variant" may or may not be used interchangeably.

[0176] The term "(serum) albumin fragment" refers to a polypeptide having one or more (several) amino acids deleted from the amino and / or carboxyl termini and / or internal regions of a serum albumin protein, which retains the ability to bind to FcRn. A fragment may comprise or consist of one uninterrupted sequence derived from human serum albumin, or it may comprise or consist of two or more sequences derived from human serum albumin.

[0177] In certain embodiments, the at least one additional moiety comprising a serum albumin protein is a portion, fragment, derivative or variant of a serum albumin protein.

[0178] In certain embodiments, the at least one serum albumin protein-containing domain comprises or consists of human serum albumin (HSA) or a portion, fragment, derivative or variant of human serum albumin.

[0179] The sequence of HSA unique ID P02768 (SEQ ID NO: 109) is depicted below:

[0180] MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFH DNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCE KPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHHDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0181] Thus, in one embodiment, the HSA protein comprised in the polypeptides of the present technology comprises or consists of amino acids 25 to 609 of the protein sequence of HSA unique ID P02768 (SEQ ID NO: 23). In another embodiment, the HSA protein comprised in the polypeptides of the present technology comprises or consists of amino acids 25 to 609 of the protein sequence of HSA unique ID P02768 with the following mutations: E529Q, T551M, and K597P (for increased FcRn binding), see SEQ ID NO: 110.

[0182] In other specific embodiments, the polypeptides as disclosed herein comprise at least one domain that specifically binds to a serum albumin protein, such as human serum albumin (AAA98797 as defined in SEQ ID NO: 22, P02768-1 as defined in SEQ ID NO: 23, or HSA(25-609)(E529Q, T551M, K597P) as defined in SEQ ID NO: 110), or a (polymorphic) variant or isoform thereof.

[0183] In yet other specific embodiments, the polypeptides as disclosed herein comprise at least one domain comprising a serum albumin protein and at least one domain that specifically binds to a serum albumin protein, such as human serum albumin (AAA98797 as defined in SEQ ID NO: 22, P02768-1 as defined in SEQ ID NO: 23, or HSA(25-609)(E529Q, T551M, K597P) as defined in SEQ ID NO: 110), or a (polymorphic) variant or isoform thereof.

[0184] In other embodiments, the polypeptides of the present technology comprise (i) at least one domain that specifically binds to a serum albumin protein and / or variants thereof, such as human serum albumin (e.g., "human serum albumin (1)" as defined in SEQ ID NO: 22 or "human serum albumin (2) (HSA (25-609))" as defined in SEQ ID NO: 23, or HSA (25-609) (E529Q, T551M, K597P) as defined in SEQ ID NO: 110), or a (polymorphic) variant or isoform thereof.

[0185] In another specific embodiment, (i) at least one domain specifically binding to a serum albumin protein contained in the polypeptide of the present invention specifically binds to an amino acid residue on the serum albumin protein that is not involved in the binding of the serum albumin protein to FcRn.

[0186] In another specific embodiment, (i) at least one domain that specifically binds to a serum albumin protein comprised in the polypeptide of the present technology specifically binds to domain II of human serum albumin.

[0187] In certain embodiments, the (i) at least one domain that specifically binds to a serum albumin protein comprised in the polypeptides of the present technology is a peptide or protein comprising between 5 and 500 amino acids.

[0188] In certain embodiments of the technology, at least one domain that specifically binds to a serum albumin protein is such that it binds (at least) a non-linear epitope comprising one or more amino acid residues within one or more of the following stretches of amino acid residues within the primary sequence of human serum albumin: positions 298-311 (and in particular one or more of Met298, Pro299, Ala300, Asp301, Leu302, Pro303, Ser304, Leu305, Ala306, and Glu311); positions 334 to 341 (and in particular one or more of Tyr334, Arg337, His338, Pro339, and / or Asp340) and / or positions 374-381 (and in particular one or more of Phe374, Asp375, Phe 377, Lys378, and Val381), wherein the amino acid residues in human serum albumin are according to Meloun et al., FEBS Letters, 1975, 58, pp. 134-137.

[0189] In yet another specific embodiment, the polypeptides of the present invention comprise (i) at least one domain that specifically binds to a serum albumin protein selected from (affibrioles), scFv, Fab, designed ankyrin repeat proteins Albumin binding domain (ABD), Examples of preferred albumin binding domains comprised in the polypeptides of the present technology comprise or consist of polypeptides as defined in SEQ ID NOs: 7-21, 61-69 and 102-104, preferably polypeptides as defined in SEQ ID NOs: 7-21 and 61-69. In a preferred embodiment, (i) at least one domain that specifically binds to a serum albumin protein comprised in the polypeptides of the present technology is an ISVD, more preferably a VHH, even more preferably comprises or consists of SEQ ID NOs: 7-21 and 61-69, even more preferably comprises or consists of ALB23002 (SEQ ID NO: 20), Alb23002-A (SEQ ID NO: 21), HSA006A06 (SEQ ID NO: 65), ALBX0000 (SEQ ID NO: 64), ALB11002 (SEQ ID NO: 13), and T023500029 (SEQ ID NO: 69), even more preferably comprises or consists of HSA006A06 (SEQ ID NO: 65), ALB11002 (SEQ ID NO: 13), and ALB23002 (SEQ ID NO: 20), even more preferably SEQ ID NO: 20 (Alb23002).

[0190] The albumin binding domain (ABD) is described in, for example, Hopp J. et al., “The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion protein”, Protein Eng Des Sel., 2010, 23(11): 827-34.

[0191] In certain embodiments, at least one serum albumin binding domain in the polypeptides of the present invention is cross-reactive between (at least) human serum albumin and cynomolgus monkey serum albumin, and preferably between human serum albumin and / or cynomolgus monkey serum albumin on the one hand and at least one, preferably two, of rat serum albumin and porcine serum albumin on the other hand. For convenience, stretches of amino acids that are hypothesized to be part of putative epitopes of the polypeptides of the present invention are highlighted in the sequence of serum albumin. Without being limited to any particular mechanism or hypothesis, it is hypothesized that the polypeptides of the present invention are (substantially) capable of binding to corresponding stretches of amino acid residues (one or more amino acid residues therein) present in the amino acid sequences of those mammalian serum albumin proteins with which the polypeptides of the present invention are cross-reactive.

[0192] Typically, a polypeptide of the present technology comprising at least one serum albumin binding moiety can be considered to have cross-reactivity between human serum albumin and serum albumin from one of the other species mentioned above when it binds to human serum albumin with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM and also binds to serum albumin from those species mentioned above with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, both again as determined using SPR.

[0193] In certain additional specific embodiments, at least one serum albumin binding domain specifically binds to an amino acid residue on human serum albumin that is not involved in the binding of human serum albumin to human FcRn.

[0194] Thus, (i) at least one domain that specifically binds to a serum albumin protein may preferably be an albumin-binding ISVD as described herein. In certain other specific embodiments, the present technology provides a polypeptide as described herein, characterized in that at least one ISVD that specifically binds to serum albumin is a (single) domain antibody, VHH, VHH, humanized VHH, or camelized VH. Therefore, according to a particularly preferred embodiment of the present technology, the at least one albumin-specific binding domain comprised in the polypeptide of the present technology is at least one ISVD that specifically binds to (human) serum albumin.

[0195] The term "immunoglobulin single variable domain" (ISVD) has been described in this specification. When the at least one domain specifically binding to a serum albumin protein contained in the polypeptide of the present technology is an albumin-binding ISVD, it preferably comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively).

[0196] In certain additional specific embodiments, at least one serum albumin binding domain is an ISVD that binds serum albumin and consists essentially of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein CDR1 is SFGMS (SEQ ID NO: 1), CDR2 is SISGSGSDTLYADSVKG (SEQ ID NO: 2), and CDR3 is GGSLSR (SEQ ID NO: 3), with the CDRs being determined according to the Kabat definition; and / or wherein CDR1 is GFTFRSFGMS (SEQ ID NO: 4), CDR2 is SISGSGSDTL (SEQ ID NO: 5), and CDR3 is GGSLSR (SEQ ID NO: 6), with the CDRs being determined according to the AbM definition (Kontermann et al. 2010). Preferred CDRs and FR regions of the albumin binding ISVDs comprised in the polypeptides of the present technology are provided in Table A-6. Thus, in one embodiment, the polypeptides of the present technology comprise an albumin binding (alb binding) ISVD comprising the CDR and FR regions as described in Table A-6.

[0197] Therefore, the CDR regions are preferably as follows (according to AbM numbering):

[0198] a) comprising the amino acid sequence of SEQ ID NO: 4 or a CDR1 having 3, 2 or 1 amino acid differences with SEQ ID NO: 4, or comprising the amino acid sequence of SEQ ID NO: 77 or a CDR1 having 3, 2 or 1 amino acid differences with SEQ ID NO:

[0199] b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or having 3, 2 or 1 amino acid differences from SEQ ID NO: 5; and

[0200] c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or 6, or having 3, 2 or 1 amino acid differences with SEQ ID NO: 3 or 6,

[0201] and / or

[0202] a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 80 or having 3, 2 or 1 amino acid differences from SEQ ID NO: 80;

[0203] b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 81 or having 3, 2 or 1 amino acid differences from SEQ ID NO: 81; and

[0204] c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 82 or having 3, 2 or 1 amino acid differences from SEQ ID NO: 82;

[0205] and / or

[0206] a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 90 or having 3, 2 or 1 amino acid differences from SEQ ID NO: 90;

[0207] b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 91 or having 3, 2 or 1 amino acid differences from SEQ ID NO: 91; and

[0208] c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 92 or a CDR3 having 3, 2 or 1 amino acid differences with SEQ ID NO: 92.

[0209] The above CDR sequences were determined according to the AbM numbering.

[0210] In a preferred embodiment, the at least one albumin-binding ISVD (i) comprised in the polypeptides of the present technology comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the CDR sequences are determined according to AbM numbering.

[0211] In other embodiments, at least one albumin binding ISVD (i) comprised in the polypeptides of the present technology comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 77; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the CDR sequences are determined according to AbM numbering.

[0212] In other embodiments, at least one albumin binding ISVD (i) comprised in the polypeptides of the present technology comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 80; a CDR2 comprising the amino acid sequence of SEQ ID NO: 81; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 82, wherein the CDR sequences are determined according to AbM numbering.

[0213] In other embodiments, at least one albumin binding ISVD (i) comprised in the polypeptides of the present technology comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 90; a CDR2 comprising the amino acid sequence of SEQ ID NO: 91; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 92, wherein the CDR sequences are determined according to AbM numbering.

[0214] If the CRD sequence is determined according to Kabat numbering, then at least one albumin binding ISVD (i) comprised in the polypeptides of the present technology will preferably comprise the following CDR regions:

[0215] a) comprises the amino acid sequence of SEQ ID NO: 1 or a CDR1 which has 3, 2 or 1 amino acid differences with SEQ ID NO: 1,

[0216] b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or having 3, 2 or 1 amino acid differences from SEQ ID NO: 2; and

[0217] c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or having 3, 2 or 1 amino acid differences with SEQ ID NO: 3,

[0218] and / or

[0219] a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 78 or having 3, 2 or 1 amino acid differences from SEQ ID NO: 78;

[0220] b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 79 or having 3, 2 or 1 amino acid differences from SEQ ID NO: 79; and

[0221] c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 82 or having 3, 2 or 1 amino acid differences from SEQ ID NO: 82;

[0222] and / or

[0223] a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 93 or having 3, 2 or 1 amino acid differences from SEQ ID NO: 93;

[0224] b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 94 or having 3, 2 or 1 amino acid differences from SEQ ID NO: 94; and

[0225] c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 92 or a CDR3 having 3, 2 or 1 amino acid differences with SEQ ID NO: 92.

[0226] In some embodiments, at least one albumin-binding ISVD (i) comprised in the polypeptides of the present technology comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; 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 the CDR sequences are determined according to Kabat numbering.

[0227] In other embodiments, the at least one albumin binding ISVD (i) comprised in the polypeptides of the present technology comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 78; a CDR2 comprising the amino acid sequence of SEQ ID NO: 79; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 82, wherein the CDR sequences are determined according to Kabat numbering.

[0228] In other embodiments, the at least one albumin binding ISVD (i) comprised in the polypeptides of the present technology comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 93; a CDR2 comprising the amino acid sequence of SEQ ID NO: 94; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 92, wherein the CDR sequences are determined according to Kabat numbering.

[0229] A specific example of an ISVD that specifically binds to HSA is an ISVD comprising four framework regions (FR1 to FR4, respectively) and three complementarity determining regions, wherein the at least one ISVD that specifically binds to the serum albumin protein has:

[0230] a) a degree of sequence identity with any of the sequences as defined in SEQ ID NOs: 7 to 21 or 61 to 69 (wherein any C-terminal extension that may be present and the CDRs are not taken into account for determining the degree of sequence identity) of at least 85%, preferably at least 90%, more preferably at least 95%; and / or

[0231] b) differs from any of the sequences as defined in SEQ ID NOs: 7 to 21 or 61 to 69 in no more than 7, preferably no more than 5 (such as only 3, 2 or 1) amino acids.

[0232] In certain additional specific embodiments, the polypeptides according to the present technology comprise at least one domain that specifically binds to a serum albumin protein, said domain being at least one ISVD that specifically binds to human serum albumin and essentially consisting of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0233] a. CDR1 comprises the amino acid sequence of SEQ ID NO: 1 according to Kabat numbering or has 3, 2 or 1 amino acid differences from SEQ ID NO: 1;

[0234] b. CDR2 comprises the amino acid sequence of SEQ ID NO: 2 according to Kabat numbering or has 3, 2 or 1 amino acid differences from SEQ ID NO: 2; and

[0235] c. CDR3 comprises the amino acid sequence of SEQ ID NO: 3 according to Kabat numbering or has 3, 2 or 1 amino acid differences from SEQ ID NO: 3.

[0236] In certain additional specific embodiments, the polypeptides according to the present technology contain at least one domain that specifically binds to a serum albumin protein, said domain being at least one ISVD that specifically binds to human serum albumin and essentially consisting of four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein:

[0237] a. CDR1 comprises the amino acid sequence of SEQ ID NO: 4 according to AbM numbering or has 3, 2 or 1 amino acid differences from SEQ ID NO: 4;

[0238] b. CDR2 comprises the amino acid sequence of SEQ ID NO: 5 according to AbM numbering or has 3, 2 or 1 amino acid differences from SEQ ID NO: 5; and

[0239] c. CDR3 comprises the amino acid sequence of SEQ ID NO: 6 according to AbM numbering or has 3, 2 or 1 amino acid differences from SEQ ID NO: 6.

[0240] In certain additional specific embodiments, the at least one ISVD that specifically binds to (human) serum albumin comprised in the polypeptides of the present technology has:

[0241] a. the degree of sequence identity to SEQ ID NO: 20 (wherein the CDRs and any C-terminal extensions that may be present are not taken into account when determining the degree of sequence identity) is at least 85%, preferably at least 90%, more preferably at least 95%; and / or

[0242] b. has no more than 7, preferably no more than 5 (such as only 3, 2 or 1) "amino acid differences" (as defined herein and not taking into account CDRs and any C-terminal extension that may be present) from the sequence of SEQ ID NO: 20.

[0243] Compared to the sequence of SEQ ID NO: 20, at least one serum albumin binding portion in the polypeptides of the present technology preferably further contains (at least): one or more humanizing substitutions;

[0244] and / or

[0245] one or more mutations (i.e., amino acid substitutions, deletions or additions, particularly substitutions) that reduce binding of a pre-existing antibody; and may optionally contain one or more additional mutations as described herein (e.g., to improve the chemical stability of the FcRn binding polypeptide).

[0246] In certain additional specific embodiments, the at least one ISVD that specifically binds to (human) serum albumin comprised in the polypeptides of the present technology has:

[0247] a. the degree of sequence identity to SEQ ID NO: 21 (wherein the CDRs and any C-terminal extensions that may be present are not taken into account when determining the degree of sequence identity) is at least 85%, preferably at least 90%, more preferably at least 95%; and / or

[0248] b. has no more than 7, preferably no more than 5 (such as only 3, 2 or 1) "amino acid differences" (as defined herein and not taking into account CDRs and any C-terminal extension that may be present) from the sequence of SEQ ID NO: 21.

[0249] Compared to the sequence of SEQ ID NO: 21, at least one serum albumin binding portion in the polypeptides of the present technology preferably further contains (at least): one or more humanizing substitutions;

[0250] and / or

[0251] one or more mutations (i.e., amino acid substitutions, deletions or additions, particularly substitutions) that reduce binding of a pre-existing antibody; and may optionally contain one or more additional mutations as described herein (e.g., to improve the chemical stability of the FcRn binding polypeptide).

[0252] In certain additional specific embodiments, the at least one ISVD that specifically binds to (human) serum albumin comprised in the polypeptides of the present technology has:

[0253] a. the degree of sequence identity to SEQ ID NO: 65 (wherein the CDRs and any C-terminal extensions that may be present are not taken into account when determining the degree of sequence identity) is at least 85%, preferably at least 90%, more preferably at least 95%; and / or

[0254] b. has no more than 7, preferably no more than 5 (such as only 3, 2 or 1) "amino acid differences" (as defined herein and not taking into account CDRs and any C-terminal extension that may be present) from the sequence of SEQ ID NO: 65.

[0255] Compared to the sequence of SEQ ID NO: 65, at least one serum albumin binding portion in the polypeptides of the present technology preferably further contains (at least): one or more humanizing substitutions;

[0256] and / or

[0257] one or more mutations (i.e., amino acid substitutions, deletions or additions, particularly substitutions) that reduce binding of a pre-existing antibody; and may optionally contain one or more additional mutations as described herein (e.g., to improve the chemical stability of the FcRn binding polypeptide).

[0258] In certain additional specific embodiments, the at least one ISVD that specifically binds to (human) serum albumin comprised in the polypeptides of the present technology has:

[0259] a. the degree of sequence identity to SEQ ID NO: 64 (wherein the CDRs and any C-terminal extensions that may be present are not taken into account when determining the degree of sequence identity) is at least 85%, preferably at least 90%, more preferably at least 95%; and / or

[0260] b. has no more than 7, preferably no more than 5 (such as only 3, 2 or 1) "amino acid differences" (as defined herein and not taking into account CDRs and any C-terminal extension that may be present) from the sequence of SEQ ID NO: 64.

[0261] Compared to the sequence of SEQ ID NO: 64, at least one serum albumin binding portion in the polypeptides of the present technology preferably further contains (at least): one or more humanizing substitutions;

[0262] and / or

[0263] one or more mutations (i.e., amino acid substitutions, deletions or additions, particularly substitutions) that reduce binding of a pre-existing antibody; and may optionally contain one or more additional mutations as described herein (e.g., to improve the chemical stability of the FcRn binding polypeptide).

[0264] In certain additional specific embodiments, the at least one ISVD that specifically binds to (human) serum albumin comprised in the polypeptides of the present technology has:

[0265] a. the degree of sequence identity to SEQ ID NO: 13 (wherein the CDRs and any C-terminal extensions that may be present are not taken into account when determining the degree of sequence identity) is at least 85%, preferably at least 90%, more preferably at least 95%; and / or

[0266] b. has no more than 7, preferably no more than 5 (such as only 3, 2 or 1) "amino acid differences" (as defined herein and not taking into account CDRs and any C-terminal extension that may be present) from the sequence of SEQ ID NO: 13.

[0267] Compared to the sequence of SEQ ID NO: 13, at least one serum albumin binding portion in the polypeptides of the present technology preferably further contains (at least): one or more humanizing substitutions;

[0268] and / or

[0269] one or more mutations (i.e., amino acid substitutions, deletions or additions, particularly substitutions) that reduce binding of a pre-existing antibody; and may optionally contain one or more additional mutations as described herein (e.g., to improve the chemical stability of the FcRn binding polypeptide).

[0270] In certain additional specific embodiments, the at least one ISVD that specifically binds to (human) serum albumin comprised in the polypeptides of the present technology has:

[0271] a. a degree of sequence identity with SEQ ID NO: 69 (wherein any CDRs and any C-terminal extensions that may be present are not taken into account when determining the degree of sequence identity) of at least 85%, preferably at least 90%, more preferably at least 95%; and / or

[0272] b. has no more than 7, preferably no more than 5 (such as only 3, 2 or 1) "amino acid differences" (as defined herein and not taking into account CDRs and any C-terminal extension that may be present) from the sequence of SEQ ID NO: 69.

[0273] Compared to the sequence of SEQ ID NO: 69, at least one serum albumin binding portion in the polypeptides of the present technology preferably further contains (at least): one or more humanizing substitutions;

[0274] and / or

[0275] one or more mutations (i.e., amino acid substitutions, deletions or additions, particularly substitutions) that reduce binding of a pre-existing antibody; and may optionally contain one or more additional mutations as described herein (e.g., to improve the chemical stability of the FcRn binding polypeptide).

[0276] For suitable humanizing substitutions (and suitable combinations thereof), reference is made, for example, to WO 09 / 138519 (or the prior art cited therein) and WO 08 / 020079 (or the prior art cited therein), and to Tables A-3 to A-8 of WO 08 / 020079 (which are lists showing possible humanizing substitutions). Some preferred, but non-limiting, examples of such humanizing substitutions are Q108L and A14P, or suitable combinations thereof. Such humanizing 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).

[0277] For suitable mutations (and suitable combinations of such mutations) that may reduce the binding of pre-existing antibodies, reference is made, for example, to WO 2012 / 175741 and WO 2015 / 173325, and also to, for example, WO 2013 / 024059 and WO 2016 / 118733.

[0278] Amino acid sequence modifications of the polypeptides or ISVDs described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the polypeptide or ISVD. Amino acid sequence variants of the polypeptides or ISVDs described herein can be prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the polypeptide or ISVD or by peptide synthesis.

[0279] Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within polypeptides as described herein or ISVDs. Any combination of deletion, insertion, and substitution may be performed to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid changes may also change the post-translational processes of the binding molecule, such as by changing the quantity or position of glycosylation sites. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted in the CDR, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids may be substituted in the framework region (FR). Substitution is preferably a conservative substitution as described herein. Additionally or alternatively, 1, 2, 3, 4, 5 or 6 amino acids may be inserted or deleted in each CDR (depending, of course, on their length), while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids may be inserted or deleted in each FR.

[0280] International application WO 2006 / 122787 (the contents of which are incorporated herein by reference) describes a variety of ISVDs directed against (human) serum albumin. These ISVDs include an ISVD called Alb-1 (SEQ ID NO: 52 in WO 2006 / 122787) and humanized variants thereof, such as Alb-8 (SEQ ID NO: 62 in WO 2006 / 122787). Again, these can be used to extend the half-life of biologics according to the present technology.

[0281] WO 2012 / 175400 (the contents of which are incorporated herein by reference) describes a further improved form of Alb-1, designated Alb-23.

[0282] In one embodiment, the polypeptides of the present technology comprise a serum albumin binding ISVD selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 (described in WO 2006 / 122787), and Alb-23. In one embodiment, the serum albumin binding moiety is Alb-8 or Alb-23, or a variant thereof, as set forth on pages 7-9 of WO 2012 / 175400. In one embodiment, the serum albumin binding ISVD is selected from the albumin binders described in WO 2012 / 175741, WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235, and WO 2018 / 134234, the contents of which are incorporated herein by reference. Table A-3 also shows some preferred serum albumin binders. Polypeptides comprising at least one of these albumin binding ISVDs were generated and tested for their beneficial PK properties, as described in the Examples below.

[0283] Table A-3: Serum albumin binding ISVD sequences ("ID" refers to SEQ ID NO as used herein)

[0284]

[0285]

[0286]

[0287] In some preferred embodiments, at least one ISVD that specifically binds to a serum albumin protein comprised in the polypeptides of the present technology has a sequence selected from SEQ ID NOs: 7 to 21 or 61 to 69 (Table A-3).

[0288] In certain embodiments of the present technology, the polypeptides of the present technology comprise at least one serum albumin binding ISVD having the full-length amino acid sequence of ALB23002 (SEQ ID NO: 20, see Table A-3).

[0289] In certain embodiments of the present technology, the polypeptides of the present technology comprise at least one serum albumin binding ISVD having the full-length amino acid sequence of Alb223 (SEQ ID NO: 21, see Table A-3).

[0290] In some embodiments, the amino acid sequence of at least one ISVD that specifically binds to a serum albumin protein contained in the polypeptides of the present technology has greater than 90%, such as greater than 95% or greater than 99%, sequence identity to Alb-23002 (SEQ ID NO: 20), to Alb223 (SEQ ID NO: 21), to Alb23002 (E1D) (SEQ ID NO: 61), to ALBX00001 (SEQ ID NO: 63), to ALBX00002 (SEQ ID NO: 64), to Alb82 (SEQ ID NO: 13), to T023500029 (SEQ ID NO: 62), to HSA006A06 (SEQ ID NO: 65), or to HSA006A06-A (SEQ ID NO: 66).

[0291] In some embodiments, the amino acid sequence of at least one ISVD that specifically binds to a serum albumin protein contained in the polypeptides of the present technology has greater than 90% (such as greater than 95% or greater than 99%) sequence identity to ALB23002 (SEQ ID NO:20), Alb23002-A (SEQ ID NO:21), HSA006A06 (SEQ ID NO:65), ALBX00002 (SEQ ID NO:64), ALB11002 (SEQ ID NO:13), and T023500029 (SEQ ID NO:69).

[0292] Also in a preferred embodiment, the amino acid sequence of the human serum albumin binding ISVD may have greater than 90%, such as greater than 95% or greater than 99% sequence identity with SEQ ID NO: 20, wherein optionally the CDRs are defined as SEQ ID NOs: 1 to 3 (according to Kabat) or SEQ ID NOs: 4 to 6 (according to AbM) as defined above. In particular, the human serum albumin binding ISVD preferably has the amino acid sequence of SEQ ID NO: 20.

[0293] When such a human serum albumin binding ISVD has 2 or 1 amino acid difference in at least one CDR relative to the corresponding reference CDR sequence (as defined above as SEQ ID NOs: 1 to 3 (according to Kabat) or SEQ ID NOs: 4 to 6 (according to AbM)), the ISVD preferably has at least half the binding affinity for human serum albumin compared to construct ALB23002, preferably at least the same binding affinity, wherein the binding affinity is measured using the same method (such as SPR).

[0294] When such a human serum albumin-binding ISVD has a C-terminal position, it may exhibit a C-terminal alanine (A) or glycine (G) stretch and is preferably selected from SEQ ID NOs: 9, 10, 12, 14, 15, 16, 21 (see Table A-3). In a preferred embodiment, the human serum albumin-binding ISVD has another position different from the C-terminal position (i.e., it is not the C-terminal ISVD of the polypeptides of the present technology) and is selected from SEQ ID NOs: 17 to 19 (see Table A-3). Thus, in one embodiment, the human serum albumin-binding ISVD is located at the N-terminus of the polypeptide. In other embodiments, the human serum albumin-binding ISVD is located at the C-terminus of the polypeptide.

[0295] In certain embodiments, a polypeptide comprising an ISVD as described herein having one or more CDRs having 1, 2, 3, or 4 amino acid differences binds serum albumin with about the same affinity as measured by surface plasmon resonance compared to the binding of an amino acid sequence or polypeptide comprising a CDR without the 4, 3, 2, or 1 amino acid differences.

[0296] When comparing two immunoglobulin single variable 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; it is understood that two immunoglobulin single variable domains may contain one, two or more such amino acid differences.

[0297] In a preferred embodiment, at least one ISVD that specifically binds to a serum albumin protein comprises or consists of the amino acid sequence of Alb-23002 (SEQ ID NO: 20). In another preferred embodiment, at least one ISVD that specifically binds to a serum albumin protein comprises or consists of the amino acid sequence of Alb23002 (E1D) (SEQ ID NO: 61). In another preferred embodiment, at least one ISVD that specifically binds to a serum albumin protein comprises or consists of the amino acid sequence of ALBX00002 (SEQ ID NO: 64). In another preferred embodiment, at least one ISVD that specifically binds to a serum albumin protein comprises or consists of the amino acid sequence of T023500029 (SEQ ID NO: 62). In another preferred embodiment, at least one ISVD that specifically binds to a serum albumin protein comprises or consists of the amino acid sequence of Alb82 (SEQ ID NO: 13). In another preferred embodiment, at least one ISVD that specifically binds to a serum albumin protein comprises or consists of the amino acid sequence of Alb223 (SEQ ID NO: 21). In another preferred embodiment, at least one ISVD that specifically binds to a serum albumin protein comprises or consists of the amino acid sequence of HSA006A06 (SEQ ID NO: 65). In another preferred embodiment, at least one ISVD that specifically binds to a serum albumin protein comprises or consists of the amino acid sequence of HSA006A06-A (SEQ ID NO: 66). In another embodiment, at least one ISVD that specifically binds to a serum albumin protein comprises or consists of the amino acid sequence of ALB-1 (SEQ ID NO: 67).

[0298] In other embodiments, the amino acid sequence of at least one ISVD that specifically binds to a serum albumin protein has greater than 90% (such as greater than 95% or greater than 99%) sequence identity with T023500029 (SEQ ID NO: 62). In other embodiments, the amino acid sequence of at least one ISVD that specifically binds to a serum albumin protein has greater than 90% (such as greater than 95% or greater than 99%) sequence identity with Alb-23002 (SEQ ID NO: 20). In other embodiments, the amino acid sequence of at least one ISVD that specifically binds to a serum albumin protein has greater than 90% (such as greater than 95% or greater than 99%) sequence identity with Alb23002 (E1D) (SEQ ID NO: 61). In other embodiments, the amino acid sequence of at least one ISVD that specifically binds to a serum albumin protein has greater than 90% (such as greater than 95% or greater than 99%) sequence identity with ALBX00002 (SEQ ID NO: 64). In other embodiments, the amino acid sequence of at least one ISVD that specifically binds to a serum albumin protein has greater than 90% (such as greater than 95% or greater than 99%) sequence identity with Alb 82 (SEQ ID NO: 13). In other embodiments, the amino acid sequence of at least one ISVD that specifically binds to a serum albumin protein has greater than 90% (such as greater than 95% or greater than 99%) sequence identity with Alb223 (SEQ ID NO: 21). In other embodiments, the amino acid sequence of at least one ISVD that specifically binds to a serum albumin protein has greater than 90% (such as greater than 95% or greater than 99%) sequence identity with HSA006A06 (SEQ ID NO: 65). In other embodiments, the amino acid sequence of at least one ISVD that specifically binds to a serum albumin protein has greater than 90% (such as greater than 95% or greater than 99%) sequence identity with HSA006A06-A (SEQ ID NO: 66). In other embodiments, the amino acid sequence of at least one ISVD that specifically binds to a serum albumin protein has greater than 90% (such as greater than 95% or greater than 99%) sequence identity to ALB-1 (SEQ ID NO: 67).

[0299] When the ISVD that binds human serum albumin has 2 or 1 amino acid difference in at least one CDR relative to the corresponding reference CDR sequence (e.g., as defined above as SEQ ID NOs: 4 to 6 (according to AbM numbering)), the ISVD preferably has at least half the binding affinity and preferably at least the same binding affinity for human serum albumin compared to construct ALB23002 (SEQ ID NO: 20), wherein the binding affinity is measured using the same method (such as surface plasmon resonance).

[0300] When such a human serum albumin binding ISVD in the polypeptides of the present technology has a C-terminal position, it may exhibit a C-terminal alanine (A) or a C-terminal glycine (G) extension, and is preferably selected from SEQ ID NO: 9, 10, 12, 14, 15, 16, 17, 18, 19 and 21 (see Table A-3).

[0301] In certain embodiments, a polypeptide as described herein comprising an ISVD as defined herein having one or more CDRs having 1, 2, 3 or 4 amino acid differences binds serum albumin with about the same affinity as compared to the binding of an amino acid sequence or polypeptide comprising a CDR as defined herein without the 4, 3, 2 or 1 amino acid differences, as measured by surface plasmon resonance.

[0302] Compared to the sequence of SEQ ID NO: 20, at least one serum albumin binding ISVD comprised in the polypeptides of the present technology preferably further comprises (at least): one or more humanizing substitutions;

[0303] and / or

[0304] One or more mutations (ie, amino acid substitutions, deletions or additions, particularly substitutions) that reduce binding of a pre-existing antibody; and may optionally contain one or more additional mutations as described herein (eg, to improve the chemical stability of the polypeptide).

[0305] For suitable humanizing substitutions (and suitable combinations thereof), reference is made, for example, to WO 09 / 138519 (or the prior art cited therein) and WO 08 / 020079 (or the prior art cited therein), and to Tables A-3 to A-8 of WO 08 / 020079 (which are lists showing possible humanizing substitutions). Some preferred, but non-limiting, examples of such humanizing substitutions are Q108L and A14P, or suitable combinations thereof. Such humanizing 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).

[0306] For suitable mutations (and suitable combinations of such mutations) that may reduce the binding of pre-existing antibodies, reference is made, for example, to WO 2012 / 175741 and WO 2015 / 173325, and also to, for example, WO 2013 / 024059 and WO 2016 / 118733.

[0307] A method that can be used to identify certain residues or regions of a polypeptide or ISVD as described herein as preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells 1989 (Science 244: 1081-1085). Here, residues or target residue groups within a binding molecule (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to influence the interaction of the amino acid with the epitope. Amino acid positions that exhibit functional sensitivity to the substitution are then refined by introducing more or other variants at or at the substitution site. Thus, while the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is performed at the target codon or region, and the expressed binding molecule variants are screened for the desired activity.

[0308] Preferably, amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues to polypeptides containing 100 or more residues.

[0309] The variant of another type is an amino acid substitution variant. These variants preferably (at least) 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues are replaced by different residues in amino acid sequence, ISVD or polypeptide. The most interesting site for carrying out substitution mutagenesis includes CDR, particularly hypervariable region, but also considers that FR changes. For example, if the CDR sequence comprises 6 amino acids, it is envisioned that one, two or three of these amino acids are replaced. Similarly, if the CDR sequence comprises 15 amino acids, it is envisioned that one, two, three, four, five or six of these amino acids are replaced.

[0310] In general, if amino acids are substituted in one or more or all CDRs, it is preferred that the resulting "substituted" sequence be at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, and 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, i.e., the degree of identity between the CDR and the "substituted" sequence. For example, a CDR with 5 amino acids is preferably 80% identical to its substituted sequence, as long as at least one amino acid is substituted. Thus, the amino acid sequences of CDRs of an ISVD or polypeptide may have different degrees of identity with their substituted sequences; for example, CDR1 may have 80% identity, while CDR3 may have 90%.

[0311] Preferred amino acid substitutions are conservative substitutions. Such conservative substitutions preferably refer to substitutions in which one amino acid in the following groups (a) to (e) is substituted with another amino acid residue in the same group: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Further preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu. However, any substitution (including non-conservative substitutions) is contemplated so long as the polypeptide retains its ability to specifically bind to an epitope on FcRn as described herein, at an acidic pH (such as pH 5.0 to 6.8) with an affinity as described herein (e.g., at 10 -6 with 10 -11 K between M D) has the ability to specifically bind to FcRn and / or its CDRs have at least 60%, more preferably 65%, even more preferably 70%, particularly preferably 75%, and more particularly preferably 80% identity with the "original" CDR sequence.

[0312] In some embodiments, the ISVD is a (single) domain antibody, a V HH , humanized V HH or Camelized V H , preferably

[0313] Thus, in certain embodiments, the polypeptides according to the present technology comprise at least one ISVD that binds human serum albumin selected from the group consisting of SEQ ID NOs: 7 to 21 or 61 to 69; and at least one IgG Fc domain, as described herein.

[0314] According to specific embodiments, the polypeptides of the present technology (such as the FcRn targeting polypeptides of the present technology) are also preferably such that they compete with a polypeptide having the amino acid sequence of SEQ ID NOs: 7 to 21, or 61 to 69, or 102-104 for binding to human serum albumin and / or such that they "cross-block" (as defined below) binding to human serum albumin with a polypeptide having the amino acid sequence of SEQ ID NOs: 7 to 21, or 61 to 69, or 102-104.

[0315] The terms "cross-block," "cross-blocked," and "cross-blocking" are used interchangeably herein to refer to the ability of an immunoglobulin single variable domain or polypeptide to interfere with the binding of a ligand to its target (such as a natural ligand to its receptor(s)). Competition binding assays can be used to determine the extent to which an immunoglobulin single variable domain or polypeptide of the present technology is able to interfere with the binding of another compound, such as a natural ligand, to its target and, therefore, whether it can be said to cross-block according to the present technology. A particularly suitable quantitative cross-blocking assay uses a FACS or ELISA-based method or Alphascreen to measure competition between labeled (e.g., His-tagged or biotinylated) immunoglobulin single variable domain or polypeptide according to the present technology and other binding agents for their binding to the target. Suitable FACS, ELISA, or Alphascreen displacement-based assays for determining whether a binding molecule cross-blocks or is capable of cross-blocking a polypeptide are well known. It will be understood that these assays can be used with any of the immunoglobulin single variable domains or other binding agents described herein. Thus, typically, a cross-blocking polypeptide according to the present technology is a polypeptide that will bind to the target, e.g., in a cross-blocking assay as described above, such that during the assay and in the presence of a second polypeptide or in the presence of the natural ligand, the recorded displacement of the immunoglobulin single variable domain or polypeptide according to the present technology is between 60% and 100% (e.g., in an ELISA / Alphascreen-based competition assay) or between 80% and 100% (e.g., in a FACS-based competition assay) of the maximum theoretical displacement of the potential cross-blocker to be tested (e.g., displacement of a cold (e.g., unlabeled) immunoglobulin single variable domain or polypeptide that needs to be cross-blocked) present in an amount of 0.01 mM or less.

[0316] In certain embodiments, the polypeptides of the present invention are such that at least one ISVD they comprise binds to substantially the same amino acid residues and / or epitopes on (human) serum albumin as the amino acid residues and / or epitopes bound by the polypeptides having the amino acid sequences of SEQ ID NOs: 7 to 21, or 61 to 69, and even more preferably such that they share substantially the same amino acid interactions with the polypeptides having the amino acid sequences of SEQ ID NOs: 7 to 21, or 61 to 69. To this end, according to specific but non-limiting aspects, the polypeptides according to the present invention preferably comprise at least one ISVD having CDRs identical to the sequences of SEQ ID NOs: 7 to 21, or 61 to 69, or preferably comprise in their CDRs only mutations (such as conservative amino acid substitutions) compared to the sequences of SEQ ID NOs: 7 to 21, or 61 to 69 that still allow them to perform the same or substantially the same amino acid interactions with (human) serum albumin as the polypeptides having the sequences of SEQ ID NOs: 7 to 21, or 61 to 69.

[0317] In further specific embodiments, (i) at least one domain that specifically binds to a serum albumin protein comprised in the polypeptides of the present technology is at least one ISVD that specifically binds to domain II of serum albumin, such as domain II of human serum albumin.

[0318] According to a specific embodiment of the present technology, at least one domain specifically binding to serum albumin protein contained in the polypeptide of the present technology is at least one ankyrin repeat sequence (DARPin sequence) that specifically binds to (human) serum albumin.

[0319] In certain embodiments, the polypeptides of the present technology comprise at least one serum albumin binding domain which is an ankyrin repeat sequence, such as, but not limited to, those having the sequences SEQ ID NOs: 17 to 31 and SEQ ID NOs: 43 to 52 (as disclosed and specifically described on pages 15-27 of WO 2012 / 069654), SEQ ID NO: 50 (as disclosed in WO 2016 / 156596), SEQ ID NOs: 9 to 11 (as disclosed and specifically described on pages 9-11 of WO 2018 / 054971), and SEQ ID NOs: 3 and 4 (as disclosed and specifically described on pages 5-12 of WO 2020 / 24517).

[0320] Polypeptides comprising at least one of these albumin-binding ankyrin repeat sequences are generated and tested for their beneficial PK properties.

[0321] Thus, in other embodiments, the polypeptides of the present technology comprising (i) at least one domain that specifically binds to a serum albumin protein is at least one DARPin that specifically binds to serum albumin. For example, the at least one DARPin may comprise or consist of SEQ ID NO: 102, or a polypeptide having at least 90% (such as at least 95%, or at least 97%, or at least 99%) sequence identity to SEQ ID NO: 102.

[0322] According to a specific embodiment of the present invention, the at least one albumin-binding domain contained in the polypeptide of the present invention is at least one affitin (also known as ).

[0323] In a specific embodiment, at least one serum albumin binding Affitin is, for example but not limited to, a sequence having SEQ ID NO: 38 and SEQ ID NOs: 45 to 86 (as disclosed and specifically described on pages 6 to 16 of WO 2022 / 171852).

[0324] Polypeptides comprising at least one of these albumin-binding affitins are generated and tested for their beneficial PK properties.

[0325] Thus, in other embodiments, the (i) at least one domain that specifically binds to a serum albumin protein contained in the polypeptides of the present technology is at least one affitin that specifically binds to serum albumin. For example, the at least one affitin may comprise or consist of SEQ ID NO: 103, or a polypeptide having at least 90% (such as at least 95%, or at least 97%, or at least 99%) sequence identity to SEQ ID NO: 103.

[0326] According to a specific embodiment of the present technology, the at least one albumin-specific binding domain comprised in the polypeptide of the present technology is at least one ABD of a bacterial receptor protein that specifically binds to (human) serum albumin.

[0327] Streptococcal protein G is a bifunctional receptor present on the surface of certain streptococcal strains and is capable of binding both IgG and serum albumin (Bjorck et al., Mol Immunol 24:1113, 1987). The structure is highly repetitive, with several structurally and functionally different domains (Guss et al., EMBO J 5:1567, 1986), more precisely three Ig binding motifs and three serum albumin binding domains (Olsson et al., Eur J Biochem 168:319, 1987). The structure of one of the three serum albumin binding domains has been determined, showing a three-helix bundle domain (Kraulis et al., FEBS Lett 378:190, 1996). This motif is named ABD (albumin binding domain) and is 46 amino acid residues in size. In the literature, it is subsequently also designated as G148-GA3. Other bacterial albumin-binding proteins, besides protein G from Streptococcus, have also been identified that contain domains similar to the albumin-binding triple-helical domain of protein G. Examples of such proteins are the PAB, PPL, MAG, and ZAG proteins. Studies on the structure and function of such albumin-binding proteins have been conducted and reported, for example, by Johansson and colleagues (Johansson et al., J Mol Biol 266:859-865, 1997; Johansson et al., J Biol Chem 277:8114-8120, 2002), who introduced the name "GA module" (protein G-associated albumin-binding module) for the triple-helical protein domain responsible for albumin binding. In addition, Rozak et al. have reported the generation of artificial variants of the GA module that were selected and studied for their specificity and stability in different species (Rozak et al., Biochemistry 45:3263-3271, 2006; He et al., Protein Science 16:1490-1494, 2007). More recently, variants of the G148-GA3 domain have been developed with various optimized features. Such variants are disclosed, for example, in PCT Publication Nos. WO 2009 / 016043, WO 2012 / 004384, WO 2014 / 04897, and WO 2015 / 091957.

[0328] Polypeptides comprising at least one of these ABDs are produced and tested for their beneficial PK properties.

[0329] Thus, in other embodiments, (i) at least one domain that specifically binds to a serum albumin protein comprised in the polypeptides of the present technology 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 SEQ ID NO: 104, or a polypeptide having at least 90% (such as at least 95%, or at least 97%, or at least 99%) sequence identity to SEQ ID NO: 104.

[0330] Table A-4. Serum albumin binding protein sequences ("ID" refers to SEQ ID NO as used herein)

[0331]

[0332] In other embodiments, the polypeptides of the present technology comprise (i) at least one domain that specifically binds to a serum albumin protein selected from the group consisting of the albumin binding units described in any one of the following applications, all of which are incorporated by reference: WO 2023 / 147042, WO 2022 / 026643, WO 2021 / 119531, WO 2021 / 119531, WO 2020 / 229842, WO 2020 / 172528, WO 2020 / 099871, WO 2017 / 201488, WO 2014 / 111550, WO 2013 / 167883, WO 2013 / 043071, WO 2012 / 072731, WO 2012 / 022703, WO 2012 / 020143、WO2011 / 144751、WO 2011 / 006915、WO 2010 / 094723、WO 2010 / 094722、WO 2008 / 096158、WO2005 / 118642、WO 2004 / 003019, US20140186365 and US20130129727.

[0333] 5.4 Second domain: IgG Fc domain or fragment thereof

[0334] As described above, the polypeptides of the present technology comprise (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin binding ISVD), and (ii) at least one IgG Fc domain or a fragment thereof, preferably an FcRn binding fragment thereof.

[0335] As used in this specification, the terms "Fc", "Fc domain", "Fc region" or "Fc fragment" are used interchangeably and are defined as a portion of the heavy chain constant region that begins at the hinge region just upstream of the N-terminus of the papain cleavage site (i.e., residue 216 in IgG, with the first residue of the heavy chain constant region taken as 114) and ends at the C-terminus of the heavy chain. Thus, a complete Fc, Fc domain, Fc region or Fc fragment comprises at least a hinge region or a portion thereof, a CH2 domain and a CH3 domain. For example, an Fc domain may comprise at least one hinge region or a portion thereof, two CH2 domains and two CH3 domains. A sequence alignment of portions of exemplary human IgG1, IgG2, IgG3 and IgG4 Fc domains (CH2 and CH3 domains, residues 231 to 447, Eu numbering) is shown in Figure 32 of WO 2021 / 016571. The term encompasses natural / wild-type Fc and Fc variants as described herein, and includes molecules in monomeric or multimeric (e.g., dimer) form, whether digested from intact antibodies or produced by other means (such as recombinant technology). See, e.g., Ying et al., JBC (2013) 288: 25154-164; and Yang et al., JBC (2019) 294: 10638-48.

[0336] Thus, the terms "Fc domain," "Fc region," or "Fc" refer to the C-terminal non-antigen binding region of an immunoglobulin heavy chain that contains at least a portion of the constant region. Traditionally, the term Fc domain refers to a protease (e.g., papain) cleavage product encompassing the paired CH2, CH3, and hinge regions of an antibody. In the context of the present disclosure, the terms "Fc domain," "Fc region," or "Fc" refer to any polypeptide (or nucleic acid encoding such a polypeptide) comprising all or part of the CH2, CH3, and hinge regions of an immunoglobulin polypeptide, regardless of the means of production. Thus, in some embodiments, the Fc domain comprises CH2-CH3 and hinge-CH2-CH3 from N-terminus to C-terminus. Although the boundaries of the Fc region may vary, the generally defined human IgG heavy chain Fc region comprises residues E216, C226, or A231 at its carboxyl terminus, where numbering is according to the EU index as in Kabat.

[0337] The term includes native (i.e., wild-type) Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may be present or absent without affecting the structure or stability of the Fc region. Unless otherwise indicated herein, the numbering of amino acid residues in IgG or Fc regions is according to the EU numbering system of antibodies, also referred to as the EU index, as described in Edelman, GM et al. Proc. Natl. Acad. USA, 63, 78-85 (1969) and in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0338] In certain embodiments, the term "Fc domain," "Fc region," or "Fc" refers to the constant region of an immunoglobulin IgG heavy chain, which comprises the hinge region (starting at Cys226), the IgG CH2 domain, and the CH3 domain. In certain embodiments, the Fc region starts at the hinge region and extends to the C-terminus of the IgG heavy chain.

[0339] The original immunoglobulin source of native Fc is typically human origin, and can be any immunoglobulin G, such as IgG1, IgG2, IgG3 or IgG4, particularly IgG1 and IgG4. In a preferred embodiment, the Fc domain is human origin and from IgG1 or IgG4, preferably from IgG4. Native Fc molecules are composed of monomeric polypeptides, which can be connected into dimer or multimeric form by covalent (i.e., disulfide bond) and non-covalent association. Depending on classification (e.g., IgG, IgA and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), the number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules is within the range of 1 to 4. An example of native Fc is the disulfide bonded dimer produced by papain digestion of IgG. As used herein, term "native Fc" is general for monomeric, dimer and multimeric form.

[0340] The Fc domain contained in the polypeptide of the present invention preferably comprises two disulfide-bonded chains (chain 1 and chain 2). The chains can be identical (homodimers) or different (heterodimers). Thus, the polypeptide of the present invention can comprise two polypeptides bonded by a disulfide bridge. The polypeptide of the present invention can comprise more than two polypeptides, such as four polypeptides (more than two chains, such as four chains, see, for example, Table A-1).

[0341] In certain specific embodiments, the Fc region comprises the Fc region of human IgG1, IgG2, IgG3, or IgG4. In certain specific embodiments, the Fc region comprises the CH2 and CH3 domains of IgG, including the Fc domain as a single monomeric Fc chain. In certain specific embodiments, the Fc region comprises the CH2 and CH3 domains of IgG4. In certain other specific embodiments, the Fc region comprises the CH2 and CH3 domains of IgG1. In certain other particularly preferred embodiments, the Fc region comprises the hinge region of IgG1 and the CH2 and CH3 domains of IgG4.

[0342] In certain embodiments, the IgG CH2 domain starts at Ala 231. In certain other embodiments, the CH3 domain starts at Gly 341. It should be understood that the C-terminal lysine residue of human IgG may optionally be absent. It should also be understood that conservative amino acid substitutions in the Fc region that do not affect the desired structure and / or stability of the Fc are considered within the scope of the present technology.

[0343] In certain embodiments, the polypeptides of the present technology comprise at least one native Fc domain of immunoglobulin G. In these embodiments, at least one variant Fc domain of immunoglobulin G specifically binds to FcRn, whose K D 0, 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, or 100 nM (e.g., at a pH between 5.0 and 6.8). In further specific embodiments, the polypeptides of the present technology comprise at least one IgG native Fc domain and specifically bind to FcRn at a pH between about 5.0 and 6.8, preferably at a pH of about 6.0, with a K D The value is less than about 75nM, such as less than 50nM, 25nM, 20nM, 17nM, 15nM, 10nM, 5nM, 2.5nM, 1nM (at a pH between 5.0 and 6.8). In yet other specific embodiments, the polypeptides of the present technology comprise at least one IgG native Fc domain and specifically bind to FcRn at a pH between about 5.0 and 6.8, preferably at a pH of about 6.0, with a K D The value is between about 250nM and 1nM, such as between 100nM and 1nM, preferably between 75nM and 1nM, such as between 50nM and 1nM, most preferably between 25nM and 1nM, such as about 20nM, such as about 17nM. Preferably, the K D is measured by Kinexa, BLI or SPR, eg, as determined by SPR.

[0344] In certain embodiments, in addition to comprising a domain of a serum albumin protein and / or a domain that specifically binds to a serum albumin protein, the polypeptides of the present technology further comprise a single Fc domain that is a native Fc domain of IgG. In these embodiments, a native Fc domain of immunoglobulin G specifically binds to FcRn, and its K D In another specific embodiment, an IgG native Fc domain specifically binds to FcRn at a pH between about 5.0 and 6.8, preferably at a pH of about 6.0, with a K of less than about 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, or 100 nM. D The value is less than about 75 nM, such as less than 50 nM, 25 nM, 20 nM, 17 nM, 15 nM, 10 nM, 5 nM, 2.5 nM, 1 nM (at a pH between 5.0 and 6.8).

[0345] In certain embodiments, in addition to comprising a domain of a serum albumin protein and / or a domain that specifically binds to a serum albumin protein, the polypeptides of the present technology further comprise a single Fc monomer chain that is a native Fc monomer chain of IgG. In these embodiments, a native monomeric Fc chain of immunoglobulin G specifically binds to FcRn, and its K D In another specific embodiment, an IgG native monomeric Fc chain specifically binds to FcRn at a pH between about 5.0 and 6.8, preferably at a pH of about 6.0, with a K of less than about 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, or 100 nM (e.g., at a pH between 5.0 and 6.8). D The value is less than about 75 nM, such as less than 50 nM, 25 nM, 20 nM, 17 nM, 15 nM, 10 nM, 5 nM, 2.5 nM, 1 nM (at a pH between 5.0 and 6.8).

[0346] In certain embodiments, one or more amino acid modifications can be introduced into the native Fc region contained in the polypeptides of the present technology, thereby generating Fc variants. Thus, in some specific embodiments, the Fc domain is a variant Fc domain.

[0347] In certain specific embodiments, in addition to comprising a domain of a serum albumin protein and / or a domain that specifically binds to a serum albumin protein, the polypeptides of the present invention further comprise: at least one variant Fc domain of immunoglobulin G, and specifically bind to FcRn, whose K D 0, 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, or 100 nM (e.g., at a pH between 5.0 and 6.8). In yet other specific embodiments, the polypeptides of the present technology comprise at least one IgG variant Fc domain and bind to FcRn with a K of less than about 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, or 100 nM (e.g., at a pH between 5.0 and 6.8). D The value is between about 250nM and 1nM (such as between 100nM and 1nM, preferably between 75nM and 1nM, such as between 50nM and 1nM, most preferably between 25nM and 1nM, such as about 20nM, such as about 17nM). Preferably, the K D is measured by Kinexa, BLI or SPR, eg, as determined by SPR.

[0348] In certain other specific embodiments, the polypeptides of the present technology, in addition to comprising a domain of a serum albumin protein and / or a domain that specifically binds to a serum albumin protein, further comprise at least one variant Fc domain that does not detectably, selectively or specifically bind to FcRn, or exhibits no binding or substantially no binding to FcRn, neither at a pH between 5.0 and 6.8 nor at neutral or physiological pH (such as at a pH of 7.4).

[0349] In certain embodiments, in addition to comprising a domain of a serum albumin protein and / or a domain that specifically binds to a serum albumin protein, the polypeptides of the present technology further comprise a single Fc domain that is a variant Fc domain of IgG. In these embodiments, an immunoglobulin G variant Fc domain specifically binds to FcRn, and its K D In certain embodiments, an IgG variant Fc domain specifically binds to FcRn at a pH between about 5.0 and 6.8, preferably at a pH of about 6.0, with a K of less than about 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, or 100 nM (e.g., at a pH between 5.0 and 6.8). In certain embodiments, an IgG variant Fc domain specifically binds to FcRn at a pH between about 5.0 and 6.8, preferably at a pH of about 6.0, with a K of less than about 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, or 100 nM DThe value is less than about 75 nM, such as less than 50 nM, 25 nM, 20 nM, 17 nM, 15 nM, 10 nM, 5 nM, 2.5 nM, 1 nM (at a pH between 5.0 and 6.8).

[0350] In certain embodiments, in addition to comprising a domain of a serum albumin protein and / or a domain that specifically binds to a serum albumin protein, the polypeptides of the present technology further comprise a single monomeric Fc chain that is a variant monomeric Fc chain of IgG. In these embodiments, an immunoglobulin G variant monomeric Fc chain specifically binds to FcRn, and its K D In another specific embodiment, an IgG variant monomer Fc chain specifically binds to FcRn at a pH between about 5.0 and 6.8, preferably at a pH of about 6.0, with a K of less than about 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, or 100 nM (e.g., at a pH between 5.0 and 6.8). D The value is less than about 75 nM, such as less than 50 nM, 25 nM, 20 nM, 17 nM, 15 nM, 10 nM, 5 nM, 2.5 nM, 1 nM (at a pH between 5.0 and 6.8).

[0351] The terms "Fc", "Fc domain", "Fc region" or "Fc fragment" as used in this specification also encompass "Fc variants", "modified Fc" or "modified Fc domains", i.e., molecules or sequences modified from native / wild-type Fc but still comprising a binding site for FcRn. Fc variants or modified Fc domains may also be shorter or longer than native Fc (e.g., shorter or longer than the sequence spanning residues 216 to 447 of human IgG, Eu numbering); for example, Fc variants or modified Fc may lack certain N-terminal and / or C-terminal amino acid residues of native Fc, or may contain additional amino acid residues at the N-terminus and / or C-terminus compared to native Fc. The modified Fc domain itself does not include the antigen binding domain of an antibody or antibody variant, or the target binding domain of an immunoadhesin. The term encompasses molecules or sequences humanized from non-human native Fc. In addition, native Fc contains regions that can be removed because they provide structural features or biological activities that are not required for FcRn antagonists (e.g., antibody-like binding polypeptides) as described in WO 2021 / 016571. Thus, the term encompasses molecules or sequences that lack one or more native Fc sites or residues, or in which one or more Fc sites or residues have been modified to affect or participate in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity when expressed in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors (FcγRs), or (7) antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0352] As used herein, "Fc variant domain," "Fc variant region," "Fc variant," or "variant Fc" refers to a protein comprising at least one amino acid modification in a native Fc domain (as defined herein). The modification may be an addition, deletion, or substitution.

[0353] The Fc variant may comprise a human Fc region sequence (eg, a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (eg, addition, deletion, and / or substitution) at one or more amino acid positions.

[0354] In certain embodiments, the present technology contemplates Fc variants that possess some, but not all, effector functions, making them desirable candidates for applications in which the in vivo half-life of the polypeptide comprising the Fc region is important.

[0355] Certain effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), may be unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays may be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that the Fc lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom et al., Proc. Nat. Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom et al., Proc. Nat. Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, e.g., Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (CellTechnology, Inc., Mountain View, CA); and CYTOTOX Non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo (e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat. Acad. Sci. USA 95:652-656 (1998)). C1q binding assays can also be performed to confirm that the Fc cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg et al., Blood 101: 1045-1052 (2003); and Cragg et al., Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova et al., Int. Immunol. 18(12): 1759-1769 (2006)).

[0356] Fc regions with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Patent No. 6,737,056 ). Such Fc variants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine ( U.S. Patent No. 7,332,581 ).

[0357] Certain Fc variants with improved or diminished binding to FcRs have been described (see, eg, US Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0358] In some embodiments, alterations are made in the Fc region that result in reduced CIq binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0359] Fc domains with improved binding to the neonatal Fc receptor (FcRn) in accordance with the present technology include Fc variants with substitutions at, for example, but not limited to, one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424.

[0360] In certain embodiments, it may be desirable to produce cysteine-engineered Fc fusion proteins in which one or more residues in the Fc region of an antibody are substituted with cysteine ​​residues. In specific embodiments, the substituted residues are present at accessible sites of the Fc. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the Fc and can be used to conjugate the Fc to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates, as further described herein.

[0361] Further variants of suitable Fc domains and suitable formats of Fc domain constructs are well known in the art and are described in, inter alia, published patent applications EP 2654790, US10239944, US20120251531, US 9133274, WO2014 / 065945, WO 2015 / 150447 and WO 2021 / 016571.

[0362] Specific suitable formats of polypeptides according to certain embodiments of the present technology comprising at least one serum albumin protein or a binding agent to a serum albumin protein and at least one Fc domain will become clear from the examples as further described herein.

[0363] Thus, in specific embodiments, in addition to at least one domain comprising a serum albumin protein and / or at least one domain that specifically binds to a serum albumin protein, the polypeptides of the present invention further comprise at least one IgG Fc domain. Thus, in specific embodiments, the Fc domains contained in the polypeptides of the present invention specifically bind to human FcRn (SEQ ID NO: 24) or its (polymorphic) variants or isoforms at a pH between about 5.0 and 6.8. However, in other specific embodiments, the polypeptides of the present invention comprise at least one IgG Fc domain that does not specifically bind to FcRn (neither at a pH between about 5.0 and 6.8 nor at a pH of about 7.4). The Fc domains contained in the polypeptides of the present invention may also comprise any mutation described herein.

[0364] In certain embodiments, the polypeptides according to the present technology are preferably such that, when bound or otherwise associated with an FcRn molecule, the binding of the FcRn molecule to serum albumin and / or IgG is not (significantly) affected, reduced or inhibited. In these specific embodiments, in a cross-blocking assay (as described herein), when the FcRn-binding polypeptide is bound or otherwise associated with an FcRn molecule, the shift of serum albumin is less than 40%, such as less than 30%, less than 20%, less than 10%, or substantially no shift is detected (e.g., in an ELISA or Alphascreen-based competition assay). In this specific embodiment, in a cross-blocking assay (as described herein), when the FcRn-binding polypeptide is bound or otherwise associated with an FcRn molecule, the shift of IgG is less than 40%, such as less than 30%, less than 20%, less than 10%, or substantially no shift is detected (e.g., in an ELISA or Alphascreen-based competition assay). Preferably, as described herein, the Fc domain comprised in the polypeptides of the present technology is such that, when bound to or otherwise associated with an FcRn molecule, the binding of the FcRn molecule to serum albumin and / or IgG is not (significantly) affected, reduced or inhibited, as described herein.

[0365] In certain embodiments, the IgG Fc domains included in the polypeptides of the present invention may include one or more amino acid mutations (e.g., substitutions) that change the effector functions (e.g., ADCC or CDC functions) of the Fc domains compared to the corresponding wild-type molecules. In certain embodiments, the IgG Fc domains included in the polypeptides of the present invention may include one or more amino acid mutations (e.g., substitutions) that provide one or more desired biochemical characteristics, such as the ability to maintain monomericity, the ability to non-covalently dimerize, the increased ability to localize to the target site, and the glycosylation pattern, compared to the corresponding wild-type molecules. For example, the modified Fc domains may have reduced glycosylation (e.g., N- or O- connected glycosylation). Exemplary amino acid substitutions that confer reduced or altered glycosylation are disclosed in WO 2005 / 018572. In some embodiments, the Fc domains are modified to eliminate glycosylation (e.g., "agly" antibodies).

[0366] In a preferred embodiment, the IgG Fc domain contained in the polypeptide of the present technology is in the form of a dimer and comprises at least one hinge region or a portion thereof, two CH2 domains and two CH3 domains, i.e., two polypeptide fragments, see e.g. Figure 1 、 Figure 3 and Figure 6. The IgG Fc domain included in the polypeptide of the present invention is preferably a dimer, more preferably a heterodimer. In the preferred embodiment, at least one and preferably two of the CH3 domains included in the Fc domain contain a "knob-into-hole" mutation. Therefore, in the preferred embodiment, the Fc domain is a heterodimer. These mutations are used to engineer the interface between the first polypeptide and the second polypeptide (in this specific case, the CH3 domain) for hetero-oligomerization using the "knob-into-hole" technology, as described, for example, in US 8216805, WO 1996 / 27011, Ridgway, JB et al., "'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization", Protein engineering, 1996, 9, 7: 617-21 or in Merchant et al., "Anefficient route to human bispecific IgG", Nature Biotechnology, 1998, 16: 677–681. The preferred interface comprises at least a portion of the CH3 domain of the Fc domain. A "protrusion" is constructed by replacing a small amino acid side chain from the interface of a first polypeptide (in this case, the first CH3) with a larger side chain (e.g., tyrosine or tryptophan). Optionally, a compensatory "cavity" of the same or similar size as the protrusion is generated at the interface of a second polypeptide (in this case, the second CH3) by replacing a large amino acid side chain with a smaller side chain (e.g., alanine or threonine). When a protrusion or cavity of appropriate positioning and size exists at the interface of the first or second polypeptide, it is only necessary to engineer the corresponding cavity or protrusion at the adjacent interface. See, e.g., US 8216805, WO 1996 / 27011, Ridgway, JB et al., “'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization”, Protein engineering, 1996, 9, 7:617-21 or Merchant et al., “Anefficient route to human bispecific IgG”, Nature Biotechnology, 1998, 16:677–681 for more details.In a preferred embodiment, the amino acid HY in the CH3 domain of the Fc domain is mutated to RF, such as at positions 435 and 436 (H435R and Y436F in the CH3 domain, as described by Jendeberg, L. et al. (1997, J. Immunological Meth., 201: 25-34). Thus, in this preferred embodiment, the Fc domain is a heterodimer.

[0367] Thus, in certain embodiments, the polypeptides as described herein comprise a native (i.e., wild-type) Fc domain of human IgG, such as preferably a native Fc domain of human IgG1 (e.g., Uniprot sequence P0DOX5) or a native Fc domain of human IgG4 (e.g., Uniprot sequence P01861). Polypeptides comprising at least one such native Fc domain were generated and tested for their beneficial PK properties, as described in the Examples below.

[0368] In certain embodiments, the polypeptides according to the present technology comprise variant Fc domains having altered binding properties for Fc ligands relative to an unmodified parent Fc molecule. For example, the polypeptides described herein can comprise an Fc region in which one or more of amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 are substituted with a different amino acid residue, such that the variant Fc region has an altered affinity for an effector ligand (e.g., an Fc receptor or the C1 component of complement), as described in U.S. Pat. Nos. 5,624,821 and 5,648,260, both to Winter et al.

[0369] In certain embodiments, the polypeptides of the present technology comprise Fc variant domains with reduced effector function, particularly so-called "FALA" or "LALA" Fc mutants in which residues 234 and 235 are substituted with alanine. Additional optional mutations include substitution of arginine residue 409 with lysine and deletion of lysine residue 447. Polypeptides comprising at least one Fc domain with the above mutations were generated and tested for their beneficial PK properties, as described in the Examples below.

[0370] In certain embodiments, polypeptides according to the present technology comprise Fc variant domains that exhibit improved binding to the FcRn receptor compared to a native Fc domain. Such Fc variants include those having substitutions at one or more of Fc region residues 259, 308, 428, and 434. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry, 2001, 276(9):6591-6604).

[0371] In certain specific embodiments, the polypeptides according to the present technology comprise an Fc variant domain in which methionine 428 is substituted with leucine and asparagine 434 is substituted with serine. In certain specific embodiments, the polypeptides according to the present technology comprise an Fc variant domain (YTE, see e.g., Robbie GJ et al., “A novel investigational Fc-modified humanized monoclonal antibody, motavizumab-YTE, has an extended half-life in healthy adults”, Antimicrob Agents Chemother., 2013 Dec; 57(12): 6147-53) having the following mutations: M252Y, S254T, and T256E.

[0372] Polypeptides comprising at least one Fc domain having the above mutations were generated and tested for their beneficial PK properties.

[0373] In certain embodiments, polypeptides according to the present technology may comprise an Fc variant domain that exhibits reduced or no binding to an FcRn receptor compared to a native Fc domain. Such Fc variants include those having substitutions at one or more of Fc region residues 253, 310, and 453.

[0374] In certain embodiments, polypeptides according to the present technology comprise an Fc variant domain wherein isoleucine 428 is substituted with alanine, histidine 310 is substituted with alanine, and histidine 453 is substituted with alanine, optionally in combination with substitution of histidine 453 with alanine.

[0375] Polypeptides comprising at least one Fc domain are generated and tested for beneficial PK properties.

[0376] An example of an IgG Fc domain or FcRn binding fragment thereof that may be included in the polypeptide of the present technology is a dimeric form, i.e., it comprises at least one hinge region or a portion thereof, two CH2 domains and two CH3 domains, i.e., two polypeptide fragments, see e.g. Figure 1 、 Figure 3 and Figure 6 Preferably, the Fc domain or FcRn binding fragment thereof is a dimer, more preferably a heterodimer, as defined above.

[0377] In one embodiment, the Fc domain comprised in the polypeptides of the present technology comprises two identical chains, for example, as depicted in SEQ ID NO: 112, 113, 115 or 181, preferably 113 or 181.

[0378] As described above, an Fc domain from any IgG subtype can be used to generate an Fc domain or FcRn binding fragment thereof contained in a polypeptide of the present invention. In some embodiments, the Fc domain or FcRn binding fragment thereof is derived from human IgG1, IgG2, IgG3 or IgG4, preferably from IgG1 or IgG4, more preferably from IgG4, and comprises substitutions relative to wild-type sources as described herein. For example, the Fc domain contained in the polypeptide of the present invention comprises or consists of two polypeptides as defined in SEQ ID NO: 113, 115 or 181, preferably as defined in SEQ ID NO: 113 or 181, more preferably as defined in SEQ ID NO: 181 (e.g., two identical chains, wherein each chain comprises SEQ ID NO: 113, 115 or 181, preferably 181 or consists of). In other preferred embodiments, the Fc domain comprised in the polypeptides of the present technology comprises or consists of two different polypeptides as defined in SEQ ID NOs: 116 and 117, or as defined in SEQ ID NOs: 186 and 187, or as defined in SEQ ID NOs: 188 and 189, or as defined in SEQ ID NOs: 198 and 199, or as defined in SEQ ID NOs: 186 and 190. In these cases, the two chains forming the Fc domain are different (knob-in-hole).

[0379] In certain other embodiments, the Fc domain or its FcRn binding fragment is an artificial Fc derived from more than one IgG subtype. In other embodiments, the Fc domain or its FcRn binding fragment comprises a chimeric hinge (i.e., a hinge comprising a hinge portion of a hinge domain derived from a different antibody isotype, such as an upper hinge domain from an IgG4 molecule and a middle hinge domain from an IgG1). In certain embodiments, the Fc domain or its FcRn binding fragment is an IgG1 Fc region. In certain embodiments, the Fc domain or its FcRn binding fragment is a human IgG Fc region. In certain embodiments, the Fc domain or its FcRn binding fragment is a human IgG1 Fc region. In certain embodiments, the Fc domain or its FcRn binding fragment is an IgG4 Fc region. In certain embodiments, the Fc domain or its FcRn binding fragment is a human IgG4 Fc region. In certain embodiments, the Fc domain or its FcRn binding fragment is a chimeric Fc region.

[0380] In certain embodiments, the Fc domain comprises amino acid changes, substitutions, insertions and / or deletions that confer the desired characteristics. Useful Fc domains FcRn binding or fragments thereof included in the FcRn antagonists of the present technology are described in WO 2015 / 100299 and WO 2019 / 110823.

[0381] In other embodiments, at least one IgG Fc domain or fragment thereof in the polypeptides encompassed by the present technology is an Fc domain or fragment thereof that competes with a wild-type IgG1 Fc region for FcRn binding. Preferably, the Fc domain or fragment thereof that competes with a wild-type IgG1 Fc region for FcRn binding competes with the wild-type IgG1 Fc region for FcRn binding. For example, the Fc domain or fragment thereof that competes with a wild-type IgG1 Fc region for FcRn binding specifically binds to FcRn with increased affinity relative to FcRn binding by the wild-type IgG1 Fc region. In other embodiments, the Fc domain or fragment thereof that competes with a wild-type IgG1 Fc region for FcRn binding has increased FcRn binding affinity at both acidic pH and extracellular physiological pH compared to FcRn binding by the wild-type IgG1 Fc region. In another embodiment, the Fc domain or fragment thereof that competes with a wild-type IgG1 Fc region for FcRn binding specifically binds to FcRn with reduced pH dependence relative to FcRn binding by the wild-type IgG1 Fc region. In further embodiments, the affinity of the Fc domain or fragment thereof that competes with a wild-type IgG1 Fc region for FcRn is altered (increased or decreased) compared to a wild-type IgG1 Fc region.

[0382] In some embodiments, the Fc domain or fragment thereof comprised in the polypeptides of the present technology comprises at least one, preferably all, of the following amino acids at the following positions:

[0383] a) tyrosine (Y) at amino acid position 252,

[0384] b) Threonine (T) at amino acid position 254,

[0385] c) glutamic acid (E) at amino acid position 256,

[0386] d) lysine (K) at amino acid position 433,

[0387] e) phenylalanine (F) at amino acid position 434, and / or

[0388] f) tyrosine (Y) at amino acid position 436;

[0389] According to EU numbering.

[0390] Non-limiting examples of amino acid sequences that can be used for Fc domains or fragments thereof are listed in Table 1 of WO 2015 / 100299 (SEQ ID NOs: 167-169 in this specification). In certain embodiments, the amino acid sequence of the Fc domain or its FcRn-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 167. In certain embodiments, the amino acid sequence of the Fc domain or its FcRn-binding fragment comprises or consists of the amino acid sequence set forth in SEQ ID NO: 167, 168, or 169. In one embodiment, if the Fc domain is in the form of a dimer, i.e., comprises at least one hinge region or a portion thereof, two CH2 domains, and two CH3 domains, i.e., two polypeptide fragments, the Fc domain may comprise two polypeptides comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 167. Thus, if the Fc domain comprises at least one hinge region or a portion thereof, two CH2 domains, and two CH3 domains, i.e., two polypeptide fragments, the Fc domain may comprise two polypeptides comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 168. Thus, if the Fc domain comprises at least one hinge region or a portion thereof, two CH2 domains, and two CH3 domains, i.e., two polypeptide fragments, the Fc domain may comprise two polypeptides comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 169. In these embodiments, at least one and preferably both CH3 domains contained in the Fc domain included in the FcRn antagonists of the present technology may comprise a knob-to-hole mutation as defined above. Additionally or alternatively, amino acids HY in the CH3 domain of the Fc domain may be mutated to RF, such as at positions 435 and 436 (H435R and Y436F in the CH3 domain, as described by Jendeberg, L. et al. (1997, J. Immunological Meth., 201: 25-34)).

[0391] In certain embodiments, the Fc domain is a homodimer, wherein the amino acid sequence of each polypeptide contained in the Fc domain consists of SEQ ID NO: 167. In certain embodiments, the Fc domain is a homodimer, wherein the amino acid sequence of each polypeptide contained in the Fc domain consists of SEQ ID NO: 168. In certain embodiments, the Fc domain is a homodimer, wherein the amino acid sequence of each polypeptide contained in the Fc domain consists of SEQ ID NO: 169.

[0392] In certain embodiments, the amino acid sequence of the Fc domain of the variant Fc region comprises the amino acid sequence set forth in SEQ ID NO:167.

[0393] In certain embodiments, the amino acid sequence of the Fc domain of the variant Fc region comprises the amino acid sequence set forth in SEQ ID NO:168.

[0394] In certain embodiments, the amino acid sequence of the Fc domain of the variant Fc region comprises the amino acid sequence set forth in SEQ ID NO:169.

[0395] In certain embodiments, the polypeptides of the present technology comprise a variant Fc domain or fragment thereof that does not comprise an N-linked glycan at EU position 297. In certain embodiments, the polypeptides of the present technology comprise a variant Fc region that comprises a defucosylated N-linked glycan at EU position 297. In certain embodiments, the polypeptides of the present technology comprise a variant Fc domain or fragment thereof that has an N-linked glycan with bisected GlcNac at EU position 297.

[0396] Additional Fc domains or fragments thereof contained in the polypeptides of the present technology are described in WO 2021 / 016571.

[0397] In some embodiments, the Fc domain fragments contained in the polypeptides of the present invention may include amino acid substitutions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434 or Y436 and any combination thereof. Unless otherwise indicated, all Fc residue positions described herein are according to the EU numbering system. In some embodiments, the Fc domain or fragment thereof may include a double amino acid substitution at any two amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434 and Y436. In some embodiments, the Fc domain or fragment thereof may comprise a triple amino acid substitution at any three amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436. In some embodiments, the Fc domain or fragment thereof may comprise a quadruple amino acid substitution at any four amino acid positions selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, N434, and Y436. In some embodiments, it may be desirable for the Fc domain or fragment thereof to comprise an amino acid substitution at any amino acid position selected from M252, I253, S254, T256, K288, T307, K322, E380, L432, or Y436, and any combination thereof, wherein amino acid position N434 is not substituted (i.e., amino acid position N434 is wild type).

[0398] For example, in other embodiments, the Fc domain or fragment thereof may comprise an amino acid substitution selected from the group consisting of M252Y (i.e., tyrosine at amino acid position 252), T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, N434F, N434P, N434Y, Y436H, Y436N or Y436W, and any combination thereof.

[0399] In some embodiments, the Fc domain or fragment thereof may comprise a double amino acid substitution selected from: M252, wherein the substitution is M252Y; T256, wherein the substitution is T256D or T256E; K288, wherein the substitution is K288D or K288N; T307, wherein the substitution is T307A, T307E, T307F, T307M, T307Q or T307W; E380, wherein the substitution is E380C; N434, wherein the substitution is N434F, N434P or N434Y; Y436, wherein the substitution is Y436H, Y436N or Y436W. In some embodiments, the Fc domain or fragment thereof may comprise a triple amino acid substitution selected from the group consisting of: M252, wherein the substitution is M252Y; T256, wherein the substitution is T256D or T256E; K288, wherein the substitution is K288D or K288N; T307, wherein the substitution is T307A, T307E, T307F, T307M, T307Q, or T307W; E380, wherein the substitution is E380C; N434, wherein the substitution is N434F, N434P, or N434Y; and Y436, wherein the substitution is Y436H, Y436N, or Y436W. In some embodiments, the Fc domain or fragment thereof may comprise a quadruple amino acid substitution selected from: M252, wherein the substitution is M252Y; T256, wherein the substitution is T256D or T256E; K288, wherein the substitution is K288D or K288N; T307, wherein the substitution is T307A, T307E, T307F, T307M, T307Q, or T307W; E380, wherein the substitution is E380C; N434, wherein the substitution is N434F, N434P, or N434Y; Y436, wherein the substitution is Y436H, Y436N, or Y436W. In some embodiments, it may be desirable for the Fc domain or fragment thereof to comprise an amino acid substitution at any amino acid position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N or Y436W, and any combination thereof, wherein amino acid position N434 is not substituted with phenylalanine (F) or tyrosine (Y). In some embodiments, it may be desirable for the Fc domain or fragment thereof to comprise an amino acid substitution at any amino acid position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N or Y436W, and any combination thereof, wherein amino acid position N434 is not substituted with tyrosine (Y).In some embodiments, it may be desirable for the Fc domain or fragment thereof to comprise an amino acid substitution at any amino acid position selected from M252Y, T256D, T256E, K288D, K288N, T307A, T307E, T307F, T307M, T307Q, T307W, E380C, Y436H, Y436N or Y436W, and any combination thereof, wherein amino acid position N434 is not substituted (i.e., amino acid position N434 is wild type).

[0400] In certain embodiments, the Fc domain or its fragment may include an amino acid replacement selected from M252, T256, T307 or N434 and any combination thereof. In certain embodiments, the Fc domain or its fragment may include a dual amino acid replacement at any two amino acid positions selected from M252, T256, T307 and N434. In certain embodiments, the Fc domain or its fragment may include a triple amino acid replacement at any three amino acid positions selected from M252, T256, T307 and N434. In certain embodiments, the Fc domain or its fragment may include a quadruple amino acid replacement at amino acid positions M252, T256, T307 and N434. In some embodiments, it is desirable that the Fc domain or its fragment include an amino acid replacement selected from M252, T256 or T307 and any combination thereof, wherein amino acid position N434 is not substituted (that is, amino acid position N434 is wild type).

[0401] In exemplary embodiments, the Fc domain or fragment thereof may comprise an amino acid substitution selected from the group consisting of: M252, wherein the substitution is M252Y; T256, wherein the substitution is T256D or T256E; T307, wherein the substitution is T307Q or T307W; or N434, wherein the substitution is N434F or N434Y, and any combination thereof. In certain embodiments, the Fc domain or fragment thereof may comprise a double amino acid substitution at any two amino acid positions selected from the group consisting of: M252, wherein the substitution is M252Y; T256, wherein the substitution is T256D or T256E; T307, wherein the substitution is T307Q or T307W; or N434, wherein the substitution is N434F or N434Y. In certain embodiments, the Fc domain or fragment thereof may comprise a triple amino acid substitution at any three amino acid positions selected from the group consisting of: M252, wherein the substitution is M252Y; T256, wherein the substitution is T256D or T256E; T307, wherein the substitution is T307Q or T307W; or N434, wherein the substitution is N434F or N434Y. In certain embodiments, the Fc domain or fragment thereof may comprise a quadruple amino acid substitution at amino acid positions selected from the group consisting of: M252, wherein the substitution is M252Y; T256, wherein the substitution is T256D or T256E; T307, wherein the substitution is T307Q or T307W; or N434, wherein the substitution is N434F or N434Y. In some embodiments, it may be desirable for the Fc domain or fragment thereof to comprise an amino acid substitution selected from the group consisting of M252Y, T256D, T256E, T307Q, or T307W, and any combination thereof, wherein amino acid position N434 is not substituted with phenylalanine (F) or tyrosine (Y). In some embodiments, it may be desirable for the Fc domain or fragment thereof to comprise an amino acid substitution selected from the group consisting of M252Y, T256D, T256E, T307Q, or T307W, and any combination thereof, wherein amino acid position N434 is not substituted with tyrosine (Y).

[0402] In some embodiments, it may be desirable for the Fc domain or fragment thereof to comprise an amino acid substitution selected from M252Y, T256D, T256E, T307Q, or T307W, and any combination thereof, wherein amino acid position N434 is not substituted (i.e., amino acid position N434 is wild-type).

[0403] In certain embodiments, the Fc domain or fragment thereof may comprise an amino acid substitution selected from T256D or T256E and / or T307W or T307Q, and further comprise an amino acid substitution selected from N434F or N434Y or M252Y. In some embodiments, it may be desirable that the Fc domain or fragment thereof comprise an amino acid substitution selected from T256D or T256E and / or T307W or T307Q, and further comprise an amino acid substitution M252Y, wherein amino acid position N434 is not substituted with phenylalanine (F) or tyrosine (Y). In some embodiments, it may be desirable that the Fc domain or fragment thereof comprise an amino acid substitution selected from T256D or T256E and / or T307W or T307Q, and further comprise an amino acid substitution M252Y, wherein amino acid position N434 is not substituted with tyrosine (Y). In some embodiments, it may be desirable for the Fc domain or fragment thereof to comprise an amino acid substitution selected from T256D or T256E and / or T307W or T307Q, and further comprise the amino acid substitution M252Y, wherein amino acid position N434 is not substituted with tyrosine (Y) (i.e., amino acid position N434 is wild type).

[0404] In some embodiments, the Fc domain or fragment thereof may comprise the amino acid substitutions shown in Figure 33 of WO 2021 / 016571. For example, the Fc domain or fragment thereof may comprise the double amino acid substitution M252Y / N434Y (YY); or a triple amino acid substitution selected from M252Y / T307W / N434Y (YWY), M252Y / T256D / N434Y (YDY), and T256D / 307W / N434Y (DWY).

[0405] In some embodiments, the Fc domain or fragment thereof may comprise a quadruple amino acid substitution selected from the group consisting of M252Y / T256D / T307Q / N434F (YDQF), M252Y / T256D / T307W / N434F (YDWF), M252Y / T256D / T307Q / N434Y (YDQY), M252Y / T256E / T307Q / N434Y (YEQY), M252Y / T256D / T307W / N434Y (YDWY) and M252Y / T256E / T307W / N434Y (YEWY).

[0406] In other embodiments, the Fc domain or fragment thereof comprises a combination of the following four amino acid residues:

[0407] a) tyrosine (Y) at amino acid position 252,

[0408] b) aspartic acid (D) or glutamic acid (E) at amino acid position 256,

[0409] c) tryptophan (W) or glutamine (Q) at amino acid position 307, and

[0410] d) phenylalanine (F) or tyrosine (Y) at amino acid position 434;

[0411] According to Eu numbering.

[0412] In certain embodiments, the amino acid sequence of the Fc domain or fragment thereof comprises or consists of the amino acid sequence shown in SEQ ID NO: 170 having at least one amino acid substitution listed above.

[0413] In certain embodiments, the amino acid sequence of the Fc domain of the variant Fc region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 171 with at least one of the amino acid substitutions listed above.

[0414] In certain embodiments, the amino acid sequence of the Fc domain of the variant Fc region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 172 with at least one of the amino acid substitutions listed above.

[0415] In certain embodiments, the amino acid sequence of the Fc domain of the variant Fc region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 164 with at least one of the amino acid substitutions listed above.

[0416] Therefore, if the Fc domain is in the form of a dimer, i.e., comprises at least one hinge region or a portion thereof, two CH2 domains, and two CH3 domains, i.e., two polypeptide fragments, the Fc domain may comprise two polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO: 170 having at least one amino acid substitution listed above. Therefore, if the Fc domain comprises at least one hinge region or a portion thereof, two CH2 domains, and two CH3 domains, i.e., two polypeptide fragments, the Fc domain may comprise two polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO: 171 having at least one amino acid substitution listed above. Therefore, if the Fc domain comprises at least one hinge region or a portion thereof, two CH2 domains, and two CH3 domains, i.e., two polypeptide fragments, the Fc domain may comprise two polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO: 172 having at least one amino acid substitution listed above. Thus, if the Fc domain comprises at least one hinge region or a portion thereof, two CH2 domains, and two CH3 domains, i.e., two polypeptide fragments, the Fc domain may comprise two polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO: 164 having at least one of the amino acid substitutions listed above. In these embodiments, at least one, and preferably both, CH3 domains contained in the Fc domain included in the FcRn antagonists of the present technology may comprise a knob-in-hole mutation as defined above. Additionally or alternatively, the amino acid HY in the CH3 domain of the Fc domain may be mutated to RF, such as at positions 435 and 436 (H435R and Y436F in the CH3 domain, as described by Jendeberg, L. et al. (1997, J. Immunological Meth., 201: 25-34)).

[0417] In other embodiments, the amino acid sequence of the Fc domain or fragment thereof comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 170, 171, 172, or 164, having at least one amino acid substitution as listed above, and further comprises a linker sequence at the N- and / or C-terminal region of the sequence, for example, as set forth in Table A-2. In preferred embodiments, the linker comprises or consists of SEQ ID NOs: 38, 39, 40, 41, 42, or 200. In further preferred embodiments, the linker comprises the N-terminal region of an Fc domain or FcRn-binding fragment as set forth in any one of SEQ ID NOs: 170, 171, 172, or 164 (having at least one amino acid substitution as listed above). In further preferred embodiments, the linker comprises or consists of SEQ ID NOs: 38 or 200.

[0418] In other embodiments, the Fc domain or fragment thereof comprises a combination of the following four amino acid residues:

[0419] a) tyrosine (Y) at amino acid position 252,

[0420] b) aspartic acid (D) or glutamic acid (E) at amino acid position 256,

[0421] c) tryptophan (W) or glutamine (Q) at amino acid position 307, and

[0422] d) phenylalanine (F) or tyrosine (Y) at amino acid position 434;

[0423] According to Eu numbering.

[0424] In other embodiments, the Fc domain or fragment thereof comprises a combination of amino acid residues selected from:

[0425] a) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434;

[0426] b) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434;

[0427] c) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434;

[0428] d) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 434;

[0429] e) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434; and

[0430] f) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and phenylalanine (F) at amino acid position 434.

[0431] In certain embodiments, the Fc domain can be mutated to reduce effector function using techniques known in the art. In some embodiments, the Fc modified herein also has a changed binding affinity to Fc-gamma receptors (FcγR). FcγR belongs to a family comprising several members (e.g., FcγRI, FcγRIla, FcγRIllb, FcγRIIla and FcγRHIb). In some embodiments, compared with wild-type Fc domains, the Fc modified herein has enhanced FcRn binding affinity, but has reduced FcγRIIla binding affinity. In certain embodiments, variant Fc has increased affinity to FcγRIIla (referred to as CD 16a in this application).

[0432] In certain embodiments of the present technology, the polypeptides disclosed herein comprise (i) at least one domain comprising a serum albumin protein and / or at least one domain that specifically binds to a serum albumin protein and (ii) partial or full-length IgG. In further specific embodiments, the polypeptides disclosed herein comprise (i) at least one domain comprising a serum albumin protein and / or at least one domain that specifically binds to a serum albumin protein and (ii) partial or full-length IgG4. In yet further specific embodiments, the polypeptides disclosed herein comprise (i) at least one domain comprising a serum albumin protein and / or at least one domain that specifically binds to a serum albumin protein and (ii) partial or full-length IgG1.

[0433] In further specific embodiments, the polypeptides disclosed herein comprise (i) at least one domain comprising a serum albumin protein and (ii) a full-length IgG (such as full-length IgG1 or full-length IgG4).

[0434] In further specific embodiments, the polypeptides disclosed herein comprise (i) at least one domain that specifically binds to a serum albumin protein and (ii) a full-length IgG (such as full-length IgG1 or full-length IgG4).

[0435] In further specific embodiments, the polypeptides according to the present technology comprise at least one Fc domain that specifically binds to FcRn with an affinity (K A ) is at least ten times higher than the affinity of the same polypeptide for FcRn at a neutral or physiological pH of 7.4. In yet other specific embodiments, the affinity (K) of a polypeptide according to the present technology for binding to FcRn at an acidic pH between 5.0 and 6.8 is A ) has an affinity that is at least fifty times greater, such as at least one hundred times greater, than the same polypeptide for FcRn at a neutral or physiological pH of 7.4.

[0436] In certain embodiments, at physiological pH, such as at a pH of 7.4, the at least one Fc domain is expressed at a pH below 10. 4 Liter / mol K A Values ​​for binding to FcRn.

[0437] In certain specific embodiments, the present technology provides polypeptides as described herein, characterized in that the at least one Fc domain does not detectably, selectively or specifically bind to FcRn, or exhibits no or substantially no binding to FcRn at neutral or physiological pH, such as at a pH of 7.4.

[0438] The dissociation constant (K) of the polypeptides of the present invention for FcRn at acidic pH, preferably at a pH between 5.0 and 6.8 is D ) is greater than the dissociation constant (K) of the same polypeptide for FcRn at neutral or physiological pH of about 7.4. D ) is at least three times better (i.e., a lower value). In another specific embodiment, the polypeptides of the present technology have a dissociation constant (K) for FcRn at an acidic pH, preferably at a pH between 5.0 and 6.8. D ) is at least ten times higher / better than the dissociation constant of the same polypeptide for FcRn at a neutral or physiological pH of about 7.4. In yet other specific embodiments, the polypeptides of the present technology have a dissociation constant (K) for FcRn at an acidic pH, preferably at a pH between 5.0 and 6.8. D ) is at least fifty times higher / better, such as at least one hundred times higher, than the dissociation constant of the same polypeptide for FcRn at a neutral or physiological pH of about 7.4.

[0439] Thus, the present technology relates to polypeptides comprising at least one Fc domain that specifically binds to FcRn at an acidic pH between about 6.0 and 7.4, preferably at a pH of about 6.0, with an average K D Values ​​between 1 nM and 250 nM, such as the average K D A value of 250 nM or less is even more preferred, with an average K D Preferably, K is 200 nM, 150 nM, 100 nM, 50 nM or even less, such as less than 40, 30, 20, 10, 5, 1 nM, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 pM or even less, such as less than 10 pM. D is determined by Kinexa, BLI or surface plasmon resonance (SPR), for example, as determined by SPR. Preferably, the average K is measured by performing SPR on the recombinant protein. D .

[0440] In certain embodiments, at an acidic pH between 5.0 and 6.8, polypeptides according to the present technology specifically bind to FcRn with an association rate constant (k on ) is selected from at least about 10 2 M -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 as measured by surface plasmon resonance or BLI.

[0441] In certain embodiments, at an acidic pH between 5.0 and 6.8, a polypeptide according to the present technology specifically binds to FcRn with a dissociation rate constant (k off ) is selected from up to about 10 -1 s -1 , up to about 10 -2 s -1 , up to about 10 -3 s -1 , up to about 10 -4 s -1 , up to about 10 -5 s -1 and at most about 10 -6 s -1 , preferably as measured by surface plasmon resonance or BLI.

[0442] In certain embodiments, at an acidic pH between 5.0 and 6.8, a polypeptide according to the present technology specifically binds to FcRn with a dissociation rate constant (k off ) is selected from up to about 10 -1 s -1 , up to about 10 -2 s -1 , up to about 10 -3 s -1 , up to about 10 -4 s -1 , up to about 10 -5 s-1 and at most about 10 -6 s -1 The dissociation rate constant (k) of these amino acid sequences and polypeptides towards FcRn at acidic pH, preferably at a pH between 5.0 and 6.8 is off ) is greater than the dissociation rate constant (k) of the same amino acid sequence and polypeptide to FcRn at neutral or physiological pH of 7.4. off In another specific embodiment, at an acidic pH between 5.0 and 6.8, the dissociation rate constant (k) of the polypeptide according to the present technology for binding to FcRn is at least three times lower. off ) is greater than the dissociation rate constant (k) of the same polypeptide from FcRn at neutral or physiological pH of 7.4. off In yet another specific embodiment, at an acidic pH between 5.0 and 6.8, the dissociation rate constant (k) for binding of a polypeptide according to the present technology to FcRn is at least ten times lower. off ) is greater than the dissociation rate constant (k) of the same polypeptide from FcRn at neutral or physiological pH of 7.4. off ) is at least fifty times lower, such as at least one hundred times lower.

[0443] In certain specific embodiments, the present technology provides polypeptides as described herein, characterized in that the at least one Fc domain binds to FcRn at a neutral or physiological pH of 7.4 with a dissociation rate constant (k off ) than the dissociation rate constant (k) of the at least one Fc domain for binding to FcRn at an acidic pH between 5.0 and 6.8 off ) is at least three times higher, such as at least ten times, such as at least fifty times, such as at least one hundred times higher.

[0444] 5.5 Polypeptides of the present invention

[0445] Thus, as explained in detail above, the polypeptides of the present technology comprise or consist of (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin binding ISVD) and (ii) at least one IgG Fc domain or a fragment thereof, preferably an FcRn binding fragment thereof.

[0446] In certain embodiments, the present technology provides polypeptides comprising at least one domain that is or specifically binds to a serum albumin protein and an IgG Fc domain, such that the molecular weight of the polypeptide is at least 30 kDa, particularly, between about 30 kDa and 250 kDa, more particularly between about 65 kDa and 220 kDa, such as between about 65 kDa and 200 kDa, such as between about 65 kDa and 180 kDa, between about 65 kDa and 170 kDa, such as between about 65 kDa and 160 kDa, particularly between about 65 kDa and 150 kDa, more particularly between about 65 kDa and 130 kDa, most particularly between about 65 kDa and 120 kDa. In certain embodiments, the present technology provides polypeptides comprising at least one domain that is or specifically binds to a serum albumin protein and an IgG Fc domain, such that the molecular weight of the polypeptide is preferably about 120 kDa, 110 kDa, 100 kDa, 90 kDa, 85 kDa, 80 kDa, 75 kDa, 70 kDa, such as about 65 kDa.

[0447] By specifically binding to the FcRn receptor and / or otherwise being directed against FcRn, the polypeptides of the present technology are particularly suitable for extending the in vivo half-life of a therapeutic target or therapeutic molecule of interest to which they are appropriately linked, conjugated or fused (as demonstrated by the Examples further described herein).

[0448] As used herein, the term "half-life" may generally be defined as described in paragraph o) on page 57 of WO 2008 / 020079 and, as mentioned therein, refers to the time it takes 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 sequestration of the sequence or compound by natural mechanisms. The in vivo half-life of the polypeptides and / or fusion proteins of the present technology may be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to those skilled in the art and may generally be described, for example, in paragraph o) on page 57 of WO 2008 / 020079. As also mentioned in paragraph o) on page 57 of WO 2008 / 020079, a method such as t 1 / 2 -α, t 1 / 2 -β and area under the curve (AUC) are used to represent the half-life. In this respect, it should be noted that the term "half-life" as used herein refers in particular to t 1 / 2 -β or terminal half-life (where t 1 / 2-α and / or AUC or both may not be taken into account). For example, reference is made to 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). Reference is also made to "Pharmacokinetics", M Gibaldi and D Perron, published by Marcel Dekker, 2nd revised edition (1982). Similarly, the term "increase in half-life" or "increased half-life" is also as defined in WO 2008 / 020079, page 57, paragraph o), and refers in particular to t 1 / 2 -β increases, regardless of t 1 / 2 - Whether alpha and / or AUC or both are increased.

[0449] The half-life in a mammalian species depends primarily on the binding properties (such as affinity) of the polypeptides and / or fusion proteins of the present technology to serum albumin from said mammalian species and the half-life of naive serum albumin in said species, among other factors.

[0450] The half-life of a polypeptide according to the present technology, or a fusion protein, construct, or compound comprising the same (and further described herein) can generally be defined as the time it takes for the serum concentration of the amino acid sequence, compound, or polypeptide to decrease by 50% in vivo, e.g., due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms. In particular, the half-life can be as defined in WO 2009 / 068627.

[0451] The in vivo half-life of a polypeptide according to the present technology, or a fusion protein, construct or compound comprising the same (and further described herein) can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the skilled person, and may, for example, generally involve the following steps: suitably administering to a warm-blooded animal (i.e., a human or another suitable mammal, such as a mouse, rabbit, rat, pig, dog or primate, e.g., a monkey from the genus Macaca (e.g., and in particular, a cynomologus monkey (Macaca fascicularis) and / or a rhesus monkey (Macaca mulatta) and a baboon (Papio ursinus)) a suitable dose of an amino acid sequence, compound or polypeptide of the present technology; collecting a blood or other sample from the animal; determining the level or concentration of the amino acid sequence, compound or polypeptide of the present technology in the blood sample; and calculating from the data thus obtained (plotting) the time at which the level or concentration of the amino acid sequence, compound or polypeptide of the present technology is reduced by 50% compared to the initial level at the time of administration. Reference is made, for example, to the Experimental section below, and to standard handbooks, such as Kenneth, A et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinete analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi and D Perron, published by Marcel Dekker, 2nd revised edition (1982). Parameters such as t1 / 2-α, t1 / 2-β and the area under the curve (AUC) can be used to express the half-life. For example, reference is made to the experimental section below, as well as to standard manuals such as Kenneth et al. 1996 (Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists) and Peters et al., 1996 (Pharmacokinetic Analysis: A Practical Approach). Reference is also made to Gibaldi and Perron 1982 (Pharmacokinetics, Dekker M, 2nd revised edition). In the present specification, "an increase in half-life" refers to an increase in any one of these parameters, such as any two of these parameters or essentially all three of these parameters. The term "increase in half-life" or "increased half-life" refers in particular to t 1 / 2-β increases, regardless of t 1 / 2 - Whether alpha and / or AUC or both are increased.

[0452] In the context of the present technology, the term "clearance" or "clearance rate" (systemic plasma or serum clearance) is defined as the rate of drug elimination divided by the plasma concentration of the drug (the rate at which a substance is cleared from the plasma compartment of the blood). The clearance rate of a substance is the volume of plasma that contains the same amount of substance as is removed from the plasma per unit time. As described, for example, in the Examples, clearance or clearance rate can be measured by collecting blood at regular intervals and analyzing its composition. For example, blood can be extracted at different time points and serum can be prepared. The presence of a polypeptide in a serum sample can be analyzed, for example, by ELISA. PK parameters such as clearance can be determined using the Plasma Data Module in Phoenix (Version 8.2.2.227. Certara) obtained from non-compartmental analysis. For further details, see the Examples. The following formula can be used to calculate the clearance:

[0453]

[0454] Thus, in particular embodiments, polypeptides according to the present technology, such as the FcRn-binding polypeptides of the present technology (including fusion proteins, constructs, and compounds comprising such polypeptides) will have an increased or prolonged half-life and / or a decreased or reduced clearance rate compared to known polypeptides (described in the prior art) that bind to and / or are otherwise directed against FcRn (such as an Fc domain by itself or an Fc domain linked to a domain that does not specifically bind to a serum albumin protein).

[0455] Furthermore, the polypeptides according to the present technology (such as the FcRn binding polypeptides of the present technology), if fused to another moiety (such as one or more therapeutic moieties), will have increased half-life and / or decreased clearance compared to the other moiety by itself (such as the other one or more therapeutic moieties by themselves).

[0456] Typically, the half-life of polypeptides according to the present technology, such as the FcRn binding polypeptides of the present technology (and fusion proteins, constructs, and compounds comprising such polypeptides), is preferably at least 1.5-fold, preferably at least 2-fold, such as at least 5-fold, for example at least 10-fold or more than 20-fold or greater, than the half-life of known polypeptides (described in the prior art) that bind to FcRn (such as the Fc domain by itself or an Fc domain linked to a domain that does not specifically bind to a serum albumin protein) (as measured in humans or suitable animals such as mice or cynomolgus monkeys).

[0457] Furthermore, the half-life of polypeptides according to the present technology, such as the FcRn-binding polypeptides of the present technology (and fusion proteins, constructs, and compounds comprising such polypeptides), is preferably increased by at least 30%, at least 50%, at least 75%, for example at least 100% of the half-life, or by more than 200%, such as more than 300%, more than 400%, more than 500% or more, compared to the half-life of known polypeptides (described in the prior art) that bind to and / or are otherwise directed against FcRn, such as the Fc domain by itself or an Fc domain linked to a domain that does not specifically bind to a serum albumin protein (as measured in humans or suitable animals such as mice or cynomolgus monkeys).

[0458] Furthermore, the clearance or clearance rate as defined herein of polypeptides according to the present technology, such as the FcRn-binding polypeptides of the present technology (and fusion proteins, constructs, and compounds comprising such polypeptides), is preferably reduced or decreased by at least about 10%, such as at least about 20%, or at least about 25%, or at least about 30%, or at least about 50%, or at least about 75%, or at least about 80%, or at least about 90% or more, compared to the clearance or clearance rate of known polypeptides (described in the prior art) that bind to and / or otherwise target FcRn, such as the Fc domain by itself or an Fc domain linked to a domain that does not specifically bind to a serum albumin protein (as measured in humans or suitable animals such as mice or cynomolgus monkeys).

[0459] Furthermore, the clearance or clearance rate as defined herein of the polypeptides according to the present technology, such as the FcRn-binding polypeptides of the present technology (and fusion proteins, constructs, and compounds comprising such polypeptides), is preferably reduced or decreased by at least 1.1 fold, such as at least 1.2 fold, or at least 1.3 fold, or at least 1.5 fold, or at least 2 fold, or at least 2.5 fold, or at least 3 fold, or at least 4 fold, or at least 5 fold, or at least 7 fold, or at least 8 fold, or at least 9 fold, or at least 10 fold or more compared to the clearance or clearance rate of known polypeptides (described in the prior art) that bind to and / or otherwise target FcRn, such as the Fc domain by itself or an Fc domain linked to a domain that does not specifically bind to a serum albumin protein (as measured in humans or suitable animals such as mice or cynomolgus monkeys).

[0460] Furthermore, polypeptides of the present technology comprising at least one domain comprising a serum albumin protein or specifically binding to a serum albumin protein and at least one IgG Fc domain, such as the FcRn binding polypeptides according to the present technology (including fusion proteins, constructs, and compounds comprising the same), if fused to another moiety (such as one or more therapeutic moieties), will have an increased half-life compared to the other moiety by itself (as is), such as the other one or more therapeutic moieties by themselves (as is).

[0461] Typically, the half-life of a polypeptide, construct or fusion protein described herein comprising a drug and / or therapeutic moiety is preferably at least 1.1-fold, such as at least 1.2-fold or at least 1.5-fold, preferably at least 2-fold, such as at least 3-fold or at least 5-fold, for example at least 10-fold or greater than 20-fold, such as greater than 50-fold, greater than 100-fold, greater than 500-fold, preferably greater than 1000-fold, greater than the half-life of the corresponding other moiety itself, such as the drug and / or therapeutic moiety itself (as measured in humans or a suitable animal such as a mouse or cynomolgus monkey).

[0462] Typically, the clearance rate as defined herein of a polypeptide, construct or fusion protein described herein comprising a drug and / or therapeutic moiety is at least 1.1-fold, such as at least 1.2-fold or at least 1.5-fold, preferably at least 2-fold, such as at least 3-fold, or at least 4-fold, or at least 5-fold, for example at least 10-fold or greater than 20-fold, such as greater than 50-fold, greater than 100-fold or more, slower than the corresponding other moiety itself, such as the half-life of the drug and / or therapeutic moiety itself (as measured in humans or suitable animals such as mice or cynomolgus monkeys).

[0463] As described above, in one aspect, polypeptides of the present technology (such as FcRn binding polypeptides according to the present technology) can be used to increase the half-life and / or reduce the clearance rate of (one or more) immunoglobulin single variable domains (ISVDs), such as domain antibodies, single domain antibodies, "dAbs", VHHs or VHH (such as VHH, humanized VHH or camelized VH, such as camelized human VH).

[0464] Furthermore, the half-life (defined as t 1 / 2β) is preferably more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, and for example about one day, two days, one week, about 13 days, about two weeks, about 16 days or about 17 days, and up to and even more than the half-life of serum albumin (i.e., about 19 days in humans) or up to and even more than the half-life of IgG (i.e., about 23 days for wild-type IgG and up to 90 days for engineered IgG in humans), such as 3 months, 4 months, 5 months to 6 months or more.

[0465] Furthermore, the half-life (defined as t 1 / 2 β) is preferably more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, and for example about one day, two days, one week, about 13 days, two weeks, about 16 days, or about 17 days, or about 18 days, or about 20 days, or about 23 days, or about 25 days, or about 30 days or longer), or up to and even exceeding the half-life of serum albumin, or up to and exceeding the half-life of IgG, or longer.

[0466] In addition, the clearance rate of the polypeptides provided by the present technology and fusion proteins, constructs and compounds comprising the same (as further described herein) in mice is preferably less than 1 mL / hr / kg, preferably less than 0.8 mL / hr / kg, more preferably less than 0.6 mL / hr / kg, such as less than 0.5 mL / hr / kg or less than 0.3 mL / hr / kg, or less than 0.2 mL / hr / kg, such as about 0.16 mL / hr / kg, or about 0.15 mL / hr / kg, or about 0.1 mL / hr / kg, or about 0.09 mL / hr / kg, or about 0.08 mL / hr / kg, or even less than the clearance rate of serum albumin and / or less than the clearance rate of IgG.

[0467] The polypeptides according to various embodiments of the present technology preferably also include:

[0468] Their serum half-life in humans (expressed as t 1 / 2β) more than 6 hours, preferably more than 12 hours, more preferably more than 24 hours, even more preferably more than 72 hours; for example, about one week, two weeks, or about 16 days, or about 17 days, or about 18 days, or about 20 days, or about 23 days, or about 25 days, or about 30 days or more, and up to and even exceeding the half-life of serum albumin (i.e., about 19 days in humans) or up to and exceeding the half-life of IgG (i.e., about 23 days for wild-type IgG and up to 90 days for engineered IgG in humans); and / or such that: when they are linked to a therapeutic moiety or entity, they confer a serum half-life in humans (expressed as t 1 / 2 β) is more than 6 hours, preferably more than 12 hours, more preferably more than 24 hours, even more preferably more than 72 hours; for example, about one week, two weeks and up to and even more than the half-life of serum albumin (i.e., about 19 days in humans) or up to and more than the half-life of IgG (i.e., about 23 days for wild-type IgG in humans and up to 90 days for engineered IgG), such as 3 months, 4 months, 5 months to 6 months or more.

[0469] Among other factors, the half-life in mammalian species other than humans depends primarily on the binding properties (such as affinity) of the polypeptide of the present invention to serum albumin and / or FcRn from said mammalian species and the half-life of naive serum albumin and IgG in said species. According to a preferred embodiment of the present invention, when the FcRn-binding polypeptide of the present invention cross-reacts (as defined herein) between human serum albumin and serum albumin from another mammalian species or between human FcRn and FcRn from another mammalian species, then the half-life of the polypeptide of the present invention (and / or a compound of the present invention comprising the polypeptide) as determined in said species is preferably at least 5%, such as at least 10%, more preferably at least 25%, for example about 50%, about 100%, such as about 125%, about 150% to about 200% or more of the half-life of serum albumin or IgG, respectively, in said species.

[0470] The polypeptides according to various embodiments of the present technology preferably also include:

[0471] Their serum half-life in mice (expressed as t 1 / 2) is more than 6 hours, preferably more than 12 hours, more preferably more than 24 hours, even more preferably more than 72 hours; for example, about one week, two weeks, or about 16 days, or about 17 days, or about 18 days, or about 20 days, or about 23 days, or about 25 days, or about 30 days or more, and up to and even exceeding the half-life of serum albumin or up to and exceeding the half-life of IgG; and / or such that: when they are linked to a therapeutic moiety or entity, they confer a serum half-life (expressed as t) in mice to the resulting polypeptide constructs of the present technology. 1 / 2 ) is more than 6 hours, preferably more than 12 hours, more preferably more than 24 hours, even more preferably more than 72 hours; for example, about one week, two weeks, or about 16 days, or about 17 days, or about 18 days, or about 20 days, or about 23 days, or about 25 days, or about 30 days or more, and up to and even exceeding the half-life of serum albumin or up to and exceeding the half-life of IgG, such as 3 months, 4 months, 5 months to 6 months or more.

[0472] The half-life of the polypeptides and / or fusion proteins described herein is preferably 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, preferably at least 2-fold, preferably at least 3-fold, at least 4-fold, such as at least 5-fold, for example at least 10-fold or more than 20-fold greater than the half-life of therapeutic constructs disclosed in the prior art comprising a therapeutic moiety and a known half-life extending moiety (as measured in humans or suitable animals such as mice or cynomolgus monkeys).

[0473] Thus, the present technology provides improved polypeptides that can be used in a variety of applications, including but not limited to extending the in vivo half-life and / or reducing the clearance rate of (existing or future) therapeutic compounds, as described herein. In certain embodiments, the polypeptides of the present technology have high affinity for both serum albumin and FcRn.

[0474] In a specific embodiment, the present technology provides a polypeptide as described herein, characterized in that the polypeptide further comprises a therapeutic moiety, which preferably comprises a (single) domain antibody, As mentioned, in one aspect, the polypeptides according to the present technology can be used to increase the protein structure of (one or more) immunoglobulin single variable domains (ISVDs) such as domain antibodies, single domain antibodies, "dAbs", VHH or VHH. VHH (such as V HH , humanized V HH or Camelized V H Such as Camel V H ) half-life and / or reduced clearance (ie, improved PK parameters).

[0475] In certain embodiments, the polypeptides of the present technology comprise at least one ISVD that binds serum albumin with high affinity / specificity, at least one IgG Fc domain, and at least one second ISVD that binds serum albumin with high affinity / specificity, and a therapeutically relevant antigen other than FcRn and serum albumin.

[0476] It will be appreciated (as also demonstrated in the Examples section) that the albumin binding domains, such as an ISVD that binds serum albumin and, optionally, an ISVD that binds a therapeutic target other than FcRn and albumin, can be positioned in any order in the polypeptides of the present technology. More specifically, in one embodiment, the ISVD that binds serum albumin is positioned at the N-terminus and the ISVD that binds another antigen is positioned at the C-terminus. In another embodiment, the ISVD that binds the other antigen is positioned at the N-terminus and the ISVD that binds serum albumin is positioned at the C-terminus.

[0477] Thus, in one embodiment, the albumin and / or albumin-binding domain (such as an albumin-binding ISVD) contained in the polypeptides of the present technology may be located at the N-terminal portion of the polypeptide, for example, linked (directly or through a linker, as disclosed herein) to the N-terminal portion of the Fc domain (i.e., if the Fc domain is a dimer, then linked to the N-terminal portion of either chain of the Fc domain). In another embodiment, the albumin and / or albumin-binding domain (such as an albumin-binding ISVD) contained in the polypeptides of the present technology may be located at the C-terminal portion of the polypeptide, for example, linked (directly or through a linker, as disclosed herein) to the C-terminal portion of the Fc domain (i.e., if the Fc domain is a dimer, then linked to the C-terminal portion of either chain of the Fc domain). The polypeptides of the present technology may also comprise two albumin and / or albumin binding domains, such as two albumin binding ISVDs (see, e.g., SEQ ID NOs: 7-21 or 61-69), one linked (directly or through a linker, as disclosed herein) to the N-terminal portion of the Fc domain (i.e., if the Fc domain is a dimer, to the N-terminal portion of either chain of the Fc domain) and the other linked (directly or through a linker, as disclosed herein) to the C-terminal portion of the Fc domain (i.e., if the Fc domain is a dimer, to the C-terminal portion of either chain of the Fc domain). The polypeptides of the present technology may also comprise two albumin and / or albumin binding domains, such as two albumin binding ISVDs (see, e.g., SEQ ID NOs: 7-21 or 61-69), both linked (directly or through a linker, as disclosed herein) to the N-terminal portion of the Fc domain (i.e., if the Fc domain is a dimer, to the N-terminal portion of either chain of the Fc domain). The polypeptides of the present technology may also comprise two albumin and / or albumin binding domains, such as two albumin binding ISVDs (see, e.g., SEQ ID NOs: 7-21 or 61-69), both of which are linked (directly or through a linker, as disclosed herein) to the C-terminal portion of the Fc domain (i.e., if the Fc domain is a dimer, to the C-terminal portion of either chain of the Fc domain). The polypeptides of the present technology may also comprise more than two albumin and / or albumin binding domains, such as two albumin binding ISVDs (see, e.g., SEQ ID NOs: 7-21 or 61-69), such as three or four albumin and / or albumin binding domains, such as two albumin binding ISVDs.

[0478] In addition to albumin and / or albumin binding domains, the polypeptides of the present technology may comprise other groups or moieties or binding units as described herein, such as therapeutic moieties, drugs, vaccines and / or imaging agents. Thus, one or more groups or moieties or binding units as described herein, such as therapeutic moieties, drugs, vaccines and / or imaging agents comprised in the polypeptides of the present technology, may be linked (directly, or with the aid of a linker as described herein) to the N-terminal portion of the Fc domain (i.e., if the Fc domain is a dimer, to the N-terminal portion of either chain of the Fc domain). In other embodiments, one or more groups or moieties or binding units as described herein, such as therapeutic moieties, drugs, vaccines and / or imaging agents comprised in the polypeptides of the present technology, may be linked (directly, or via a linker as disclosed herein) to the C-terminal portion of the Fc domain (i.e., if the Fc domain is a dimer, to the C-terminal portion of either chain of the Fc domain). If there is more than one group or moiety or binding unit as described herein, such as a therapeutic moiety, drug, vaccine and / or imaging agent comprised in a polypeptide of the present technology, they may be located at the N-terminal and / or C-terminal portion of the Fc domain (e.g., if it is a dimer, at the N-terminal and / or C-terminal portion of one or both chains of the Fc domain).

[0479] For example, the Fc domain contained in the polypeptides of the present technology can have one or more albumin or albumin binding domains linked (directly or via a linker, as described herein) to the N-terminal and / or C-terminal region of the Fc domain (e.g., if it is a dimer, to the N-terminal and / or C-terminal region of one or both chains of the Fc domain), and can additionally have one or more groups or moieties or binding units as described herein, such as a therapeutic moiety, drug, vaccine and / or imaging agent, which are also linked (directly or via a linker, as described herein) to the N-terminal and / or C-terminal region of the Fc domain (e.g., if it is a dimer, to the N-terminal and / or C-terminal region of one or both chains of the Fc domain). For specific examples, see Figure 1 、 Figure 3 and Figure 6 and Table A-1.

[0480] If an albumin and / or albumin binding domain or further group or moiety or binding unit as described herein (such as a therapeutic moiety, drug, vaccine and / or imaging agent) is linked (covalently linked) to the N-terminus of the Fc domain (or in the case of a dimer, to the N-terminus of one or both chains of the Fc domain), it may be linked via a peptide linker, such as a hinge linker, preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NO: 38 or 200, even more preferably comprising or consisting of SEQ ID NO: 38.

[0481] If an albumin and / or albumin binding domain or further group or moiety or binding unit as described herein (such as a therapeutic moiety, drug, vaccine and / or imaging agent) is linked (covalently linked) to the C-terminus of the Fc domain (or in the case of a dimer, to the C-terminus of one or both chains of the Fc domain), it may be linked via a peptide linker, such as a GS linker, preferably comprising or consisting of SEQ ID NOs: 25-37, more preferably comprising or consisting of SEQ ID NOs: 29 or 36.

[0482] For example, the polypeptides of the present technology can comprise a therapeutic moiety covalently linked, preferably via a hinge linker (e.g., preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38 or 200), to the N-terminus of a first chain of the heterodimeric Fc region; and an albumin or an albumin binding domain, such as an albumin binding ISVD, affitin, DARPIN or ABC protein, preferably selected from the group consisting of SEQ ID NOs: 7-21, 61-69 and 102-104, even more preferably selected from the group consisting of SEQ ID NOs: 7-21 and 61-69, linked, preferably via a peptide linker (such as a GS linker, preferably selected from the group consisting of SEQ ID NOs: 25-37), to the N-terminus of a second chain of the heterodimeric Fc region, wherein the Fc domain is preferably an IgG 4 "FALA" Fc region with a knob-to-hole mutation.

[0483] For example, the polypeptides of the present technology can comprise a therapeutic moiety covalently linked, preferably via a hinge linker (e.g., preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38 or 200); and an albumin or an albumin binding domain, such as an albumin binding ISVD, affitin, DARPIN or an ABC protein, preferably selected from the group consisting of SEQ ID NOs: 7-21, 61-69 and 102-104, even more preferably selected from the group consisting of SEQ ID NOs: 7-21 and 61-69, linked, preferably via a peptide linker (such as a GS linker, preferably selected from the group consisting of SEQ ID NOs: 25-37), to the C-terminus of the first chain of the heterodimeric Fc region, wherein the Fc domain is preferably an IgG 4 "FALA" Fc region with a knob-to-hole mutation.

[0484] For example, the polypeptides of the present technology can comprise (i) a therapeutic moiety covalently linked, preferably via a hinge linker (e.g., preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38 or 38), to the N-terminus of a first chain of the heterodimeric Fc region, (ii) a second therapeutic moiety covalently linked, preferably via a hinge linker (e.g., preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38), to the N-terminus of a second chain of the heterodimeric Fc region, and (iii) albumin or an albumin binding domain, such as an albumin binding ISVD, affitin, DARPIN or ABC protein, preferably selected from the group consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38 or 38). NO:7-21, 61-69 and 102-104, even more preferably an albumin binding domain selected from SEQ ID NO:7-21 and 61-69, which is linked, preferably via a peptide linker (such as a GS linker, preferably selected from SEQ ID NO:25-37), to the C-terminus of the first chain of the heterodimeric Fc region, wherein the Fc domain is preferably an IgG1 Fc region or an IgG4 FALA region with a knob-to-hole mutation.

[0485] For example, the polypeptides of the present technology can comprise (i) a therapeutic moiety covalently linked, preferably via a hinge linker (e.g., preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38 or 38), to the N-terminus of a first chain of the heterodimeric Fc region, (ii) a second therapeutic moiety covalently linked, preferably via a hinge linker (e.g., preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38), to the N-terminus of a second chain of the heterodimeric Fc region, and (iii) albumin or an albumin binding domain, such as an albumin binding ISVD, affitin, DARPIN or ABC protein, preferably selected from the group consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38 or 38). NO:7-21, 61-69 and 102-104, even more preferably an albumin binding domain selected from SEQ ID NO:102-104, which is linked, preferably via a peptide linker (such as a GS linker, preferably selected from SEQ ID NO:25-37), to the C-terminus of the second chain of the heterodimeric Fc region, wherein the Fc domain is preferably an IgG1 Fc region or an IgG4 FALA region with a knob-to-hole mutation.

[0486] For example, the polypeptides of the present technology may comprise (i) a therapeutic moiety covalently linked, preferably via a hinge linker (e.g., preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38 or 38), to the N-terminus of the first chain of the heterodimeric Fc region, (ii) a second therapeutic moiety covalently linked, preferably via a hinge linker (e.g., preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38), to the N-terminus of the second chain of the heterodimeric Fc region, (iii) albumin or an albumin binding domain, such as an albumin binding ISVD, affitin, DARPIN or ABC protein, preferably selected from the group consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 38), NO: 7-21, 61-69 and 102-104, even more preferably selected from SEQ ID NO: 7-21 and 61-69, an albumin binding domain, which is linked, preferably via a peptide linker (such as a GS linker, preferably selected from SEQ ID NO: 25-37), to the C-terminus of the first chain of the heterodimeric Fc region, and (iv) a second albumin or albumin binding domain, such as an albumin binding ISVD, affitin, DARPIN or ABC protein, preferably selected from SEQ ID NO: 7-21, 61-69 and 102-104, even more preferably selected from SEQ ID NO: 7-21 and 61-69, which is linked, preferably via a peptide linker (such as a GS linker, preferably selected from SEQ ID NO: 25-37), to the C-terminus of the first chain of the heterodimeric Fc region, wherein the Fc domain is preferably an IgG1 Fc region with a knob-to-hole mutation or an IgG4 FALA region.

[0487] For example, the polypeptides of the present technology may comprise (i) a therapeutic moiety covalently linked, preferably via a hinge linker (e.g., preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NO: 38 or 200, even more preferably comprising or consisting of SEQ ID NO: 38), to the N-terminus of the first chain of the heterodimeric Fc region, (ii) a second therapeutic moiety covalently linked, preferably via a hinge linker (e.g., preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NO: 38 or 200, even more preferably comprising or consisting of SEQ ID NO: 38), to the N-terminus of the second chain of the heterodimeric Fc region, and (iii) an albumin or albumin binding domain, preferably selected from the group consisting of SEQ ID NOs: 22, 23 and 110, even more preferably selected from the group consisting of SEQ ID NO: 22, 23 and 110. NO:23 and 110, which is linked, preferably via a peptide linker (such as a GS linker, preferably selected from SEQ ID NO:25-37), to the C-terminus of the second chain of the heterodimeric Fc region, wherein the Fc domain is preferably an IgG1 Fc region with a knob-to-hole mutation or an IgG4 FALA region.

[0488] For example, the polypeptides of the present technology can comprise (i) a therapeutic moiety covalently linked, preferably via a hinge linker (e.g., preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38 or 38), to the N-terminus of a first chain of the heterodimeric Fc region, (ii) a second therapeutic moiety covalently linked, preferably via a hinge linker (e.g., preferably a short G1 hinge linker, preferably comprising or consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38), to the N-terminus of a second chain of the heterodimeric Fc region, and (iii) albumin or an albumin binding domain, such as an albumin binding ISVD, affitin, DARPIN or ABC protein, preferably selected from the group consisting of SEQ ID NOs: 38-42 and / or 200, more preferably comprising or consisting of SEQ ID NOs: 38 or 200, even more preferably comprising or consisting of SEQ ID NOs: 38 or 38). NO:7-21, 61-69 and 102-104, even more preferably an albumin binding domain selected from SEQ ID NO:7-21 and 61-69, which is linked, preferably via a peptide linker (such as a GS linker, preferably selected from SEQ ID NO:25-37), to the C-terminus of the first chain of the heterodimeric Fc region, wherein the Fc domain is preferably an IgG4 FALA Fc (YTE) region with a knob-to-hole mutation.

[0489] The present technology further provides polypeptides comprising or consisting essentially of: (i) at least one domain comprising a serum albumin protein or a domain that specifically binds to a serum albumin protein and (ii) an Fc domain of immunoglobulin G (IgG), and optionally further comprising one or more other groups, residues, moieties, or binding units. As will be clear to one skilled in the art from the further disclosure herein, such other groups, residues, moieties, binding units, or amino acid sequences may or may not provide additional functionality to the polypeptides of the present technology (and / or the compounds or constructs in which they are present), and may or may not alter the properties of the polypeptides of the present technology.

[0490] In certain embodiments, the present technology provides polypeptides as described herein, characterized in that the polypeptide further comprises a therapeutic moiety as described in detail herein.

[0491] In certain embodiments, at least one therapeutic moiety comprises or consists essentially of a therapeutic protein, polypeptide, compound, factor, or other entity.

[0492] In certain embodiments, at least one domain comprising a serum albumin protein or at least one domain that specifically binds to a serum albumin protein and at least one IgG Fc domain or fragment thereof are directly linked to each other or are linked via a linker or spacer to form a polypeptide according to the present technology. In preferred embodiments, at least one domain comprising a serum albumin protein is directly linked to at least one IgG Fc domain or fragment thereof or is linked via a linker as defined herein (e.g., selected from the linkers depicted in Table A-2). Preferably, the linker is a 9GS linker, or a 35GS linker, or a G1 short hinge or short hinge linker as defined herein.

[0493] In certain embodiments, (i) at least one domain comprising a serum albumin protein or at least one domain that specifically binds to a serum albumin protein and (ii) at least one IgG Fc domain or fragment thereof are directly linked to each other or linked via a linker or spacer to form a polypeptide according to the present technology. Preferred linkers are depicted in Table A-2. Further preferred linkers are 9GS linkers, or 35GS linkers, or short hinge linkers as defined herein (e.g., SEQ ID NO: 38 or 200).

[0494] For example, (i) at least one domain comprising a serum albumin protein or specifically binding to a serum albumin protein can be linked (directly or via a linker) to (ii) the N-terminal portion of an IgG Fc domain or fragment thereof, e.g., via a hinge region or portion thereof (e.g., SEQ ID NOs: 38-42 and 200). For example, (i) at least one domain comprising a serum albumin protein or specifically binding to a serum albumin protein can be linked (directly or via a linker) to (ii) the C-terminal portion of an IgG Fc domain or fragment thereof, e.g., via a peptide linker (see, e.g., SEQ ID NOs: 25-37, preferably a 9GS or 35GS linker).

[0495] In the context of the present application, "linked via a linker" or "covalently linked via a linker" means that the linker is directly attached to the N-terminal or C-terminal region of the Fc domain or fragment thereof (such as the Fc domains defined by SEQ ID NOs: 112-113, 115-117, 164, 167-172, 181, 186-190 and 198-199) (ii), and the N-terminal or C-terminal region of at least one domain (i) comprising a serum albumin protein or specifically binding to a serum albumin protein. For example, (i) the C-terminal region of at least one domain comprising a serum albumin protein or a domain that specifically binds to a serum albumin protein (as defined by, e.g., SEQ ID NOs: 22, 23, 109, 110, 7-21, 61-69) can be linked to the N-terminal region of an IgG Fc domain or a fragment thereof (as defined by, e.g., SEQ ID NOs: 112-113, 115-117, 164, 167-172, 181, 186-190 and 198-199) (ii), wherein the linker (as defined by, e.g., SEQ ID NOs: 38-42 and 200) is directly linked to the C-terminus of the at least one domain comprising a serum albumin protein or a domain that specifically binds to a serum albumin protein (i) and directly to the N-terminus of the Fc domain or a fragment thereof (ii). For example, (i) the N-terminal region of at least one domain comprising a serum albumin protein or a domain that specifically binds to a serum albumin protein (as defined, e.g., by SEQ ID NOs: 22, 23, 109, 110, 7-21, 61-69) can be linked to the C-terminal region of an IgG Fc domain or a fragment thereof (as defined, e.g., by SEQ ID NOs: 112-113, 115-117, 164, 167-172, 181, 186-190 and 198-199) (ii), wherein the linker (as defined, e.g., by SEQ ID NOs: 25-37) is directly linked to the N-terminus of the at least one domain comprising a serum albumin protein or a domain that specifically binds to a serum albumin protein (i) and directly to the C-terminus of the Fc domain or a fragment thereof (ii).

[0496] In another embodiment, the linker is contained in the N-terminal region of the Fc domain (eg, SEQ ID NO: 112).

[0497] In other preferred embodiments, the polypeptide comprises (i) at least one domain comprising a serum albumin protein or specifically binding to a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, further comprising additional groups, residues, moieties or binding units, as described below, such as a therapeutic moiety (iii), as defined in detail below. In a specific embodiment, the at least one IgG Fc domain or fragment thereof (ii) and the at least one domain comprising a serum albumin protein or at least one domain specifically binding to a serum albumin protein (i) and the additional groups, residues, moieties or binding units (iii) are directly linked to each other or linked via a linker or spacer to form a polypeptide according to the present technology as defined in detail herein.

[0498] The additional group, residue, part or binding unit can be connected by a linker, meaning that the linker is directly attached to the N-terminal or C-terminal region of the Fc domain or its fragment. For example, the additional group, residue, part or binding unit can be connected (directly or via a linker) to the N-terminal portion of ii) the Fc domain of IgG or its fragment, for example, through the hinge region or a portion thereof, see, for example, SEQ ID NO: 38-42 or 200. For example, the additional group, residue, part or binding unit can be connected (directly or via a linker) to the C-terminal portion of ii) the Fc domain of immunoglobulin G (IgG), for example, through a peptide linker, see, for example, SEQ ID NO: 25-37. In some cases, the additional group, residue, part or binding unit is not directly connected to at least one domain (i) comprising a serum albumin protein or specifically binding to a serum albumin protein. In some cases, the additional group, residue, part or binding unit is not connected to at least one domain (i) comprising a serum albumin protein or specifically binding to a serum albumin protein by a linker (e.g., as shown in Table A-2).

[0499] Figure 1 、 Figure 3 and Figure 6 Various orientations of the polypeptides of the present application are shown, comprising (i) at least one domain comprising a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof. See also Table A-1. For example, in compound TP009 ( Figure 1), at least one domain comprising a serum albumin protein or specifically binding to a serum albumin protein (i) is located at the N-terminal portion (ii) (chain 2) of the IgG Fc domain or a fragment thereof, and is connected to the dimeric Fc domain via a hinge region, see, for example, SEQ ID NO: 38. For example, in construct TP006 ( Figure 1 ), at least one domain (i) that specifically binds to a serum albumin protein is located at the C-terminal portion of the IgG Fc domain or a fragment thereof (ii) (chain 1), and is linked to the Fc domain (polypeptide (ii)) via a peptide linker (35GS (SEQ ID NO: 36)). For example, in construct TPP-66153 ( Figure 6 ), at least one domain comprising a serum albumin protein (i) is located at the C-terminal part of the IgG Fc domain or a fragment thereof (ii) (chain 2), and is connected to the Fc domain (polypeptide (ii)) via a peptide linker (35GS (SEQ ID NO: 36)).

[0500] In some embodiments, the polypeptides of the present invention do not comprise a protease-cleavable linker, such as a mouse or human matrix metalloproteinase (MMP) linker. In particular, the polypeptides of the present invention do not comprise a matrix metalloproteinase (MMP) linker, such as GPLGMWSR (SEQ ID NO: 125) or GPLGVR (SEQ ID NO: 126). In some embodiments, the polypeptides of the present invention do not comprise a mouse lower hinge sequence, such as CPPCKCPAPNLLGGP (SEQ ID NO: 131).

[0501] In some embodiments, the polypeptide of the present technology does not comprise or consist of one of the polypeptides of Table A-5, as disclosed in Table S1 of Fu-Yao Jiang, "A lesion-selective albumin-CTLA41g as a safe and effective treatment for collagen-induced arthritis", Inflammation and Regeneration, Vol. 43, No. 13, February 16, 2023.

[0502] Table A-5: Polypeptides that are not and / or not comprised in the polypeptides of the present technology.

[0503]

[0504]

[0505] -IMGT, International Immunogenetics Information System (https: / / www.imgt.org / ).

[0506] -N / A, not applicable.

[0507] Preferably, the polypeptides of the present technology do not comprise or consist of a polypeptide as depicted in Table A-5a.

[0508] Table A-5a: Polypeptides that are not and / or not comprised in the polypeptides of the present technology.

[0509]

[0510]

[0511] -IMGT, International Immunogenetics Information System (https: / / www.imgt.org / ).

[0512] -N / A, not applicable.

[0513] Therefore, in one embodiment, the present technology provides a polypeptide comprising (i) at least one domain comprising or specifically binding to a serum albumin protein and (ii) an immunoglobulin G (IgG) Fc domain or a fragment thereof, wherein the polypeptide does not comprise or consist of the polypeptide disclosed in Fu-YaoJiang "A lesion-selective albumin-CTLA4lg as a safe and effective treatment for collagen-induced arthritis", Inflammation and Regeneration, Vol. 43, No. 13, February 16, 2023, in particular in Table S1 (Supplementary Information).

[0514] Therefore, in one embodiment, the present technology provides a polypeptide comprising (i) at least one domain comprising or specifically binding to a serum albumin protein and (ii) an immunoglobulin G (IgG) Fc domain or a fragment thereof, wherein the polypeptide does not comprise or consist of a polypeptide selected from the group consisting of mCTLA4Ig, mAlb-CTLA4Ig, hCTLA4Ig, hAlb-CTLA4Ig, mAlb-CTLA4 ECD, mlg-CTLA4 ECD, mAlb-MMP-CTLA4Ig, Ab lock-mCTLA4Ig, VpreB-mCTLA4Ig, as described in Table A-5.

[0515] In one embodiment, the present technology provides a polypeptide comprising (i) at least one domain comprising a serum albumin protein or specifically binding to a serum albumin protein and (ii) an immunoglobulin G (IgG) Fc domain or a fragment thereof, wherein the polypeptide does not comprise the protein CTLA4 Ile38-Ser160 (protein ID: NP_033973.2) and / or the protein CTLA4 Ala37-Asp161 (protein ID: NP_005205.2). In another embodiment, the present technology provides a polypeptide comprising (i) at least one domain comprising a serum albumin protein or specifically binding to a serum albumin protein and (ii) an immunoglobulin G (IgG) Fc domain or a fragment thereof, wherein the polypeptide does not comprise the CTLA4 protein.

[0516] In another embodiment, the present technology provides a polypeptide comprising (i) at least one domain comprising a serum albumin protein or specifically binding to a serum albumin protein and (ii) an immunoglobulin G (IgG) Fc domain or a fragment thereof, wherein the polypeptide does not comprise protein albumin Glu25 Ala609 (Protein ID: NP_033784.2) connected to protein CTLA4 Ile38-Ser160 (Protein ID: NP_033973.2) by a linker and / or connected to protein CTLA4 Ala37-Asp161 (Protein ID: NP_005205.2) by a linker, wherein the linker is selected from CPPCKCPAPNLLGGP (SEQ ID NO: 131), CPPCPAPELLGGP (SEQ ID NO: 132), GPLGMWSRAAQPA (SEQ ID NO: 111), GPLGMWSRGAQPA (SEQ ID NO: 135) and GPLGMWSR (SEQ ID NO: 125). In another embodiment, the polypeptides of the present technology do not comprise albumin Glu25 Ala609 (Protein ID: NP_033784.2) directly linked to protein CTLA4 Ile38-Ser160 (Protein ID: NP_033973.2) and / or directly linked to protein CTLA4 Ala37-Asp161 (Protein ID: NP_005205.2). In another embodiment, the polypeptides of the present technology do not comprise a serum albumin protein linked to protein CTLA4 Ile38-Ser160 (Protein ID: NP_033973.2) and / or linked to protein CTLA4 Ala37-Asp161 (Protein ID: NP_005205.2) via a linker selected from the group consisting of CPPCKCPAPNLLGGP (SEQ ID NO: 131), CPPCPAPELLGGP (SEQ ID NO: 132), GPLGMWSRAAQPA (SEQ ID NO: 111), GPLGMWSRGAQPA (SEQ ID NO: 135), and GPLGMWSR (SEQ ID NO: 125). In another embodiment, the polypeptides of the present technology do not comprise a serum albumin protein directly linked to protein CTLA4 Ile38-Ser160 (Protein ID: NP_033973.2) and / or linked to protein CTLA4 Ala37-Asp161 (Protein ID: NP_005205.2).In another additional embodiment, the polypeptides of the present technology do not comprise a serum albumin protein linked to the CTLA4 protein via a linker selected from the group consisting of CPPCKCPAPNLLGGP (SEQ ID NO: 131), CPPCPAPELLGGP (SEQ ID NO: 132), GPLGMWSRAAQPA (SEQ ID NO: 111), GPLGMWSRGAQPA (SEQ ID NO: 135), and GPLGMWSR (SEQ ID NO.: 125). In another additional embodiment, the polypeptides of the present technology do not comprise a serum albumin protein directly linked to the CTLA4 protein.

[0517] In another embodiment, the polypeptides of the present technology do not comprise:

[0518] - protein albumin Glu25-Ala609 (Protein ID: NP_033784.2) linked to protein CTLA4 Ile38-Ser160 (Protein ID: NP_033973.2) via the linker CPPCKCPAPNLLGGP (SEQ ID NO: 131);

[0519] - protein albumin Asp25-Leu609 (Protein ID: NP_000468.1) linked to protein CTLA4 Ala37-Asp161 (Protein ID: NP_005205.2) via the linker CPPCPAPELLGGP (SEQ ID NO: 132);

[0520] - protein albumin Glu25 Ala609 (Protein ID: NP_033784.2) linked to protein CTLA4 Ilee38-Ser160 (Protein ID: NP_033973.2) via the linker CPPCKCPAPNLLGGP (SEQ ID NO: 131); and / or

[0521] - Protein albumin Glu25-Ala609 (Protein ID: NP_033 784.2) linked to protein CTLA4 Ile38-Ser160 (Protein ID: NP.033973.2) via the linker GPLGMWSRAAQPA (SEQ ID NO: 111).

[0522] In another embodiment, the polypeptides of the present technology do not comprise the protein albumin Glu25-Ala609 (protein ID: NP_033784.2) (amino acids 25-609 of SEQ ID NO: 180). In another embodiment, the polypeptides of the present technology do not comprise or consist of the polypeptide hAlb-CTLA4Ig consisting of albumin Asp25-Leu609 (protein ID: NP_000468.1), core hinge-lower hinge / upper CH2: CPPCPAPELLGGP, CTLA4Ala37-Asp161 (protein ID: NP_005205.2), and IgG, hinge-CH2-CH3 (IMGT accession number: J00228) (IMGT, international ImMunoGeneTics information system (https: / / www.imgt.org / )).

[0523] In preferred embodiments, the polypeptides of the present technology comprise (i) at least one domain comprising or specifically binding to a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the albumin (if present) is human albumin, and wherein the polypeptide does not comprise a polypeptide comprising or consisting of CTLA4 Ala37-Aspl61 (Protein ID: NP_005205.2) and / or a polypeptide comprising or consisting of IgG1 hinge-CH2-CH3 (IMGT Accession No.: J00228, wherein IMGT refers to the International Immunogenetics Information System (https: / / www.imgt.org / )).

[0524] In another embodiment, the polypeptides of the present technology comprise (i) at least one domain that comprises or specifically binds to a serum albumin protein, (ii) an IgG Fc domain or a fragment thereof, and (iii) a therapeutic moiety, wherein the albumin (if present) is human albumin, and wherein the therapeutic moiety is not linked to the albumin directly or via a peptide linker such as CPPCPAPELLGGP (SEQ ID NO: 132).

[0525] In another embodiment, the polypeptides of the present technology comprise (i) at least one domain that comprises or specifically binds to a serum albumin protein, (ii) an IgG Fc domain or a fragment thereof, and (iii) a therapeutic moiety, wherein the therapeutic moiety is not linked to the albumin directly or via a peptide linker, wherein the linker is preferably not a cleavable linker, such as a MMP cleavable linker, e.g., GPLGMWSRAAQPA (SEQ ID NO: 111) or, and / or wherein the linker is not CPPCKCPAPNLLGGP (SEQ ID NO: 131) and / or CPPCPAPELLGGP (SEQ ID NO: 132).

[0526] In one embodiment, the polypeptides of the present technology do not comprise a polypeptide comprising or consisting of the following components from N-terminus to C-terminus:

[0527] Albumin-hinge-eCTLA4-Fc

[0528] For example, Fu-Yao Jiang: "A lesion-selective albumin-CTLA4lg as a safe and effective treatment for collagen-induced arthritis", Inflammation and Regeneration, Vol. 43, No. 13, February 16, 2023 Figure 2 Depicted in B.

[0529] In another embodiment, the polypeptides of the present technology do not comprise or consist of albumin linked (directly or via a peptide linker, as described herein) to the N-terminus of a CTLA4 protein.

[0530] In another embodiment, the polypeptides of the present technology do not comprise mouse (Mus musculus) CTLA4 protein. In another embodiment, the polypeptides of the present technology do not comprise any of the following proteins:

[0531] - CTLA4 Ile38-Ser160 (Protein ID: NP_033973.2); and

[0532] -CTLA4 Ala37-Asp161 (Protein ID: NP_005205.2).

[0533] In one embodiment, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin-binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn-binding fragment thereof, wherein the polypeptide does not comprise an Fc region comprising or consisting of efgartigimod (CAS Reg. No. 1821402-21-4). In particular, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin-binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn-binding fragment thereof, wherein the polypeptide does not comprise an Fc region comprising or consisting of SEQ ID NO: 167, SEQ ID NO: 168, and / or SEQ ID NO: 169. Thus, the polypeptides of the present technology do not include polypeptides comprising or consisting of SEQ ID NO: 167, SEQ ID NO: 168, and / or SEQ ID NO: 169. In other embodiments, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn binding fragment thereof, wherein the polypeptide does not include an Fc region comprising or consisting of SEQ ID NO.: 170, SEQ ID NO: 171, SEQ ID NO: 172, and / or SEQ ID NO: 164.

[0534] In other embodiments, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn binding fragment thereof, wherein the polypeptide does not comprise an Fc domain comprising amino acid W at EU position 366.

[0535] In particular, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin-binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn-binding fragment thereof, wherein the polypeptide does not comprise an Fc region comprising or consisting of SEQ ID NO: 163, SEQ ID NO: 179, and / or SEQ ID NO: 183. Thus, the polypeptides of the present technology do not comprise a polypeptide comprising or consisting of SEQ ID NO: 163, SEQ ID NO: 179, and / or SEQ ID NO: 183.

[0536] In other embodiments, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn binding fragment thereof, wherein the polypeptide does not comprise an Fc domain comprising amino acids S, A, and V at EU positions 366, 368, and 407, respectively.

[0537] In particular, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin-binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn-binding fragment thereof, wherein the polypeptide does not comprise an Fc region comprising or consisting of SEQ ID NO: 184, SEQ ID NO: 185, and / or SEQ ID NO: 211. Thus, the polypeptides of the present technology do not comprise a polypeptide comprising or consisting of SEQ ID NO: 184, SEQ ID NO: 185, and / or SEQ ID NO: 211.

[0538] In other embodiments, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn binding fragment thereof, wherein the polypeptide does not comprise an Fc domain comprising amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively. In other embodiments, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn binding fragment thereof, wherein the polypeptide does not comprise an Fc domain comprising amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436, respectively.

[0539] In other embodiments, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn binding fragment thereof, wherein the polypeptide does not comprise an IgG1 Fc domain comprising amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively. In other embodiments, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn binding fragment thereof, wherein the polypeptide does not comprise an IgG1 Fc domain comprising amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436, respectively.

[0540] In other embodiments, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn binding fragment thereof, wherein the polypeptide does not comprise a human IgG1 Fc domain comprising amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively. In other embodiments, the present technology provides a polypeptide comprising or consisting of: (i) at least one domain comprising a serum albumin protein and / or a domain with high affinity / specific binding to a serum albumin protein (such as a serum albumin binding ISVD), and (ii) at least one IgG Fc domain or fragment thereof, preferably an FcRn binding fragment thereof, wherein the polypeptide does not comprise a human IgG1 Fc domain comprising amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436, respectively.

[0541] In other embodiments, the polypeptides of the present technology do not comprise a variant IgG Fc region comprising a first Fc domain and a second Fc domain that form a dimer, wherein the first Fc domain and / or the second Fc domain contain amino acids Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively.

[0542] In other embodiments, the polypeptides of the present technology do not comprise a variant IgG Fc region comprising a first Fc domain and a second Fc domain that form a dimer, wherein the first Fc domain and / or the second Fc domain contain amino acids Y, T, E, K and F at EU positions 252, 254, 256, 433, 434 and 436, respectively.

[0543] In other embodiments, the polypeptides of the present technology do not comprise a variant IgG Fc region comprising a first Fc domain and a second Fc domain that form a dimer, wherein both the first Fc domain and the second Fc domain contain amino acids Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively.

[0544] In other embodiments, the polypeptides of the present technology do not comprise a variant IgG Fc region comprising a first Fc domain and a second Fc domain that form a dimer, wherein both the first Fc domain and the second Fc domain contain amino acids Y, T, E, K and F at EU positions 252, 254, 256, 433, 434 and 436, respectively.

[0545] In other embodiments, the polypeptides of the present technology do not comprise a variant IgG Fc region comprising a first Fc domain and a second Fc domain that form a dimer, wherein the first Fc domain and / or the second Fc domain contains amino acids Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively, and wherein the first Fc domain and / or the second Fc domain is an IgG1 Fc domain.

[0546] In other embodiments, the polypeptides of the present technology do not comprise a variant IgG Fc region comprising a first Fc domain and a second Fc domain that form a dimer, wherein the first Fc domain and / or the second Fc domain contains amino acids Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively, and wherein the first Fc domain and / or the second Fc domain is a human IgG Fc domain.

[0547] In other embodiments, the polypeptides of the present technology do not comprise a variant IgG Fc region, comprising a first Fc domain and a second Fc domain that form a dimer, wherein the first Fc domain and / or the second Fc domain contains amino acids Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively, and wherein the first Fc domain and / or the second Fc domain is a human IgG1 Fc domain.

[0548] In other embodiments, the polypeptides of the present technology do not comprise a variant IgG Fc region comprising a first Fc domain and a second Fc domain that form a dimer, wherein the first Fc domain and / or the second Fc domain contain amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively, and wherein both the first Fc domain and the second Fc domain are IgG1 Fc domains.

[0549] In other embodiments, the polypeptides of the present technology do not comprise a variant IgG Fc region comprising a first Fc domain and a second Fc domain that form a dimer, wherein the first Fc domain and / or the second Fc domain contains amino acids Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively, and wherein both the first Fc domain and the second Fc domain are human IgG Fc domains.

[0550] In other embodiments, the polypeptides of the present technology do not comprise a variant IgG Fc region comprising a first Fc domain and a second Fc domain that form a dimer, wherein the first Fc domain and / or the second Fc domain contain amino acids Y, T, E, K and F at EU positions 252, 254, 256, 433 and 434, respectively, and wherein both the first Fc domain and the second Fc domain are human IgG1 Fc domains.

[0551] In one embodiment, the polypeptides of the present technology do not comprise an IgG1 Fc domain. In a preferred embodiment, the polypeptides of the present technology comprise an IgG4 Fc domain.

[0552] In other embodiments, the Fc domain contained in the polypeptides of the present technology is a variant Fc domain that does not bind FcRn with a higher affinity at pH 6.0 and / or pH 7.4 compared to the corresponding wild-type Fc region. In other embodiments, the polypeptides of the present technology do not contain an Fc domain, and the Fc domain contains a combination of amino acids selected from the group consisting of:

[0553] (i) Q and L at EU positions 250 and 428, respectively;

[0554] (ii) P and A at EU positions 308 and 434, respectively;

[0555] (iii) P and Y at EU positions 308 and 434, respectively; or

[0556] (iv) Y, E and Y at EU positions 252, 286 and 434, respectively.

[0557] In other embodiments, the polypeptides of the present technology do not comprise an Fc domain comprising a combination of amino acid substitutions selected from the group consisting of:

[0558] (i) M252Y, S254T, T256E, H433K, and N434F;

[0559] (ii) T250Q and M428L;

[0560] (iii) V308P and N434A;

[0561] (iv) V308P and N434Y; or

[0562] (v) M252Y, N286E and N434Y.

[0563] In one embodiment, the polypeptide of the present invention technology does not include such Fc district, the Fc district includes methionine (M) at position 252 to tyrosine (Y) substitution, serine (S) at position 254 to threonine (T) substitution and threonine (T) at position 256 to glutamate (E) substitution, according to the EU numbering system numbering. Referring to U.S. Patent No. 7,658,921. This type of mutant Fc domain is referred to as "YTE mutant". Therefore, in one embodiment, the polypeptide of the present invention technology does not include YTE mutant Fc domain. In one embodiment, the polypeptide of the present invention technology does not include such Fc domain, the Fc domain includes one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389 and 428-436 according to the EU numbering system numbering.

[0564] In a specific embodiment, the polypeptides of the present technology do not comprise full-length antibodies. As used herein, the term "antibody" includes full-length antibodies. Full-length antibodies comprise four polypeptide chains; two heavy chains and two light chains, typically linked by disulfide bonds. The light chain consists of a variable domain V L and a constant domain C L The heavy chain contains a variable domain V H and three to four constant domains (C HIn one embodiment, the polypeptides of the present technology do not comprise or consist of rozanolixizumab (UCB7665), nipocalimab (M281), orilanolimab (ALXN1830 / SYNT001), or batoclimab (IMVT-1401 / RVT1401 / HBM9161), which are all anti-FcRn antibodies. Nicalimab comprises a light chain (SEQ ID NO: 218) and a heavy chain (SEQ ID NO: 219) sequence. Lolixizumab comprises a light chain (SEQ ID NO: 212) and a heavy chain (SEQ ID NO: 213) sequence. Orilanolimab comprises a light chain (SEQ ID NO: 214) and a heavy chain (SEQ ID NO: 215) sequence. Batolimumab comprises light chain (SEQ ID NO: 216) and heavy chain (SEQ ID NO: 217) sequences.

[0565] In some embodiments, the polypeptides of the present technology do not comprise at least one polypeptide as defined in any one of SEQ ID NOs: 220-260.

[0566] In one embodiment, the polypeptide of the present invention comprises (i) at least one domain comprising a serum albumin protein or specifically binding to a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the Fc domain is a native Fc domain. In some embodiments, the Fc region comprises or consists of a human IgG1 or IgG4 Fc region. Preferably, the Fc domain is a variant Fc domain as described herein, such as a so-called "FALA" or "LALA" Fc mutant in which residues 234 and 235 are substituted with alanine. In other embodiments, the Fc variant domain comprises the following mutations M252Y, S254T, and T256E (YTE, see, e.g., Robbie GJ et al., "A novel investigational Fc-modified humanized monoclonal antibody, motavizumab-YTE, has an extended half-life in healthy adults", Antimicrob Agents Chemother., December 2013; 57(12): 6147-53).

[0567] Preferably, the polypeptide of the present technology comprises (i) at least one domain comprising a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the Fc domain comprises or consists of two identical polypeptides as defined in SEQ ID NO: 113, 115 or 181, preferably 113 or 181. In another preferred embodiment, the polypeptide of the present technology comprises (i) at least one domain comprising a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the Fc domain comprises or consists of two different polypeptides selected from the group consisting of:

[0568] - SEQ ID NO: 116 and 117;

[0569] - SEQ ID NO: 186 and 187;

[0570] - SEQ ID NO: 188 and 189;

[0571] - SEQ ID NOs: 198 and 199; and

[0572] - SEQ ID NO: 186 and 190.

[0573] In one embodiment, the polypeptide of the present technology comprises (i) at least one domain that specifically binds to a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the Fc domain comprises or consists of two identical polypeptides as defined in SEQ ID NO: 113, 115 or 181, preferably 113 or 181. In another preferred embodiment, the polypeptide of the present technology comprises (i) at least one domain that specifically binds to a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the Fc domain comprises or consists of two different polypeptides selected from the group consisting of:

[0574] - SEQ ID NO: 116 and 117;

[0575] - SEQ ID NO: 186 and 187;

[0576] - SEQ ID NO: 188 and 189;

[0577] - SEQ ID NOs: 198 and 199; and

[0578] - SEQ ID NO: 186 and 190.

[0579] In another embodiment, the polypeptides of the present technology comprise (i) at least one domain that specifically binds to a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the at least one domain that specifically binds to a serum albumin protein is an albumin binding ISVD, preferably selected from the group consisting of SEQ ID NOs: 7-21 and 61-69, more preferably wherein the albumin binding domain is selected from the group consisting of a polypeptide comprising or consisting of ALB23002 (SEQ ID NO: 20), Alb23002-A (SEQ ID NO: 21), HSA006A06 (SEQ ID NO: 65), ALBX00002 (SEQ ID NO: 64), ALB11002 (SEQ ID NO: 13) and T023500029 (SEQ ID NO: 69), even more preferably selected from the group consisting of HSA006A06 (SEQ ID NO: 65), ALB11002 (SEQ ID NO: 13) and ALB23002 (SEQ ID NO: 20).

[0580] In another embodiment, the polypeptides of the present technology comprise (i) at least one domain that specifically binds to a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the at least one domain that specifically binds to a serum albumin protein is an albumin binding ISVD, preferably selected from the group consisting of SEQ ID NOs: 7-21 and 61-69, more preferably wherein the albumin binding domain is selected from the group consisting of a polypeptide comprising or consisting of ALB23002 (SEQ ID NO: 20), Alb23002-A (SEQ ID NO: 21), HSA006A06 (SEQ ID NO: 65), ALBX00002 (SEQ ID NO: 64), ALB11002 (SEQ ID NO: 13), and T023500029 (SEQ ID NO: 69), even more preferably selected from the group consisting of HSA006A06 (SEQ ID NO: 65), ALB11002 (SEQ ID NO: 13), and ALB23002 (SEQ ID NO: 20), and wherein the Fc domain comprises NO: 113, 115 or 181, preferably two identical polypeptides as defined in 113 or 181 or consisting thereof, or wherein the Fc domain comprises or consists of two different polypeptides selected from the group consisting of:

[0581] - SEQ ID NO: 116 and 117;

[0582] - SEQ ID NO: 186 and 187;

[0583] - SEQ ID NO: 188 and 189;

[0584] - SEQ ID NOs: 198 and 199; and

[0585] - SEQ ID NO: 186 and 190.

[0586] In one embodiment, (i) at least one domain comprising or specifically binding to a serum albumin protein and (ii) the IgG Fc domain comprised in the polypeptide of the present technology are connected via a non-cleavable peptide linker, preferably a GS linker as defined herein, even more preferably a linker selected from the group consisting of SEQ ID NOs: 25 to 37, even more preferably a 9GS linker (SEQ ID NO: 29) or a 35GS linker (SEQ ID NO: 36).

[0587] In one embodiment, the polypeptides of the present technology comprise a single albumin binding ISVD, preferably selected from the group consisting of SEQ ID NOs: 7-21 and 61-69, more preferably wherein the albumin binding domain is selected from the group consisting of a polypeptide comprising or consisting of ALB23002 (SEQ ID NO: 20), Alb23002-A (SEQ ID NO: 21), HSA006A06 (SEQ ID NO: 65), ALBX00002 (SEQ ID NO: 64), ALB11002 (SEQ ID NO: 13), and T023500029 (SEQ ID NO: 69), even more preferably selected from the group consisting of HSA006A06 (SEQ ID NO: 65), ALB11002 (SEQ ID NO: 13), and ALB23002 (SEQ ID NO: 20).

[0588] In another embodiment, the polypeptide of the present technology comprises (i) at least one domain that specifically binds to a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the at least one domain that specifically binds to a serum albumin protein is a DARPin, an affitin or a protein ABD, preferably selected from the group consisting of SEQ ID NOs: 102 to 104. In another embodiment, the polypeptide of the present technology comprises (i) at least one domain that specifically binds to a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the at least one domain that specifically binds to a serum albumin protein is a DARPin, an affitin or a protein ABD, preferably selected from the group consisting of SEQ ID NOs: 102 to 104, and wherein the Fc domain comprises or consists of two identical polypeptides as defined in SEQ ID NOs: 113, 115 or 181, preferably 113 or 181, or wherein the Fc domain comprises or consists of two different polypeptides selected from the group consisting of:

[0589] - SEQ ID NO: 116 and 117;

[0590] - SEQ ID NO: 186 and 187;

[0591] - SEQ ID NO: 188 and 189;

[0592] - SEQ ID NOs: 198 and 199; and

[0593] - SEQ ID NO: 186 and 190.

[0594] In another embodiment, the Fc region comprised in the polypeptide of the present technology comprises or consists of two identical polypeptides as defined in SEQ ID NO: 115 or 181, preferably 181, or comprises or consists of two different polypeptides selected from the group consisting of:

[0595] - SEQ ID NO: 186 and 187;

[0596] - SEQ ID NO: 188 and 189;

[0597] - SEQ ID NOs: 198 and 199; and

[0598] - SEQ ID NO: 186 and 190.

[0599] In another embodiment, the polypeptides of the present technology comprise (i) at least one domain that specifically binds to a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the at least one domain that specifically binds to a serum albumin protein is an albumin binding ISVD, preferably selected from the group consisting of SEQ ID NOs: 7-21 and 61-69, more preferably wherein the albumin binding domain is selected from the group consisting of a polypeptide comprising or consisting of ALB23002 (SEQ ID NO: 20), Alb23002-A (SEQ ID NO: 21), HSA006A06 (SEQ ID NO: 65), ALBX00002 (SEQ ID NO: 64), ALB11002 (SEQ ID NO: 13), and T023500029 (SEQ ID NO: 69), even more preferably selected from the group consisting of HSA006A06 (SEQ ID NO: 65), ALB11002 (SEQ ID NO: 13), and ALB23002 (SEQ ID NO: 20), and wherein the Fc domain comprises NO: 115 or 181, preferably two identical polypeptides as defined in 181 or consisting thereof, or comprising or consisting of two different polypeptides selected from the following:

[0600] - SEQ ID NO: 186 and 187;

[0601] - SEQ ID NO: 188 and 189;

[0602] - SEQ ID NOs: 198 and 199; and

[0603] - SEQ ID NO: 186 and 190.

[0604] In another embodiment, the polypeptide of the present technology comprises (i) at least one domain that specifically binds to a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the at least one domain that specifically binds to a serum albumin protein is a DARPin, an affitin or a protein ABD, preferably selected from the group consisting of SEQ ID NOs: 102 to 104. In another embodiment, the polypeptide of the present technology comprises (i) at least one domain that specifically binds to a serum albumin protein and (ii) an IgG Fc domain or a fragment thereof, wherein the at least one domain that specifically binds to a serum albumin protein is a DARPin, an affitin or a protein ABD, preferably selected from the group consisting of SEQ ID NOs: 102 to 104, and wherein the Fc domain comprises or consists of two identical polypeptides as defined in SEQ ID NOs: 115 or 181, preferably 181, or comprises or consists of two different polypeptides selected from the group consisting of:

[0605] - SEQ ID NO: 186 and 187;

[0606] - SEQ ID NO: 188 and 189;

[0607] - SEQ ID NOs: 198 and 199; and

[0608] - SEQ ID NO: 186 and 190.

[0609] In a preferred embodiment, the polypeptide of the present invention comprises or consists of a polypeptide as described in Table A-1. In another preferred embodiment, the polypeptide of the present invention comprises or consists of a polypeptide as described in Table A-11.

[0610] 5.6 Other groups, residues, parts or binding units

[0611] Thus, the polypeptides of the present technology can generally be prepared by a method comprising at least one step of suitably linking (directly or via a linker, as described herein) one or more domains (i.e., a domain comprising a serum albumin protein or a serum albumin binding domain and an IgG Fc domain or a fragment thereof) to each other, and optionally additionally to one or more additional groups, residues, moieties or binding units as mentioned above, directly or via one or more suitable linkers.

[0612] For example, such additional groups, residues, parts or binding units may be one or more additional immunoglobulins, so as to form a (fusion) protein or (fusion) polypeptide. In a preferred, but non-limiting aspect, the one or more other groups, residues, parts or binding units are immunoglobulin single variable domains. Even more preferably, the one or more other groups, residues, parts or binding units are selected from domain antibodies, immunoglobulin single variable domains suitable for use as domain antibodies, single domain antibodies, immunoglobulin single variable domains (ISVDs) suitable for use as single domain antibodies, "dAbs", immunoglobulin single variable domains suitable for use as dAbs, VHHs, humanized VHHs, camelized VHs or VHH. Alternatively, such groups, residues, moieties or binding units may be, for example, chemical groups, residues, moieties, which themselves may or may not have biological and / or pharmacological activity. For example, but not limited to, such groups may be linked to one or more domains in the polypeptides of the present technology to provide "derivatives" of the polypeptides of the present technology, as further described herein. The polypeptides of the present technology may also contain additional groups that have certain functions, such as labels, toxins, one or more linkers, binding sequences, etc. These additional functions include both amino acid-based and non-amino acid-based groups.

[0613] It will be understood that the terms "fusion protein," "fusion polypeptide construct," "compound of the present technology," "polypeptide construct," and "polypeptide" can be used interchangeably herein (unless the context clearly dictates otherwise).

[0614] In some embodiments, the domains comprised in the polypeptides of the present technology are antibody-based scaffolds and / or non-antibody-based scaffolds as disclosed herein.

[0615] The polypeptides of the present invention technology can also be prepared by such a method, which generally comprises at least the following steps: providing a nucleic acid encoding a polypeptide of the present invention technology, expressing the nucleic acid in a suitable manner, and recovering the expressed polypeptide of the present invention technology. Such a method can be carried out in a manner known per se, which is clear 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 a polypeptide of the present invention technology starting from a polypeptide comprising at least one domain of the present invention technology is also referred to herein as "formatting" the polypeptide of the present invention technology. Based on the disclosure herein, examples of ways in which the polypeptides of the present invention technology can be formatted and examples of such formats will be clear to those skilled in the art.

[0616] Therefore, the present technology also relates to polypeptides or fusion proteins comprising a polypeptide of the present technology, as defined herein (comprising (i) at least one domain comprising a serum albumin protein or specifically binding to serum albumin and (ii) an IgG Fc domain or a fragment thereof), and one or more other groups, residues, parts or binding units, directly or through one or more suitable linkers. As described herein, PK parameters (such as half-life or clearance) are improved by the presence of a polypeptide of the present technology in a fusion protein. The half-life of the one or more other groups, residues, parts or binding units is increased by the presence of the polypeptide of the present technology in a fusion protein as described herein compared to the half-life of the one or more other groups, residues, parts or binding units themselves (as is). The clearance rate of the one or more other groups, residues, parts or binding units is reduced or decreased by the presence of a polypeptide of the present technology in a fusion protein as described herein compared to the clearance rate of the one or more other groups, residues, parts or binding units themselves (as is).

[0617] Suitable linkers for use in the molecules of the present invention will be clear to the skilled person, and can generally be any linker used in the art for connecting amino acid sequences or any other molecule contained in the fusion protein. Preferably, the linker is suitable for constructing a protein or polypeptide intended for pharmaceutical use. Some particularly preferred linkers include linkers used in the art for connecting antibody fragments or antibody domains. For example, the linker can be a suitable amino acid or amino acid sequence, and in particular an amino acid sequence having between 1 and 50, preferably between 1 and 30, such as between 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, for example type (Gly x Ser y ) z, such as (Gly4Ser)3 or (Gly3Ser2)3, as described in WO 1999 / 42077, and the GS30, GS15, GS9 and GS7 linkers described by Ablynx in the applications described herein (see, for example, WO 2006 / 040153 and WO2006 / 122825), and hinge-like regions, such as the hinge region of a naturally occurring heavy chain antibody or similar sequences (such as described in WO 1994 / 04678). Examples of linkers are also provided in Table A-2. Polyethylene glycol (PEG) in any of the variants described below can also be used as a linker in the fusion proteins of the present technology. Other suitable linkers for use in the molecules of the present technology are described, for example, in (Kjeldsen T. et al. "Dually reactive long recombinant linkers for bioconjugations as an alternative to PEG", ACS Omega, 2020, 5: 19827-19833). As described herein, polar protein sequences with PEG-like properties, sometimes referred to as “recombinant PEG”, have been described in recent years by Alvarez (“Improving protein pharmacokinetics by genetic fusion to simple amino acid sequences”, J.Biol.Chem., 2004, 279: 3375-3381), Amunix (a mixed sequence of GEDSTAP residues, referred to as “ELNN polypeptides”, see, for example, US2014 / 0301974 A1), XL protein (PAS repeats), Novo Nordisk (GQAP-like repeats), SOBI, etc. Other suitable linkers for use in the molecules of the present invention are, for example, cleavable linkers, i.e., linkers having a trigger in their structure that can be efficiently cleaved. For example, Su, Z. et al. (“Antibody–drug conjugates: Recent advances in linker chemistry”, Acta Pharmaceutica Sinica B, 2021, 11(12):3889-3907) reviews linkers that can be included in antibody-drug conjugates and can also be used in molecules of the present invention. For example, suitable linkers for use in molecules of the present invention are APN-maleimide linkers (3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propiolanitrile, MAPN) or bismaleimido-PEG3 (BM(PEG)3) linkers (BM(PEG)3(1,11-bismaleimido-triethylene glycol)).Alternatively, a bifunctional linker can be used. For example, an APN-maleimide linker (806536, Sigma-Aldrich) can be used. Some other particularly preferred linkers are polyalanine (such as AAA), and linkers GS30 (SEQ ID NO: 85 in WO 06 / 122825) and GS9 (SEQ ID NO: 84 in WO 06 / 122825).

[0618] Other suitable linkers generally include organic compounds or polymers, particularly those in proteins suitable for pharmaceutical use. For example, poly(ethylene glycol) moieties are used to link antibody domains, see for example WO 04 / 081026.

[0619] It is within the scope of the present technology that the length, degree of flexibility and / or other properties of the one or more linkers used (but not critical, as it is typically used for linkers used in ScFv fragments) may have some impact on the properties of the final polypeptide of the present technology, including but not limited to affinity, specificity or avidity for FcRn or one or more other antigens. Based on the disclosure herein, the skilled artisan will be able to determine the optimal linker or links for use in a specific polypeptide of the present technology, optionally after some limited routine experimentation.

[0620] The following are also within the scope of the present technology: the one or more linkers used impart one or more other advantageous properties or functions to the polypeptides of the present technology, and / or provide one or more sites for forming derivatives and / or for attaching functional groups (e.g., as described herein for the ISVDs, Nanobody VHHs or derivatives of the polypeptides of the present technology). For example, linkers containing one or more charged amino acid residues can provide improved hydrophilic properties, while linkers that form or contain small epitopes or tags can be used for detection, identification and / or purification purposes. Likewise, based on the disclosure herein, a skilled person will be able to determine the optimal linker for use in a specific polypeptide of the present technology, optionally after some limited routine experiments.

[0621] Finally, when two or more linkers are used in the polypeptide of the present invention, these linkers can be the same or different. Likewise, based on the disclosure herein, the skilled person will be able to optionally determine the best linker for use in a specific polypeptide of the present invention after some limited routine experiments.

[0622] It will be appreciated that the order (i.e., the orientation or configuration of the binding domains or building blocks) in the polypeptides of the present technology, such as, for example, a first domain (e.g., a serum albumin protein or a domain that specifically binds to a serum albumin protein), a second binding domain (e.g., an IgG Fc domain or a fragment thereof), a third binding domain (e.g., one or more additional groups, residues, moieties, or binding units, or domains that bind to a therapeutically relevant target, or a therapeutic moiety), etc., in the polypeptides, can be selected according to the needs of those skilled in the art and the relative affinities that may depend on the positions of these binding domains in the polypeptide. Whether the polypeptide includes one or more linkers to interconnect the binding domains and optional other groups, residues, or moieties is a matter of design choice. However, some orientations, with or without linkers, may provide preferred binding properties compared to other orientations. However, the present technology encompasses all different possible orientations. In preferred embodiments, the third binding domain (e.g., one or more additional groups, residues, moieties or binding units, or a domain that binds to a therapeutically relevant target, or a therapeutic moiety) is not directly linked or not linked via a linker as defined herein to the first domain as defined herein (e.g., a serum albumin protein or a domain that specifically binds to a serum albumin protein).

[0623] In one embodiment, the present technology provides a polypeptide or fusion protein comprising (i) at least one domain comprising a serum albumin protein or specifically binding to a serum albumin protein, (ii) an IgG Fc domain, and (ii) one or more groups, residues, moieties or binding units, optionally attached via one or more linkers, wherein the one or more other groups, residues, fragments or binding units target the molecules of the present technology to a target molecule on a cell, organ or tissue ("targeting moiety"). A targeting moiety as defined herein is any group, residue, moiety or binding unit that is capable of being directed by its binding to a target. An amino acid sequence (such as an ISVD, an antibody, an antigen binding domain or a fragment thereof such as a VHH domain or a VH / VL domain, or a general antigen binding protein or polypeptide or a fragment thereof) that "(specifically) binds", "can (specifically) bind", "has affinity" and / or "has specificity" for a specific antigenic determinant, epitope, antigen or protein, or for a specific non-protein molecule (such as a nucleic acid (such as DNA or RNA) or a glycan (or at least a part, fragment or epitope thereof) is said to be "for" or "directed against" the antigenic determinant, epitope, antigen or protein. Specific binding of an antigen binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competition assays, and their different variants known per se in the art; as well as other techniques mentioned herein.

[0624] In one embodiment, the present technology provides a polypeptide or fusion protein comprising (i) at least one domain comprising a serum albumin protein or specifically binding to a serum albumin protein, (ii) an IgG Fc domain, and (ii) one or more groups, residues, moieties or binding units, optionally attached via one or more linkers, wherein the one or more other groups, residues, moieties or binding units are capable of exerting therapeutic activity in an animal or human ("therapeutic moiety or precursor thereof"). A therapeutic moiety as defined herein is any group, residue, moiety or binding unit capable of exerting therapeutic activity in an animal and / or human. A therapeutic moiety may also be in the form of a precursor, which is then activated to exert its therapeutic activity. For example, a therapeutic moiety according to the present technology can be any therapeutic agent (such as a drug, protein, peptide, gene, compound, or any other pharmaceutically active ingredient that can be used to treat and / or prevent certain disease conditions). For example, a therapeutic moiety can be a therapeutic antibody or a therapeutic ISVD. Non-limiting examples of therapeutic moieties that may be present in a polypeptide or fusion protein of the present technology are as follows:

[0625] - Epidermal Growth Factor Receptor (EGFR) binding molecules as described, for example, in WO 2005 / 044858, WO 2007 / 042289 or WO 2016 / 097313.

[0626] - von Willebrand Factor (vWF) binding molecules as described, for example, in WO 2006 / 122825.

[0627] - human epidermal growth factor receptor 2 (HER-2 or receptor tyrosine-protein kinase erbB-2) binding molecules as described, for example, in WO 2009 / 068625.

[0628] - an interleukin-6 receptor (IL-6R) binding molecule as described, for example, in WO 2010 / 115995 or WO 2010 / 115998.

[0629] - Neonatal Fc receptor (FcRn) binding molecules as described, for example, in WO 2008 / 074867 or WO 2009 / 080764.

[0630] - polymeric immunoglobulin receptor (pIgR) binding molecules, such as are also described in WO 2008 / 074867 or WO 2009 / 080764.

[0631] - Vascular endothelial growth factor receptor 1 (VEGF-R1 , also known as FIt-I) binding molecules as described, for example, in WO 2008 / 142165.

[0632] - Platelet-derived growth factor receptor beta (PDGF-R[beta]) binding molecules as described, for example, in WO 2008 / 142165.

[0633] - Fibroblast Growth Factor Receptor 4 (FGF-R4) binding molecules as described, for example, in WO 2008 / 142165.

[0634] - Tumor necrosis factor-alpha (TNF-alpha) binding molecules as described, for example, in WO 2006 / 122786, WO 2015 / 173325, WO 2017 / 081320, WO 2021 / 110816, WO 2021 / 110817 or WO 2022 / 129572.

[0635] - Insulin-like growth factor 1 receptor (IGF-IR) binding molecules as described, for example, in WO 2007 / 042289.

[0636] - Vascular endothelial growth factor (VEGF) binding molecules as described, for example, in WO 2008 / 101985.

[0637] - Receptor promoter nuclear factor kappa-beta ligand (RANK-L) binding molecules as described, for example, in WO 2008 / 142164 or WO 2015 / 173325.

[0638] - interleukin 23 (IL-23) binding molecules as described, for example, in WO 2009 / 068627, WO 2011 / 135026, WO 2011 / 161263, WO 2015 / 173325, WO 2017 / 072299 or WO 2021 / 110816.

[0639] - Respiratory syncytial virus (RSV) fusion (F) protein binding molecules, for example as described in WO 2009 / 147248 or WO 2010 / 139808.

[0640] - Influenza H5N1 hemagglutinin (HA) binding molecules as described, for example, in WO 2009 / 147248.

[0641] - Rabies virus G protein binding molecules as described, for example, in WO 2009 / 147248.

[0642] - CXC chemokine receptor type 4 (CXCR4) binding molecules as described, for example, in WO 2009 / 138519, WO 2011 / 083141, WO 2011 / 161266, WO 2015 / 044386, WO 2015 / 173325 or WO 2016 / 156570.

[0643] - CXC chemokine receptor type 7 (CXCR7) binding molecules as described, for example, in WO 2009 / 138519, WO 2011 / 117423, WO 2012 / 130874 or WO 2015 / 173325.

[0644] - sclerostin binding molecules, as described, for example, in WO 2010 / 130830

[0645] - Dickkopf-1 (Dkk-1) binding molecules as described, for example, in WO 2010 / 130832.

[0646] - HER-3 binding molecules as described, for example, in WO 2011 / 144749 or WO 2015 / 173325.

[0647] -c-Met binding molecules as described, for example, in WO 2012 / 042026, WO 2013 / 045707 or WO 2015 / 173325.

[0648] - Amyloid beta (Aβ or A-β) binding molecules as described, for example, in WO 2011 / 107507 or WO 2015 / 173325.

[0649] - CXC chemokine receptor type 2 (CXCR2) binding molecules as described, for example, in WO 2012 / 062713 or WO 2013 / 168108.

[0650] - Immunoglobulin E (IgE) binding molecules as described, for example, in WO 2012 / 175740, WO 2014 / 087010 or WO 2015 / 173325.

[0651] - Interleukin-17 (IL-17) A, IL-17F and / or IL-17A / F binding molecules, as described, for example, in WO 2012 / 156219.

[0652] - Hepatocyte Growth Factor (HGF) binding molecules as described, for example, in WO 2013 / 110531.

[0653] - Pseudomonas aeruginosa PcrV binding molecules as described, for example, in WO 2013 / 128031.

[0654] - P2X7 binding molecules as described, for example, in WO 2013 / 178783.

[0655] - an interleukin-3 receptor (CD123) binding molecule as described, for example, in WO 2015 / 044386 or WO 2018 / 091606.

[0656] - Interleukin-3 receptor alpha (IL-3Rα) binding molecules as described, for example, in WO 2015 / 044386.

[0657] - Voltage-gated pot...

Claims

1. A polypeptide comprising (i) at least one domain comprising a serum albumin protein or specifically binding to a serum albumin protein and (ii) an Fc domain of immunoglobulin G (IgG), wherein the polypeptide does not comprise or consist of one of the polypeptides depicted in the following table: -IMGT, International Immunogenetics Information System (https: / / www.imgt.org / ) -N / A, not applicable.

2. The polypeptide according to claim 1, characterized in that The at least one domain comprising a serum albumin protein is human serum albumin or a portion or variant of human serum albumin.

3. The polypeptide according to claim 1, characterized in that The at least one domain that specifically binds to the serum albumin protein specifically binds to an amino acid residue on the serum albumin protein, which is not involved in the binding of serum albumin to FcRn.

4. The polypeptide according to claim 1 or 3, characterized in that The at least one domain that specifically binds to a serum albumin protein specifically binds to domain II of human serum albumin.

5. The polypeptide according to claim 1, 3 and 4, characterized in that The at least one domain that specifically binds to a serum albumin protein is selected from the group consisting of an affibody molecule, a scFv, a Fab, a designed ankyrin repeat protein (DARPin), an albumin binding domain (ABD), an affitin, and an immunoglobulin variable domain sequence (ISVD).

6. The polypeptide according to claims 1 and 3 to 5, characterized in that The at least one domain that specifically binds to a serum albumin protein is at least one ISVD that specifically binds to serum albumin.

7. The polypeptide according to claims 1 and 3 to 6, characterized in that The at least one domain that specifically binds to a serum albumin protein is at least one ISVD that specifically binds to human serum albumin, wherein the ISVD is a (single) domain antibody, a VHH, a humanized VHH or a VH.

8. The polypeptide of any one of claims 1 to 7, wherein the Fc domain of IgG is the Fc region of immunoglobulin G type 1 (IgG1), immunoglobulin G type 2 (IgG2), immunoglobulin G type 3 (IgG3), or immunoglobulin G type 4 (IgG4).

9. The polypeptide of any one of claims 1 to 8, wherein the Fc domain is a native Fc domain of immunoglobulin G or a variant Fc domain of IgG or a fragment thereof.

10. The polypeptide of any one of claims 1 to 9, further comprising a therapeutic moiety.

11. The polypeptide of any one of claims 1 to 10, wherein the therapeutic moiety comprises at least one ISVD that specifically binds to a therapeutic target.

12. The polypeptide according to any one of claims 1 to 11, characterized in that Its serum half-life in humans is at least 5%, such as 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.

13. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 12.

14. A nucleic acid or a nucleic acid sequence encoding the polypeptide according to any one of claims 1 to 12.

15. A vector comprising the nucleic acid or nucleic acid sequence according to claim 14.

16. A host cell or a (non-human) host organism transformed or transfected with a nucleic acid or a nucleic acid sequence as claimed in claim 14 or with a vector as claimed in claim 15.

17. A method or process for producing a polypeptide according to any one of claims 1 to 12, said method or process comprising at least the following steps: a. expressing the nucleic acid sequence in a suitable host cell or (non-human) host organism or in another suitable expression system; optionally followed by: b. Isolate and / or purify the polypeptide according to any one of claims 1 to 12.

18. The polypeptide of any one of claims 1 to 12 for use in treating a subject in need thereof.

19. A kit comprising the polypeptide according to any one of claims 1 to 12, the nucleic acid or nucleic acid sequence according to claim 14, the vector according to claim 15, or the host cell or (non-human) host organism according to claim 16.

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