Albumin binding polypeptides
阅读说明：本技术 白蛋白结合多肽 (Albumin binding polypeptides ) 是由 卡罗琳·埃克布拉德 J·菲尔德维什 安炅勋 于 2013-09-25 设计创作，主要内容包括：本申请涉及白蛋白结合多肽。本公开内容涉及具有对白蛋白的结合亲和力的一类工程化的多肽。特别地,本发明涉及对酶促裂解具有高度耐受性的白蛋白结合多肽。本公开内容提供了包含白蛋白结合基序的白蛋白结合多肽,该基序由氨基酸序列GVSDFYKKLI X<Sub>a</Sub>KAKTVEGVE ALKX<Sub>b</Sub>X<Sub>c</Sub>I组成。(The present application relates to albumin binding polypeptides. The present disclosure relates to a class of engineered polypeptides having binding affinity for albumin. In particular, the present invention relates to albumin binding polypeptides that are highly resistant to enzymatic cleavage. The present disclosure provides albumin binding polypeptides comprising an albumin binding motif consisting of amino acid sequence GVSDFYKKLI X a KAKTVEGVE ALKX b X c And I, forming.)
1. An albumin binding polypeptide comprising an albumin binding motif [ BM ], said motif consisting of the amino acid sequence:
GVSDFYKKLI XaKAKTVEGVE ALKXbXcI
wherein, independently of each other,
Xaselected from D and E;
Xbselected from D and E; and
Xcselected from A and E.
2. Albumin binding polypeptide according to claim 1, in which said sequence is SEQ ID No. 1.
3. Albumin binding polypeptide according to any preceding claim, in which the albumin binding motif forms part of a triple helix bundle protein domain.
4. Albumin binding polypeptide according to claim 3, comprising the amino acid sequence:
LAX3AKX6X7ANX10ELDX14Y-[BM]-LX43X44LP
wherein
[ BM ] is an albumin binding motif as defined in any one of claims 1-2,
and, independently of each other,
X3selected from C, E, Q and S;
X6selected from C, E and S;
X7selected from A and S;
X10selected from A, R and S;
X14selected from A, C, K and S;
X43selected from A and K; and
X44selected from A, E and S.
5. Albumin binding polypeptide according to claim 4, the amino acid sequence of which comprises a sequence satisfying one definition selected from:
i) the sequence is selected from SEQ ID NO 9-16;
ii) the sequence is an amino acid sequence having 93% or more identity to a sequence selected from SEQ ID NO 9-16, with the proviso that the amino acid at the position corresponding to position 23 in SEQ ID NO 9-16 is K.
6. Albumin binding polypeptide according to claim 5, having an amino acid sequence selected from SEQ ID NO 17-24, such as SEQ ID NO 17.
7. Albumin binding polypeptide according to any preceding claim, which isThe albumin binding polypeptide binds albumin such that the interacting KDA value of at most 1x10-9M, e.g. at most 1x10-10M, e.g. at most 1x10-11M, e.g. at most 1x10-12M, e.g. at most 1x10-13M, e.g. at most 1x10-14M。
8. A fusion protein or conjugate comprising:
i) a first portion consisting of an albumin binding polypeptide according to any preceding claim; and
ii) a second portion consisting of a polypeptide having a desired biological activity.
9. The fusion protein or conjugate of claim 8, wherein the second moiety having a desired biological activity is a therapeutically active polypeptide.
10. The fusion protein or conjugate of any one of claims 8-9, wherein the second moiety having a desired biological activity is a binding polypeptide capable of selective interaction with a target molecule.
11. Albumin binding polypeptide, fusion protein or conjugate according to any preceding claim, further comprising a label.
12. Albumin binding polypeptide, fusion protein or conjugate according to claim 11, wherein the label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and particles.
13. A polynucleotide encoding the albumin binding polypeptide or fusion protein according to any one of claims 1-10.
14. A method of making a polypeptide according to any one of claims 1-10, the method comprising expressing a polynucleotide according to claim 13.
Technical Field
The present invention relates to a class of engineered polypeptides having binding affinity for albumin. In particular, the present invention relates to albumin binding polypeptides (albumin binding polypeptides) that are highly resistant to enzymatic cleavage.
Background
Serum albumin
Serum albumin is the most abundant Protein in mammalian serum (40 g/l; about 0.7mM in humans) and one of its functions is to bind molecules such as lipids and bilirubin (Peters T, Advances in Protein Chemistry37:161,1985). The half-life of serum albumin is directly proportional to the size of the animal, with, for example, Human Serum Albumin (HSA) having a 19-day half-life and rabbit serum albumin having a half-life of about 5 days (McCurdy TR et al, J LabClin Med 143:115,2004). Human serum albumin is widely distributed in the body, particularly in the intestinal and blood compartments, where it is primarily involved in maintaining permeability. Structurally, albumin is a single chain protein comprising three homology domains and a total of 584 or 585 amino acids (Dugaiczyk L et al, Proc Natl Acad Sci USA 79:71,1982). Albumin contains 17 disulfide bonds and a single reactive C34 thiol group, but lacks both N-linked and O-linked carbohydrate moieties (Peters,1985, supra; Nicholson JP et al, Br J Anaesth 85:599,2000). The lack of glycosylation simplifies recombinant expression of albumin. This property of albumin, together with the fact that its three-dimensional structure is known (He XM and Carter DC, Nature358: 2091992), makes it an attractive candidate for use in recombinant fusion proteins. Such fusion proteins typically combine a therapeutic protein (which is rapidly cleared from the body following administration of the protein itself) and a plasma protein (which exhibits natural slow clearance) in a single polypeptide chain (Sheffield WP, Curr Drug targets cardiovacs Haematol disorder 1:1,2001). Such fusion proteins may provide clinical benefits in requiring less frequent injections and higher levels of therapeutic protein in vivo.
Fusion or association with HSA results in extending the in vivo half-life of the protein
Serum albumin lacks any enzymatic or immunological function and therefore, should not exhibit undesirable side effects when conjugated to a biologically active polypeptide. In addition, HSA is a natural carrier involved in the endogenous transport and delivery of many natural and therapeutic molecules (Sellers EM and Koch-Weser MD, "Albumin Structure, Function and Uses", EdSerosenoer VM et al, Pergamon, Oxford, p.159, 1977). Several strategies have been reported to covalently couple proteins directly to serum albumin or to proteins that allow association with serum albumin in vivo. Examples of the latter process have been described, for example, in WO 91/01743. This document describes, inter alia, the use of albumin binding peptides or proteins derived from streptococcus (streptococcus) protein G for extending the half-life of other proteins. The idea is to fuse bacterially derived albumin binding peptides/proteins with therapeutically interesting peptides/proteins that have been shown to have rapid clearance in blood. The fusion protein thus produced binds serum albumin in vivo and benefits from its longer half-life, which extends the net half-life of the fused therapeutically interesting peptide/protein.
Association with HSA results in reduced immunogenicity
In addition to the effect on the in vivo half-life of the biologically active protein, it has been proposed that the non-covalent association of albumin with a fusion between the biologically active protein and the albumin binding protein acts to reduce the immune response of the biologically active protein. Thus, in WO2005/097202, the use of this principle to reduce or eliminate an immune response to a biologically active protein is described.
Albumin binding domains of bacterial receptor proteins
Streptococcal protein G is a bifunctional receptor present on the surface of certain strains of Streptococcus (Streptococcus) and is capable of binding to both IgG and serum albumin (SEt al, Mol Immunol 24:1113,1987). This structure is a high repetition of several structurally and functionally distinct domains (Guss et al, EMBO J5: 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 triple-helix bundle domain (Kraulis et al, FEBS Lett 378:190,1996). This motif is designated ABD (albumin binding domain) and is 46 amino acid residues in size. This motif is subsequently also referred to in the literature as G148-GA 3.
Other bacterial albumin binding proteins besides protein G from streptococcus have also been identified, which 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. Johansson and his colleagues conducted and reported studies on the structure and function of such albumin-binding proteins (Johansson et al, J Mol Biol 266: 859-. Furthermore, Rozak et al have reported the creation of artificial variants of GA modules that were selected and studied for different species specificities and stabilities (Rozak et al, Biochemistry45: 3263-. In the present disclosure, the terminology established in the Johansson et al and Rozak et al articles for GA modules from different bacterial species will be followed.
Recently, variants of the G148-GA3 domain with various optimization characteristics were developed. Such variants are disclosed, for example, in PCT publications WO2009/016043 and WO 2012/004384.
Clostripain
Clostripain, also known as the intracellular protease Arg-C, is a two-chain protease that can be isolated from Clostridium histolyticum. Clostripain was shown to have proteolytic and amidase/esterase activities (Mitchell, et al (1968), J Biol Chem 243: 4683-4692). Clostripain activity has been reported to be optimal in the pH range 7.6-7.9.
Clostripain cleaves preferentially at the carboxyl group of arginine residues (Labrou et al (2004), Eur Jbiochem 271(5): 983-92; Keil (1992), "Specificity of proteolysis", Springer-Verlag, page 335), but cleavage of the lysyl bond has also been reported. Clostripain has been shown to accept substrates containing Lys instead of Arg, but the reaction rate is low compared to reactions with Arg containing substrates. For example, clostripain has been reported to cleave glucagon at Arg-Arg, Arg-Ala and Lys-Tyr sites. The relative initial rates of hydrolysis of these three bonds were 1, 1/7 and 1/300(Labouesses (1960), Bull Soc Chim Biol 42: 1293-304).
Clostripain cleavage is often used in biochemical and biotechnological applications. Applications of clostripain cleavage include peptide mapping, sequence analysis, cell separation, hydrolysis/condensation of amide bonds, and peptide synthesis.
Clostripain, for example, can be used to cleave a tag (e.g., His) for protein purification and/or detection6c-Myc, Flag and GST tag). In addition, clostripain cleavage can be used during the preparation of an amidated therapeutic polypeptide from a precursor polypeptide, thereby increasing the resistance of the therapeutic polypeptide to proteolytic degradation by endogenous proteases following administration to an animal or human subject.
As is apparent from the different sections described in this background, the provision of polypeptide molecules with high affinity for albumin and exhibiting high tolerance to enzymatic cleavage, in particular of clostripain, is a key factor in the development of various biomedical, biotechnological and other applications, and there is therefore a need in the art for such polypeptide molecules.
Disclosure of the invention
In a first aspect of the present invention, the need for new polypeptides with relatively high albumin affinity and high tolerance to clostripain cleavage is met by providing albumin polypeptides comprising an albumin Binding Motif (BM) consisting of the amino acid sequence:
GVSDFYKKLI XaKAKTVEGVE ALKXbXcI
wherein, independently of each other,
Xaselected from D and E;
Xbselected from D and E; and
Xcselected from A and E.
In one embodiment of the polypeptide according to this aspect of the invention, XaIs D.
In one embodiment of the polypeptide according to this aspect of the invention, XbIs D.
In one embodiment of the polypeptide according to this aspect of the invention, XcIs A.
In view of all the above combinations, it is clear that the sequence of the albumin binding motif BM is selected from the group consisting of SEQ ID NO: 1-8. In one embodiment of the polypeptide according to this aspect of the invention, the sequence of BM is SEQ ID NO 1.
In order to provide albumin binding polypeptides comprising an Albumin Binding Domain (ABD) or a variant thereof, which peptides are highly resistant to clostripain cleavage, the inventors investigated various variants of PEP07843(SEQ ID NO: 27). The inventors have shown that the substitution of the arginine residue (R) at the position corresponding to
Thus, in view of previous studies showing that clostripain cleaves peptides at the carboxyl group of Lys residues, the discovery that the Arg-to-Lys substitution mutations discussed above stabilize and improve the tolerance of albumin binding polypeptides to clostripain cleavage was surprising and unexpected.
In one embodiment according to this aspect of the invention, an albumin binding polypeptide is provided wherein the albumin binding motif forms part of a three-helix bundle protein domain. For example, BM may substantially constitute or form part of two alpha helices with interconnected loops in the three-helix bundle protein domain.
In a particular embodiment of the invention, such triple-helix bundle protein domain is selected from the group consisting of the triple-helix domains of bacterial receptor proteins. Non-limiting examples of such bacterial receptor proteins may be selected from the group consisting of albumin binding receptor proteins from Streptococcus (Streptococcus), Streptococcus digestus (Peptostreptococcus) and Finegoldia species, such as for example selected from the group consisting of protein G, MAG, ZAG, PPL and PAB.
In a particular embodiment of the invention, BM forms part of a domain of protein G, such as for example the domain of protein G from streptococcus strain G148. In a different variant of this embodiment, the triple helix bundle protein domain of which the BM forms part is selected from the group consisting of domain GA1, domain GA2 and domain GA3 of protein G from streptococcus strain G148, in particular domain GA 3.
In alternative embodiments, the BM forms part of one or more of the 5 triple-helical domains of bacterial receptor protein a from Staphylococcus aureus (Staphylococcus aureus); that is, the triple helix bundle protein domain is selected from the group consisting of protein a domains A, B, C, D and E. In other similar embodiments, the BM forms part of protein Z derived from domain B of protein a from staphylococcus aureus.
In embodiments of the invention in which the BM "forms" part "of the" three-helix bundle protein domain, this is understood to mean that the sequence of the BM is "inserted" or "grafted" into the sequence of the original three-helix bundle domain such that the BM replaces a similar structural motif in the original domain. For example, without wishing to be bound by theory, BM is thought to constitute two of the three helices of a triple helix bundle, and may therefore replace such a double helix motif in any triple helix bundle. As will be appreciated by those skilled in the art, the replacement of two helices of the three-helix bundle domain by two BM helices must be performed so as not to affect the basic structure of the polypeptide. That is, according to this embodiment of the invention, the overall folding of the C.alpha.backbone of the polypeptide will be substantially identical to the three-helix bundle protein domain of which it forms a part, e.g., identical components having the same order of secondary structure, etc. Thus, according to the invention the BM "forms" part "of the" triple-helix bundle domain, if the polypeptide according to this embodiment of the invention has the same fold as the original domain, meaning that the basic structural properties are common, which properties for example result in the same CD spectrum. Other relevant parameters are known to those skilled in the art.
In one embodiment of this aspect of the invention, the albumin binding polypeptide is a triple-helix bundle protein domain comprising the albumin binding motif as defined above and further sequences constituting the remainder of the triple-helix configuration. Accordingly, in one embodiment there is provided an albumin binding polypeptide comprising the amino acid sequence:
LAX3AKX6X7ANX10ELDX14Y-[BM]-LX43X44LP
wherein
[ BM ] is an albumin binding motif as defined above,
and, independently of each other,
X3selected from C, E, Q and S;
X6selected from C, E and S;
X7selected from A and S;
X10selected from A, R and S;
X14selected from A, C, K and S;
X43selected from A and K; and
X44selected from A, E and S.
In a particular embodiment of this albumin binding polypeptide, X3Is E.
In a particular embodiment of this albumin binding polypeptide, X6Is E.
In a particular embodiment of this albumin binding polypeptide, X7Is A.
In a particular embodiment of this albumin binding polypeptide, X10Is A.
In a particular embodiment of this albumin binding polypeptide, X14Is S.
In a particular embodiment of this albumin binding polypeptide, X43Is A.
In a particular embodiment of this albumin binding polypeptide, X44Is A.
As will be appreciated by the skilled person, the function of any polypeptide, such as the albumin binding capacity of a polypeptide according to the invention, depends on the tertiary structure of the polypeptide. It is therefore possible to make minor changes to the amino acid sequence of a polypeptide without affecting its function. Thus, the present invention comprises modified variants of BM that retain albumin binding characteristics and are highly resistant to clostripain cleavage. For example, it may be possible that an amino acid residue belonging to a certain functional group of amino acid residues (e.g. hydrophobic, hydrophilic, polar, etc.) can be exchanged for another amino acid residue from the same functional group.
As described in detail in the experimental section below, the inventors have identified individual albumin binding polypeptide sequences. These sequences constitute separate embodiments of the albumin binding polypeptide according to the first aspect of the invention. The sequences of these individual albumin binding polypeptides are shown in figure 1 and as SEQ ID NOs 9-16.
Thus, in one embodiment of the invention according to this first aspect, an albumin binding polypeptide is provided comprising an amino acid sequence selected from the group consisting of SEQ ID Nos. 9-16. The invention also encompasses albumin binding polypeptides comprising an amino acid sequence having 93% or greater identity to a sequence selected from SEQ ID NOs 9-16, with the proviso that the amino acid at the position corresponding to position 23 in SEQ ID NOs 9-16 is K. In some embodiments, the polypeptide of the invention may comprise a sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs 9-16, provided that the amino acid at the position corresponding to position 23 in SEQ ID NOs 9-16 is K.
In a particular embodiment. The albumin binding polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:9 and sequences having 93% or greater identity thereto, with the proviso that the amino acid at the position corresponding to position 23 in SEQ ID NO:9 is K. In some embodiments, the polypeptide of the invention may comprise a sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID No. 9, provided that the amino acid at the position corresponding to position 23 in SEQ ID No. 9 is K.
The term "% identity", as used throughout the specification, may be calculated as follows. The query sequence (querysequence) is aligned to the target sequence by using the CLUSTAL W algorithm (Thompson et al, Nucleic Acids Research,22:4673-4680 (1994)). The comparison is performed over the window corresponding to the shortest aligned sequence. The shortest aligned sequence may be the target sequence in some cases. In other cases, the query sequence may constitute the shortest aligned sequence. The amino acid sequences at each position are compared and the percentage of positions in the query sequence that have the same correspondence in the target sequence is reported as% identity.
The terms "albumin binding" and "binding affinity for albumin" as used in this specification refer to the properties of a polypeptide that can be detected, for example, by using surface plasmon resonance technology (surface plasmon resonance technology), such as in a Biocore instrument. For example, as described in the examples below, albumin binding affinity can be detected in an assay in which albumin or a fragment thereof is immobilized on a sensor chip (sensor chip) of a device) And passing the sample containing the polypeptide to be detected over the chip. Optionally, the polypeptide to be detected is immobilized on a sensor chip of the device and a sample comprising albumin or a fragment thereof is passed over the chip. In this aspect, the albumin can be a serum albumin from a mammal, such as human serum albumin. The results obtained by such experiments can then be interpreted by the skilled person to establish an at least qualitative measure of the binding affinity of the polypeptide for albumin. If quantitative measures are desired, e.g. determining K for the interactionDValue, surface plasmon resonance methods may also be used. The binding value may be defined, for example, in the Biacore2000 instrument (Biacore AB). Albumin is suitably immobilized on the sensor chip of the device and a sample of the polypeptide whose affinity is to be determined is prepared by serial dilution and injected in random order. KDValues can then be calculated from the results of the 1:1Langmuir binding model using, for example, BIAevaluation 4.1 software, or other suitable software provided by the device manufacturer (Biacore AB).
The albumin binding polypeptide according to this first aspect of the invention binds to albumin such that the relative K of interactionDThe value is at most 1x10-9M is 1 nM. In some embodiments, the interacting KDThe value is at most 1x10-10M, e.g. at most 1x10-11M, e.g. at most 1x10-12M, e.g. at most 1x10-13M, e.g. at most 1x10-14M。
In one embodiment of the invention, the albumin to which the albumin binding polypeptide binds is selected from the group consisting of human serum albumin, rat serum albumin, cynomolgus serum albumin and mouse serum albumin.
In a particular embodiment, the albumin to which the albumin binding polypeptide binds is human serum albumin.
The present invention also includes the albumin binding polypeptides described above, which further include one or more amino acids located on one or both sides of the albumin binding motif. These amino acid residues may play a role in enhancing the binding of albumin by the polypeptide, but may equally well be used for other purposes, such as one or more of preparation, purification, in vivo or in vitro stabilization, coupling or detection of the polypeptide, and any combination thereof.
Thus in one embodiment, amino acids directly before or after the alpha helix at the N-or C-terminus of an amino acid sequence as defined herein may affect the stability of the conformation. One example of an amino acid residue that may contribute to improved conformational stability is a serine residue located at the N-terminus of the amino acid sequence. The N-terminal serine residue may in some cases form a standard S-X-X-E capping box (capping box) by hydrogen bonding between the gamma oxygen involved in the serine side chain and the polypeptide backbone NH of the glutamic acid residue. This N-terminal capping may contribute to the stabilization of the first alpha-helix of the triple-helical domain, which in some embodiments includes an albumin binding motif.
Thus, in one embodiment of this aspect of the invention, an albumin binding polypeptide is provided which further comprises at least one serine residue flanking the N-terminus of the polypeptide sequence as defined herein. In other words, one or more serine residues precede the amino acid sequence. In addition, the albumin binding polypeptide may further comprise one, two or three or more serine residues at either or both of the N-terminal or C-terminal sides of the polypeptide.
In one embodiment of this aspect of the invention, an albumin binding polypeptide is provided which further comprises a glutamic acid residue flanking the N-terminus of the polypeptide sequence as defined herein.
It is to be understood that one, two, three, four or any suitable number of amino acid residues may precede an amino acid sequence as defined herein. Thus, a single serine residue, a single glutamate residue, or a combination of both, such as a glutamate-serine (GS) combination or a glutamate-serine (GSs) combination, may precede the amino acid sequence.
Thus, in one embodiment, an albumin binding polypeptide is provided which further comprises the amino acid GS on the N-terminal side of the polypeptide sequence as defined herein.
In a particular embodiment, albumin binding polypeptides are provided which further comprise the amino acid GSS on the N-terminal side of the polypeptide sequence as defined herein.
In particular, the invention comprises the sequences of the individual albumin binding polypeptides shown as SEQ ID NOs 17-24 in FIG. 1, such as SEQ ID NO 17. These sequences constitute separate embodiments of the albumin binding polypeptide according to the above embodiments of the first aspect of the invention.
In yet another embodiment, the added amino acid residue comprises glutamic acid flanking the N-terminus of the polypeptide sequence as defined herein.
Similarly, when such triple-helical domains are present, C-terminal capping may be utilized to improve the stability of the third alpha-helix in the triple-helical domain including the albumin binding motif.
A proline residue may at least partially function as a capping residue when present on the C-terminal side of an amino acid sequence as defined herein. In this case, the lysine residue following the proline residue at the C-terminal side may contribute to further stabilization of the third helix of the albumin-binding polypeptide by hydrogen bonding between the amino group of the lysine residue and the carbonyl group of an amino acid located two or three residues before the lysine in the polypeptide backbone.
Thus, in one embodiment, an albumin binding polypeptide is provided, which further comprises a lysine residue on the C-terminal side of the polypeptide sequence according to any one or more of the above definitions.
As discussed above, the added amino acids may be involved in the preparation of albumin binding polypeptides. In particular, when the albumin binding polypeptide according to the embodiment wherein there is a proline at the C-terminus is prepared by chemical peptide synthesis, one or more optional amino acid residues after the C-terminal proline may provide an advantage. Such added amino acid residues may, for example, prevent the formation of undesirable substances, such as diketopiperazines in the dipeptide synthesis stage. An example of such an amino acid residue is glycine.
Thus, in another embodiment, an albumin binding polypeptide is provided, which further comprises a glycine residue on the C-terminal side of the polypeptide sequence according to any one or more of the above definitions.
In one embodiment, the added amino acids include glycine residues at the C-terminal side of the polypeptide, either directly after proline residues or after the addition of lysine and/or glycine residues as specified above.
Alternatively, when a C-terminal proline residue is present, polypeptide preparation may benefit from amidation of the C-terminal proline residue of the amino acid sequence as defined herein. In this case, the C-terminal proline includes an added amine group at the carboxyl carbon. In one embodiment of the polypeptides described herein, particularly those whose C-terminus is terminated by proline or other amino acid known to be racemic during peptide synthesis, the addition of glycine to the C-terminus or amidation of proline as described above, when present, may also counter the potential problem of racemization of the C-terminal amino acid residue. If a polypeptide amidated in this way is intended to be prepared by recombinant means rather than by chemical synthesis, then amidation of the C-terminal amino acid may be performed by some method known in the art, for example by using an amidated PAM enzyme.
The person skilled in the art knows the methods for carrying out C-terminal modifications, for example by means of different types of preformed matrices (pre-master substrates) for peptide synthesis.
Thus, the added amino acid residues may comprise one or more amino acid residues added for the purpose of chemical coupling, e.g. coupling to a chromatography resin to obtain an affinity matrix or coupling to a chelating moiety for complexing with a metal radionuclide. An example of this is the addition of a cysteine residue at the very first or very last position, i.e.the N-or C-terminus, of a polypeptide chain. Such added amino acid residues may also include a "tag" for polypeptide purification or detection, e.g., hexahistaminyl (His) for interaction with an antibody specific for the tag6) A tag, or a glutathione S-transferase tag (GST-tag), or a "Myc" ("c-Myc") tag or a "FLAG" tag. The state of the artThe person is aware of other alternatives.
The "added amino acid residues" discussed above may also constitute one or more polypeptide domains having any desired function, e.g. the same binding function as the first albumin binding domain, or another binding function, or a therapeutic function, or a cytotoxic function, or an enzymatic function, or a fluorescent function, or a mixture thereof. According to the invention, the polypeptide "units" linked in such polypeptides may be linked by covalent coupling using known methods of organic chemistry, or expressed as one or more fusion polypeptides in a system for recombinant expression of the polypeptides, or linked directly in any other way or mediated by a linker comprising a number of amino acids.
In another embodiment, the added amino acid residues include cysteine residues at the N-and/or C-terminus of the polypeptide. Such cysteine residues may be directly before and/or after the amino acid sequence as defined herein, or may be before and/or after any other added amino acid residue as described above. Site-directed conjugation of sulfhydryl groups for albumin binding polypeptides can be obtained by adding cysteine residues to the polypeptide chain. Alternatively, selenocysteine residues may be introduced at the C-terminus of the polypeptide chain to facilitate site-specific conjugation (Cheng et al, Nat Prot 1:2, 2006).
Thus, in a further embodiment of this aspect of the invention, an albumin binding polypeptide is provided, which further comprises a cysteine residue flanking the N-terminus of the polypeptide sequence, according to any one or more of the above definitions.
In another embodiment, an albumin binding polypeptide is provided, which further comprises a cysteine residue at the C-terminal side of the polypeptide sequence according to any one or more of the above definitions.
In one embodiment of this aspect of the invention, albumin binding polypeptides are provided that comprise no more than two cysteine residues, for example no more than one cysteine residue.
Furthermore, the present invention also encompasses multimers of polypeptides having affinity for albumin, i.e. polypeptide chains comprising at least two albumin binding polypeptides or fragments thereof as monomer units. It may be of interest to obtain an even stronger binding of albumin than is possible with a polypeptide according to the invention, for example in an albumin purification process or a therapeutic process that utilizes the albumin binding function. In this case, providing multimers of the polypeptides, such as dimers, trimers or tetramers, may provide the necessary affinity effects. According to the invention, the polymer can be composed of a suitable number of polypeptides. According to the invention, the polypeptide domains that form monomers in such multimers may all have the same amino acid sequence, but it is equally possible that they have different amino acid sequences. As described above, the linked polypeptide "units" in the multimers according to the invention may be linked by covalent coupling using known methods of organic chemistry, or expressed as one or more fusion polypeptides in a system for recombinant expression of the polypeptides, or linked directly in any other way or mediated by a linker comprising a number of amino acids.
Furthermore, "heterologous" fusion polypeptides or proteins, or conjugates, or multimers thereof, wherein the albumin binding polypeptide according to the invention constitutes a first domain, or first moiety, and the second and further moieties have a function other than binding to albumin, are also encompassed and fall within the scope of the invention. The second and further part or parts of the fusion polypeptide or conjugate in such a protein suitably have the desired biological activity.
Accordingly, in a second aspect of the invention there is provided a fusion protein or conjugate comprising a first portion consisting of an albumin binding polypeptide according to the first aspect and a second portion consisting of a polypeptide having a desired biological activity.
Non-limiting examples of such desired biological activities include therapeutic activity, binding activity, and enzymatic activity. In one embodiment, the second moiety having the desired biological activity is a therapeutically active polypeptide.
Non-limiting examples of therapeutically active polypeptides are biomolecules, e.g. selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokinesA molecule of the group consisting of a daughter, a cytokine and a lymphokine. Non-limiting examples of therapeutically active biomolecules that may prove useful in fusions or conjugates with albumin binding polypeptides are selected from the group consisting of IL-2, GLP-1, BNP (Alb-beta-natriuretic peptide), IL-1-RA (interleukin-1 receptor antagonist), KGF (keratinocyte growth factor),
Further non-limiting examples of suitable biomolecules are non-human biologically active proteins, such as proteins selected from the group consisting of bacterial toxins (e.g. pseudomonas exotoxin and superantigens of staphylococci and streptococci), enzymes (e.g. ribonucleases and beta-lactamases) and activation proteins (e.g. streptokinase).
In another embodiment, the invention provides fusion proteins or conjugates in which the second moiety having the desired biological activity is a binding polypeptide capable of selective interaction with a target molecule. The second and any further moieties are selected from binding moieties capable of selective interaction (binding) with a target molecule, typically a target molecule other than albumin, although albumin is not excluded.
Such binding polypeptides may for example be selected from the group consisting of: antibodies and fragments and domains thereof that substantially retain antibody binding activity; microbodies, maxybodies, high affinity multimers, and other small disulfide-bonded proteins; a binding protein derived from a scaffold selected from the group consisting of: staphylococcal protein a and its domain, other triple-helical domains, lipocalins, ankyrin repeat domains, cellulose binding domains, gamma crystals, green fluorescent protein, human cytotoxic T lymphocyte-associated
In some embodiments, the target molecule for binding the target binding polypeptide may be selected from the group consisting of: amyloid beta (a β) peptide of alzheimer's disease; amyloid peptides associated with other diseases; toxins, such as bacterial toxins and snake venom; blood coagulation factors, such as von willebrand factor; interleukins, such as IL-13; myostatin; proinflammatory factors such as TNF- α, TNF- α receptor, IL-1, IL-8, and IL-23; complement factors, such as C3 and C5; hypersensitivity mediators, such as histamine and IgE; tumor-associated antigens, such as CD19, CD20, CD22, CD30, CD33, CD40, CD52, CD70, cMet, HER1, HER2, HER3, HER4, CAIX (carbonic anhydrase IX), CEA, IL-2 receptor, MUC1, PSMA, TAG-72, and other biomolecules such as G-CSF, GM-CSF, growth factor (GH), insulin, and somatostatin.
As understood by the skilled person, the albumin binding polypeptide according to the first aspect may be useful in a fusion protein or as a conjugate partner to any other moiety. Thus, the above list of therapeutically active polypeptides, binding polypeptides and target molecules should not be construed as limiting in any way.
Other possibilities for producing fusion polypeptides or conjugates are also contemplated. Thus, the albumin binding polypeptide according to the first aspect of the invention may be covalently coupled to a second or further moiety or moieties which exhibit a further function in addition to or instead of target binding. One example is the fusion between one or more albumin binding polypeptides and an enzymatically active polypeptide as a reporter or effector moiety. Examples of reporter enzymes that can be coupled to the albumin binding polypeptide to form fusion proteins are known to those skilled in the art and include, for example, beta galactosidase, alkaline phosphatase, horseradish peroxidase, and carboxypeptidase. Other options for the second and further one or more moieties of the fusion protein or conjugate according to the invention include, again without limitation, fluorescent polypeptides such as green fluorescent protein, red fluorescent protein, luciferase and variants thereof.
In one embodiment of this aspect of the invention, fusion proteins or conjugates are provided wherein the additional moiety is comprised of a polypeptide having other desired biological activity, which may be the same or different from that of the second moiety. In a particular embodiment, the second moiety may be selected from the group consisting of a therapeutically active polypeptide, a human endogenous enzyme, a hormone, a growth factor, a chemokine, a cytokine, a lymphokine, IL-2, GLP-1, BNP, an IL-1 receptor antagonist, KGF, a polypeptide,GH. G-CSF, CTLA-4, myostatin, factor VII, factor VIII, factor IX and factor X, and non-human biologically active proteins, are selected from the group consisting of bacterial toxins, enzymes and activating proteins, and further moieties may comprise binding polypeptides capable of selectively interacting with a target molecule as defined above. In another particular embodiment, the second and further moieties each comprise a binding polypeptide capable of selective interaction with a target molecule as defined above.
With regard to the description herein of fusion proteins or conjugates incorporating albumin binding polypeptides according to the invention, it should be noted that the designation of the first, second and further moieties is for clarity reasons to distinguish between one or more albumin binding polypeptides according to the invention on the one hand and moieties exhibiting other functions on the other hand. These designations are not intended to refer to the actual order of the different domains in the polypeptide chains of the fusion protein or conjugate. Thus, for example, the first moiety may be present without limitation at the N-terminus, at the middle or at the C-terminus of the fusion protein or conjugate.
In one embodiment of the conjugate according to the present disclosure, the second moiety is conjugated to the albumin binding polypeptide via a lysine or cysteine residue added to the N-or C-terminus of the albumin binding polypeptide or via a lysine or cysteine residue at the position in the albumin binding polypeptide where the lysine or cysteine residue occurs. For example, if the albumin binding polypeptide comprises the 46 amino acid sequence disclosed above, conjugation may be at a position selected from X3、X6And X14Is performed. If the conjugation site is one of the amino acid sequences of the albumin binding polypeptide, e.g. position X of the 46-mer14Without the need to add additional amino acids to the albumin binding polypeptide for the purpose of enabling conjugation of the second moiety. Thus, in one embodiment of this aspect, conjugates are provided wherein the second moiety is via X, which is present in the corresponding disclosed 46-mer14The thiol group of any cysteine residue at a position of said first moiety at position is conjugated to the first moiety.
In a related aspect, there is provided an albumin binding polypeptide, fusion protein or conjugate as defined in the present disclosure, further comprising an organic molecule, for example a cytotoxic agent. Non-limiting examples of cytotoxic agents that may be fused or conjugated to the albumin binding polypeptide according to the first aspect, or combined with the fusion protein or conjugate according to the second aspect, are selected from the group consisting of a spinotoxin, an auristatin, doxorubicin, maytansinoids, paclitaxel, ecteinascidins, geldanamycin, methotrexate and derivatives thereof and combinations thereof. The use of direct albumin conjugates has been previously attempted to treat various disorders. Such direct albumin conjugates are used, for example, with doxorubicin in cancer (Kratz et al, J MedChem 45: 5523-4833, 2002) and with methotrexate in rheumatoid arthritis (Wunder et al, JImmunol 170: 4793-4801, 2003). It will be appreciated that albumin binding polypeptides by themselves or as part of a fusion protein or conjugate, provide an indirect means of constructing albumin complexes through its high albumin binding capacity and may therefore provide an alternative treatment option to the above-mentioned attempts.
The above aspects also comprise polypeptides, wherein the albumin binding polypeptide according to the first aspect, or the albumin binding polypeptide according to the second aspect as comprised in a fusion protein or conjugate, is provided with a labeling group, e.g. for polypeptide detection purposes, e.g. a label selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and particles. In particular, the present disclosure comprises radiolabeled polypeptides consisting of albumin binding polypeptides, radio-chelates of fusion proteins or conjugates as described herein and radionuclides, e.g. radioactive metals.
In embodiments wherein the labeled albumin binding polypeptide comprises an albumin binding polypeptide according to the first aspect of the disclosure and a label, the labeled polypeptide may for example be used for indirectly labeling serum albumin. Due to the strong association between the tagged polypeptide and serum albumin, the tagged polypeptide can be used, for example, to study vascular permeability and blood pool.
In other embodiments, the labeled albumin binding polypeptide is present as part of a fusion protein or conjugate that also includes a second moiety having the desired biological activity. The label may in some cases be conjugated only to the albumin binding polypeptide, and in some cases to both the albumin binding polypeptide and the second portion of the conjugate or fusion protein. Furthermore, it is also possible that the label may be conjugated only to the second moiety instead of the albumin binding moiety. Thus, in yet another embodiment, an albumin binding polypeptide is provided comprising a second moiety, wherein the label is conjugated to the second moiety only. When referring to a labelled polypeptide, it is to be understood that reference to all aspects of the polypeptide as described herein includes fusion proteins and conjugates comprising the albumin binding polypeptide and the second moiety and optionally further moieties. Thus, the labeled polypeptide may comprise only the albumin binding polypeptide and, for example, a therapeutic radionuclide, which may be chelated to or covalently coupled to the albumin binding polypeptide, or the albumin binding polypeptide, the therapeutic radionuclide and a second moiety, such as a small molecule, having a desired biological activity, for example, a therapeutic effect.
In embodiments where the albumin binding polypeptide, fusion protein, or conjugate is radiolabeled, such radiolabeled polypeptide may include a radionuclide. Most radionuclides have metallic properties and metals are typically not capable of forming stable covalent bonds with elements present in proteins and peptides. For this reason, the labeling of proteins and peptides with radiometals is performed using chelating agents, i.e. polydentate ligands, which form non-covalent compounds called chelates with the metal ions. In embodiments of albumin binding polypeptides, fusion proteins or conjugates, incorporation of the radionuclide is achieved by providing a chelating environment through which the radionuclide can coordinate, chelate or complex with the polypeptide.
An example of a chelating agent is a polyamine polycarboxylic acid type chelating agent. Two such classes of polyamine polycarboxylic acid chelants can be divided into: macrocyclic and acyclic chelating agents.
In one embodiment, the albumin binding polypeptide, fusion protein or conjugate comprises a chelating environment provided by a polyamine polycarboxylic acid chelator conjugated to the albumin binding polypeptide via an amine group of a thiol group of a cysteine residue or a lysine residue.
The most commonly used macrocyclic chelating agents for radioisotopes of indium, gallium, yttrium, bismuth, radioactive actinium and radioactive lanthanum are different derivatives of DOTA (1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid). In one embodiment, the chelating environment for the albumin binding polypeptide, fusion protein or conjugate is provided by DOTA or a corresponding derivative. More specifically, in one embodiment, the chelating polypeptides encompassed by the present disclosure are obtained by reacting the
Furthermore, 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA) and derivatives thereof may be used as chelating agents. Thus, in one embodiment. Albumin binding polypeptides, fusion proteins or conjugates are provided wherein the polyamine polycarboxylic acid chelating agent is 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid or a derivative thereof.
The most commonly used acyclic polyamine polycarboxylic acid chelating agents are different derivatives of DTPA (diethylenetriaminepentaacetic acid). Thus, polypeptides having a chelating environment provided by diethylenetriaminepentaacetic acid or corresponding derivatives are also encompassed by the present disclosure.
In a third aspect of the invention, there is provided a polynucleotide encoding an albumin binding polypeptide or fusion protein as described herein.
Also encompassed is a method of making an albumin binding polypeptide or fusion protein as described above, the method comprising expressing a polynucleotide; an expression vector comprising the polynucleotide and a host cell comprising the expression vector.
Also encompassed is a method of producing a polypeptide comprising culturing the host cell under conditions that allow expression of the polypeptide from the expression vector, and isolating the polypeptide.
The albumin binding polypeptides of the present disclosure may optionally be prepared using amino acids and/or amino acid derivatives having protected reactive side chains by non-biological peptide synthesis including:
stepwise coupling of amino acids and/or amino acid derivatives to form a polypeptide having protected reactive side chains according to the first aspect,
removing the protecting group from the reactive side chain of the polypeptide, and
folding of polypeptides in aqueous solution.
The present application provides the following:
1) an albumin binding polypeptide comprising an albumin binding motif [ BM ], said motif consisting of the amino acid sequence:
GVSDFYKKLI XaKAKTVEGVE ALKXbXcI
wherein, independently of each other,
Xaselected from D and E;
Xbselected from D and E; and
Xcselected from A and E.
2) The albumin binding polypeptide of 1), wherein the sequence is SEQ ID NO 1.
3) Albumin binding polypeptide according to any of the preceding 1) -2), in which the albumin binding motif forms part of a triple helix bundle protein domain.
4) Albumin binding polypeptide according to claim 3), comprising the amino acid sequence:
LAX3AKX6X7ANX10ELDX14Y-[BM]-LX43X44LP
wherein
[ BM ] is an albumin binding motif as defined in any one of 1) to 2),
and, independently of each other,
X3selected from C, E, Q and S;
X6selected from C, E and S;
X7selected from A and S;
X10selected from A, R and S;
X14selected from A, C, K and S;
X43selected from A and K; and
X44selected from A, E and S.
5) Albumin binding polypeptide according to claim 4), the amino acid sequence of which comprises a sequence satisfying one definition selected from:
i) the sequence is selected from SEQ ID NO 9-16;
ii) the sequence is an amino acid sequence having 93% or more identity to a sequence selected from SEQ ID NO 9-16, with the proviso that the amino acid at the position corresponding to position 23 in SEQ ID NO 9-16 is K.
6) The albumin binding polypeptide according to 5), having an amino acid sequence selected from the group consisting of SEQ ID NO 17-24, such as SEQ ID NO 17.
7) Albumin binding polypeptide according to any one of the preceding 1) -6), in which the albumin binding polypeptide binds to albumin such that the interacting KDA value of at most 1x10-9M, e.g. at most 1x10-10M, e.g. at most 1x10-11M, e.g. at most 1x10-12M, e.g. at most 1x10-13M, e.g. at most 1x10-14M。
8) A fusion protein or conjugate comprising:
i) a first part consisting of the albumin binding polypeptide according to any one of the preceding 1) -7); and
ii) a second portion consisting of a polypeptide having a desired biological activity.
9) The fusion protein or conjugate of claim 8), wherein the second moiety having the desired biological activity is a therapeutically active polypeptide.
10) The fusion protein or conjugate of any one of claims 8) -9), wherein the second moiety having the desired biological activity is a binding polypeptide capable of selective interaction with a target molecule.
11) The albumin binding polypeptide, fusion protein or conjugate of any one of the preceding 1) -10), further comprising a label.
12) The albumin binding polypeptide, fusion protein or conjugate of claim 11), wherein said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and particles.
13) A polynucleotide encoding the albumin binding polypeptide or fusion protein according to any one of claims 1) -10).
14) A method of making a polypeptide according to any one of claims 1) -10), the method comprising expressing a polynucleotide according to 13).
While the invention has been described with reference to various exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or molecule to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to any particular embodiment contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.
Brief Description of Drawings
FIG. 1 is a list of amino acid sequences of examples of albumin binding motifs (SEQ ID NOS: 1-8), examples of albumin binding polypeptides according to the invention (SEQ ID NOS: 9-24) and control polypeptides (SEQ ID NOS: 25(PEP12381), 26(PEP12379), 27(PEP07843) and 28(PEP06923)) comprised in albumin binding polypeptides of the invention.
FIGS. 2A-2B show the superposition of the four CD spectra of the indicated albumin binding polypeptides PEP12379(SEQ ID NO:26), PEP12380(SEQ ID NO:17), PEP12381(SEQ ID NO:25) and PEP07843(SEQ ID NO:27) before and after heat treatment.
Figure 3 shows the results of SDS-PAGE analysis of samples in which the albumin binding polypeptide was incubated with clostripain for up to 22 hours.
Figure 4 shows the results of LC/MS analysis of samples of the indicated albumin binding polypeptides incubated with 0.2U clostripain per mg of polypeptide for up to 22 hours.
FIG. 5 shows the results of LC/MS analysis of samples incubated with the indicated albumin binding polypeptide for up to 22 hours with clostripain per mg of polypeptide.
FIG. 6 shows a representative LC/MS chromatogram of a sample of PEP12380(SEQ ID NO:17) incubated for up to 22 hours with clostripain, a polypeptide of 5U per mg.
FIG. 7 shows thatThe binding assay performed in the device was used to investigate the binding of the indicated albumin binding polypeptide PEP12379(SEQ ID NO:26) to human serum albumin.
FIG. 8 shows that
FIG. 9 shows thatThe binding assay performed in the device was used to investigate the binding of the indicated albumin binding polypeptide PEP12381(SEQ ID NO:25) to human serum albumin.
FIG. 10 shows that
FIG. 11 shows that
The invention will now be further elucidated by the following non-limiting description of experiments in accordance with the implementations. Conventional chemical and molecular biological methods are used throughout unless otherwise indicated.
Examples
The aim of the studies described below is to enable cleavage of a fusion protein comprising the albumin binding domain and the enzyme clostripain (ArgC), without cleaving the protein within the albumin binding domain sequence. Herein, the inventors designed three variants of the albumin binding polypeptide PEP07843(SEQ ID NO:27) and showed that the inventive variant PEP12380(SEQ ID NO:17) featuring Arg-to-Lys substitutions demonstrated unexpected superiority with respect to protease stability and albumin binding activity over the other variants tested.
As used herein, the term PEPXXXX refers to an albumin binding polypeptide having 46 amino acid residues, as defined in relation to the first aspect of the invention, and also having a GSS extension on the N-terminal side. Thus, unless otherwise indicated, the numbering of amino acid positions refers to the positions of amino acid residues in the above-described 46 amino acid polypeptide.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：对几种病原细菌有抑菌活性的苏云金菌素的前体肽