CN103379915A - HSA-related compositions and methods of use - Google Patents

HSA-related compositions and methods of use Download PDF

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CN103379915A
CN103379915A CN2011800674634A CN201180067463A CN103379915A CN 103379915 A CN103379915 A CN 103379915A CN 2011800674634 A CN2011800674634 A CN 2011800674634A CN 201180067463 A CN201180067463 A CN 201180067463A CN 103379915 A CN103379915 A CN 103379915A
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hsa
domain iii
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C·高
C·乔杜里
X·姚
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MedImmune LLC
MedImmune Vaccines Inc
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MedImmune Vaccines Inc
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Abstract

Provided are human serum albumin (HSA) compositions with improved properties over native HSA.

Description

Compositions related and the using method of HSA
The cross reference of 1 related application
The application requires the PCT application number of submission on February 15th, 2011: the priority of PCT/US2011/24855 is combined in this with it by reference in full with it.
Quoting of 2 sequence tables
In conjunction with the sequence table of submitting to the application by EFS-Web with text, described sequence table name is called " MED0554_PCT2_ST25 " to the application, be created on August 7th, 2011, and size is 45,056 bytes by reference.
3 background of invention
Neonatal Fc receptor (FcRn) prolongs IgG and both life-spans of human serum haemproteins (HSA) by the mechanism that relies on pH, under the acid pH of endosome specifically in conjunction with these two kinds of molecules and make their recirculatioies get back to cell surface, thereby make these two kinds of molecular transfer leave the lysosome degradation pathway of acquiescence.Confirmed that the FcRn binding ability is intrinsic for albuminous Domain III.
4 summary of the inventions
This disclosure provides the compositions related and using method of HSA.This disclosure provides the chimeric polyeptides that comprises human serum albumin (HSA) part and the compositions that comprises the chimeric polyeptides that makes up with pharmaceutical carrier, and described human serum albumin partly comprises neonatal Fc Rn binding fragment and heterologous polypeptide or its bioactive fragment.Also disclosed the construct for generation of such chimeric polyeptides.In addition, the method for making chimeric polyeptides and their construct of coding has been instructed in this disclosure.This disclosure also provides the polypeptide that comprises human serum albumin (HSA) part, this HSA partly comprises HSA Domain III or its neonatal Fc receptor (FcRn) binding fragment, wherein this HSA Domain III comprises one to 18 aminoacid replacement, thus with respect to the part of HSA wherein do not comprise the contrast polypeptide of described aminoacid replacement and increase this polypeptide to the affinity of FcRn and one or both of serum half-life.This disclosure also provides the chimeric polyeptides that comprises human serum albumin (HSA) part, this HSA partly comprises HSA Domain III or its neonatal Fc receptor (FcRn) binding fragment, and heterologous protein, wherein this chimeric polyeptides keeps the functional activity of this heterologous protein and can be combined with FcRn, and this HSA Domain III comprises at least one aminoacid replacement, thus with respect to the part of HSA wherein do not comprise the contrast chimeric polyeptides of described aminoacid replacement and increase this chimeric polyeptides to the affinity of FcRn and one or both of serum half-life.In addition, disclose the method for using chimeric polyeptides at this, for example, be used for increasing the serum half-life of protein.Also disclosed method and carrier for generation of the adenovirus library, described adenovirus library is used for a large amount of different polypeptide groups of screening.Such method is used for screening and identification increases HSA Domain III aminoacid replacement to one or both of the affinity of FcRn or serum half-life.
In certain embodiments, chimeric polyeptides with respect to the contrast polypeptide that does not comprise HSA part have increase to the affinity of FcRn and the serum half-life of increase.In certain embodiments, chimeric polyeptides has the serum half-life of increase.In certain embodiments, chimeric polyeptides have increase in the serum half-life of FcRn affinity and increase both.In certain embodiments, chimeric polyeptides under acid pH (for example, pH is about 5.5), have increase to the FcRn affinity.In other embodiments, chimeric polyeptides (for example, pH is about 5.5) under acid pH has the FcRn of increase, and (for example, pH is about 7.4) chimeric polyeptides is basically constant to the affinity of FcRn under neutral pH.
All combinations of any aforementioned aspect of this disclosure expectation and embodiment, and with any embodiment that in detailed description, proposes and the combination of example.
5 Brief Description Of Drawings
For illustrating the present invention, described in the drawings for some embodiment of the present invention.Yet, the present invention is not limited to accurate arrangement and the mechanism of the embodiment that describes among the figure.
Fig. 1 provides kinetics and the equilibrium analysis of the people FcRn of being combined with human serum albumin (HSA) Domain III.What present is combination, Dissociation and the equilibrium association constant that the SPR of the people FcRn of being combined with fixed structure territory III under pH5.5 derives herein.Figure 1A represents the PAGE gel of Coomassie brilliant blue (Coomassie) dyeing, prove successful expression and purification (indicating such as arrow) from the Domain III of the HSA of Pichia sp. (Pichia Pastoris).Figure 1B represent by be fixed in that Domain III on the CM5 chip injects that a series of FcRn concentration produce in conjunction with sensing figure.Association reaction when Fig. 1 C is illustrated in balance with respect to the plot of the FcRn concentration of stable state affinity models fitting.
Fig. 2 provides the diagram of various constructs design, and the purification of the IgG that merges about the IgG that merges with HSA with Domain III and the information of sign.Fig. 2 A represents the recombinate heavy chain of IgG-HSA or IgG-Domain III fusion rotein and the DNA construct of YTE variant.Fig. 2 B is illustrated under reduction and the non-reduced condition, and the SDS PAGE of the fusion rotein of purification (5 μ g/ swimming lane) analyzes.Fig. 2 C represents the analytical size exclusion chromatography of the IgG fusion rotein of purification.
The equilibrium constant that Fig. 3 provides and the SPR of the IgG that merges with HSA and the people FcRn of being combined with the IgG that Domain III merges derives.Response units (Req) in the time of will being infused in balance with respect to each FcRn of people FcRn concentration is mapped, and with data and stable state affinity models fitting, in order to calculate fixing IgG(figure A), the IgG(figure B that merges with HSA) and the IgG(figure C that merges with Domain III) K DSensing figure in the illustration shows with respect to the quality (resonance units) of the FcRn that is incorporated into fixed ligands on Y-axis after the blank deduction in the time on the X-axis.
Fig. 4 provides and indicated the epi-position on HSA for FcRn is the evidence of comformational epitope.Under pH5.5, will hatch with the agarose gel (S) processed with three kinds of different modes-HA, S-IgG or S-Tris and people FcRn.The FcRn of elution of bound is also quantitative by carry out immunoblotting with anti--β2-microglobulin antibody.The position that has shown molecular weight marker (M is in kD).Swimming lane 1 contains the people FcRn of 20 μ g, and this amount is added in each adsorption sample.
Fig. 5 has shown and has been illustrated in yeast cells (saccharomyces cerevisiae (S.Cerevisiae)) lip-deep HSA and Domain III has kept the FcRn binding ability.Fig. 5 A represents to use resisting-HSA antibody of FITC combination, with the HSA on the brewing yeast cell of the derivable pYDl cell surface display of galactose Plasmid Transformation or the Flow cytometry of Domain III.Continue the time of appointment with the galactose inducing cell.Figure B represent to use flow cytometry by anti--visual biotinylated people FcRn of Streptavidin phycoerythrin be illustrated in HSA on the brewing yeast cell of inducing 48 hours or the combination of Domain III.To be used as with the yeast cells that scfv transforms the background fluorescence contrast of FITC and phycoerythrin.Experiment is expressed as fluorescence intensity (logarithmic scale) with respect to the rectangular histogram of cell number.
Fig. 6 provides the aminoacid sequence comparison from the Domain III of different plant species (people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse).Comparison among the figure A to D comprises chicken, and the comparison among the figure EH forecloses chicken.The amino acid residue solid marks of between different plant species, guarding, and conservative cysteine residues with dashed lines labelling.The amino acid residue of shade is not guarded between species.Should be noted in the discussion above that the aminoacid numbering in Fig. 6 only is directed to people's Domain III, rather than show for the numbering with respect to the Domain III of the ripe HSA of total length.
Fig. 7 shows that HSA and the fusion of the IgG with wild type make the serum persistency increase to the level similar to IgG-YTE variant finding.With the % of remaining injected sample in serum past (1 to the 240 hour) mapping along with the time.
Fig. 8 has described the plasmid figure of scFv-Fc cell surface display library entry vector.Figure A has described the plasmid figure of pENDisplay carrier, and it comprises the scFv-Fc-GPI-anchor box (anchor cassette) that may be operably coupled to promoter (being the CMV promoter herein) and with polyadenylic acid (polyA) sequence (being BGH polyA sequence herein) termination.Sfi I is positioned at the both sides of scFv part to promote the clone of different scFv sequences from Not I restriction endonuclease sites.AttLl and attL2 site are positioned at the both sides of scFv-Fc-GPI-anchor expression cassette.Figure B has described the plasmid figure of pENDisplay-OriP carrier, and it is based on the carrier shown in the figure A, but combines OriP sequence (referring to Fig. 9 C) after the polyA afterbody of scFv-Fc-GPI-anchor box.
Fig. 9 provides the representative of EBNA-1 and OriP.The aminoacid sequence of EBNA-1 and nucleotide sequence are provided at respectively among figure A and the B.The sequence of OriP is in providing figure C.
Figure 10 provides the sketch map of the representative general adenovirus expression carrier of expressing interested one or more protein.The carrier of describing comprises: the interested DNA sequence of one or more interested protein of encoding; OriP sequence and optional EBNA-1 coding region.Randomly, these elements on either side are the att recombination site that possible be used for vector construction.In this situation that makes El and/or E3 gene delection, be the adenoviral gene group and be the ITR sequence in a side of these parts in a side of these parts.Indicated 3 ' and 5 ' ITR sequence.For the ease of making up, breed and selecting, carrier also provides the sequence that is used for copying at bacterial cell (for example escherichia coli (E.coli) starting point) and antibiotic selection (for example amicillin resistance), and these parts are positioned as so that they can be in conjunction with the adenovirus of rescuing.
Figure 11 has shown that being illustrated in the lip-deep HSA of mammalian cell (293F cell) has kept the FcRn binding ability.Figure A has described the mammal expression construct of called after pEN-HSA-GPI, and it comprises CMV promoter (thick line), signal sequence (thick dashed line), N end Flag label (fine dotted line), both sides is the (G of HSA part (shade wire frame) and DAF-GPI sequence 4S) 3Joint (fine line).Figure B represent to use the FITC combination anti--HSA antibody (figure A), with producing after the adenovirus infection of pEN-HSA-GPI 16 hours and 24 hours or the Flow cytometry of the HSA on the 293-F cell surface of the control plasmid that contrasts the scFv-Fc fusion rotein with encoding.Figure C and D represent to use flow cytometry to be respectively 25 μ g/ml and 5 μ g/ml by anti--visual biotinylated people FcRn(of Streptavidin phycoerythrin) and be illustrated in the combination of the HSA on the 293F cell.Experiment is expressed as fluorescence intensity (logarithmic scale) with respect to the rectangular histogram of cell number.
Figure 12 has shown biotinylated people FcRn and wild type HSA(HSA-wt) and the variation that is illustrated in the bind profile of two the HSA mutant libraries (HSA-DIII-lib1 and HSA-DIII-lib2) on the 293F cell.Figure A represents with the Flow cytometry of wild type with the HSA on the 293-F cell surface of sudden change HSA-DIII library infection.Figure B represents the Flow cytometry by the HSA of the visual biotinylation people FcRn that is combined in HSA on the cell surface of anti--Streptavidin phycoerythrin.Experiment is expressed as fluorescence intensity (logarithmic scale) with respect to the rectangular histogram of cell number.
Figure 13 has shown expression wild type HSA(HSA-wt, figure A) and the FACS sorting of the cell of two HSA mutant libraries (being respectively HSA-DIII-lib1 and the HSA-DIII-lib2 of figure B and figure C) compose, these cells are by biotinylated people FcRn(10 μ g/ml) dyeing, detect with anti--Streptavidin phycoerythrin.
Figure 14 has shown before sorting and the first round and second is taken turns the variation of biotinylated people FcRn and the bind profile of 293F cell after the sorting, and described 293F cell is at their cell surface expression wild type HSA(HSA-wt), the HSA-DIII-lib1 mutant library.Figure A is illustrated in before the sorting and the first round and second is taken turns the Flow cytometry of the HSA on the 293F cell surface of expressing wild type, HSA-DIII-lib1 after the sorting.Figure B and C represent to use flow cytometry to be respectively l μ g/ml and 0.1 μ g/ml by anti--visual biotinylated people FcRn(of Streptavidin phycoerythrin) and the on the same group combination of cell.Experiment represents with the rectangular histogram of fluorescence intensity (logarithmic scale) with respect to cell quantity.
Figure 15 has shown that FcRn and the combination that is illustrated in the mutant HSA on the cell surface are that pH is dependent.Figure A and B are presented at pH5.5(0.1 μ g/ml FcRn, scheme A) and pH7.2(10 μ g/ml, figure B) be used under the 293F cell lip-deep anti--Flow cytometry of the biotinylated people FcRn that the Streptavidin phycoerythrin detects, described 293F cellular expression contrast scFv-Fc fusion rotein, wild type HSA and three kinds of representative sudden changes (referring to table 5).Figure C show to use the FITC combination anti--HSA antibody is in the Flow cytometry of the lip-deep HSA of these cells.
Figure 16 shows that the HSA sudden change as separating by the measured great majority of flow cytometry has higher affinity for FcRn.The mutant of wild type HSA and a group selection is analyzed for the biotinylated people FcRn that is combined under the variable concentrations by flow cytometry.The data mapping is the MFI with respect to FcRn concentration.
Figure 17 has described position (the PDB accession number: 1BM0) of the many variants on the analytic structure (solved structure) at HSA.Most of representation is strip-chart, and wherein residue L463, E495, T508, I523 and K534 represent with bar shaped and indicate with arrow.Comprise residue 492-536 ring 6 and 7 and spiral 7 and 8 lived by circle.Most focus and preferred point are found in this zone.
Figure 18 has shown the half-life curve of several combination mutant HSA in the people FcRn transgenic mice.These results also are summarised in the table 11 of example 8.14.
6 detailed description of the invention
6.1 foreword
Neonatal Fc receptor (FcRn) prolongs IgG and both life-spans of human serum haemproteins (HSA) by the mechanism that relies on pH, under the acid pH of endosome specifically in conjunction with these two kinds of molecules and make their recirculatioies get back to cell surface, thereby make these two kinds of molecular transfer leave the lysosome degradation pathway of acquiescence.Confirmed that the FcRn binding ability is intrinsic for albuminous Domain III.As confirming that at this FcRn binding fragment that adds HSA can be used for increasing the serum half-life of protein and/or the FcRn binding affinity of therapeutic agent, described therapeutic agent is antibody, antibody surrogate thing, protein, protein scaffolds and peptide for example.Particularly, as confirming at this, under acid pH (for example, pH is about 5.5), the FcRn binding affinity increases, and under neutral pH (for example, pH is about 7.4), affinity is substantially constant.The chimeric polyeptides that comprises the FcRn binding structural domain variant of this disclosure can increase serum half-life or the FcRn binding affinity of protein, even considerably beyond wild type FcRn binding structural domain.HSA variant polypeptide of the present invention can be as be combined the support that maybe can be coupled to therapeutic agent with the treatment target.
This disclosure provides the variant of Domain III.This type of variant of Domain III can use separately maybe and can be used in the situation of other HSA sequence, in order to increase serum half-life and/or the FcRn binding affinity of heterologous protein and/or nonprotein agent.
Chimeric polyeptides disclosed here and HSA variant have a lot of purposes.Should be understood that protein is sometimes relative with other molecules to be removed rapidly from animal or human body.Fast the removing meeting weakens the protein of zoologizeing in the model and the ability of other molecules gradually, and they are used for the treatment of the ability of purpose can to weaken gradually effective use.In some cases, protein is removed fast, thereby makes it not have therapeutic effect.In other cases, protein is eliminated with the speed that needs frequent drug administration.Frequent drug administration has increased the relevant cost for the treatment of, and has also increased the risk of Incompliance scheme.In some cases, protein is eliminated with the speed that needs give larger dose.The activating agent of larger dose can increase the risk of side effect, comprises immunoreation.
The chimeric polyeptides of this disclosure helps by increasing serum half-life and/or affinity solution and the quick or relative fast protein of FcRn being removed relevant problem with variant HSA polypeptide.Equally, non-proteinaceous matter can be combined with the variant HSA of this disclosure polypeptide to increase serum half-life and/or to the affinity of FcRn.
6.2 term
Before continuing that the present invention is described in further detail, it should be understood that the present invention is not restricted to specific compositions or processing step, because these compositionss or processing step can change equally.Unless must be noted that in the context and clearly point out, comprise plural indicant such as employed singulative " a kind of " in this description and claims, " one " and " being somebody's turn to do ".
Unless otherwise defined, all technology used herein and scientific terminology have with this area that the present invention relates under the common identical meaning of understanding of technical staff.For example, the Concise Dictionary of Biomedicine and Molecular Biology(" biomedical and molecular biology concise dictionary "), Juo, Pei-Show, second edition, 2002, CRC publishing house; The Dictionary of Cell and Molecular Biology(" cell and molecular biology dictionary "), the third edition, 1999, academic press (Academic Press); And the Oxford Dictionary Of Biochemistry And Molecular Biology(" Biochemistry and Molecular Biology oxford dictionary "), revised edition, 2000, Oxford University Press (Oxford University Press) provides the common dictionary of the many terms that use in the present invention for the technical staff.
Aminoacid at this can be by their the common trigram symbol that oneself knows or by by IUPAC-IUB Biochemical Nomenclature Commission(IUPAC-IUB biochemical nomenclature commission) one-letter symbol recommended is mentioned.Similarly, nucleotide can be mentioned by their universally recognized single-letter code." aminoacid replacement " expression is with another amino acid replacement of aminoacid of the specific location in parent's peptide sequence as used in this.For example, replace the variant polypeptide that L463N refers to that wherein the leucine at 463 places is replaced with asparagine in the position.
Except as otherwise noted, the aminoacid numbering of the variable domain of antibody, complementary determining region (CDR) and framework region (FR) is followed the Kabat definition, this definition is as listing in people such as Kabat, Sequences of Proteins of Immunological Interest(" protein sequence with immunology importance "), the 5th edition, NIH, public health service section, Bei Saisida city, the Maryland State, (1991).Use this numbering system, actual linear aminoacid sequence can contain less or other aminoacid, and it is corresponding to shortening or the insertion of FR or the CDR of variable domain.For example, heavy chain variable domain can be included in the residue that single amino acids after the residue 52 of H2 inserts (according to the residue 52a of Kabat) and insertion heavy chain FR residue 82 after (for example, according to residue 82a, 82b and the 82c of Kabat, etc.).For a kind of given antibody, can determine that with the comparison in the homology zone of the sequence of " standard " Kabat numbering the Kabat of residue numbers by the sequence at antibody.The high specific of framework residue is to often inserting " interval " residue, to be used for the Fv zone in numbering system.In addition, because between planting or allele difference, can be different because of antibody chain in the identity of some independent residue at period place, any given Kabat position.
As used herein, term " antibody (antibody and antibodies) " is also referred to as immunoglobulin, contain monoclonal antibody (comprising full length monoclonal antibodies), polyclonal antibody, the multi-specific antibody that is formed by at least two different epi-position binding fragments (for example, bispecific antibody), people's antibody, humanized antibody, camelization antibody, chimeric antibody, scFv (scFv), single-chain antibody, single domain antibody, domain antibodies, the Fab fragment, F (ab') 2 fragments, (for example show desirable bioactive antibody fragment, antigen-binding portion thereof), the Fv(dsFv that disulfide bond connects), and antiidiotype (anti-Id) antibody (comprising that (for example) is for the anti-Id antibody of antibody of the present invention), intrabody, and any above epi-position binding fragment.Particularly, antibody comprises the immunocompetence fragment of immunoglobulin molecules and immunoglobulin molecules,, contains the molecule of at least one antigen binding site that is.Immunoglobulin molecules (for example can be any isotype, IgG, IgE, IgM, IgD, IgA and IgY), hypotype (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or allotype is (for example, Gm, for example, G1m(f, z, a or x), G2m(n), G3m(g, b or c), Am, Em and Km(1,2 or 3)).Antibody can be derived from any mammal, includes but not limited to people, monkey, pig, horse, rabbit, Canis familiaris L., cat, mice etc., or other animals, such as birds (for example chicken).
As used herein, term " total length HSA " refers to ripe total length human serum albumin's protein or refers to the nucleotide sequence of encoding such proteins.Total length HSA albumen is about 585 aminoacid (removing N end presequence (pro-sequence) and front former sequence (prepro-sequence) afterwards).Ripe total length HSA(is also referred to as the ripe HSA of total length) protein lists in SEQ ID NO:2.In certain embodiments, total length HSA refers to not have the ripe total length form of the HSA of presequence.The sequence of front former HSA (before removing N end presequence and front former sequence) is 609 aminoacid and lists in GenBank accession number NP_000468.In addition because allele difference, the identity of the residue that some is independent can from be presented on SEQIDNO:2 in those are different.The allelic variation that appears in the HSA Domain III comprises: at the residue 410 R → C of place; At the residue 466 K → E of place; At the residue 479 E → K of place; At the residue 494 D → N of place; At the residue 501 E → K of place; At the residue 505 E → K of place; At the residue 533 V → M of place; At the residue 536 K → E of place; At the residue 541 K → E of place; At the residue 550 D → A of place or D → G; At the residue 560 K → E of place; At the residue 563 D → N of place; At the residue 565 E → K of place; At the residue 570 E → K of place; At the residue 573 K → E of place; At the residue 574 K → E of place; At residue 572 to the 585 GKKLVAASQAALGL → PTMRIRERK of place; And at residue 575 to 585 LVAASQAALGL of place → TCCCKSSCLRLITSHLKASQ PTMRIRERK, such as the numbering of carrying out with respect to the position among the ripe HSA of total length.
As used herein, term " Domain III of HSA " refers to cross over the conventional structure territory III of HSA of the aminoacid 381-385 of the ripe HSA of total length, is about 205 aminoacid, or the nucleotide sequence of a kind of like this protein of coding.The Domain III of HSA also is called Domain III or simple DIII for short at this.The aminoacid sequence of Domain III polypeptide is listed in SEQ ID NO:l.As above pointed because allele difference, the identity of the residue that some is independent can from be presented on SEQ ID NO:l in those are different.
As used herein, to refer to comprise be not the polypeptide of at least two parts of allos to term " chimeric polyeptides " each other.For example, chimeric polyeptides is also referred to as fused polypeptide or fusion rotein, comprises at least one the HSA part that is engaged to the heterologous protein part.This HSA part and heterologous protein part self can with (for example) Fc or other partial fusion.This HSA part and heterologous protein part can engage via covalently or non-covalently interacting.For example, HSA part and heterologous protein part chemical bond or merge (for example translation fusion in the frame) in the mode of restructuring each other.
As used herein, term " heterologous protein " refers to not to be protein all or part of of HSA.Although use generic term " heterologous protein " at this, expect that this term contains the biologically active peptide of different length and total length or the protein of total length basically, comprise antibody and antibody fragment.Preferred heterologous protein can use or studies for therapeutic purposes.The Exemplary types of heterologous protein includes but not limited to enzyme, cytokine and somatomedin.
As used herein, the HSA polypeptide comprises the modified forms of various bioactive fragments and variant, fusion rotein and wild type HSA polypeptide.The modified forms of such bioactive fragment or variant, fusion rotein and HSA polypeptide has the aminoacid sequence of the substantive sequence identity of at least a portion and natural HSA albumen, and the FcRn that keeps at least natural HSA albumen is in conjunction with activity.In certain embodiments, the bioactive fragment of HSA polypeptide, variant or fusion rotein comprise with the HSA polypeptide and have at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% conforming aminoacid sequence.As used herein, term " fragment " should be understood to comprise and shows FcRn in conjunction with bioactive fragment or the biological activity variant of activity.The bioactive fragment that is fit to can be used for preparing chimeric polyeptides, and such chimeric polyeptides can use in described any method herein.
As used herein, term " sudden change ", " mutant " etc. refer to molecule, refer in particular to the HSA polypeptide, and it is by using the well-known process or any other conventional method that are used for direct mutagenesis to experience one or more amino acid whose disappearance, interpolation or replacement.
6.3HSA Domain III
In some aspects, this disclosure provides human serum albumin (HSA) variant polypeptide, it comprises HSA Domain III or its neonatal Fc receptor (FcRn) binding fragment, wherein said variant polypeptide can be combined with FcRn, and wherein said HSA Domain III comprises one to 18 aminoacid replacement, thereby increases the serum half-life of described variant polypeptide with respect to the contrast HSA polypeptide that lacks described aminoacid replacement or increase affinity to FcRn.
In certain embodiments, one to 18 aminoacid replacement increases the HSA variant polypeptide to the affinity of FcRn.In certain embodiments, the serum half-life of one to 18 aminoacid replacement increase HSA variant polypeptide.In certain embodiments, one to 18 aminoacid replacement increases the HSA variant polypeptide to the affinity of FcRn and the serum half-life of HSA variant polypeptide.In certain embodiments, under acid pH (for example, pH is about 5.5), one to 18 aminoacid replacement increases the HSA variant polypeptide to the affinity of FcRn.In certain embodiments, under acid pH (for example, pH is about 5.5), one to 18 aminoacid replacement increases the HSA variant polypeptide to the affinity of FcRn, and under neutral pH (for example, pH is about 7.4), basically do not change the HSA variant polypeptide to the affinity of FcRn.
In certain embodiments, the HSA variant is combined with FcRn, and has dissociation rate or the association rate different from described contrast HSA polypeptide.For example, in certain embodiments, the HSA variant is combined with FcRn, and has faster association rate and/or slower dissociation rate.In other embodiments, association rate is slow and/or dissociation rate is very fast.
In certain embodiments, this disclosure provides chimeric polyeptides, and it comprises the HSA part, and described HSA partly comprises Domain III or its FcRn bound fraction and heterologous protein, and wherein said chimeric polyeptides has kept the functional activity of heterologous protein.In certain embodiments, HSA partly comprises whole HSA polypeptide or comprises the bioactive fragment of HSA Domain III, or its neonatal Fc receptor (FcRn) binding fragment.In certain embodiments, HSA partly comprises the HSA Domain III, or at least a portion of another domain of its neonatal Fc receptor (FcRn) binding fragment and HSA, for example, at least a portion of HSA domain I or at least a portion of HSA domain II, or at least a portion of HSA domain I and II.As being numbered with respect to the position in the ripe HSA of total length, HSA domain I as used herein comprises residue 1-197; The HSA domain II comprises residue 189-385; The HSA Domain III comprises residue 381-585.
In certain embodiments, chimeric polyeptides with respect to the contrast polypeptide that does not comprise HSA part have increase to the affinity of FcRn and the serum half-life of increase.In certain embodiments, chimeric polyeptides has the affinity to FcRn of increase.In certain embodiments, chimeric polyeptides has the serum half-life of increase.In certain embodiments, chimeric polyeptides have increase to the affinity of FcRn and the serum half-life of increase.In certain embodiments, chimeric polyeptides has the affinity to FcRn of increase under acid pH (for example, pH is about 5.5).In other embodiments, chimeric polyeptides (for example, pH is about 5.5) under acid pH has the FcRn of increase, and chimeric polyeptides (for example, pH is about 7.4) under neutral pH is basically constant to the affinity of FcRn.
In addition, as said, in certain embodiments, polypeptide (for example, HSA variant or chimeric polyeptides) the Domain III of HSA part comprise one to 18 aminoacid replacement, thereby do not comprise the contrast chimeric polyeptides of described aminoacid replacement and increase to the serum half-life of the affinity of FcRn and chimeric polyeptides one or both with respect to HSA part wherein.In certain embodiments, one to 18 aminoacid replacement increases chimeric polyeptides to the affinity of FcRn.In certain embodiments, the serum half-life of one to 18 aminoacid replacement increase chimeric polyeptides.In certain embodiments, one to 18 aminoacid replacement increases chimeric polyeptides to the affinity of FcRn and the serum half-life of chimeric polyeptides.
In certain embodiments, the Domain III of the HSA of polypeptide (for example, HSA variant polypeptide or chimeric polyeptides) part comprises l, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17 or 18 aminoacid replacement.Equally, in the situation of the HSA variant polypeptide that comprises Domain III or its FcRn bound fraction, Domain III comprises l, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17 or 18 aminoacid replacement.In certain embodiments, aminoacid replacement not merely is single amino acids to the replacement of another residue of existing in the allele variant.
In certain embodiments, the Domain III of the HSA part of polypeptide (for example, HSA variant polypeptide or chimeric polyeptides) is included at least one aminoacid replacement of any following position that is numbered with respect to the position among the ripe HSA of total length: residue 381, residue 383, residue 391, residue 401, residue 402, residue 407, residue 411, residue 413, residue 414, residue 415, residue 416, residue 424, residue 426, residue 434, residue 442, residue 445, residue 447, residue 450, residue 454, residue 455, residue 456, residue 457, residue 459, residue 463, residue 495, residue 506, residue 508, residue 509, residue 511, residue 512, residue 515, residue 516, residue 517, residue 519, residue 521, residue 523, residue 524, residue 525, residue 526, residue 527, residue 531, residue 535, residue 538, residue 539, residue 541, residue 557, residue 561, residue 566, residue 569.
In certain embodiments, the Domain III of the HSA part of polypeptide (for example, HSA variant polypeptide or chimeric polyeptides) is included in the aminoacid replacement of any following position that is numbered with respect to the position among the ripe HSA of total length: (a) residue 383 and 413; (b) residue 401 and 523; (c) residue 407 and 447; (d) residue 407 and 447 and 539; (e) residue 407 and 509; (f) residue 407 and 526; (g) residue 411 and 535; (h) residue 414 and 456; (i) residue 415 and 569; (j) residue 426 and 526; (k) residue 442 and 450 and 459; (l) residue 463 and 508; (m) residue 508 and 519 and 525; (n) residue 509 and 527; (o) residue 523 and 538; (p) residue 526 and 557; (q) residue 541 and 561; (r) residue 463 and 523; (s) residue 508 and 523; (t) residue 508 and 524; (u) residue 463,508 and 523; (v) residue 463,508 and 524; (w) residue 508,523 and 524; (x) residue 463,508,523 and 524; (y) residue 463 and 524; (z) residue 523 and 524; And (aa) residue 463,523 and 524.
In one embodiment, the Domain III of the HSA of polypeptide (for example, HSA variant polypeptide or chimeric polyeptides) part comprises SEQ ID NO:18, wherein, and X 1Not leucine and/or X 2Not threonine and/or X 3Not isoleucine and/or X 4Not lysine.In certain embodiments, X 1Cysteine, phenylalanine, glycine, histidine, isoleucine, agedoite, serine or glutamine; X 2Cysteine, glutamic acid, isoleucine, lysine, arginine or serine; X 3Alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, agedoite, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine; And X 4Alanine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, glutamine, threonine or valine.In other embodiments, X 1Phenylalanine or agedoite; X 2Arginine or serine; X 3Aspartic acid, glutamic acid, glycine, phenylalanine, lysine or arginine; And/or X 4It is leucine.In other embodiment again, X 1Agedoite and/or X 2Arginine and or X 3Glycine and/or X 4It is leucine.
In certain embodiments, with respect to different contrasts, affinity and/or the serum half-life to the FcRn that increase have been assessed.For example, can be with respect to the characteristic of the characteristic evaluation chimeric polyeptides of the heterologous protein that lacks the HSA part, or can replace and/or lack with respect to lack amino acid the characteristic of characteristic evaluation chimeric polyeptides of the same or similar HSA part of heterologous protein.Equally, can be with respect to the characteristic evaluation HSA variant polypeptide of the HSA molecule that does not have aminoacid replacement.
In certain embodiments, chimeric polyeptides/HSA variant polypeptide is combined with FcRn with the affinity higher than described contrast polypeptide.In certain embodiments, one to 18 aminoacid replacement is at the affinity of the lower increase of acid pH (for example, pH is about 5.5) chimeric polyeptides/HSA variant polypeptide to FcRn.In certain embodiments, one to 18 aminoacid replacement at acid pH (for example, pH is about 5.5) increase down chimeric polyeptides/HSA variant polypeptide to the affinity of FcRn, but under neutral pH (for example, pH is about 7.4), basically do not change chimeric polyeptides to the affinity of FcRn.In certain embodiments, chimeric polyeptides is combined with FcRn with higher affinity under acid pH and is had a serum half-life of increase.
In certain embodiments, chimeric polyeptides/HSA variant polypeptide and FcRn in conjunction with and have dissociation rate and an association rate different with described contrast polypeptide.For example, in certain embodiments, chimeric polyeptides/HSA variant polypeptide is combined with FcRn and is had faster association rate and/or a slower dissociation rate.In other embodiments, association rate is slow and/or dissociation rate is very fast.
In certain embodiments, polypeptide (for example, HSA variant or the chimeric polyeptides with HSA part) the HSA Domain III comprise one to ten aminoacid replacement, thereby do not comprise the contrast polypeptide of described aminoacid replacement and increase this polypeptide to the affinity of FcRn and/or increase the serum half-life of this polypeptide with respect to HSA part wherein.In certain embodiments, the HSA Domain III comprises an aminoacid replacement.In certain embodiments, the HSA Domain III comprises 2,3,4,5,6,7,8,9 or 10 aminoacid replacement.In certain embodiments, the HSA Domain III comprises 11,12,13,14,15,16,17 or 18 aminoacid replacement.In certain embodiments, the HSA Domain III comprises at least one aminoacid replacement.In certain embodiments, the HSA Domain III comprises at least ten aminoacid replacement.
Exemplary aminoacid replacement comprises: (i) replace with alanine; (ii) conservative aminoacid replacement; (iii) nonconservative aminoacid replacement.What this disclosure was expected is, all aminoacid replacement in the Domain III of given polypeptide can be that these replace one of type, and expectation is that each aminoacid replacement in the Domain III of given polypeptide (for example, HSA variant and chimeric polyeptides with HSA part) can individually and be independently selected from these types.
In certain embodiments, replacing (the HSA Domain III of the numbers such as at least 1,2,3,4,5,6 replaces) is that the residue among the HSA is substituted by alanine.In certain embodiments, replacing (the HSA Domain III of the numbers such as at least 1,2,3,4,5,6 replaces) replaces one in HSA given neutral amino acid residue with another neutral amino acid residue.In certain embodiments, replacing (the HSA Domain III of l, 2,3,4,5, the numbers such as 6 replaces at least) replaces a given acidic amino acid residue in HSA with another kind of acidic amino acid residue.In certain embodiments, replacing (the HSA Domain III of l, 2,3,4,5, the numbers such as 6 replaces at least) replaces a given alkaline amino acid residue in HSA with another alkaline amino acid residue.This disclosure expectation following examples, wherein each replacement all is independently selected from aforementioned replacement type.The polypeptide (for example, HSA variant and the chimeric polyeptides with HSA part) that comprises any combination of aforementioned replacement type is special expectation.
In certain embodiments, replace (the HSA Domain III of l, 2,3,4,5, the numbers such as 6 replaces at least) with an aminoacid with another amino acid replacement in following group: lysine (K; Lys), arginine (R; Arg); Histidine (H; His).In certain embodiments, replace (the HSA Domain III of l, 2,3,4,5, the numbers such as 6 replaces at least) with an aminoacid with another amino acid replacement in following group: aspartic acid (D; Asp; Aspartic acid) and glutamic acid (E; Glu; Glutamic acid).In certain embodiments, replace (the HSA Domain III of l, 2,3,4,5, the numbers such as 6 replaces at least) with an aminoacid with another amino acid replacement in following group: agedoite (N; Asn), glutamine (Q; Gln), serine (S; Ser), threonine (T; Thr) and tyrosine (Y; Tyr).In certain embodiments, replace (the HSA Domain III of l, 2,3,4,5, the numbers such as 6 replaces at least) with an aminoacid with another amino acid replacement in following group: alanine (A; Ala), valine (V; Val), isoleucine (I; Ile), leucine (L; Leu), proline (P; Pro), phenylalanine (F; Phe), tryptophan (W; Trp), methionine (M; Met), cysteine (C; Cys) and glycine (G; Gly).In certain embodiments, replace (the HSA Domain III of l, 2,3,4,5, the numbers such as 6 replaces at least) with an aminoacid with another amino acid replacement in following group: phenylalanine, tryptophan and tyrosine.In certain embodiments, replace (the HSA Domain III of l, 2,3,4,5, the numbers such as 6 replaces at least) with an aminoacid with another amino acid replacement in following group: cysteine, serine and threonine.In certain embodiments, replace (the HSA Domain III of l, 2,3,4,5, the numbers such as 6 replaces at least) with an aminoacid with another amino acid replacement in following group: agedoite, glutamine, serine, threonine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid.In certain embodiments, replace (the HSA Domain III of l, 2,3,4,5, the numbers such as 6 replaces at least) with an aminoacid with another amino acid replacement in following group: glycine, serine, threonine, alanine, valine, leucine and isoleucine.This disclosure expectation following examples, wherein each replacement is independently selected from aforementioned replacement type.The polypeptide (for example, HSA variant and the chimeric polyeptides with HSA part) that comprises any combination of aforementioned replacement type is special expectation.
In certain embodiments, have increase to the polypeptide of the serum half-life of the affinity of FcRn and/or increase (for example, HSA variant polypeptide or the chimeric polyeptides with HSA part) the Domain III of HSA part comprise at least one aminoacid replacement that is selected from lower group, this group is comprised of the following: V381N, V381Q, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y401D, Y401E, K402A, K402G, K402I, K402L, K402V, L407F, 407H, 407M, L407N, L407Q, 407R, L407W, L407Y, Y411Q, Y411N, K413C, K413S, K413T, K414S, K414T, V415C, V415L, V415S, V415T, Q416H, Q416P, V424A, V424D, V424G, V424I, V424L, V424M, V424N, V424Q, V424W, V426D, V426E, V426F, V426H, V426L, V426N, V426P, V426Q, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, P447S, P447T, E450D, E450E, S454C, S454M, S454T, V455N, V455Q, V456N, V456Q, L457F, L457W, L457Y, Q459K, Q459R, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T506F, T506M, T506N, T506R, T506W, T506Y, T508C, T508E, T508I, T508K, T508R, T508S, F509C, F509D, F509G, F509I, F509L, F509M, F509P, F509V, F509W, F509Y, A511D, A511F, A511I, A511R, A511T, A511V, A511W, A511Y, D512F, D512M, D512Q, D512W, D512Y, T515C, T515D, T515E, T515E, T515G, T515H, T515L, T515N, T515P, T515Q, T515S, T515W, T515Y, L516C, L516F, L516S, L516T, L516W, L516Y, S517C, S517F, S517M, S517T, S517W, S517Y, K519A, K519G, K519I, K519L, K519V, R521F, R521H, R521M, R521Q, R521T, R521W, R521Y, I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W, I523Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526A, Q526C, Q526F, Q526H, Q526L, Q526M, Q526P, Q526S, Q526T, Q526V, Q526Y, T527F, T527W, T527Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535K, H535L, H535N, H535P, H535S, K538F, K538W, K538Y, A539I, A539L, A539V, K541F, K541W, K541Y, K557A, K557G, K557I, K557L, K557N, K557S, K557V, A561F, A561W, A561Y, T566F, T566W, T566Y, A569H, and A569P.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5 ...) or even all aminoacid replacement all be selected from aforementioned replacement.
In other specific embodiments, polypeptide (for example, HSA variant polypeptide or chimeric polyeptides) the Domain III of HSA part comprise at least one aminoacid replacement that is selected from lower group, this group is comprised of the following: V381N, E383G, N391V, Y401E, K402A, L407N, L407Y, Y411Q, K414S, K413S, V415T, V415C, Q416P, V424I, V424Q, V426E, V426H, G434C, E442K, R445W, P447S, E450D, S454C, V455N, V456N, L457F, Q459R, L463C, L463F, L463H, L463M, L463N, L463Q, E495D, T506Y, T508C, T508E, T508I, T508R, T508S, F509I, F509M, F509W, A511F, D512Y, T515P, T515Q, T515S, L516T, L516W, S517C, S517W, K519I, R521W, I523C, I523D, I523E, I523F, I523Q, I523H, I523K, I523L, I523N, I523P, I523G, I523R, I523Y, K524A, K524F, K524I, K524L, K524M, K524T, K524V, K525V, Q526T, Q526M, Q526Y, T527Y, E531I, H535N, H535P, K538Y, A539I, K541F, K557G, A561F, T566W, and A569P.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5 ...) or even all aminoacid replacement all be selected from aforementioned replacement.
In other specific embodiments, polypeptide (for example, HSA variant polypeptide or chimeric polyeptides) the Domain III of HSA part comprise at least one aminoacid replacement that is selected from lower group, this group is comprised of the following: L407N, L407Y, V415T, V424I, V424Q, V426E, V426H, P447S, V455N, V456N, L463N, E495D, T506Y, T508R, F509M, F509W, A511F, D512Y, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T527Y, H535P and K557G.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5 ...) or even all aminoacid replacement all be selected from aforementioned replacement.
In other specific embodiments, polypeptide (for example, HSA variant polypeptide or chimeric polyeptides) the Domain III of HSA part comprise at least one aminoacid replacement that is selected from lower group, this group is comprised of the following: L407Y, V415T, V424I, V424Q, P447S, V455N, V456N, L463N, E495D, T506Y, T508R, S517W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T527Y, H535P and K557G.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5 ...) or even all aminoacid replacement all be selected from aforementioned replacement.
In certain embodiments, the Domain III of the HSA of polypeptide (for example, HSA variant polypeptide or chimeric polyeptides) part is included in the aminoacid replacement of the HSA Domain III at following residue place: residue 463 and 508; Or residue 463 and 523; Or residue 463 and 524; Or residue 508 and 524; Or residue 463,508 and 523; Or residue 463,508 and 524; Or 508,523 and 524 or residue 463,508,523 and 524, wherein the replacement at residue 463 places is selected from L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q; Replacement at residue 508 places is selected from T508C, T508E, T508I, T508K, T508R, T508S; Replacement at residue 523 places is selected from I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W, I523Y; And the replacement at residue 524 places is selected from K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V.
In certain embodiments, the Domain III of the HSA of polypeptide (for example HSA variant polypeptide or chimeric polyeptides) part comprises and is selected from lower group the aminoacid replacement in the HSA Domain III, and this group is comprised of the following: (a) E383G/K413S; (b) Y401E/I523G; (c) L407N/P447S; (d) L407N/P447S/A539I; (e) L407N/F509M; (f) L407Y/Q526T; (g) Y411Q/H535N; (h) K414S/V456N; (i) V415T/A569P; (j) V426H/Q526Y; (k) E442K/E450D/Q459R; (l) L463N/T508R; (m) T508R/K519I/K525V; (n) F509I/T527Y; (o) I523Q/K538Y; (p) Q526M/K557G; (q) K541F/A561F; (r) L463N/K524L; (s) T508R/I523G; (t) T508R/K524L; (u) L463N/T508R/I523G; (v) L463N/T508R/K524L; (w) T508R/I523G/K524L; (x) L463N/T508R/I523G/K524L; (y) L463N/I523G; (z) I523G/K524L; (aa) L463N//I523G/K524L.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5 ...) or even all aminoacid replacement all be selected from aforementioned replacement.
In certain embodiments, the Domain III of the HSA of polypeptide (for example HSA variant polypeptide or chimeric polyeptides) part comprises and is selected from lower group the aminoacid replacement in the HSA Domain III, and this group is comprised of the following: (a) L407N/P447S; (b) L407N/P447S/A539I; (c) L407N/F509M; (d) Y411Q/H535N; (e) K414S/V456N; (f) V426H/Q526Y; (g) L463N/T508R; (h) F509I/T527Y; (i) I523Q/K538Y; (j) Q526M/K557G; (k) K541F/A561F; (l) L463N/K524L; (m) T508R/I523G; (n) T508R/K524L; (o) L463N/T508R/I523G; (p) L463N/T508R/K524L; (q) T508R/I523G/K524L; (r) L463N/T508R/I523G/K524L; (s) L463N/I523G; (t) I523G/K524L and (u) L463N//I523G/K524L.
In certain embodiments, the Domain III of the HSA of polypeptide (for example HSA variant polypeptide or chimeric polyeptides) part comprises and is selected from lower group the aminoacid replacement in the HSA Domain III, and this group is comprised of the following: (a) L463N/T508R; (b) L463N/K524L; (c) T508R/I523G; (d) T508R/K524L; (e) L463N/T508R/I523G; (f) L463N/T508R/K524L; (g) T508R/I523G/K524L; (h) L463N/T508R/I523G/K524L.
In certain embodiments, the Domain III of the HSA of polypeptide (for example HSA variant polypeptide or chimeric polyeptides) part comprises and is selected from lower group the aminoacid replacement in the HSA Domain III, and this group is comprised of the following: (a) L463N/K508R; (b) T508R/I523G; (c) T508R/K524L; (d) L463N/T508R/I523G.
In addition, fragment or variant can use oneself technology known of this area chemically synthetic, for example conventional Merrifield solid phase f-Moc or t-Boc chemical method.Can produce these fragments or variant (with recombination form or by chemosynthesis) and to its test to identify for example can play a role equally with natural HSA albumen or with natural HSA albumen basic simlarity those fragments or the variant that play a role.
In certain embodiments, the present invention expectation modifies to be used for such purpose to the structure of HSA polypeptide, as strengthening treatment or prevention effects or stability (for example, serum half-life, external shelf-life and to the resistance of protein degradation in the body).The HSA polypeptide that this class is modified has the identical or essentially identical biological activity of HSA polypeptide with natural existence the (that is, natural or wild type).As said, the HSA polypeptide of modifying can be bonded to other treatment part (for example, protein or non-proteinaceous matter).Can produce the polypeptide of modification by for example aminoacid replacement, disappearance or interpolation.For example, can reasonable expectation be, for example, separately with isoleucine or valine displacement leucine, replace aspartic acid with glutamic acid, replace threonine with serine, or will the biological activity of gained molecule not had significant impact with displacement one seed amino acid (for example, conserved amino acid replaces) like the amino acids relevant on the structure.Conservative substitution is to occur in the related amino acid whose family of its side chain those.
In certain embodiments, at least one the described aminoacid replacement in the HSA Domain III is to be conservative residue in a plurality of species.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III all is residue conservative in a plurality of species.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is to be conservative residue in from the serum albumin protein of following species: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, illiteracy account for gerbil jird, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is residue conservative in from the serum albumin protein of following species: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is to be conservative residue in serum albumin protein, and these serum albumin protein are from the species (for example ape and monkey) with respect to people's high conservative.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is to be conservative residue in from the serum albumin protein of nonmammalian.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is to be conservative residue in serum albumin protein, and these serum albumin protein are from the species (for example ape and monkey) with respect to people's high conservative.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is to be conservative residue in from the serum albumin protein of nonmammalian.In certain embodiments, the described aminoacid replacement of in the HSA Domain III at least one is to be conservative residue in serum albumin protein, and these serum albumin protein are from the great majority of people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and Malaysia and China.In certain embodiments, the described aminoacid replacement of in the HSA Domain III at least one is to be conservative residue in serum albumin protein, these serum albumin protein are from least two species that are selected from lower group, and this group is comprised of the following: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of in the HSA Domain III at least one is to be conservative residue in serum albumin protein, these serum albumin protein are from least three species that are selected from lower group, and this group is comprised of the following: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of in the HSA Domain III at least one is to be conservative residue in serum albumin protein, these serum albumin protein are from least four species that are selected from lower group, and this group is comprised of the following: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of in the HSA Domain III at least one is to be conservative residue in serum albumin protein, these serum albumin protein are from least five species that are selected from lower group, and this group is comprised of the following: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of in the HSA Domain III at least one is to be conservative residue in serum albumin protein, these serum albumin protein are from two to five species that are selected from lower group, and this group is comprised of the following: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of in the HSA Domain III all is to be conservative residue in serum albumin protein, and these serum albumin protein are from the great majority of people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and Malaysia and China.In certain embodiments, the described aminoacid replacement of in the HSA Domain III all is to be conservative residue in serum albumin protein, these serum albumin protein are from least two species that are selected from lower group, and this group is comprised of the following: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of in the HSA Domain III all is to be conservative residue in serum albumin protein, these serum albumin protein are from least three species that are selected from lower group, and this group is comprised of the following: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of in the HSA Domain III all is to be conservative residue in serum albumin protein, these serum albumin protein are from least four species that are selected from lower group, and this group is comprised of the following: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of in the HSA Domain III all is to be conservative residue in serum albumin protein, these serum albumin protein are from least five species that are selected from lower group, and this group is comprised of the following: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of in the HSA Domain III all is to be conservative residue in serum albumin protein, these serum albumin protein are from two to five species that are selected from lower group, and this group is comprised of the following: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.The polypeptide (for example, HSA variant and chimeric polyeptides) that comprises any combination of aforementioned aminoacid replacement classification is also expected.
In certain embodiments, at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.In certain embodiments, at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 417, residue 442, residue 499 and residue 502.In certain embodiments, at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 392, residue 399, residue 403, residue 411, residue 412, residue 414, residue 416, residue 418, residue 420, residue 423, residue 434, residue 437, residue 438, residue 445, residue 448, residue 450, residue 453, residue 461, residue 476, residue 477, residue 484, residue 485, residue 487, residue 488, residue 494, residue 497, residue 507, residue 509, residue 514, residue 529, residue 534, residue 537, residue 540, residue 551, residue 558, residue 559, residue 567, residue 568, residue 572.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18) or even all aminoacid replacement all in aforementioned residue.The polypeptide (for example, HSA variant and chimeric polyeptides) that is included in all combinations of the aminoacid replacement in any one or a plurality of aforementioned residue is special expectation.
In certain embodiments, at least one aminoacid replacement in the HSA Domain III is arranged in any following position that the position with respect to the ripe HSA Domain III of total length is numbered: residue 383, residue 391, residue 411, residue 414, residue 416, residue 434, residue 442, residue 445, residue 450 and residue 509.
In certain embodiments, at least one aminoacid replacement in the HSA Domain III is selected from lower group, and this group is comprised of the following: E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y411Q, Y411N, K414S, K414T, Q416H, Q416P, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, E450D, E450E, F509C, F509D, F509G, F509I, F509L, F509M, F509P, F509V, F509W and F509Y.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5 ...) or even all aminoacid replacement all be selected from aforementioned replacement.
In certain embodiments, at least one aminoacid replacement in the HSA Domain III is arranged in any following position that the position with respect to the ripe HSA Domain III of total length is numbered: residue 380, residue 381, residue 384, residue 387, residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 419, residue 421, residue 422, residue 424, residue 428, residue 430, residue 431, residue 433, residue 441, residue 457, residue 458, residue 463, residue 464, residue 466, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 489, residue 491, residue 495, residue 500, residue 508, residue 510, residue 515, residue 516, residue 524, residue 525, residue 526, residue 528, residue 531, residue 535, residue 539, residue 544, residue 547, residue 576.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18) or even all aminoacid replacement all in aforementioned residue.The polypeptide (for example, HSA variant and chimeric polyeptides) that comprises all combinations of the aminoacid replacement in any one or a plurality of aforementioned residue is special expectation.
In certain embodiments, at least one aminoacid replacement in the HSA Domain III is arranged in any following position that the position with respect to the ripe HSA Domain III of total length is numbered: residue 381, residue 401, residue 424, residue 457, residue 463, residue 495, residue 508, residue 515, residue 516, residue 524, residue 525, residue 526, residue 531, residue 535 and residue 539.In certain embodiments, the Domain III of the HSA of polypeptide (for example, HSA variant polypeptide or chimeric polyeptides) part is included in the aminoacid replacement at following residue place in the HSA Domain III: residue 463 and 508; Or residue 463 and 524; Or residue 508 and 524; Or residue 463,508 and 524.
In certain embodiments, at least one aminoacid replacement in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, V381Q, Y401D, Y401E, V242A, V242G, V424I, V424L, V424N, V424Q, V424V, L457F, L457W, L457Y, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T508C, T508E, T508I, T508K, T508R, T508S, T515C, T515H, T515N, T515P, T515Q, T515S, L516F, L516S, L516T, L516W, L516Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526C, Q526M, Q526S, Q526T, Q526Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535P, A539I, A539L and A539V.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5 ...) or even all aminoacid replacement all be selected from aforementioned replacement.
In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is to be not conservative residue in a plurality of species.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is to be not conservative residue in a plurality of species.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is to be not conservative residue in serum albumin protein, and these serum albumin protein are from people, rat, Canis familiaris L., rabbit and cattle.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is to be not conservative residue in serum albumin protein, and these serum albumin protein are from people, rat, Canis familiaris L., rabbit and cattle.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is to be not conservative residue in serum albumin protein, and these serum albumin protein are from people, rat, Canis familiaris L., rabbit and cattle.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is to be not conservative residue in serum albumin protein, and these serum albumin protein are from people, rat, Canis familiaris L., rabbit and cattle.The polypeptide (for example, HSA variant and the chimeric polyeptides with HSA part) that comprises any combination of aforementioned aminoacid replacement classification is also expected.
In certain embodiments, at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 382, residue 385, residue 390, residue 397, residue 400, residue 402, residue 415, residue 429, residue 432, residue 435, residue 439, residue 440, residue 443, residue 444, residue 446, residue 447, residue 459, residue 471, residue 472, residue 478, residue 479, residue 483, residue 490, residue 492, residue 493, residue 503, residue 511, residue 517, residue 518, residue 519, residue 521, residue 538, residue 541, residue 542, residue 546, residue 549, residue 550, residue 552, residue 554, residue 556, residue 560, residue 562, residue 563, residue 565, with residue 566.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18) or even all aminoacid replacement all in aforementioned residue.The polypeptide (for example, HSA variant and chimeric polyeptides with HSA part) that is included in any combination of the aminoacid replacement in any one or a plurality of aforementioned residue is special expectation.
In certain embodiments, at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 402, residue 415, residue 447, residue 459, residue 511, residue 517, residue 519, residue 521, residue 538, residue 541 and residue 566.
In certain embodiments, at least one aminoacid replacement in the HSA Domain III is selected from lower group, and this group is comprised of the following: K402A, K402G, K402I, K402L, K402V, V415C, V415S, V415T, P447S, P447T, Q459K, Q459R, L463N, L463Q, A511F, A511W, A511Y, S517C, S517F, S517M, S517T, S517W, S517Y, K519A, K519G, K519I, K519L, K519V, R521F, R521W, R521Y, K538F, K538W, K538Y, K541F, K541W, K541Y, T566F, T566W, and T566Y.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5 ...) or even all aminoacid replacement all be selected from aforementioned replacement.
In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is that the surface can reach residue.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is that the surface can reach residue.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is that the surface can reach and is conservative residue in a plurality of species.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is that the surface can reach and is the residue of guarding in a plurality of species.In certain embodiments, at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.In certain embodiments, at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 417, residue 442, residue 499 and residue 502.In certain embodiments, at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 383, residue 391 and residue 442.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18) or even all aminoacid replacement all in aforementioned residue.The polypeptide (for example, HSA variant and chimeric polyeptides) that comprises any combination of aforementioned aminoacid replacement classification is special expectation.In certain embodiments, at least one aminoacid replacement in the HSA Domain III is selected from lower group, and this group is comprised of the following: E383A, E383G, E3831, E383L, E383V, N391A, N391G, N3911, N391L, N391V, E442K, E442R.In certain embodiments, the more than one aminoacid replacement in Domain III (for example, 2,3,4,5 ...) or even all aminoacid replacement all be selected from aforementioned replacement.
In certain embodiments, at least one aminoacid replacement in the HSA Domain III is not only R410C; K466E; E479K; D494N; E501K; E505K; V533M; K536E536; K541E; D550A or D550G; K560E; D563N; E565K; E570K; K573E; Or the replacement of K574E.
In certain embodiments, polypeptide (for example, HSA variant and the chimeric polyeptides with HSA part) comprises the HSA Domain III, and it comprises with SEQ ID NO:l and has at least 80%, 85% or at least 90% conforming aminoacid sequence.In certain embodiments, the HSA Domain III comprises with SEQ ID NO:1 and has at least 95% conforming aminoacid sequence.In certain embodiments, the HSA Domain III comprises with SEQ ID NO:1 and has at least 98% conforming aminoacid sequence.In certain embodiments, the appropriate section that partly comprises with SEQ ID NO:2 of HSA has at least 80%, 85% or at least 90% conforming aminoacid sequence.In certain embodiments, the appropriate section that partly comprises with SEQ ID NO:2 of HSA has at least 95% conforming aminoacid sequence.In certain embodiments, the appropriate section that partly comprises with SEQ ID NO:2 of HSA has at least 98% conforming aminoacid sequence.
In certain embodiments, what this disclosure was expected is, except the one or more aminoacid replacement in the HSA Domain III, polypeptide (for example, HSA variant and the chimeric polyeptides with HSA part) can comprise one or more aminoacid replacement in the HSA part beyond the Domain III.
In certain embodiments, what this disclosure was expected is, except the one or more aminoacid replacement in the HSA Domain III, polypeptide (for example, HSA variant and the chimeric polyeptides with HSA part) also can be included in one or more aminoacid deletion in the Domain III and/or insertion (for example, l, 2,3,4,5,6,7,8).Should be noted in the discussion above that when HSA partly contains one or more aminoacid deletion and/or inserts, can use the residue of these insertions of letter representation or disappearance, thereby not disturb the numbering with respect to the residue of natural HSA.For example, if between residue 414 and 415, insert amino acid residue, this residue can be expressed as 414a.In certain embodiments, the amino acid residue of insertion be inserted in the surface can and the ring in to increase the size of this ring.In certain embodiments, the amino acid residue of insertion is inserted in the spiral to increase the big or small of this spiral and/or to change its structure.
In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the ring 2 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the ring 2 of HSA Domain III.In certain embodiments, the HSA Domain III comprises one to five (1,2,3,4 or 5) aminoacid replacement, and wherein said one is substituted in the ring 2 of HSA Domain III to five amino acid.In certain embodiments, the HSA Domain III is included in one to five (1,2,3,4 or 5) aminoacid replacement in the ring 2 of HSA Domain III, and further comprises the not one or more other aminoacid replacement in the HSA Domain III in ring 2.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the ring 3 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the ring 3 of HSA Domain III.In certain embodiments, the HSA Domain III comprises one to five (1,2,3,4 or 5) aminoacid replacement, and wherein said one is substituted in the ring 3 of HSA Domain III to five amino acid.In certain embodiments, the HSA Domain III is included in one to five (1,2,3,4 or 5) aminoacid replacement in the ring 3 of HSA Domain III, and further comprises the one or more other aminoacid replacement in the HSA Domain III in ring 3 not.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the ring 6 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the ring 6 of HSA Domain III.In certain embodiments, the HSA Domain III comprises one to 18 (1,2,3,4,5,6, etc.) aminoacid replacement, and wherein said one to 18 aminoacid replacement is in the ring 6 of HSA Domain III.In certain embodiments, the HSA Domain III comprises to 18 (1,2,3,4,5,6 in the ring 6 of HSA Domain III, etc.) aminoacid replacement, and further comprise the not one or more other aminoacid replacement in the HSA Domain III in ring 6.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the spiral 7 of HSA Domain III.In certain embodiments, all the described aminoacid replacement in the HSA Domain III are all in the spiral 7 of HSA Domain III.In certain embodiments, the HSA Domain III comprises one to six (1,2,3,4,5 or 6) aminoacid replacement, and wherein said one to six aminoacid replacement is all in the spiral 7 of HSA Domain III.In certain embodiments, the HSA Domain III is included in one to six (1,2,3,4,5 or 6) aminoacid replacement in the spiral 7 of HSA Domain III, and further comprises the one or more other aminoacid replacement in the HSA Domain III in spiral 7 not.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the ring 7 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the ring 7 of HSA Domain III.In certain embodiments, the HSA Domain III comprises one to three (1,2 or 3) aminoacid replacement, and wherein said one to three aminoacid replacement is in the ring 7 of HSA Domain III.In certain embodiments, the HSA Domain III is included in one to three (1,2 or 3) aminoacid replacement in the ring 7 of HSA Domain III, and further comprises the one or more other aminoacid replacement in the HSA Domain III in ring 7 not.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the spiral 8 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the spiral 8 of HSA Domain III.In certain embodiments, the HSA Domain III comprises one to 18 (1,2,3,4,5,6, etc.) aminoacid replacement, and wherein said one to 18 aminoacid replacement is all in the spiral 8 of HSA Domain III.In certain embodiments, the HSA Domain III is included in to six (1,2,3,4,5,6 in the spiral 8 of HSA Domain III, etc.) aminoacid replacement, and further comprise the not one or more other aminoacid replacement in the HSA Domain III in spiral 8.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the ring 8 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the ring 8 of HSA Domain III.In certain embodiments, the HSA Domain III comprises one to five (1,2,3,4 or 5) aminoacid replacement, and wherein said one is substituted in the ring 8 of HSA Domain III to five amino acid.In certain embodiments, the HSA Domain III is included in one to five (1,2,3,4 or 5) aminoacid replacement in the ring 8 of HSA Domain III, and further comprises the not one or more other aminoacid replacement in the HSA Domain III in ring 8.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the ring 9 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the ring 9 of HSA Domain III.In certain embodiments, the HSA Domain III comprises one to four aminoacid replacement, and wherein said one to four (1,2,3,4) aminoacid replacement is in the ring 9 of HSA Domain III.In certain embodiments, the HSA Domain III is included in one to five (1,2,3,4) aminoacid replacement in the ring 9 of HSA Domain III, and further comprises the not one or more other aminoacid replacement in the HSA Domain III in ring 9.As mentioned above, the possible aminoacid replacement at each position place is independently selected from above-mentioned any replacement type (for example, alanine replaces, conservative replaces, etc.).The polypeptide (for example, HSA variant and chimeric polyeptides) that comprises any combination of aforementioned aminoacid replacement classification is also expected.
In certain embodiments, the described aminoacid replacement in the HSA Domain III is not in the ring 2 of HSA Domain III.In certain embodiments, the described aminoacid replacement in the HSA Domain III is not in the ring 3 of HSA Domain III.In certain embodiments, the described aminoacid replacement in the HSA Domain III is not in the ring 6 of HSA Domain III.In certain embodiments, described aminoacid replacement is not in the spiral 7 of HSA Domain III.In certain embodiments, described aminoacid replacement is not in the ring 7 of HSA Domain III.In certain embodiments, described aminoacid replacement is not in the spiral 8 of HSA Domain III.In certain embodiments, the described aminoacid replacement in the HSA Domain III is not in the ring 8 of HSA Domain III.In certain embodiments, the described aminoacid replacement in the HSA Domain III is not in the ring 9 of HSA Domain III.
In certain embodiments, the described aminoacid replacement in the HSA Domain III be not selected from two rings of HSA Domain III of lower group at least, and this group is comprised of ring 2,3,6,7,8 and 9.In certain embodiments, the described aminoacid replacement in the HSA Domain III be not selected from three rings of HSA Domain III of lower group at least, and this group is comprised of ring 2,3,6,7,8 and 9.In certain embodiments, the described aminoacid replacement in the HSA Domain III be not selected from four rings of HSA Domain III of lower group at least, and this group is comprised of ring 2,3,6,7,8 and 9.In certain embodiments, the described aminoacid replacement in the HSA Domain III is not in spiral 7 or 8.
In certain embodiments, Domain III comprises at least two aminoacid replacement and described aminoacid replacement in being selected from lower group at least two rings and/or spiral of HSA Domain III, this group by encircle 2,3,6,7,8 and 9 and spiral 7 and 8 form.In certain embodiments, Domain III comprises at least three aminoacid replacement and described aminoacid replacement in being selected from lower group at least three rings and/or spiral of HSA Domain III, this group by encircle 2,3,6,7,8 and 9 and spiral 7 and 8 form.In certain embodiments, Domain III comprise at least four aminoacid replacement and and described aminoacid replacement in being selected from lower group at least four of HSA Domain III rings and/or spiral, this group by encircle 2,3,6,7,8 and 9 and spiral 7 and 8 form.In certain embodiments, Domain III comprise that five amino acid at least replaces and described aminoacid replacement in being selected from lower group at least five rings and/or spiral of HSA Domain III, this group by encircle 2,3,6,7,8 and 9 and spiral 7 and 8 form.In certain embodiments, Domain III comprises at least six aminoacid replacement and described aminoacid replacement in being selected from lower group at least six rings and/or spiral of HSA Domain III, this group by encircle 2,3,6,7,8 and 9 and spiral 7 and 8 form.In certain embodiments, Domain III comprise that five amino acid at least replaces and described aminoacid replacement the ring 2,3,6,7 of HSA Domain III, 8 and 9 each in.In certain embodiments, Domain III comprise at least six aminoacid replacement and described aminoacid replacement the ring 2,3,6,7 of HSA Domain III, 8 and 9 each in.In certain embodiments, Domain III comprises at least two aminoacid replacement and described aminoacid replacement in each of the spiral 7 of HSA Domain III and 8.
In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is encircling in 2 and is being selected from: residue 415, residue 416, residue 417, residue 418 and residue 419.In certain embodiments, the aminoacid replacement of (2,3,4,5) is encircling in 2 and is being selected from more than one: residue 415, residue 416, residue 417, residue 418 and residue 419.In certain embodiments, at least one the described aminoacid replacement in HSA Domain III ring 2 is selected from: V415C, V415S, V415T, Q416H and Q416P.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is encircling in 3 and is being selected from: residue 439, residue 440, residue 441, residue 442 and residue 443.In certain embodiments, the aminoacid replacement of (2,3,4,5) is encircling in 3 and is being selected from more than one: residue 439, residue 440, residue 441, residue 442 and residue 443.In certain embodiments, at least one the described aminoacid replacement in HSA Domain III ring 3 is selected from: E442K and E442R.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is encircling in 6 and is being selected from: residue 492, residue 493, residue 494, residue 495, residue 496, residue 497, residue 498, residue 499, residue 500, residue 501, residue 502, residue 503, residue 504, residue 505, residue 506, residue 507, residue 508 and residue 509.In certain embodiments, the aminoacid replacement of (2,3,4,5,6,7,8,9,10, etc.) is in ring 6 and be selected from more than one: residue 492, residue 493, residue 494, residue 495, residue 496, residue 497, residue 498, residue 499, residue 500, residue 501, residue 502, residue 503, residue 504, residue 505, residue 506, residue 507, residue 508 and residue 509.In certain embodiments, at least one the described aminoacid replacement in HSA Domain III ring 6 is selected from: T506F, T506W, T506Y, T508C, T508E, T508I, T508K, T508R, T508S, F509C, F509I, F509L, F509M, F509V, F509W and F509Y.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in spiral 7 and be selected from: residue 510, residue 511, residue 512, residue 513, residue 514 and residue 515.In certain embodiments, more than one the aminoacid replacement of (2,3,4,5,6) in spiral 7 and be selected from: residue 510, residue 511, residue 512, residue 513, residue 514 and residue 515.In certain embodiments, at least one the described aminoacid replacement in HSA Domain III ring 7 is selected from: A511F, A511W, A511Y, D512F, D512W, D512Y, T515C, T515H, T515N, T515P, T515Q and T515S.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is encircling in 7 and is being selected from: residue 516, residue 517 and residue 518.In certain embodiments, the aminoacid replacement of (2,3) is encircling in 7 and is being selected from more than one: residue 516, residue 517 and residue 518.In certain embodiments, at least one the described aminoacid replacement in HSA Domain III ring 7 is selected from: L516F, L516S, L516T, L516W, L516Y, S517C, S517F, S517M, S517T, S517W and S517Y.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in spiral 8 and be selected from: residue 519, residue 518, residue 519, residue 520, residue 521, residue 522, residue 523, residue 524, residue 525, residue 526, residue 527, residue 528, residue 529, residue 530, residue 531, residue 532, residue 533, residue 534, residue 535 and residue 536.In certain embodiments, the aminoacid replacement of (2,3,4,5,6,7,8,9,10, etc.) is in spiral 8 and be selected from more than one: residue 519, residue 518, residue 519, residue 520, residue 521, residue 522, residue 523, residue 524, residue 525, residue 526, residue 527, residue 528, residue 529, residue 530, residue 531, residue 532, residue 533, residue 534, residue 535 and residue 536.In certain embodiments, at least one the described aminoacid replacement in HSA Domain III spiral 8 is selected from: K519A, K519G, K519I, K519L, K519V, R521F, R521W, R521Y, I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W, I523Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526C, Q526M, Q526S, Q526T, Q526Y, T527F, T527W, T527Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, and H535P.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is encircling in 8 and is being selected from: residue 537, residue 538, residue 539, residue 540 and residue 541.In certain embodiments, the aminoacid replacement of (2,3,4,5) is encircling in 8 and is being selected from more than one: residue 537, residue 538, residue 539, residue 540, residue 541.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is encircling in 9 and is being selected from: residue 561, residue 562, residue 563, residue 564.In certain embodiments, at least one the described aminoacid replacement in HSA Domain III ring 8 is selected from: K538F, K538W, K538Y, A539I, A539L, A539V, K541F, K541W, K541Y.In certain embodiments, the aminoacid replacement of (2,3,4) is encircling in 9 and is being selected from more than one: residue 561, residue 562, residue 563, residue 564.In certain embodiments, at least one the described aminoacid replacement in HSA Domain III ring 9 is selected from: A561F, A561W and A561Y.
Expectation is insertion and disappearance in Domain III in addition, for example, and its increase or reduce the length of HSA Domain III ring.In certain embodiments, the described insertion in the HSA Domain III or disappearance change the length of ring 2.In certain embodiments, the described insertion in the HSA Domain III or disappearance change the length of ring 3.In certain embodiments, the described insertion in the HSA Domain III or disappearance change the length of ring 6.In certain embodiments, the described insertion in the HSA Domain III or disappearance change the length of spiral 7.In certain embodiments, the described insertion in the HSA Domain III or disappearance change the length of ring 7.In certain embodiments, the described insertion in the HSA Domain III or disappearance change the length of spiral 8.In certain embodiments, the described insertion in the HSA Domain III or disappearance change the length of ring 8.In certain embodiments, the described insertion in the HSA Domain III or disappearance change the length of ring 9.In certain embodiments, the described insertion in Domain III or disappearance change at least two rings being selected from lower group and/or the length of spiral, this group by encircle 2,3,6,7,8 and 9 and spiral 7 and 8 form.In certain embodiments, the described insertion in Domain III or disappearance change at least three rings being selected from lower group and/or the length of spiral, this group by encircle 2,3,6,7,8 and 9 and spiral 7 and 8 form.In certain embodiments, the described insertion in Domain III or disappearance change at least four rings being selected from lower group and/or the length of spiral, this group by encircle 2,3,6,7,8 and 9 and spiral 7 and 8 form.In certain embodiments, the described insertion in Domain III or disappearance change at least five rings being selected from lower group and/or the length of spiral, this group by encircle 2,3,6,7,8 and 9 and spiral 7 and 8 form.In certain embodiments, the length of each in the ring 2,3,6,7,8 and 9 of the described insertion in the HSA Domain III or disappearance change HSA Domain III.Should be noted that, when a plurality of rings and/or spiral change, this disclosure these rings of expectation and/or spiral all change independently, so that can increase one or more ring/spirals by insertion, and can reduce one or more ring/spirals by disappearance.
Comprise the embodiment of amino acid whose insertion or disappearance, amino acid whose insertion of this disclosure expectation or disappearance for Domain III wherein.Surpass an aminoacid, for example 2,3,4,5,6,7,8,9,10 amino acid whose insertions or disappearance are also expected.In certain embodiments, the amino acid residue of this disclosure expectation more than 10, for example 10-20,20-40,40-50, a 50-100 amino acid whose insertion or disappearance.Consistent with chimeric polyeptides and the HSA variant polypeptide of this disclosure, test the compositions that comprises insertion or disappearance and kept FcRn in conjunction with activity in order to confirm them.Preferred compositions is for providing the compositions of improved FcRn combination and/or serum half-life with respect to contrast.
For the sake of clarity, the combination of the special any aforementioned or following aspect of expectation of this disclosure and embodiment.In the situation of the chimeric polyeptides that comprises the HSA part, and in the situation of the variant HSA polypeptide that comprises the HSA part, this kind HSA partly comprises Domain III or its FcRn bound fraction.In addition, as said, the Domain III of HSA part comprises one to 18 aminoacid replacement.In certain embodiments, the Domain III of HSA part comprises l, 2,3,4,5,6,7,8,9,10,11,12,13,14, l5,16,17 or 18 aminoacid replacement.Exemplary aminoacid replacement comprises: (i) replace with alanine; (ii) conservative aminoacid replacement; (iii) nonconservative aminoacid replacement.All aminoacid replacement that this disclosure is desirably in the Domain III of given polypeptide can be one of these aminoacid replacement classifications, and each aminoacid replacement that is desirably in the Domain III of given polypeptide can individually and be independently selected from these types.In certain embodiments, the natural cysteine residues in Domain III is retained and is not substituted.In certain embodiments, the natural proline residue in Domain III is retained and is not substituted.In certain embodiments, the natural cysteine residues in Domain III and natural proline residue are retained and are not substituted.In certain embodiments, cysteine residues is not substituted (for example, not using cysteine displacement natural residue).In certain embodiments, proline residue is not substituted (for example, not using proline displacement natural residue).In other embodiments, use in 20 seed amino acids any to replace given natural residue.
The HSA variant and the chimeric polyeptides that comprise any combination of aforementioned aminoacid replacement type are also expected.
Variant and the chimeric polyeptides with HSA part contained in the present invention, described HSA partly comprise with the essentially identical sequence of aminoacid sequence described herein in aminoacid.Comprise the sequence that comprises the conserved amino acid replacement with the essentially identical aminoacid sequence of said sequence, and aminoacid deletion and/or insertion.Conserved amino acid replaces and to refer to that the first aminoacid is had the second amino acid replacement with the first amino acid whose chemistry and/or physical characteristic (for example electric charge, structure, polarity, hydrophobicity/hydrophilic) similar characteristic.Conserved amino acid replaces and comprises that a seed amino acid is used to the another kind of amino acid replacement in lower group: lysine (K), arginine (R) and histidine (H); Aspartic acid (D) and glutamic acid (E); Agedoite (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; Alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.
In certain embodiments, described HSA variant polypeptide is purification basically.In certain embodiments, described chimeric polyeptides is purification basically.In some aspects, this disclosure provides the HSA variant that comprises this disclosure or the compositions of chimeric polyeptides and pharmaceutically acceptable carrier.In certain embodiments, said composition is aseptic composite.In certain embodiments, said composition is non-pyrogen.
6.4 the Domain III mutant of combination
The present invention further expects to produce the combination mutant of the HSA part that comprises Domain III and the group of truncated mutant, and the present invention is useful especially for the identification of biological activity variant sequence.Can produce the variant that combination is derived, it has the selection potentiality with respect to naturally occurring HSA polypeptide.Equally, mutation can produce have from the significantly different cell of corresponding wild type HSA polypeptide in the variant of half-life.The protein that for example, can make change is to protein degradation or cause the destruction of interested protein or additionally other cell processes of inactivation are more stable or more unstable.Such variant can be used for changing HSA polypeptide level by the half-life of regulating them.Exist and manyly whereby can be for example produce the method in the library of potential HSA variant sequence from degenerate oligonucleotide sequence.Can in automatic dna synthesizer, carry out the chemosynthesis of degeneracy gene order, and then synthetic gene is connected to suitable gene for expressing.The genomic purpose of degeneracy is all sequences that the desirable potential peptide sequence group of coding is provided with a kind of form of mixture.Degenerate oligonucleotide synthetic be well known in the art (referring to, for example, Narang, SA(1983) Tetrahedron(" tetrahedron ") 39:3; Itakura(Ban Cang) people such as, (1981) Recombinant DNA, Proc.3rd Cleveland Sympos.(recombinant DNA, the 3rd Cleveland seminar) Macromolecules(macromole), AG Walton(pauses the Wal) compile Amsterdam(Amsterdam): like to think only your (Elsevier) pp273-289; The people such as Ban Cang, (1984) Annu.Rev.Biochem.(" bioid academic year comments ") 53:323; The people such as Ban Cang, (1984) Science(" science ") 198:1056; Ike(Ai Ke) people such as, (1983) Nucleic Acid Res.(" nucleic acids research ") 11:477).This type of technology has been used for the people such as orthogenesis (referring to for example, the Scott(Scott) of other oroteins, (1990) Science(" science ") 249:386-390; The Roberts(Robert) people such as, (1992) PNAS USA(" PNAS ") 89:2429-2433; Devlin(De Fulin) people such as, (1990) Science(" science ") 249:404-406; The people such as Cwirla, (1990) PNAS USA(" PNAS ") 87:6378-6382; And U.S. Patent number: 5,223,409,5,198,346 and 5,096,815).
Alternately, can produce combinatorial library with other mutation forms.For example, can produce by the following method and from the library, separate the HSA polypeptide variants: by screening, such as using the people such as alanine scanning mutagenesis etc. (Ruf(pressgang), (1994) Biochemistry(" biochemistry ") 33:1565-1572; Wang(king) people such as, (1994) J.Biol.Chem.(" journal of biological chemistry ") 269:3095-3099; Balint(Bahrain is special) etc. the people, (1993) Gene(" gene ") 137:109-118; The Grodberg(Goldberger) people such as, (1993) Eur.J.Biochem.(" european journal of biological chemistry ") 218:597-601; The Nagashima(Long Island) people such as, (1993) J.Biol.Chem.(" journal of biological chemistry ") 268:2888-2892; Lowman(Luo Man) people such as, (1991) Biochemistry(" biochemistry ") 30:10832-10838; And Cunningham(Cunningham's skink) people such as, (1989) Science(" science ") 244:1081-1085), by people such as linker scanning mutagenesises (Gustin(Gu Siting), (1993) Virology(" virusology ") 193:653-660; Brown(Blang) people such as, (1992) Mol.Cell Biol.(" molecular cytobiology ") 12:2644-2652; The McKnight(MacKnight) people such as, (1982) Science(" science ") 232:316); By the people such as saturation mutagenesis (Meyers(Meyers), (1986) Science(" science ") 232:613); By the people such as PCR mutation (Leung(beam), (1989) Method Cell Mol Biol(" cellular elements biological method ") 1:11-19); Or by random mutagenesis, comprise the people such as (Miller(Millers) such as chemomorphosis, (1992) A Short Course in Bacterial Genetics(" bacterial genetics concise course "), CSHL publishing house, cold spring port, New York; And Greener(jesse greener) people such as, (1994) Strategies in Mol Biol(" molecular biology strategy ") 7:32-34).Especially be very attractive method with the linker scanning mutagenesis that makes up the form that arranges for truncate (bioactive) form of identifying the HSA polypeptide.Provide at this to produce and the additive method in screening HSA polypeptide variants library, referring to for example, name is called the 8th chapters and sections of " illustration ".Particularly, chapters and sections 8.10 and 8.11 be for generation of with the ad hoc approach of screening combination HSA Domain III mutant library.
Above-mentioned any embodiment of the aminoacid replacement in the polypeptide of this disclosure all can be used for producing peptide library.In certain embodiments, produce the variant that combination is derived in the residue in the HSA Domain III.In certain embodiments, the introducing Domain III suddenlys change and it is screened in following one or more situations: (i) independent variant structure territory III construct; (ii) the variant structure territory III construct that in total length HSA situation, exists, or (iii) at truncate HSA or comprise in the situation of the chimeric polyeptides of Domain III at least.In certain embodiments, filter out variant from peptide library, these variants have the one or both to the serum half-life of the affinity of FcRn and increase of increase with respect to initial polypeptide.In certain embodiments, use in this application described standard external test (for example, flow cytometry) assessment variant.In certain embodiments, one or more variants to the affinity of FcRn that show increase have been identified.In other embodiments, the screening variant to be determining whether improved affinity to FcRn only appears at (for example, pH is about 5.5) under the acid pH, and do not appear at (for example, pH is about 7.4) under the neutral pH.
In certain embodiments, make up at least two aminoacid replacement that the variant of deriving is included in any following position that is numbered with respect to the ripe HSA of total length: residue 407, residue 415, residue 463, residue 495, residue 508, residue 509, residue 511, residue 512, residue 515, residue 516, residue 517, residue 521, residue 523, residue 524, residue 526, residue 527 and residue 557.
In certain embodiments, the combination variant of deriving comprises and is selected from least two aminoacid replacement of lower group, and this group is comprised of the following: L407N, L407Y, V415T, L463N, L463F, E495D, T508R, T508S, F509M, F509W, F509I, A511F, D512Y, D512M, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523F, I523G, I523K, I523R, K524L, Q526A, Q526M, Q526Y, T527Y and T557G.
In certain embodiments, the variant that combination is derived is included in the aminoacid replacement in the HSA Domain III of the position that is numbered with respect to the ripe HSA of total length, and these positions are selected from lower group, and this group is comprised of the following: (a) residue 383 and 413; (b) residue 401 and 523; (c) residue 407 and 447; (d) residue 407 and 447 and 539; (e) residue 407 and 509; (f) residue 407 and 526; (g) residue 411 and 535; (h) residue 414 and 456; (i) residue 415 and 569; (j) residue 426 and 526; (k) residue 442 and 450 and 459; (l) residue 463 and 508; (m) residue 508 and 519 and 525; (n) residue 509 and 527; (o) residue 523 and 538; (p) residue 526 and 557; (q) residue 541 and 561; (r) residue 463 and 523; (s) residue 508 and 523; (t) residue 508 and 524; (u) residue 463,508 and 523; (v) residue 463,508 and 524; (w) residue 508,523 and 524; (x) residue 463,508,523 and 524; (y) residue 463 and 524; (z) residue 523 and 524; And (aa) residue 463,523 and 524.
In certain embodiments, the variant that combination is derived is included in the aminoacid replacement in the HSA Domain III of a plurality of positions that are numbered with respect to the position among the ripe HSA of total length, these positions are selected from lower group, and this group is comprised of the following: (a) residue 463 and 508; (b) residue 463 and 523; (c) residue 508 and 523; (d) residue 508 and 524; (e) residue 463,508 and 523; (f) residue 463,508 and 524; (g) residue 508,523 and 524; (h) residue 463,508,523 and 524; (i) residue 463 and 523; And (j) residue 523 and 524, wherein the replacement at residue 463 places is selected from L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q; Replacement at residue 508 places is selected from T508C, T508E, T508I, T508K, T508R, T508S; Replacement at residue 523 places is selected from I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W, I523Y; And the replacement at residue 524 places is selected from K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V.
In certain embodiments, the variant that combination is derived is included in the aminoacid replacement in the HSA Domain III of a plurality of positions that are numbered with respect to the position among the ripe HSA of total length, these positions are selected from lower group, and this group is comprised of the following: (a) residue 463 and 524; (b) residue 508 and 523; (c) residue 508 and 524; And (d) residue 463,508 and 523, wherein the replacement at residue 463 places is selected from L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q; Replacement at residue 508 places is selected from T508C, T508E, T508I, T508K, T508R, T508S; Replacement at residue 523 places is selected from I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W, I523Y; And the replacement at residue 524 places is selected from K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V.
In certain embodiments, the combination variant of deriving comprises and is selected from lower group the aminoacid replacement in the HSA Domain III, and these replacements are selected from lower group, and this group is comprised of the following: (a) E383G/K413S; (b) Y401E/I523G; (c) L407N/P447S; (d) L407N/P447S/A539I; (e) L407N/F509M; (f) L407Y/Q526T; (g) Y411Q/H535N; (h) K414S/V456N; (i) V415T/A569P; (j) V426H/Q526Y; (k) E442K/E450D/Q459R; (l) L463N/T508R; (m) T508R/K519I/K525V; (n) F509I/T527Y; (o) I523Q/K538Y; (p) Q526M/K557G; (q) K541F/A561F; (r) L463N/K524L; (s) T508R/I523G; (t) T508R/K524L; (u) L463N/T508R/I523G; (v) L463N/T508R/K524L; (w) T508R/I523G/K524L; (x) L463N/T508R/I523G/K524L; (y) L463N/I523G; (z) I523G/K524L; (aa) L463N//I523G/K524L.
In certain embodiments, the combination variant of deriving comprises and is selected from lower group the aminoacid replacement in the HSA Domain III, and these replacements are selected from lower group, and this group is comprised of the following: (a) L463N/T508R; (b) L463N/K524L; (c) T508R/I523G; (d) T508R/K524L; (e) L463N/T508R/I523G; (f) L463N/T508R/K524L; (g) T508R/I523G/K524L; (h) L463N/T508R/I523G/K524L.
In certain embodiments, the combination variant of deriving comprises and is selected from lower group the aminoacid replacement in the HSA Domain III, and these replacements are selected from lower group, and this group is comprised of the following: (a) L463N/K508R; (b) T508R/I523G; (c) T508R/K524L; (d) L463N/T508R/I523G.
In certain embodiments, the variant that combination is derived comprises SEQ ID NO:18, wherein, and X 1Not leucine and/or X 2Not threonine and/or X 3Not isoleucine and/or X 4Not lysine.In certain embodiments, X 1Cysteine, phenylalanine, glycine, histidine, isoleucine, agedoite, serine or glutamine; X 2Cysteine, glutamic acid, isoleucine, lysine, arginine or serine; X 3Alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, agedoite, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine; And X 4Alanine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, glutamine, threonine or valine.In other embodiments, X 1Phenylalanine or agedoite; X 2Arginine or serine; X 3Aspartic acid, glutamic acid, glycine, phenylalanine, lysine or arginine; And/or X 4It is leucine.In other embodiment again, X 1Agedoite and/or X 2Arginine and/or X 3Glycine and/or X 4It is leucine.
In certain embodiments, produce the variant that combination is derived in the residue of in a plurality of species, guarding.In certain embodiments, can reach the variant that the generation combination is derived in the residue on the surface.In certain embodiments, in the residue that the surface can reach and guard, produce the variant that combination is derived in a plurality of species.In certain embodiments, conservative in a plurality of species and in chicken HSA, produce the variant that combination is derived in the conservative residue.
In some aspects, this disclosure provides the library that comprises multiple polypeptides, each self-contained HSA Domain III of wherein said multiple polypeptides or its FcRn binding fragment, and be included in independently of one another at least one aminoacid replacement of the residue in the described HSA Domain III in the wherein said multiple polypeptides, described residue is guarded in the serum albumin protein from people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.
In some aspects, this disclosure provides the library that comprises multiple polypeptides, each self-contained HSA Domain III of wherein said multiple polypeptides or its FcRn binding fragment, and wherein said multiple polypeptides is included at least one aminoacid replacement of the residue in the described HSA Domain III independently of one another, described residue is guarded in the serum albumin protein from people, pig, rat, mice, Canis familiaris L., rabbit, cattle, donkey, mongolian gerbil, sheep, cat and horse, and does not guard in the serum albumin from chicken.
In some aspects, this disclosure provides the library that comprises multiple polypeptides, each self-contained HSA Domain III of wherein said multiple polypeptides or its FcRn binding fragment, and wherein said multiple polypeptides is included at least one aminoacid replacement of the residue in the described HSA Domain III independently of one another, and described residue is that the surface can reach residue.
In some aspects, this disclosure provides the library that comprises multiple polypeptides, each self-contained HSA Domain III of wherein said multiple polypeptides or its FcRn binding fragment, and wherein said multiple polypeptides is included at least one aminoacid replacement of the residue in the described HSA Domain III independently of one another, described residue can reach residue for (i) surface, and (ii) guards in the serum albumin protein from people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.
In certain embodiments, described surface can reach residue in the ring 2 of HSA Domain III.In certain embodiments, described surface can reach residue in the ring 3 of HSA Domain III.In certain embodiments, described surface can reach residue in the ring 6 of HSA Domain III.In certain embodiments, described surface can reach residue in the spiral 7 of HSA Domain III.In certain embodiments, described surface can reach residue in the ring 7 of HSA Domain III.In certain embodiments, described surface can reach residue in the spiral 8 of HSA Domain III.In certain embodiments, described surface can reach residue in the ring 8 of HSA Domain III.In certain embodiments, described surface can reach residue in the ring 9 of HSA Domain III.
In certain embodiments, at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.In certain embodiments, at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 417, residue 442, residue 499 and residue 502.
In certain embodiments, at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 392, residue 399, residue 403, residue 411, residue 412, residue 414, residue 416, residue 418, residue 420, residue 423, residue 434, residue 437, residue 438, residue 445, residue 448, residue 450, residue 453, residue 461, residue 476, residue 477, residue 484, residue 485, residue 487, residue 488, residue 494, residue 497, residue 507, residue 509, residue 514, residue 529, residue 534, residue 537, residue 540, residue 551, residue 558, residue 559, residue 567, residue 568, residue 572.In certain embodiments, at least one aminoacid replacement in the HSA Domain III is arranged in any following position that the position with respect to the ripe HSA Domain III of total length is numbered: residue 380, residue 381, residue 384, residue 387, residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 419, residue 421, residue 422, residue 424, residue 428, residue 430, residue 431, residue 433, residue 441, residue 457, residue 458, residue 463, residue 464, residue 466, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 489, residue 491, residue 495, residue 500, residue 508, residue 510, residue 515, residue 516, residue 524, residue 525, residue 526, residue 528, residue 531, residue 535, residue 539, residue 544, residue 547, residue 576.
In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the ring 2 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the ring 2 of HSA Domain III.In certain embodiments, the HAS Domain III is included at least one the described aminoacid replacement (in the ring 2 of HSA Domain III) in the HSA Domain III, and further is included in the not one or more other aminoacid replacement in ring 2 in the HSA Domain III.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the ring 3 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the ring 3 of HSA Domain III.In certain embodiments, the HSA Domain III is included at least one aminoacid replacement in the ring 3 of HSA Domain III, and further comprises the not one or more other replacement in the HSA Domain III in ring 3.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the ring 6 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the ring 6 of HSA Domain III.In certain embodiments, the HSA Domain III is included at least one aminoacid replacement in the ring 6 of HSA Domain III, and further comprises the not one or more other aminoacid replacement in the HSA Domain III in ring 6.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the spiral 7 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the spiral 7 of HSA Domain III.In certain embodiments, the HSA Domain III is included at least one aminoacid replacement in the spiral 7 of HSA Domain III, and further comprises the not one or more other aminoacid replacement in the HSA Domain III in spiral 7.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the ring 7 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the ring 7 of HSA Domain III.In certain embodiments, the HSA Domain III is included at least one aminoacid replacement in the ring 7 of HSA Domain III, and further comprises the not one or more other aminoacid replacement in the HSA Domain III in ring 7.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the spiral 8 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the spiral 8 of HSA Domain III.In certain embodiments, the HSA Domain III is included at least one aminoacid replacement in the spiral 8 of HSA Domain III, and further comprises the not one or more other aminoacid replacement in the HSA Domain III in spiral 8.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the ring 8 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the ring 8 of HSA Domain III.In certain embodiments, the HSA Domain III is included at least one aminoacid replacement in the ring 8 of HSA Domain III, and further comprises the not one or more other aminoacid replacement in the HSA Domain III in ring 8.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is in the ring 9 of HSA Domain III.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is all in the ring 9 of HSA Domain III.In certain embodiments, the HSA Domain III is included at least one aminoacid replacement in the HSA Domain III, and further comprises the not one or more other aminoacid replacement in the HSA Domain III in ring 9.
Any aforementioned or following aspect or among some embodiment of embodiment, aminoacid replacement can be that residue becomes alanine.Any aforementioned or following aspect or among some embodiment of embodiment, aminoacid replacement can be that conservative amino acid replaces, and wherein replaces another kind of residue with a kind of residue with similar electric charge and other characteristics.Any aforementioned or following aspect or among some embodiment of embodiment, aminoacid replacement can be that non-conservative amino acid replaces, and wherein replaces another kind of residue with a kind of residue that does not have similar electric charge and similar other characteristics.Any aforementioned or following aspect or among some embodiment of embodiment, aminoacid replacement can be that residue is become any other residue.When HSA partly comprises more than one amino acid residue, can expect to replace any one or any combination that can fall into aforementioned aminoacid replacement classification.For example, all replacements can be that to become alanine maybe can be that conservative amino acid replaces, and maybe can be that non-conservative amino acid replaces.Alternately, aminoacid replacement can comprise, for example as, become a variation of alanine, conservative amino acid replaces and non-conservation aminoacid replacement in any combination.
The gene outcome that is used for screening the combinatorial library for preparing by point mutation and truncate of broad range, and the technology that is used for thus for the gene outcome screening cDNA library with certain specific character is known in the art.Such technology will be applicable to rapid screening usually by the gene library of the combinatorial mutagenesis generation of HSA polypeptide.Being used for the most widely used technology in screening lots of genes library generally includes gene library is cloned into reproducible expression vector, transform suitable cell with the gained vector library, and express under the following conditions combination gene: wherein the detection of desirable activity promotes relatively easily to separate the carrier of the detected gene of its product of coding.In case of necessity, each illustrative described below is measured and all be applicable to a large amount of degenerate sequences that high throughput analysis produces by the combinatorial mutagenesis technology with screening.
In certain embodiments, the HSA polypeptide can comprise peptide and peptide mimics.As used herein, term " peptide mimics " comprises chemical modification peptide and peptide quasi-molecule, and it contains aminoacid, class peptide that non-natural exists, etc.Peptide mimics provides the various advantages that surpass peptide, comprises the stability that strengthens when being applied to the experimenter.The method of identifying peptide mimics is known in the art, and comprises that screening contains the data base in potential peptide mimics library.For example, cambridge structure database contains and surpasses 300,000 kinds of people such as set (Allen(Alan) with chemical compound of known crystal structure, Acta Crystal_logr.(" crystal diffraction journal ") chapters and sections B, 35:2331(1979)).But when the crystal structure time spent that does not have target molecule, can use, for example, program CONCORD produces a kind of structure (people such as Rusinko, J.Chem.Inf.Comput.Sci.(" chemical information and computer science magazine ") 29:251(1989)).Another data base, existing chemicals catalog data base (AvailableChemicals Directory) (Molecular Design Limited, lnformations Systems; San LeandroCalif.) contains about 100,000 kinds of commercially available chemical compounds and also can search for these chemical compounds in order to identify the potential peptide mimics of HSA polypeptide.
In certain embodiments, the HSA polypeptide can further comprise post translational modification.Exemplary post translational protein modification comprises phosphorylation, acetylation, methylates, ADP-ribosylation, ubiquitin, glycosylation, carbonylation, ubiquitin-like (sumoylation), biotinylation or add polypeptide side chain or hydrophobic group.Therefore, the HSA polypeptide of modification can contain non-amino acid composition, for example lipid, polysaccharide or monosaccharide and phosphate.Can test so non-amino acid composition to the impact of functional (for its biological activity, for example it is in conjunction with the ability of FcRn) of HSA polypeptide.Suppose that natural HSA polypeptide can be by glycosylation, in certain embodiments, the HSA polypeptide that uses in the chimeric polyeptides according to this disclosure is by glycosylation.In certain embodiments, glycosylated level is identical or basic identical with natural HSA polypeptide with pattern.In other embodiments, glycosylated level and/or pattern different from natural HSA polypeptide (for example, low glycosylation, excessive glycosylation, not glycosylations).
In certain embodiments of the present invention, comprise HSA polypeptide (for example, HSA variant or chimeric multistage) partly and can be incorporated in to non-proteinaceous matter.Such non-proteinaceous matter includes but not limited to nucleic acid molecules, chemical agent, organic molecule, etc., can obtain from natural origin separately (for example as natural product screening) or can chemosynthesis.In certain embodiments, the HSA part is bonded to non-proteinaceous matter with chemical mode.
In a specific embodiment of the present invention, can modify the HSA polypeptide with charged non-protein polymer.In a specific embodiment, polymer is Polyethylene Glycol (" PEG "), polypropylene glycol or polyoxyalkylene, as is in U.S. Patent number 4,640,835; 4,496,689; 4,301,144; 4,670,417; The mode of listing in 4,791,192 or 4,179,337.PEG is the water-soluble polymer of knowing, its commercially available or can be according to ring-opening polymerisation preparation (Sandler(Sandler) and the Karo(Caro of method well known in the art by ethylene glycol), Polymer Synthesis(" polymer is synthetic "), Academic Press(academic press), New York, the 3rd volume, the 138-161 page or leaf).
In certain embodiments, the fragment of HSA polypeptide or variant will preferably keep at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% the biological activity that is associated with natural HSA polypeptide.In certain embodiments, the fragment of HSA polypeptide or variant have the half-life (t1/2) that increases with respect to native protein.For the embodiment that half-life wherein increases, the half-life of the half-life of HSA fragment or variant with respect to natural HSA albumen increased at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500% or even 1000%.In certain embodiments, for example in buffer saline or serum, measured the protein half-life external.In certain embodiments, the half-life of protein is Half-life in vivo, for example in half-life of serum or the protein in other body fluid of animal.
In some aspects, comprising HSA polypeptide partly can be the fusion rotein that further comprises one or more Fusion domains.The example of knowing of such Fusion domain includes but not limited to polyhistidine, Glu-Glu, glutathione s-transferase (GST), thioredoxin, protein A, Protein G and immunoglobulin heavy chain constant region (Fc), maltose-binding protein (MBP), and these are useful especially for divide isolated fusion protein by affinity chromatograph.In order to carry out affinity purification, use the relevant substrate that is used for affinity chromatograph, for example with glutathion-, amylase-and nickel-or the resin of cobalt-combination.Fusion domain also comprises " epitope tag ", it typically is the short peptide sequence that can obtain specific antibody.The epitope tag of knowing that can obtain easily monoclonal antibody specific comprises FLAG, influenza virus hemagglutinin (HA) and c-myc label.In some cases, Fusion domain has protease cutting site, for example factor Xa or thrombin, and it allows relevant protease partly to digest fusion rotein, discharges recombiant protein from it thus.Then can isolate the protein that discharges from Fusion domain by follow-up chromatographic isolation.In certain embodiments, the HSA polypeptide can contain one or more and can make the stable modification of polypeptide.For example, such modification increases the vitro half-lives of polypeptide, increases the protein degradation of circulating half-life or the minimizing polypeptide of polypeptide.Equally, in the situation of chimeric polyeptides, aforementioned modified types can be attached in addition or alternately the heterologous protein part of chimeric polyeptides.
In certain embodiments, comprising HSA polypeptide partly can be the fusion rotein with all or part of Fc zone of immunoglobulin.In certain embodiments, fusion rotein comprises FcRn binding structural domain or its fragment of IgG.Equally, in certain embodiments, all or part of Fc zone of immunoglobulin can be as the joint that HSA partly is connected to heterologous protein.Know that as oneself each immunoglobulin heavy chain constant region comprises four or five domains.Domain is named as follows successively: CH1-hinge-CH2-CH3 (CH4).The DNA sequence of heavy chain domain has residual homology (cross-homology) at the immunoglobulin apoplexy due to endogenous wind, for example, the CH2 domain of IgG and the CH2 domain of IgA and IgD and with the CH3 domain of IgM and IgE be homology.As used herein, term " immunoglobulin fc region territory " it should be understood that the c-terminus part of immunoglobulin chain constant region, is preferably immunoglobulin heavy chain constant region or its part.For example, the immunoglobulin fc region territory can comprise: 1) CH1 domain, CH2 domain and CH3 domain; 2) CH1 domain and CH2 domain; 3) CH1 domain and CH3 domain; 4) CH2 domain and CH3 domain; Or 5) combination of two or more domains and immunoglobulin hinge region.In a preferred embodiment, the immunoglobulin fc region territory comprises at least one immunoglobulin hinge region, CH2 domain and CH3 domain, and preferably lacks the CH1 domain.In one embodiment, CH is IgG(Ig γ from wherein immunoglobulin class) (γ subclass 1,2,3 or 4).Can use the immunoglobulin of other types, such as IgA(Ig α), IgD(Ig δ), IgE(Ig ε) and IgM(Ig μ).At U.S. Patent number 5,541, discussed the selection of suitable immunoglobulin heavy chain constant region in 087 and 5,726,044 in detail.From some immunoglobulin class and subclass, select specific immunoglobulin heavy chain constant region sequence to obtain within the technical merit that specific result is considered to be in this area.The part of the DNA construct of coding immunoglobulin fc region preferably includes at least a portion in hinge arrangement territory, and the CH of preferred Fc γ 3At least a portion of any homeodomain among domain or IgA, IgD, IgE or the IgM.In addition, what can expect is that the amino acid whose replacement in immunoglobulin heavy chain constant region or disappearance may be useful in practice of the present invention.Example in upper CH2 zone, introduce aminoacid replacement with produce to the Fc receptor have the affinity of minimizing people (1997) J.IMMUNOL.(such as Fc variant (Cole(Cole) " IMMUNOLOGY KEY WORDS INDEX) 159:3613).Those of ordinary skill in the art can use the molecular biotechnology of knowing to prepare such construct.Equally, in the situation of chimeric polyeptides, aforementioned modified types can be attached in addition or alternately the heterologous protein part of chimeric polyeptides.
In some aspects, the HSA polypeptide can be support.In certain embodiments, but protein is used for the protein framework of selecting or design specificity to be combined with target.When by support Design protein, keep the amino acid residue important to the advantageous feature of framework, and other residues can change.In certain embodiments, support can have be less than or equal 50% have the amino acid residue that changes between the protein derivatives of different qualities, and more than or equal to 50% constant residue between such derivant.In certain embodiments, support can have be less than or equal 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% have the amino acid residue that changes between the protein derivatives of different qualities, and more than or equal to 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% constant residue between such derivant.In certain embodiments, support can have more than or equal to 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% have the amino acid residue that changes between the protein derivatives of different qualities, and be less than or equal 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% constant residue between such derivant.In certain embodiments, these constant residues give all variant structure territories identical whole three dimensional fold, no matter and its characteristic how.In certain embodiments, can such as among other aspects of this disclosure and the embodiment discussion modify the HSA polypeptide-scaffold.In certain embodiments, the HSA polypeptide-scaffold can be curative.In certain embodiments, the HSA polypeptide-scaffold can be exciting to target.In certain embodiments, the HSA polypeptide-scaffold can be antagonism to target.
6.5 chimeric polyeptides
The polypeptide that comprises HSA part of this disclosure comprises that the HSA variant polypeptide can be combined with any heterologous protein.In certain embodiments, heterologous protein is a kind of therapeutic agent.In certain embodiments, therapeutic agent is antibody or peptide.In certain embodiments, the heterologous protein of chimeric polyeptides partly comprises antibody or its Fab.In certain embodiments, chimeric polyeptides further comprises the constant region of IgG immunoglobulin.In certain embodiments, heterologous protein comprises the non-antibody therapeutic protein.In certain embodiments, the heterologous protein of chimeric polyeptides partly comprises somatomedin or cytokine.In certain embodiments, chimeric polyeptides further comprises epi-position.For example, for detection of and/or the epi-position of purification (for example, His label, FLAG label, etc.).
In certain embodiments, HSA is partly chemically bound to heterologous protein.In certain embodiments, HSA part recombination nodule is bonded to heterologous protein.In certain embodiments, use coding HSA part and the recombinant vector of heterologous protein to produce chimeric polyeptides.
In certain embodiments, in prokaryotic cell or eukaryotic cell, produce the HSA variant.In certain embodiments, in prokaryotic cell or eukaryotic cell, produce chimeric polyeptides.In certain embodiments, eukaryotic cell is selected from yeast cells, avian cell, insect cell or mammalian cell.
Can prepare chimeric polyeptides of the present invention by variety of way.In certain embodiments, the C end of HSA part can be connected to the N end of heterologous protein (for example, antibody or therapeutic peptide).Alternately, the C end of heterologous protein (for example, antibody or therapeutic peptide) can be connected to the N end of HSA part.In certain embodiments, HSA partly is bonded to the internal amino acid of heterologous protein.In certain embodiments, possible conformation comprises that when needed being connected truncate with sequence of light chain with the heavy chain of antibody partly keeps the functional completeness of the HSA part that connects and/or the heterologous protein that connects.In some other embodiment, HSA partly comprises HSA Domain III or its neonatal Fc receptor (FcRn) binding fragment, and at least a portion among HSA domain I or HSA domain II or HSA domain I and the II.In addition, heterologous protein can be connected to inside (non-end) residue of the exposure of HSA part or its variant.In a further embodiment, can use any combination of HSA and heterologous protein conformation, thereby so that the ratio of HSA and heterologous protein greater than 1:1(for example, 2 HSA molecules: 1 heterologous protein).
Can make HSA part and each other directly combination of heterologous protein.Alternately, can they be connected to each other via joint sequence, described joint sequence separates certain distance with the HSA part with heterologous protein, and this suitably is folded into secondary structure and tertiary structure apart from each domain of sufficient to guarantee.In certain embodiments, joint is the cleavable joint.Preferred joint (1) should adopt flexible and extendable conformation, (2) should not show the tendency that produces orderly secondary structure, described secondary structure can interact with the functional structure territory of HSA polypeptide or heterologous protein, and (3) should have minimum hydrophobicity or charging property, this can promote the interaction with the functional protein domain.
In certain embodiments, this joint length is at least 80 dusts
Figure BDA0000365918090000471
Or at least 100
Figure BDA0000365918090000472
Or at least
Figure BDA0000365918090000473
Or at least
Figure BDA0000365918090000474
Or at least
Figure BDA0000365918090000475
Or at least Or at least In certain embodiments, this joint length is about
Figure BDA0000365918090000478
To about
Figure BDA0000365918090000479
Between or approximately
Figure BDA00003659180900004710
To about
Figure BDA00003659180900004711
Between or approximately
Figure BDA00003659180900004712
To about
Figure BDA00003659180900004713
Between.
In certain embodiments, this joint is peptide linker.In certain embodiments, this joint is that peptide linker and this peptide linker have one or more following characteristics: a) it allows heterologous protein sequence and HSA albumen relative to each other to rotate; B) it has resistance to protease digestion; And c) it does not partly interact with heterologous protein sequence or HSA.Typical surface aminoacid in flexible protein zone comprises Gly, Asn and Ser.The standard of above-mentioned joint sequence is satisfied in the arrangement of aminoacid sequence that expectation contains Gly, Asn and Ser.Other approach neutral aminoacid, and for example Thr and Ala also can be used for joint sequence.In certain embodiments, each aminoacid in the peptide linker is selected from lower group, and this group is comprised of Gly, Ser, Asn, Thr and Ala.In certain embodiments, this peptide linker comprises the Gly-Ser component.In certain embodiments, peptide linker comprises one or more Gly-Gly-Gly-Gly-Ser repetitions.In certain embodiments, peptide linker comprises 1,2,3,4,5,6 or 7 Gly-Gly-Gly-Gly-Ser repetition.In certain embodiments, can use length to be about 20 amino acid whose joint sequences, in order to be provided suitably separating of functional protein domain, although also can use long or short joint sequence.Separately the length of the joint sequence of HSA polypeptide and heterologous protein can be 5 to 500 amino acid whose length, or more preferably 5 to 100 amino acid whose length.In certain embodiments, the length of joint sequence is about 5-60 or about 5-30 aminoacid.In certain embodiments, joint sequence is about 5 to about 20 aminoacid, and advantageously is about 10 to about 30 aminoacid.In other embodiments, joint HSA part can be the constant domain (for example, the Fc zone of antibody is all or part of) of antibody with the joint of heterologous protein.In certain embodiments, joint is the cleavable joint.
In certain embodiments, can use cross-linking agent and the scheme known to produce chimeric polyeptides of the present invention.For example, have a large amount of chemical cross-linking agents well known by persons skilled in the art, and they are for making HSA polypeptide and heterologous protein (for example, antibody) crosslinked.For example, cross-linking agent is the isodigeranyl functional cross-link agent, can be used for connecting molecule in the mode of substep.The isodigeranyl functional cross-link agent provides the ability of the more special coupling method that is designed for conjugated protein, thereby reduces the appearance of unwanted side reaction, for example homologous protein polymer.A large amount of isodigeranyl functional cross-link agent known in the art comprises 4-(N-maleimide ylmethyl) cyclohexane extraction-1-carboxylic acid succinimide ester (SMCC); between maleimide benzoyl-N-hydroxysuccinimide eater (MBS); N-succinimido (4-iodo acetyl group)-Aminobenzoate (STAB); 4-(to maleimide phenyl) butanoic acid succinimide ester (SMPB); 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC); 4-succinimido oxygen carbonyl-a-methyl-a (2-pyridine radicals disulfide group) toluene (SMPT); N-succinimido 3-(2-pyridine radicals disulfide group) propionic ester (SPDP); succinimido 6-[3-(2-pyridine radicals disulfide group)-propanoic acid] alkyl caproate (LC-SPDP).Those cross-linking agent with N-hydroxy-succinamide part can be used as N-hydroxy thiosuccinimide analog and obtain, and it has higher water solublity usually.In addition, but those cross-linking agent that have disulfide bond in connection chain can synthesize alkyl derivative, thereby reduce the amount of cracking in the fittings body.Except the isodigeranyl functional cross-link agent, also there are many other cross-linking agent, comprise with bi-functional cross-linking agent and photosensitive crosslinker.Suberic acid two butanimides (DSS), dimaleimide base hexane (BMH) and heptan two imidic acid dimethyl ester dihydrochloride (DMP) be the example of useful same bi-functional cross-linking agent, and two-[B-(4-azidosalicylamides base) ethyl] disulphide (BASED) and N-succinimido-6 (4'-azido-2'-Nitrobenzol amido) alkyl caproate (SANPAH) are the examples that can be used for the photosensitive crosslinker among the present invention.The up-to-date summary of albumen coupling technology is referring to the people such as Means (1990) Bioconjugate Chemistry.(" bioconjugate chemistry ") 1:2-12, be combined in by reference this.
Above included a kind of useful especially isodigeranyl functional cross-link agent type contains the primary amine reaction group, N-hydroxy-succinamide (NHS), or its water-soluble analogues N-hydroxy thiosuccinimide (sulfo-NHS).Primary amine under alkaline pH (lysine ε group) is unprotonated, and it reacts by the nucleophillic attack on NHS or sulfo-NHS ester.This reaction causes the formation of amido link and as the release of by-product NHS or sulfo-NHS.The another kind of reactive group that is used as the part of isodigeranyl functional cross-link agent is sulfhydryl reactive group.Common sulfhydryl reactive group comprises maleimide, halogen and pyridine disulphide.Particularly, to neutral (pH6.5-7.5) condition, maleimide and free sulfhydryl groups (cysteine residues) in minutes react in subacidity.Halogen (iodo acetyl group functional group) under physiological pH with--SH radical reaction.These reactive groups all cause forming stable thioether bond.The third component of isodigeranyl functional cross-link agent is spacerarm or bridge.Bridge is the structure that connects two reactive terminal groups.The most obvious attribute of bridge is the impact on steric hindrance.In some cases, long bridge can easier leap connect two distances that the complex biological molecule is essential.
The method of using heterobifunctional agent to prepare protein conjugate is two step processes, comprises amine reaction and sulfydryl reaction.For the reaction of first step amine, selected protein should contain primary amine.This primary amine can be lysine ε amine or hold uncle's α amine (primary alpha amine) of discovery at the N of most protein.Described protein should not contain free sulfhydryl groups.Two kinds of albumen of therein combination all contain in the situation of free sulfhydryl groups, a kind of protein can be modified, so that can use, for example, NEM is blocked all sulfydryls (referring to the people such as Partis (1983) J.Pro.Chem.(" protein chemistry magazine ") 2:263, is combined in by reference this).Can use ellman's reagent (Ellman's Reagent) to calculate sulfydryl quantity in the specific protein (referring to for example, the people such as Elman (1958) Arch.Biochem.Biophys.(" biochemistry and biophysics's collected papers ") people (1979) Anal.Biochem. " analytical biochemistry " such as 74:443 and Riddles) 94:75 is combined in this by reference).
In certain embodiments, can produce chimeric polyeptides of the present invention: Bodansky below with reference to the standard protein chemical technology of describing in the document by using, M.(Bo Dansiji) Principles of Peptide Synthesis(" peptide composition principle "), Springer Verlag publishing company (Springer Verlag), Berlin (1993) and Grant G.A.(Grant) (editor), Synthetic Peptides:A User's Guide(" synthetic peptide: users' guidebook "), W.H.Freeman and Company(freeman company), New York (1992).In addition, automatic peptide synthesizer is commercially available (for example, Advanced ChemTech Model396; Milligen/Biosearch9600).At any aforementioned cross-linking method for HSA being chemically bound to heterologous protein, can use cleavable domain or cleavable joint.Cracking allows heterologous protein is separated with the HSA polypeptide.For example, behind the chimeric polyeptides penetration cell, the cracking of cleavable joint will allow HSA to separate from heterologous protein.
In certain embodiments, chimeric polyeptides of the present invention can be produced as the fusion rotein that contains HSA part and heterologous protein (for example, antibody or therapeutic peptide), and it is expressed as the polypeptide chain of an adjacency.This type of chimeric polyeptides is referred to here as the polypeptide with the recombination form combination.In preparation during this type of fusion rotein, gene fusion construct, it comprises coding HSA part and heterologous protein and the optional described HSA of leap partly and the nucleic acid of the peptide linker sequence of heterologous protein.Use recombinant DNA technology generation translation product is that the fusion gene of desirable fusion rotein is well known in the art.The coded sequence of gene and control region thereof both can be redesigned to change this protein product functional characteristic, preparation protein amount or produce therein the cell type of this protein.Can greatly change the coded sequence of gene--for example, by the described gene fusion of part to heterogeneic coded sequence being produced the novel heterozygous genes of encoding fusion protein.At PCT application PCT/US87/02968, PCT/US89/03587 and PCT/US90/07335 and Traunecker(Te Laoneike) etc. people (1989) Nature(" nature ") example for generation of the method for fusion rotein has been described among the 339:68, by reference they are combined in this.In essence, carry out joint to the various dna fragmentations of the different peptide sequences of encoding according to routine techniques, adopt the flush end or staggered the end so that suitable end to be provided that are used for connection, digestion with restriction enzyme, take the circumstances into consideration to add sticky end, alkaline phosphatase treatment is to avoid undesirable joint to be connected with enzyme.Alternately, can pass through routine techniques, comprise that automatic dna synthesizer synthesizes fusion gene.In other method, can use anchor primer that genetic fragment is carried out pcr amplification, it produces the complementation outstanding (complementary overhang) between two consecutive gene fragments, described consecutive gene fragment can anneal subsequently to produce chimeric gene sequence (referring to, for example, Ausubel(Ao Subaier) etc. Current Protocols in Molecular Biology(" modern molecular biology experimental technique "), the people edits John Wiley﹠amp; Sons(John Willie father and son publishing company): 1992).As known in the art, can use various expression systems to produce the chimeric polyeptides (vide infra equally) of being encoded by fusion gene with recombination form.
The chimeric polyeptides of restructuring combination comprises that the N that wherein HSA partly is bonded to heterologous protein holds or the embodiment of C end.
In certain embodiments, as described in the U.S. Patent Publication No. 2003/0166877, be tested and appraised the candidate's t cell epitope in the join domain of crossing over chimeric polyeptides and change the immunogenicity that aminoacid in the bonding pad reduces chimeric polyeptides.
The term chimeric protein is used to refer to the protein that comprises HSA part (for example HSA variant polypeptide) and heterologous protein, and no matter whether these parts are connected to each other (for example, chemical bond or restructuring combination).In itself, term chimeric protein, fusion rotein and use interchangeably in conjunction with albumen.
Exemplary heterologous protein classification includes but not limited to enzyme, somatomedin and cytokine.In certain embodiments, heterologous protein is antibody.
Heterologous protein for the chimeric polyeptides that comprises the HSA part can be therapeutic protein, or its fragment, for example somatomedin, enzyme, bone morphogenetic protein and soluble receptor fragment.Exemplary heterologous polypeptide comprises somatomedin, for example hepatocyte growth factor (HGF), nerve growth factor (NGF), epidermal growth factor (EGF; The member of the EFG family of somatomedin), fibroblast growth factor (FGF; The member of the FGF family of somatomedin), transforming growth factor (for example TGF-α, TGF-β, TGF-β 2, TGF-β 3), VEGF (VEGF; For example, VEGF-2), interferon (for example, INF-α, INF-β), interleukin (for example, IL-1, IL-2), cytokine and insulin.Other exemplary heterologous proteins comprise enzyme.Other exemplary heterologous polypeptides comprise bone morphogenetic protein (BMP; The member of the BMP family of protein), erythropoietin (EPO), myogenesis inhibin (myostatin) and tumor necrosis factor (for example, TNF-α).Other exemplary heterologous polypeptides comprise the extracellular domain of transmembrane receptor, comprise cell receptor and any variant thereof any naturally occurring extracellular domain of (comprising mutant, fragment and peptide mimics form).
Heterologous protein for the chimeric polyeptides that comprises the HSA part can be therapeutic protein or its fragment, comprises antibody or its antigen-binding portion thereof.Exemplary antibody and antibody fragment include but not limited to,
Figure BDA0000365918090000511
Figure BDA0000365918090000512
Figure BDA0000365918090000513
Figure BDA0000365918090000514
Figure BDA0000365918090000516
Figure BDA0000365918090000517
Figure BDA0000365918090000518
Figure BDA0000365918090000519
Figure BDA00003659180900005110
Figure BDA00003659180900005111
Figure BDA00003659180900005112
Figure BDA00003659180900005113
Figure BDA00003659180900005114
Figure BDA00003659180900005115
Figure BDA00003659180900005116
Etc..
6.6HSA associated nucleic acid and expression
In some aspects, this disclosure provides the nucleic acid construct that comprises nucleotide sequence, any polypeptide of described nucleotide sequence coded disclosure (for example, HSA variant and the chimeric polyeptides with HSA part).In addition, the present invention utilizes such nucleic acid to produce chimeric polyeptides or HSA variant polypeptide (for example, the HSA part comprises its bioactive fragment, variant and fusions).In some specific embodiment, nucleic acid may further include the DNA of coding heterologous protein (for example, antibody or therapeutic peptide), for the preparation of restructuring chimeric protein of the present invention.All these nucleic acid are referred to as HSA nucleic acid.
In some aspects, this disclosure provides nucleic acid construct, it comprises the nucleotide sequence of (i) encoding human serum albumin (HSA) part, described HSA partly comprises HSA Domain III or its FcRn binding fragment, described HSA Domain III comprises one to 18 aminoacid replacement, it may be operably coupled to the nucleotide sequence of (ii) coding heterologous protein, wherein said nucleic acid construct coding keeps the functional activity of heterologous protein and the chimeric polyeptides that can be combined with FcRn, and wherein said chimeric polyeptides does not comprise with respect to HSA part wherein that the contrast chimeric polyeptides of described aminoacid replacement has the half-life of increase and/or to the affinity of FcRn.
In certain embodiments, be combined with FcRn with the affinity higher than described contrast chimeric polyeptides by the chimeric polyeptides of nucleic acid construct coding.In certain embodiments, the serum half-life that is had increase by the chimeric polyeptides of nucleic acid construct coding with respect to described contrast chimeric polyeptides.In certain embodiments, the chimeric polyeptides by the nucleic acid construct coding has above-mentioned two specific characters.
In certain embodiments, (i) comprise encoding human serum albumin (HSA) nucleotide sequence partly, described HSA partly comprises HSA Domain III or its FcRn binding fragment, and described HSA Domain III comprises one to 18 (1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17 or 18) aminoacid replacement.
In certain embodiments, be in a plurality of species, to be conservative residue by at least one the described aminoacid replacement in the HSA Domain III of nucleic acid construct coding.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III all is to be conservative residue in a plurality of species.In certain embodiments, the described aminoacid of at least one in the HSA Domain III is to be conservative residue in from the serum albumin protein of following species: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, illiteracy account for gerbil jird, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is to be conservative residue in from the serum albumin protein of following species: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is that the surface can reach residue.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is that the surface can reach residue.In certain embodiments, the described aminoacid replacement of at least one in the HSA Domain III is that the surface can reach and is conservative residue in a plurality of species.In certain embodiments, the described aminoacid replacement of all in the HSA Domain III is that the surface can reach residue and be conservative residue in a plurality of species.
In certain embodiments, (i) comprise coding and have the nucleotide sequence of at least 90% conforming HSA Domain III with SEQ ID NO:l.In certain embodiments, (i) comprise coding and have the nucleotide sequence of at least 95% conforming HSA Domain III with SEQ ID NO:l.In certain embodiments, (i) comprise coding and have the nucleotide sequence of at least 98% conforming HSA Domain III with SEQ ID NO:l.
In certain embodiments, at least one described aminoacid replacement is in the ring 2 of HSA Domain III.In certain embodiments, at least one described aminoacid replacement is in the ring 3 of HSA Domain III.In certain embodiments, at least one described aminoacid replacement is in the ring 6 of HSA Domain III.In certain embodiments, at least one described aminoacid replacement is in the spiral 7 of HSA Domain III.In certain embodiments, at least one described aminoacid replacement is in the ring 7 of HSA Domain III.In certain embodiments, at least one described aminoacid replacement is in the spiral 8 of HSA Domain III.In certain embodiments, at least one described aminoacid replacement is in the ring 8 of HSA Domain III.In certain embodiments, at least one described aminoacid replacement is in the ring 9 of HSA Domain III.
In certain embodiments, (ii) comprise the nucleotide sequence of the heterologous protein of encoding, described heterologous protein comprises antibody or its Fab.In certain embodiments, (ii) comprise the nucleotide sequence of the heterologous protein of encoding, described heterologous protein comprises human cytokines.
In certain embodiments, the nucleotide sequence constant region of IgG immunoglobulin of further encoding.
In certain embodiments, (ii) comprise the nucleotide sequence of the heterologous protein of encoding, described heterologous protein comprises somatomedin or cytokine.In certain embodiments, nucleic acid construct further comprises the nucleotide sequence of the joint of encoding.
Nucleic acid can be strand or double-stranded DNA or RNA molecule.In certain embodiments, this disclosure relates to the nucleotide sequence that coding and the same area of HSA sequence (for example, SEQ ID NO:1 and 2) have the separation or reorganization of at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% conforming HSA part.In a further embodiment, the HSA nucleotide sequence can be that separate, restructuring and/or merge with heterologous nucleotide sequence or in the DNA library.
In certain embodiments, HSA nucleic acid also comprises such nucleotide sequence, its under the rigorous condition of height with above-mentioned natural HSA nucleotide sequence or its complementary sequence hybridization.Those of ordinary skill in the art will readily appreciate that, can change the suitable rigorous condition that promotes DNA hybridization.For example, can under 6.0x sodium chloride/sodium citrate (SSC), hybridize under about 45 ℃, then under 50 ℃, wash with 2.0x SSC.For example, can be from be chosen in the salinity this washing step at the low preciseness of the about 2.0x SSC under 50 ℃ to the high preciseness of the about 0.2x SSC under 50 ℃.In addition, can be from the low rigorous condition under room temperature (about 22 ℃) to the temperature that is increased in about 65 ℃ high rigorous condition this washing step.Temperature and salt both can change, or temperature or salinity can keep constant, and another variable can change.In one embodiment, the invention provides such nucleic acid, it is at room temperature hybridized under the low rigorous condition of 6 * SSC, then at room temperature washs with 2 * SSC.
Because the degeneracy of genetic code, the isolating nucleic acids different from natural HSA nucleic acid also within the scope of the invention.For example, many aminoacid are named by more than one triplet.Specify the codon of same amino acid or synonym (for example, CAU and CAC are the synonym of histidine) can cause " silence " sudden change, it does not affect the aminoacid sequence of protein.Yet, be contemplated that the dna sequence polymorphism of the aminoacid sequence variation that really causes theme protein will be present in the mammalian cell.Those skilled in the art should be understood that, because natural allele changes, may reside in the individuality of given species in these variations of one or more nucleotide (up to the nucleotide of about 3%-5%) of the nucleic acid of encode specific protein matter.Any amino acid polymorphism that changes with all such nucleotide and produce all within the scope of the present invention.
In certain embodiments, restructuring HSA nucleic acid can be may be operably coupled to one or more regulatory nucleotide sequences in the expression construct.Regulatory nucleotide sequence is suitable for the host cell of expressing usually.Be used for multiple main host born of the same parents permitted eurypalynous suitable expression vector and the regulating and controlling sequence that is fit to is known in the art.Typically, described one or more regulatory nucleotide sequence can include but not limited to promoter sequence, targeting sequencing or signal sequence, ribosome binding site, transcriptional initiation sequence and terminator sequence, translation initiation and terminator sequence and enhancer or activation sequence.The present invention's expectation composing type or inducible promoter as known in the art.Promoter can be naturally occurring promoter or the hybrid promoter that makes up an above promoter component.Expression construct may reside on the episome (for example plasmid) in the cell, or expression construct can be inserted in the chromosome.In a preferred embodiment, expression vector contains selectable marker gene with the selection of the host cell of permission conversion.Selectable marker gene in this area be know and will change along with the host cell that uses.In some aspects, the present invention relates to comprise the expression vector of nucleotide sequence, described nucleotide sequence coded HSA polypeptide and may be operably coupled at least one regulating and controlling sequence.Regulating and controlling sequence is art-recognized and is selected as instructing the expression of the polypeptide of coding.Therefore, the term regulating and controlling sequence comprises promoter, enhancer and other expression control elements.Exemplary regulating and controlling sequence is described in the Goeddel(Godel); Gene Expression Technology:Methods in Enzymology(" gene expression technique: the method in the zymetology "), the academic press is in San Diego, CA city (1990).Should be understood that the design of expression vector can be depended on such factor, such as the selection of the host cell that will transform and/or wish the type of the protein of expressing.And, also should consider carrier copy number, control copy number ability and by the expression of any other protein (for example antibiotic marker) of described vector encoded.
The invention still further relates to the host cell with the recombination transfection of coding HSA polypeptide of the present invention or chimeric polyeptides.Host cell can be any protokaryon or eukaryotic cell.For example, can in bacterial cell, express HSA polypeptide or chimeric polyeptides, for example escherichia coli, insect cell (for example, using baculovirus expression system), yeast or mammalian cell.Other host cells that are fit to are well known by persons skilled in the art.
The invention further relates to the method that produces HSA polypeptide of the present invention, heterologous protein and/or chimeric polyeptides.For example, can cultivate under suitable condition the expression that allows to occur polypeptide with the host cell of the expression vector transfection of coding HSA polypeptide or chimeric polyeptides.Can be from the mixture of the cell that contains polypeptide and culture medium secretion and isolated polypeptide.Alternately, polypeptide can be retained in Cytoplasm or cell membrane partly neutralize in the cell of results, dissolving and the protein that separates in.Cell culture comprises host cell, culture medium and other by-products.The culture medium that is used for cell culture that is fit to is well known in the art.Use oneself technology that is used for protein purification of knowing of this area, comprise ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis and to polypeptide (for example use, the HSA polypeptide) immunoaffinity purification that the special antibody of defined epitope carries out can be isolated polypeptide from cell culture medium, host cell or among both.In a preferred embodiment, polypeptide is the fusion rotein that contains the domain that promotes its purification.
Can produce restructuring HSA nucleic acid by clone gene or its part being connected to carrier, described carrier is adapted at expressing among prokaryotic cell, eukaryotic cell (yeast, birds, insecticide or mammal) or both.Expression vector for generation of recombinant polypeptide comprises plasmid and other carriers.For example, the carrier that is fit to comprises the plasmid with Types Below: be used at prokaryotic cell, for example the pBR322-of the expression in escherichia coli plasmid of deriving, plasmid that pEMBL-derives, plasmid that pEX-derives, plasmid that pBTac-derives and the pUC-plasmid of deriving.Preferred mammalian expression vector contains and promotes protokaryon sequence that carrier is bred and in one or more eukaryotic transcription units of eukaryotic expression in antibacterial.The carrier that pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derive is the example that is suitable for the mammalian expression vector of transfecting eukaryotic cells.The sequence modification of in these carriers some from bacterial plasmid (for example pBR322) selected to promote copying with Drug resistance in prokaryotic cell and eukaryotic cell.Alternately, can use the derivant of virus to be used at the temporary transient marking protein of eukaryotic cell, these viruses as bovine papilloma virus (BPV-1) or Epstein-Barr viral (pHEBo, pREP derive and p205).The several different methods of using in the conversion of the preparation of these plasmids and host living beings is well known in the art.For other expression system and general restructuring programs that are fit to that are used for protokaryon and eukaryotic cell, referring to Molecular Cloning A Laboratory Manual(" molecular cloning experiment guide "), second edition, Sambrook(Pehanorm Brooker) editor, Fritsch and Maniatis(not Ritchie and the Germania base of a fruit this) (publishing house of Cold Spring Harbor Laboratory Press(cold spring harbor laboratory), 1989) the 16th and 17 chapters.In some cases, may be desirable by using baculovirus expression system to express recombinant polypeptide.The example of such baculovirus expression system comprises the carrier (the pBlueBac III that for example contains β-gal) that carrier (for example pVL1392, pVL1393 and pVL941) that PVL derives, carrier (for example pAcUWl) that pAcUW derives and pBlueBac derive.
The technology of preparation fusion gene is known.In essence, carry out joint to the various dna fragmentations of the different peptide sequences of encoding according to routine techniques, adopt the flush end or staggered the end so that suitable end to be provided that are used for connection, digestion with restriction enzyme, take the circumstances into consideration to add sticky end, alkaline phosphatase treatment is to avoid undesirable joint to be connected with enzyme.In other embodiments, can pass through routine techniques, comprise that automatic dna synthesizer synthesizes fusion gene.Alternately, can use anchor primer that genetic fragment is carried out pcr amplification, it produces the complementation outstanding (complementary overhang) between two consecutive gene fragments, described consecutive gene fragment can anneal subsequently to produce chimeric gene sequence (referring to, for example, Ausubel(Ao Subaier) etc. Current Protocols in Molecular Biology(" modern molecular biology experimental technique "), the people edits John Wiley﹠amp; Sons(John Willie father and son publishing company): 1992).
6.7 Therapeutic Method
In certain embodiments, this disclosure provides the heterologous protein treatment of the chimeric fusions that uses this disclosure can sanatory method.In certain embodiments, this disclosure provides to be increased the serum half-life of the protein among the experimenter and/or increases it to the method for the binding affinity of FcRn, comprise that with described protein bound to the HSA part, described HSA partly comprises Domain III or its neonatal Fc receptor (FcRn) binding fragment.In certain embodiments, the HSA Domain III comprises one to 18 aminoacid replacement, thereby increases this relative chimeric polyeptides to the affinity of FcRn and one or both of serum half-life with respect to the contrast polypeptide that generates.These methods relate to the aforesaid chimeric or HSA variant polypeptide for the treatment of effective dose to the individuality that it is had needs.In certain embodiments, the method comprises and gives chimeric polyeptides, and it comprises (a) HSA part or its bioactive fragment and (b) heterologous protein.These methods are especially for the treatment of animal (and more specifically human) or preventative-therapeutic.
Should be noted in the discussion above that treatable specified disease and disease depend on the heterologous protein part of chimeric protein.In addition, this disclosure expectation be that the part that the chimeric polyeptides that comprises the heterologous protein that is applicable to treat specified disease or disease can be used as therapeutic scheme is used with the therapeutic modality that one or more other chemical compounds or other are suitable for treating specified disease or disease.In addition, this disclosure expectation be, consider patient's age, body weight, health status, the seriousness of disease, etc., give chimeric polyeptides in the mode consistent with medically suitable treatment.
For instance, if heterologous protein is Humira, chimeric polyeptides can be used for for example treating rheumatoid arthritis, psoriasis, juvenile arthritis and Crohn disease.If heterologous protein is insulin, chimeric polyeptides can be used for for example insulinize.
Term " treatment (treatment) ", " treatment (treating) " etc. ordinary representation obtains desirable pharmacology and/or physiological effect as used herein.With regard to wholly or in part prevent disease, disease or its symptom, effect can be preventative, and/or just partially or completely treats disease or disease and/or because with regard to the untoward reaction of disease or disease, effect can be curative.Any treatment of mammal, particularly human diseases or disease is contained in " treatment " as used herein, and comprises: (a) prevention is may susceptible disease or disease but not yet be diagnosed as disease among the experimenter who suffers from it or the generation of disease; (b) suppress disease or disease (for example, stoping its development); Or (c) palliate a disease or disease (for example, cause disappearing of disease or disease, thereby the improvement of one or more symptoms is provided).Can easily evaluate the improvement of any disease according to standard method known in the art and technology.The population of subjects for the treatment of through the method for this disease comprises the experimenter who suffers from undesirable disease or disease and is in the experimenter who produces this disease or disease risks.
Term " treatment effective dose " or " effective dose " expression produce the dosage of desirable effect to giving object.Accurate dosage will depend on therapeutic purposes, and can by those skilled in the art with known technology determine (referring to, Lloyd(Selwyn Lloyd for example) (1999) The Art, Science and Technology of Pharmaceutical Compounding(" medicine preparing process, Science and Technology ")).
In certain embodiments, together (simultaneously) or give one or more chimeric or HSA variant polypeptides of the present invention in the different time (in order).In addition, can use chimeric or HSA variant polypeptide of the present invention with the combination of one or more other chemical compounds that are used for the treatment of same disease or symptom or therapy.For example, can treat chemical compound and one or more chimeric or HSA variant polypeptide co-administereds together with one or more.Therapeutic alliance can comprise simultaneously or alternately administration.In addition, combination can comprise acute or chronic administration.Randomly, chimeric or HSA variant polypeptide of the present invention and other chemical compound are with addition or collaborative mode effect and be used for the treatment of disease or symptom.The other chemical compound that is used for therapeutic alliance includes but not limited to micromolecule, polypeptide, antibody, antisense oligonucleotide and siRNA molecule.In addition, therapeutic alliance comprises that also method disclosed here is together with other non-drug therapies.According to the character of therapeutic alliance, can be when giving other treatment and/or afterwards, continue to give chimeric or HSA variant polypeptide of the present invention.Can give chimeric or the HSA variant polypeptide by single dose or multiple dose.In some cases, at least beginning in several days before other treatment of chimeric or HSA variant polypeptide, and in other cases is just in time before giving other treatment or begin simultaneously to give.
6.8 medication
In certain embodiments, comprise that to described experimenter's administration whole body gives HSA variant or chimeric polyeptides.In certain embodiments, comprise by the oral HSA of giving variant or chimeric polyeptides to described experimenter's administration.In certain embodiments, comprise that to described experimenter's administration intravenous gives described HSA variant or chimeric polyeptides.
In some aspects, this disclosure provides the HSA variant that comprises this disclosure or the compositions of chimeric polyeptides and pharmaceutically acceptable carrier.In certain embodiments, said composition is aseptic composite.In certain embodiments, said composition is non-pyrogen.
Various delivery systems are that oneself knows and can be used for giving with the chimeric of this disclosure or HSA variant polypeptide, for example, be encapsulated in liposome, microgranule, the microcapsule, can express the reconstitution cell, receptor mediated endocytosis of chemical compound (referring to (for example) Wu and Wu, 1987, J.Biol.Chem.262:4429-4432).Introducing method can be enteral or parenteral route, includes but not limited to the approach of Intradermal, percutaneous, intramuscular, intraperitoneal, intravenous, subcutaneous, lung, intranasal, ophthalmic, epidural, part or oral administration.In a particular embodiment, parenteral is introduced and to be comprised in intramuscular, subcutaneous, intravenous, the blood vessel and administration in the pericardium.
Can give chimeric or the HSA variant polypeptide by any easily approach, for example by infusion or bolus injection (bolus injection), by via epithelium or mucocutaneous internal layer (for example oral mucosa, rectum and intestinal mucosa, etc.) absorb, and it can be given with the other biological activating agent.Administration can be general or local.Also can be for example adopt pulmonary administration by the preparation that uses inhaler or nebulizer and have a propellant.
What in certain embodiments, may make us wishing be that chimeric or HSA variant polypeptide part of the present invention is administered to the zone (for example, muscle) that needs treatment; This can realize by for example following methods, but be not limited only to these methods: in during surgery local infusion, local application, for example, by injection, by conduit or by implant, described implant is porous, non-porous or gel-like material, comprise film, such as sialastic film, fiber or commercially available Graftskin.
In other embodiments, can in vesicle, especially in liposome, send the chimeric of this disclosure or HSA variant polypeptide (referring to the Langer(Lange), 1990, Science(" science ") 249:1527-1533).In another embodiment again, can in controlled release system, send the chimeric of this disclosure or HSA variant polypeptide.In another embodiment, can use pump (referring to Lange, 1990, with above).In another embodiment, can use the people such as polymeric material (referring to Howard(Howard), 1989, J.Neurosurg.(" neurosurgery magazine ") 71:105).In some specific embodiment, can send by intravenous the chimeric or variant polypeptide of this disclosure.
In certain embodiments, can give chimeric or the HSA variant polypeptide by venoclysis.In certain embodiments, the chimeric or HSA variant polypeptide of infusion within the time period of at least 10 minutes, at least 15 minutes, at least 20 minutes or at least 30 minutes.In other embodiments, the chimeric or HSA variant polypeptide of infusion within the time period of at least 60 minutes, 90 minutes or 120 minutes.No matter infusion the time how, the each infusion of this disclosure expectation is the part of whole therapeutic scheme, wherein gives chimeric or the HSA variant polypeptide according to periodic plan (for example, weekly, per month, etc.).
6.9 appraisal procedure
Based on the chimeric polyeptides that characterizes this disclosure to get off: the serum half-life to FcRn affinity and/or increase that (i) substantially keeps the function of heterologous protein and (ii) have increase with respect to the chimeric polyeptides that is bonded to non-modified HSA part or the contrast suitable with respect to another kind.Based on the HSA variant polypeptide that characterizes this disclosure to get off: the serum half-life to FcRn affinity and/or increase that has increase with respect to natural HSA part or the contrast suitable with respect to another kind.Can be in any bodies that one or more are fit to or in the external test characteristic of assessment chimeric polyeptides or HSA variant polypeptide.
For instance, can be with for example coming external assessment to the affinity (K of FcRn at any or many measure described in the example or other in conjunction with mensuration aAnd/or K d).Equally, can be with for example coming external assessment K at any or many measure described in the example or other in conjunction with mensuration OffAnd/or K On
Antigen and and antibody between the affinity constant of combination and specific measurement be the key factor that determines to use the effect for the treatment of, diagnosis or the research method of anti--HSA antibody." binding affinity " typically refers to the overall strength at single binding site and the noncovalent interaction between its binding partners (for example, antigen, FcRn) of molecule (for example, antibody, HSA part).Except as otherwise noted, as used herein, " binding affinity " refers to be reflected in the interactional intrinsic binding affinity of 1:1 in conjunction with between the right member (for example, antibody and antigen).Molecule X can be expressed as equilibrium dissociation constant (K usually to the affinity of its gametophyte Y dOr K D), be calculated as K Off/ K OnRatio.Referring to, Chen for example, Y.(is old) etc. the people, (1999) J.Mol Biol(" molecular biology magazine ") 293:865-881.Common methods that can oneself knows with this area comprises that those (for example BIAcore) said and that exemplify measure affinity.Low-affinity antibody is conjugated antigen and be tending towards dissociating easily lentamente usually, and the common conjugated antigen and be tending towards the combination that keeps more permanent quickly of high-affinity antibody.The several different methods of measuring binding affinity is known in the art, and wherein any method all can be used for purpose of the present invention.
In certain embodiments, chimeric polyeptides or variant HSA polypeptide have with respect to contrast and improve about 1.5,2,2.5,3,4 times or about 5 times to the FcRn affinity.In certain embodiments, chimeric polyeptides has with respect to contrast and improves greater than 5 times or even greater than 10 times affinity.In certain embodiments, chimeric polyeptides or HSA variant polypeptide have with respect to contrast and improve greater than 20,25,40 times or greater than 50 times affinity.In certain embodiments, chimeric polyeptides or HSA variant polypeptide have about 5 to 10 times, about 10 to 20 times, about 25 to 40 times, about 40 to 50 times, about 50 to 75 times or about 75 to 100 times affinity of raising with respect to contrast.When by calculating K a assessment affinity, these raisings of affinity change into Ka increase (for example, as mentioned above, 2 times, 5 times, 10 times, etc.).When by calculating K d assessment affinity, these raisings of affinity change into Kd reduction (for example, as mentioned above, 2 times, 5 times, 10 times, etc.).
In certain embodiments, to the affinity of FcRn at low pH(for example, pH about 5.5) lower increasing.In some other embodiment, the affinity of FcRn is increased under low pH, and constant in neutral pH (for example, pH about 7.2).
In further example, can in human or animal's model, measure serum half-life.The increase of the serum half-life in any animal model (including but not limited to have the transgenic animal of people FcRn) is enough to chimeric or HSA variant polypeptide are characterized by the serum half-life that has increase with respect to contrast.
In one embodiment, chimeric polyeptides or HSA variant polypeptide have in blood and are no less than 10 days, preferably are no less than about 14 days half-life, and most preferably are no less than the half-life of natural sera albuminous protein employed or its homologue 50%.In another embodiment, the half-life of chimeric polyeptides or HSA variant polypeptide increases about 1.5,2,2.5,3,4 times or about 5 times with respect to the contrast polypeptide.In certain embodiments, the half-life of chimeric polyeptides or HSA variant polypeptide increases greater than 5 times with respect to the contrast polypeptide, or even greater than 10 times.In certain embodiments, the half-life of chimeric polyeptides or HSA variant polypeptide increases greater than 20,25,40 times with respect to the contrast polypeptide, or greater than 50 times.In certain embodiments, the half-life of chimeric polyeptides or HSA variant polypeptide increases about 5 to 10 times, about 10 to 20 times, about 25 to 40 times, about 40 to 50 times, about 50 to 75 times or about 75 to 100 times with respect to the contrast polypeptide.
The mensuration that is fit to whether the assessment chimeric polyeptides keeps the heterologous protein function basically will depend on heterologous protein and natural function thereof.Yet, can (being included in the animal model) be measured in any suitable external or body in evaluation function.Exemplary function includes but not limited to the ability that (i) is combined with special receptor; (ii) induce or suppress ability via the signal transduction of signal specific transduction pathway; (iii) induce or the ability of inhibited apoptosis; (iv) induce or suppress the ability of angiogenesis; (V) ability of stimulation or inhibition cell proliferation; (vi) ability of promotion or inhibition cell differentiation; (vii) ability of promotion cell survival; And the ability that (viii) promotes or suppress another kind of rho factor secretion.
In certain embodiments, the chimeric polyeptides of this disclosure of formation biological activity heterologous protein is more effective than biological activity heterologous protein itself (for example, not merging the part to HSA).For example, the activity of chimeric polyeptides can than the activity of independent biological activity protein sequence high 2 times, 4 times, 5 times, 10 times, 25 times, 50 times, 100 times or even 1000 times, for example, many l, 2 or even 3 orders of magnitude.Therefore, the biological activity peptide sequence suppresses among the bioactive embodiment therein, the IC of chimeric polyeptides 50Can be than the IC of independent biological activity protein 50Low 10 times, low 100 times or even low 1000 times, and biological activity protein sequential induction or promote among the bioactive embodiment EC of chimeric polyeptides therein 50Can be than the EC of independent biologically active peptide 50Low 10 times, low 100 times or even low 1000 times.Among the embodiment of therein biological activity protein sequence and biomolecule (for example, nucleic acid, peptide or carbohydrate) combination, the dissociation constant K of chimeric polyeptides and its biomolecule of being combined dCan be than independent biomolecule and the K of biological activity protein dLow 10 times, low 100 times or even low 1000 times, for example, the combination of two entities surpasses dissociating of they gradually.
6.10 pharmaceutical composition
In certain embodiments, the theme of this disclosure is chimeric or HSA variant polypeptide and the preparation of pharmaceutically acceptable carrier.Can be with one or more chimeric or the HSA variant polypeptide gives separately or give as the component of pharmaceutical preparation (compositions).Chimeric or HSA variant polypeptide can be used for the administration of any easily mode for people or veterinary through preparation.In compositions, can also there be wetting agent, emulsifying agent and lubricant, such as sodium lauryl sulphate and magnesium stearate, and coloring agent, releasing agent, coating materials, sweeting agent, flavoring agent and aromatic, antiseptic and antioxidant.
Theme preparation chimeric or the HSA variant polypeptide comprises those that are fit to oral, nose, part, parenteral, rectum and/or intravaginal administration.Preparation can exist with unit dosage forms easily and can prepare by any method that pharmaceutical field is known.Can will depend on the amount that carrier material makes up to produce the active component of single dosage form the host that treats and specific administering mode and change.Can be generally with the amount that carrier material makes up to produce the active component of single dosage form the amount of the chemical compound that produces curative effect.
In certain embodiments, the method for preparing these preparations or compositions comprises therapeutic agent and carrier and one or more the optional auxiliary combinations with other types.Generally speaking, can use liquid-carrier, or the solid carrier of fine dispersion, or both prepare preparation its, and if necessary, then with the product molding.
The dosage form of oral administration can be capsule, cachet, pill, tablet, lozenge (use flavoured base, be generally sucrose and arabic gum or tragacanth), the form of powder, granule or as solution or suspension in water solublity or non-water-soluble liquid, or as oil-in-water or Water-In-Oil liquid emulsion, or as elixir or syrup, or as pastille (use inert base, for example gelatin and glycerol, or sucrose and arabic gum), and/or as collutory etc., each form contains the theme polypeptide therapeutic agent of scheduled volume as active component.Except reactive compound, suspension can contain suspending agent, for example ethoxylation isooctadecanol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline Cellulose, aluminium hydroxide oxide (aluminum metahydroxide), bentonite, agar and tragacanth, and composition thereof.
Be used for oral administration (capsule, tablet, pill, dragee, powder, granule, etc.) solid dosage forms in, one or more polypeptide therapeutic agents of the present invention can with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or following any material mixes: (1) filler or extender, such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binding agent, for example as, carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and/or arabic gum; (3) extender is such as glycerol; (4) disintegrating agent is such as agar-agar, calcium carbonate, Rhizoma Solani tuber osi or tapioca, alginic acid, some silicate and sodium carbonate; (5) solution blocker is such as paraffin; (6) absorb accelerator, such as quaternary ammonium compound; (7) wetting agent, for example as, spermol and glyceryl monostearate; (8) absorbent is such as Kaolin and bentonite; (9) lubricant is such as Talcum, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulphate and composition thereof; And (10) coloring agent.In the situation of capsule, tablet and pill, pharmaceutical composition can also comprise buffer agent.Can also use excipient, such as lactose (lactose) or lactose (milk sugar) and high molecular weight polyethylene glycol etc., with the solid composite of similar type as the filler in the gelatine capsule of soft and hard filling.The liquid dosage form that is used for oral administration comprises pharmaceutically acceptable Emulsion, microemulsion, solution, suspension, syrup and elixir.Except active component, liquid dosage form can contain this area inert diluent (for example water or other solvents), solubilizing agent and emulsifying agent commonly used, for example ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, l, 3-butanediol, oils (particularly Oleum Gossypii semen, Oleum Arachidis hypogaeae semen, Semen Maydis oil, germ oil, olive oil, Oleum Ricini and Oleum sesami), glycerol, tetrahydrofurfuryl carbinol, Polyethylene Glycol and sorbitan fatty acid esters, and composition thereof.Except inert diluent, Orally administered composition also can comprise adjuvant, for example wetting agent, emulsifying and suspending agent, sweeting agent, flavoring agent, coloring agent, aromatic and antiseptic.
The pharmaceutical composition that is fit to parenteral can comprise one or more the chimeric or HSA variant polypeptides with one or more and pharmaceutically acceptable following agent combination: sterile isotonic aqueous or non-aqueous solution, dispersion liquid, suspension or emulsion or sterilized powder, described sterilized powder can be just in time restore in aseptic injectable solution or dispersion before using, solute or suspending agent or thickening agent that described sterile injectable solution or dispersion can contain antioxidant, buffer agent, antibacterial, ooze preparation and expection receptor's blood etc.The suitable aqueous of pharmaceutical composition of the present invention and the example of non-aqueous carrier be can be used for and water, ethanol, polyhydric alcohol (such as glycerol, propylene glycol, Polyethylene Glycol etc.) comprised, and the mixture, vegetable oil (such as olive oil) and injectable organic ester, for example ethyl oleate that are fit to.For example, by using coating material (for example lecithin), by remaining on the size of required granule in the dispersion situation, and by using surfactant, can keep suitable flowability.
These compositionss can also contain adjuvant, for example antiseptic, wetting agent, emulsifying agent and dispersant.Can guarantee to prevent the effect of microorganism by comprising various antibacterial agents and antifungal (for example, metagin, chlorobutanol, sorbic acid phenol, etc.).What also make us wishing is to comprise isotonic agent in compositions, for example sugar, sodium chloride, etc.In addition, can cause that the prolongation of injectable drug form absorbs by comprising the reagent (for example aluminum monostearate and gelatin) that postpones to absorb.
In certain embodiments, the compositions of this disclosure comprises pharmaceutical composition, for non-pyrogen.In other words, in certain embodiments, compositions is basically pyrogen-free.In one embodiment, preparation of the present invention is the apyrogeneity preparation, its essentially no endotoxin and/or relevant pyrogenicsubstance.Endotoxin comprises and being limited in the microbial body and and if only if microbial destruction or the toxin that just discharges when dead.Pyrogenicsubstance also comprises the heat-staple material (glycoprotein) of inducing heating from the adventitia of antibacterial and other microorganisms.If these materials are applied to the people, can cause heating, hypotension and shock.Because potential adverse effect is even the endotoxin of low amount also must be removed from the drug solution that intravenous gives.Shi Pin ﹠amp; FAD (" FDA ") has been provided with the upper limit (the The United States Pharmacopeial Convention of 5 endotoxin units (EU) of the every dosage of per kilogram of body weight in single a hour that uses for intravenous pharmacy, Pharmacopeial Forum(American Pharmacopeia committee, Pharmacopeial Forum) 26 (1): 223(2000)).When the amount with per kilogram of body weight hundreds of or several thousand milligrams gave therapeutic protein, as having in the situation of antibody, even the harmful and dangerous endotoxin of trace also must be removed.In some specific embodiment, the endotoxin in the compositions and pyrogen level are less than 10EU/mg or less than 5EU/mg or less than 1EU/mg or less than 0.1EU/mg or less than 0.01EU/mg or less than 0.001EU/mg.
Prepare Injectable depot (Injectable depot) form by the microcapsule substrate that in biodegradable polymer (for example polylactide-PGA), forms one or more polypeptide therapeutic agents.According to the character of the ratio of medicine and polymer and the particular polymers that adopts, can control drug release rate.The example of other biodegradable polymer comprises poe and poly-anhydride.Also by being entrained in the liposome compatible with bodily tissue or the microemulsion, medicine prepares Injectable depot formulations.
In certain embodiments, the chimeric of this disclosure or HSA variant polypeptide are formulated as the pharmaceutical composition that is suitable for administration in the human vein according to conventional program.In case of necessity, compositions can also comprise that solubilizing agent and local anesthetic (for example lignocaine) are to alleviate the pain of injection site.When giving compositions by infusion, can prepare with the infusion bottle that contains sterile pharmaceutical grade water or saline.When giving compositions by injection, can provide the ampoule of sterile water for injection or saline, so that can before administration, these compositions be mixed.
Can by standard clinical techniques determine organize associated conditions or and the treatment of disease in the effective amount of the chimeric or HSA variant polypeptide of this disclosure.In addition, randomly can adopt external test to help to determine the optimal dose scope.The exact dose that adopts in the preparation also will depend on the seriousness of route of administration and disease, and should decide according to medical practitioner's judgement and each experimenter's situation.Yet the suitable dosage range that is used for intravenously administrable is generally the active chimeric or HSA variant polypeptide of the about 20-5000 milligram of per kilogram of body weight.The suitable dosage range that is used for intranasal administration is generally about 0.01pg/kg body weight to the 1mg/kg body weight.Can infer effective dose according to the dose-effect curve that is derived from external or animal model test macro.
6.11 goods and test kit
In certain embodiments, this disclosure also provides pharmaceutical pack or test kit, and it comprises one or more containers of at least a chimeric or HSA variant polypeptide that this disclosure is housed.In certain embodiments, the preparation of this disclosure comprises chimeric or the HSA variant polypeptide, it merges with recombination form or chemically binds to another part, but described part includes but not limited to heterologous protein, heterologous polypeptide, heterologous peptides, macromole, micromolecule, labelled sequence, diagnosis or detection agent, treatment part, drug moiety, radioactive metal ion, second antibody and solid carrier.In certain embodiments, the preparation of this disclosure is formulated as sterile liquid in the single dose bottle.The preparation of this disclosure can be provided at target volume and be in the borosilicate amber vial of 3cc USP Type I of 1.2ml (West Pharmaceutical Serices-Part No.6800-0675).Exemplary container includes but not limited to bottle, bottle, pre-filled syringe, IV bag, blister package (comprising one or more pills).Randomly, relevant with one or more like this containers can be by the bulletin of the form of government organs' regulation of manufacturing, use or the sale of management medicine or biological product, and described bulletin reflection is for the license of government organs to making, using or sell of mankind's diagnosis and/or administration.
In certain embodiments, also provide to comprise test kit chimeric or the HSA variant polypeptide, it is used for various purposes, for example increases the serum half-life of therapeutic agent or increases its FcRn binding affinity.In order to separate and purified reagent, test kit can contain the polypeptide that is bonded to pearl (for example agarose gel pearl).The test kit that contains polypeptide can be provided, be used for for example target in ELISA or Western blotting detection and quantification body.With regard to goods, test kit comprises container and on container or relative label or package insert.Described container holds the compositions of at least a chimeric or HSA variant polypeptide that comprises this disclosure.Can also comprise other container, it contains for example diluent and buffer agent, contrast diagnostic reagent.Label or package insert can provide the description of compositions and the explanation of or diagnostic uses external for expection.
6.12 adenovirus vector and method
Well known in the art and commercially available reagent is to be easy to obtain (referring to for example, from catalog number (Cat.No.) V493-20 and the V494-20 of hero company (Invitrogen)) for the useful dna vector of generation from the recombinant adenovirus granule of host cell.It is a kind of for strengthening the method that recombinant adenovirus produces by the dna vector that the OriP sequence is attached to for generation of adenovirus particles (being also referred to as adenovirus vector herein) that this disclosure provides.The OriP that contains adenovirus vector can further comprise for the sequence of expressing EBNA-1 albumen or alternately express the host cell of EBNA-1 albumen, for generation of the recombinant adenovirus granule.The OriP that contains adenovirus vector is particularly useful in and produces the recombinant adenovirus group, and it comprises the difference of interested DNA sequence (for example, the DNA sequence of coding HSA Domain III variant)/compound library.
Use multiple technologies known in the art, can with the OriP sequence easily through engineering approaches in any known adenovirus vector.For example, if use the Gateway recombination system, the OriP sequence should be between the att recombination site of entry vector or this adenovirus purpose carrier.When sequence is added into adenovirus vector, should carefully avoids being inserted into and to disturb in the site that adenovirus DNA copies or assemble.Alternately, or randomly, the adenovirus vector that contains the OriP sequence can be engineered to and also express EBNA-1 albumen (referring to Figure 10).By directly expressing EBNA-1 albumen from adenovirus vector, host cell does not need to express the EBNA-1 gene.
Adenovirus vector of the present invention comprises OriP sequence and adenoviral gene group sequence.Adenovirus vector of the present invention can further comprise one or more following elements:
(i) one or more recombination sites (for example, attR1 and attR2), it is used for the recombinant clone with another kind of carrier, and such site is useful on impact the clone of the DNA sequence interested of expressing from the recombinant adenovirus of instant carrier (instant vector) generation;
(ii) one or more antibiotic/medicines (for example, chloromycetin) resistant gene and/or toxin expressing gene (for example, the ccdB gene), it is useful on selects and/or anti-the selection;
(iii) cloning site (can be multiple clone site), it is useful on the interested DNA sequence of sub-clone;
(iv) interested DNA, it can comprise one or more interested genes of one or more interested protein of encoding;
(v) be used for expressing at far-ranging mammalian cell the promoter (for example, human cytomegalic inclusion disease virus (CMV) is early promoter at once) of interested gene, described promoter can be composing type maybe can be induction type;
(vi) epitope tag (for example, the His6X epi-position), its for detection of and/or the interested protein of purification.Described epitope tag may reside in 5 ' or 3 ' end of interested recombiant protein;
(vii) polyadenylation (polyA) sequence (for example, the polyA sequence of simian virus 40), it is used for effective tanscription termination and the polyadenylation of mRNA;
(viii) origin of replication, it is used for height copy at the plasmid of escherichia coli and copies and keep;
(ix) antibiotics resistance gene, it is used for the selection escherichia coli;
(x) (for example, Pacl), it is used for linearized vector to restriction enzyme sites, and described restriction endonuclease sites can be in element (viii) and side (ix); And
(xi) DNA sequence, its coding EBNA-1 protein.
DNA in the multiple higher eucaryotic cells of Epstein-Barr virus (EBV) starting point oriP effective support that plasmid DNA is synthesized is synthetic.Representational OriP sequence is provided among Fig. 9 C.This starting point is only used a kind of virus protein EBNA-1, and other all factors are provided by cell.In certain embodiments, provide EBNA-1 albumen by host cell (for example, 293E cell).In other embodiments, adenovirus vector of the present invention further comprises the DNA sequence of element (xi) coding EBNA-1 albumen.Representational EBNA-1 protein and DNA sequence are provided at respectively among Fig. 9 A and Fig. 9 B.
Special expectation be adenoviral gene group sequence (for example, adenovirus hominis 5 type sequences are with the suitable packing of encoding adenovirus and produce essential gene and other elements (for example left side and right end inverted repeat (ITR), packaging signal sequence (encapsidation signal sequence), the people 1999 such as late gene (Hitt(is uncommon special), The Development of Human Gene Therapy(" human gene therapy's development "), the T.Friedmann(Fried) compiles (cold spring port, New York: cold spring port experiment publishing house), pp.61-86; Russell(Russell), 2000, J.Gen.Virol.(" general virology magazine ") 81,2573-2604).The adenoviral gene group that adenoviral gene group sequence can be encoded complete.Alternately, adenoviral gene group sequence can be encoded except with trans one or more protein that provide and/or all proteins the controlling element and other controlling elements, thereby produces replication defect type (replication-incompetent) adenovirus.For example, (Russell(Russell), 2000, J.Gen.Virol.(" general virology magazine " can be got rid of in the E1 zone of coding El albumen (Ela and Elb) from adenovirus vector of the present invention) 81,2573-2604).So protein and/or other elements of disappearance will be provided with trans by the host for generation of adenovirus usually, for example, 293 cell lines contain the genome copy in El zone.In certain embodiments, adenoviral gene group sequence comprises the adenovirus hominis 5 type sequences or consisting essentially of corresponding to wild-type sequence 1-458 and 3513-35935.
Those skilled in the art should be understood that, some element and/or can be in conjunction with other elements will be provided in combination, for example element (iv) can binding member (V) to (vii).Should further be understood that, to provide some element at 5 ' and/or 3 ' of other elements, for example, element (x) can be used for the linearisation of adenovirus vector, when binding member (x) is used for linearisation, it should be provided at 5 ' ITR 5 ' and/or 3 ' ITR 3 '.Equally, when having element (viii) and (ix) time, it is positioned as usually so that they can not be attached in the rescue adenovirus (rescued adenovirus), for example these elements can be positioned at 5 ' ITR 5 ' and 3 ' ITR 3 '.OriP and element (i) to (vii) (if exist) are the adenoviral gene group sequence that comprises 3 ' ITR in a side, opposite side be 5 ' ITR sequence (for example, Figure 10).
Representative adenovirus vector of the present invention is provided among Figure 10, its binding member (i), (ii), (iv), (viii) to (x) and optional (xi).
7 embodiments
1. chimeric polyeptides, it comprises: (a) human serum albumin (HSA) part, described HSA partly comprises HSA Domain III or its neonatal Fc receptor (FcRn) binding fragment, and (b) heterologous protein, wherein said chimeric polyeptides has kept the functional activity of heterologous protein and can be combined with FcRn, and wherein said HSA Domain III comprises one to 18 aminoacid replacement, thereby does not comprise the contrast chimeric polyeptides of described aminoacid replacement and increase the one or both to affinity and the serum half-life of FcRn of this chimeric polyeptides with respect to the part of HSA wherein.
2. chimeric polyeptides as described in Example 1, wherein this chimeric polyeptides is combined with FcRn with the affinity higher than described contrast chimeric polyeptides.
3. such as embodiment 1 or 2 described chimeric polyeptides, wherein this chimeric polyeptides is combined with FcRn with the affinity higher than described contrast chimeric polyeptides, and wherein said affinity is measured at acid pH.
4. chimeric polyeptides as described in Example 3, wherein this acid pH is between 5.0 and 6.0.
5. chimeric polyeptides as described in Example 4, wherein this acid pH is 5.5 ± 0.2.
6. such as each described chimeric polyeptides among the embodiment 1-3, wherein this chimeric polyeptides is combined with FcRn with the affinity higher than described contrast chimeric polyeptides under acid pH, but this chimeric polyeptides is not combined with FcRn with the affinity higher than described contrast chimeric polyeptides under neutral pH.
7. chimeric polyeptides as described in Example 6, wherein this neutral pH is between 6.9 and 7.9.
8. chimeric polyeptides as described in Example 7, wherein this neutral pH is 7.4 ± 0.2.
9. such as each described chimeric polyeptides among the embodiment 1-6, wherein this chimeric polyeptides and FcRn in conjunction with and have dissociation rate or an association rate different with described contrast chimeric polyeptides.
10. chimeric polyeptides as described in Example 9, wherein this chimeric polyeptides is combined with FcRn and is had the association rate of increase and/or the dissociation rate of reduction with respect to described contrast chimeric polyeptides.
11. chimeric polyeptides as described in Example 9, wherein this chimeric polyeptides is combined with FcRn and is had the dissociation rate of increase with respect to described contrast chimeric polyeptides.
12. such as each described chimeric polyeptides among the embodiment 1-11, wherein this HSA Domain III comprises one to ten aminoacid replacement, thereby does not comprise the contrast chimeric polyeptides of described aminoacid replacement and increase the serum half-life of this chimeric polyeptides with respect to the part of HSA wherein.
13. such as each described chimeric polyeptides among the embodiment 1-12, wherein the described aminoacid replacement of at least one in the HSA Domain III is to be conservative residue in a plurality of species.
14. such as embodiment 13 described chimeric polyeptides, wherein the described aminoacid replacement of all in the HSA Domain III is to be conservative residue in a plurality of species.
15. such as each described chimeric polyeptides among the embodiment 1-13, wherein the described aminoacid replacement of at least one in the HSA Domain III is to be conservative residue in from the serum albumin protein of following species: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.
16. such as embodiment 15 described chimeric polyeptides, wherein the described aminoacid replacement of all in the HSA Domain III is to be conservative residue in from the serum albumin protein of following species: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.
17. such as each described chimeric polyeptides among the embodiment 1-15, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from listed those in table 5.
18. such as each described chimeric polyeptides among the embodiment 1-15, wherein the described aminoacid replacement of at least one in the HSA Domain III with respect to total length ripe HSA(SEQ ID NO:2) in any following position that is numbered, position: residue 381, residue 383, residue 391, residue 401, residue 402, residue 407, residue 411, residue 413, residue 414, residue 415, residue 416, residue 424, residue 426, residue 434, residue 442, residue 445, residue 447, residue 450, residue 454, residue 455, residue 456, residue 457, residue 459, residue 463, residue 495, residue 506, residue 508, residue 509, residue 511, residue 512, residue 515, residue 516, residue 517, residue 519, residue 521, residue 523, residue 524, residue 525, residue 526, residue 527, residue 531, residue 535, residue 538, residue 539, residue 541, residue 557, residue 561, residue 566, residue 569.
19. such as each described chimeric polyeptides among the embodiment 1-15, wherein this chimeric polyeptides is included in the aminoacid replacement in the HSA Domain III of a plurality of positions that are numbered with respect to the position among the ripe HSA of total length, these positions are selected from lower group, and this group is comprised of the following: (a) residue 383 and 413; (b) residue 401 and 523; (c) residue 407 and 447; (d) residue 407 and 447 and 539; (e) residue 407 and 509; (f) residue 407 and 526; (g) residue 411 and 535; (h) residue 414 and 456; (i) residue 415 and 569; (j) residue 426 and 526; (k) residue 442 and 450 and 459; (l) residue 463 and 508; (m) residue 508 and 519 and 525; (n) residue 509 and 527; (o) residue 523 and 538; (p) residue 526 and 557; (q) residue 541 and 561; (r) residue 463 and 523; (s) residue 508 and 523; (t) residue 508 and 524; (u) residue 463,508 and 523; (v) residue 463,508 and 524; (w) residue 508,523 and 524; (x) residue 463,508,523 and 524; (y) residue 463 and 524; (z) residue 523 and 524; And (aa) residue 463,523 and 524.
20. such as each described chimeric polyeptides among the embodiment 1-15, wherein this chimeric polyeptides is included in the aminoacid replacement in the HSA Domain III of a plurality of positions that are numbered with respect to the position among the ripe HSA of total length, these positions are selected from lower group, and this group is comprised of the following: (a) residue 463 and 508; (b) residue 463 and 523; (c) residue 508 and 523; (d) residue 508 and 524; (e) residue 463,508 and 523; (f) residue 463,508 and 524; (g) residue 508,523 and 524; (h) residue 463,508,523 and 524; (i) residue 463 and 523; And (j) residue 523 and 524.
21. such as each described chimeric polyeptides among embodiment 1-15 or the 18-20, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, V381Q, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y401D, Y401E, K402A, K402G, K402I, K402L, K402V, L407F, 407H, 407M, L407N, L407Q, 407R, L407W, L407Y, Y411Q, Y411N, K413C, K413S, K413T, K414S, K414T, V415C, V415L, V415S, V415T, Q416H, Q416P, V424A, V424D, V424G, V424I, V424L, V424M, V424N, V424Q, V424W, V426D, V426E, V426F, V426H, V426L, V426N, V426P, V426Q, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, P447S, P447T, E450D, E450E, S454C, S454M, S454T, V455N, V455Q, V456N, V456Q, L457F, L457W, L457Y, Q459K, Q459R, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T506F, T506M, T506N, T506R, T506W, T506Y, T508C, T508E, T508I, T508K, T508R, T508S, F509C, F509D, F509G, F509I, F509L, F509M, F509P, F509V, F509W, F509Y, A511D, A511F, A511I, A511R, A511T, A511V, A511W, A511Y, D512F, D512M, D512Q, D512W, D512Y, T515C, T515D, T515E, T515E, T515G, T515H, T515L, T515N, T515P, T515Q, T515S, T515W, T515Y, L516C, L516F, L516S, L516T, L516W, L516Y, S517C, S517F, S517M, S517T, S517W, S517Y, K519A, K519G, K519I, K519L, K519V, R521F, R521H, R521M, R521Q, R521T, R521W, R521Y, I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W, I523Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526A, Q526C, Q526F, Q526H, Q526L, Q526M, Q526P, Q526S, Q526T, Q526V, Q526Y, T527F, T527W, T527Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535K, H535L, H535N, H535P, H535S, K538F, K538W, K538Y, A539I, A539L, A539V, K541F, K541W, K541Y, K557A, K557G, K557I, K557L, K557N, K557S, K557V, A561F, A561W, A561Y, T566F, T566W, T566Y, A569H, and A569P.
22. such as each described chimeric polyeptides among embodiment 1-15 or the 18-20, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, E383G, N391V, Y401E, K402A, L407N, L407Y, Y411Q, K414S, K413S, V415T, V415C, Q416P, V424I, V424Q, V426E, V426H, G434C, E442K, R445W, P447S, E450D, S454C, V455N, V456N, L457F, Q459R, L463C, L463F, L463H, L463M, L463N, L463Q, E495D, T506Y, T508C, T508E, T508I, T508R, T508S, F509I, F509M, F509W, A511F, D512Y, T515P, T515Q, T515S, L516T, L516W, S517C, S517W, K519I, R521W, I523C, I523D, I523E, I523F, I523Q, I523H, I523K, I523L, I523N, I523P, I523G, I523R, I523Y, K524F, K524I, K524L, K524M, K524T, K524V, K525V, Q526T, Q526M, Q526Y, T527Y, E531I, H535N, H535P, K538Y, A539I, K541F, K557G, A561F, T566W, and A569P.
23. such as each described chimeric polyeptides among embodiment 1-15 or the 18-20, wherein this chimeric polyeptides is included in an aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: V381N, E383G, N391V, Y401E, K402A, L407N, L407Y, Y411Q, K414S, K413S, V415T, V415C, Q416P, V424I, V424Q, V426E, V426H, G434C, E442K, R445W, P447S, E450D, S454C, V455N, V456N, L457F, Q459R, L463C, L463F, L463H, L463M, L463N, L463Q, E495D, T506Y, T508C, T508E, T508I, T508R, T508S, F509I, F509M, F509W, A511F, D512Y, T515P, T515Q, T515S, L516T, L516W, S517C, S517W, K519I, R521W, I523C, I523D, I523E, I523F, I523Q, I523H, I523K, I523L, I523N, I523P, I523G, I523R, I523Y, K524F, K524I, K524L, K524M, K524T, K524V, K525V, Q526T, Q526M, Q526Y, T527Y, E531I, H535N, H535P, K538Y, A539I, K541F, K557G, A561F, T566W, and A569P.
24. such as embodiment 22 described chimeric polyeptides, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: L407N, L407Y, V415T, V424I, V424Q, V426E, V426H, P447S, V455N, V456N, L463N, E495D, T506Y, T508R, F509M, F509W, A511F, D512Y, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T527Y, H535P and K557G.
25. such as embodiment 23 described chimeric polyeptides, wherein said chimeric polyeptides is included in an aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: L407N, L407Y, V415T, V424I, V424Q, V426E, V426H, P447S, V455N, V456N, L463N, E495D, T506Y, T508R, F509M, F509W, A511F, D512Y, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T527Y, H535P and K557G.
26. such as each described chimeric polyeptides among embodiment 1-14 or the 18-20, wherein this chimeric polyeptides is included in the aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: (a) E383G/K413S; (b) Y401E/I523G; (c) L407N/P447S; (d) L407N/P447S/A539I; (e) L407N/F509M; (f) L407Y/Q526T; (g) Y411Q/H535N; (h) K414S/V456N; (i) V415T/A569P; (j) V426H/Q526Y; (k) E442K/E450D/Q459R; (l) L463N/T508R; (m) T508R/K519I/K525V; (n) F509I/T527Y; (o) I523Q/K538Y; (p) Q526M/K557G; (q) K541F/A561F; (r) L463N/K524L; (s) T508R/I523G; (t) T508R/K524L; (u) L463N/T508R/I523G; (v) L463N/T508R/K524L; (w) T508R/I523G/K524L; (x) L463N/T508R/I523G/K524L; (y) L463N/I523G; (z) I523G/K524L; (aa) L463N//I523G/K524L.
27. such as embodiment 26 described chimeric polyeptides, wherein this chimeric polyeptides is included in the aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: (a) L407N/P447S; (b) L407N/P447S/A539I; (c) L407N/F509M; (d) Y411Q/H535N; (e) K414S/V456N; (f) V426H/Q526Y; (g) L463N/T508R; (h) F509I/T527Y; (i) I523Q/K538Y; (j) Q526M/K557G; (k) K541F/A561F; (l) L463N/K524L; (m) T508R/I523G; (n) T508R/K524L; (o) L463N/T508R/I523G; (p) L463N/T508R/K524L; (q) T508R/I523G/K524L; (r) L463N/T508R/I523G/K524L; (s) L463N/I523G; (t) I523G/K524L and (u) L463N//I523G/K524L.
28. such as embodiment 26 described chimeric polyeptides, wherein this chimeric polyeptides is included in the aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: (a) L463N/T508R; (b) L463N/K524L; (c) T508R/I523G; (d) T508R/K524L; (e) L463N/T508R/I523G; (f) L463N/T508R/K524L; (g) T508R/I523G/K524L; (h) L463N/T508R/I523G/K524L.
29. such as embodiment 26 described chimeric polyeptides, wherein this chimeric polyeptides is included in the aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: (a) L463N/K508R; (b) T508R/I523G; (c) T508R/K524L; (d) L463N/T508R/I523G.
30. such as each described chimeric polyeptides among the embodiment 1-15, wherein the described aminoacid replacement of at least one in the HSA Domain III be in from the serum albumin protein of people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse for conservative but in the chicken serum albumin conservative residue.
31. such as embodiment 30 described chimeric polyeptides, wherein the described aminoacid replacement of all in the HSA Domain III be in from the serum albumin protein of people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse for conservative but in the chicken serum albumin conservative residue.
32. such as embodiment 15 described chimeric polyeptides, wherein described at least one aminoacid replacement in the HSA Domain III with respect to total length ripe HSA(SEQ ID NO:2) in any following position that is numbered, position: residue 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.
33. such as embodiment 32 described chimeric polyeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 383, residue 391, residue 434, residue 442, residue 445 and residue 450.
34. such as embodiment 33 described chimeric polyeptides, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, E450D and E450E.
35. such as embodiment 32 described chimeric polyeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 417, residue 442, residue 499 and residue 502.
36. such as embodiment 30 described chimeric polyeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 380, residue 381, residue 384, residue 387, residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 419, residue 421, residue 422, residue 424, residue 428, residue 430, residue 431, residue 433, residue 441, residue 457, residue 458, residue 463, residue 464, residue 466, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 489, residue 491, residue 495, residue 500, residue 508, residue 510, residue 515, residue 516, residue 524, residue 525, residue 526, residue 528, residue 531, residue 535, residue 539, residue 544, residue 547, with residue 576.
37. such as embodiment 36 described chimeric polyeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 381, residue 401, residue 424, residue 457, residue 463, residue 495, residue 508, residue 515, residue 516, residue 524, residue 525, residue 526, residue 531, residue 535 and residue 539.
38. such as embodiment 37 described chimeric polyeptides, wherein this HSA Domain III is included in the aminoacid replacement of a plurality of positions that are numbered with respect to the position among the ripe HSA of total length, these positions are selected from lower group, and this group is comprised of the following: residue 463 and 508; Residue 463 and 524; Residue 508 and 524; Residue 463,508 and 524.
39. such as embodiment 37 or 38 described chimeric polyeptides, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, V381Q, Y401D, Y401E, V424N, V424Q, L457F, L457W, L457Y, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T508C, T508E, T508I, T508K, T508R, T508S, T515C, T515H, T515N, T515P, T515Q, T515S, L516F, L516S, L516T, L516W, L516Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526C, Q526M, Q526S, Q526T, Q526Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535P, A539I, and A539L, A539V.
40. such as embodiment 32 described chimeric polyeptides, wherein all described aminoacid replacement are selected from lower group member, and this group is comprised of the following: residue 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.
41. such as embodiment 36 described chimeric polyeptides, wherein all described aminoacid replacement are selected from lower group member, and this group is comprised of the following: residue 380, residue 381, residue 384, residue 387, residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 419, residue 421, residue 422, residue 424, residue 428, residue 430, residue 431, residue 433, residue 441, residue 457, residue 458, residue 463, residue 464, residue 466, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 489, residue 491, residue 495, residue 500, residue 508, residue 510, residue 515, residue 516, residue 524, residue 525, residue 526, residue 528, residue 531, residue 535, residue 539, residue 544, residue 547, with residue 576.
42. such as embodiment 32 or 36 described chimeric polyeptides, wherein all described aminoacid replacement are selected from lower group member, and this group is comprised of the following: residue 380, residue 381, residue 384, residue 383, residue 387, residue 389, residue 391, residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 410, residue 417, residue 419, residue 421, residue 422, residue 424, residue 425, residue 428, residue 430, residue 431, residue 433, residue 441, residue 442, residue 457, residue 458, residue 463, residue 464, residue 465, residue 466, residue 467, residue 468, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 486, residue 489, residue 491, residue 495, residue 499, residue 500, residue 502, residue 508, residue 510, residue 515, residue 516, residue 520, residue 524, residue 525, residue 526, residue 528, residue 531, residue 532, residue 535, residue 536, residue 539, residue 543, residue 544, residue 547, residue 571, with residue 576.
43. such as embodiment 42 described chimeric polyeptides, wherein all described aminoacid replacement are selected from lower group member, and this group is comprised of the following: residue 381, residue 383, residue 391, residue 401, residue 424, residue 442, residue 463, residue 495, residue 506, residue 508, residue 515, residue 516, residue 524, residue 525, residue 526, residue 531, residue 535 and residue 539.
44. such as embodiment 43 described chimeric polyeptides, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, V381Q, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y401D, Y401E, V424A, V424G, V424I, V424L, V424N, V424Q, E442K, E442R, L463C, L463F, L463G, L463H, L463M, L463N, L463Q, E495D, T506F, T506W, T506Y, T508C, T508E, T508I, T508K, T508R, T508S, T515C, T515H, T515N, T515P, T515Q, T515S, L516S, L516T, L516W, L516Y, K524A, K524F, K524G, K524I, K524L, K524M, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526C, Q526M, Q526S, Q526T, Q526Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535P, A539I, A539L, and A539V.
45. such as each described chimeric polyeptides among the embodiment 1-44, wherein the described aminoacid replacement of at least one in the HSA Domain III is that the surface can reach residue.
46. such as embodiment 45 described chimeric polyeptides, wherein the described aminoacid replacement of all in the HSA Domain III is that the surface can reach residue.
47. such as each described chimeric polyeptides among the embodiment 1-12, wherein the described aminoacid replacement of at least one in the HSA Domain III is that the surface can reach and is conservative residue in a plurality of species.
48. such as embodiment 47 described chimeric polyeptides, wherein the described aminoacid replacement of all in the HSA Domain III is that the surface can reach and is conservative residue in a plurality of species.
49. such as embodiment 47 or 48 described chimeric polyeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.
50. such as embodiment 49 described chimeric polyeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 383, residue 391 and residue 442.
51. such as embodiment 49 described chimeric polyeptides, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: E383A, E383G, E3831, E383L, E383V, N391A, N391G, N391I, N391L, N391V and E442K, E442R.
52. such as embodiment 49 described chimeric polyeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 417, residue 442, residue 499 and residue 502.
53. such as each described chimeric polyeptides among the embodiment 1-52, wherein this HSA Domain III comprises with SEQ ID NO:l and has at least 90% conforming aminoacid sequence.
54. such as embodiment 53 described chimeric polyeptides, wherein this HSA Domain III comprises with SEQ IDNO:l and has at least 95% conforming aminoacid sequence.
55. such as embodiment 53 described chimeric polyeptides, wherein this HSA Domain III comprises with SEQ IDNO:l and has at least 98% conforming aminoacid sequence.
56. such as each described chimeric polyeptides among the embodiment 1-52, wherein this HSA partly comprises with SEQ ID NO:2 and has at least 90% conforming aminoacid sequence.
57. such as embodiment 56 described chimeric polyeptides, wherein this HSA partly comprises with SEQ ID NO:2 and has at least 95% conforming aminoacid sequence.
58. such as embodiment 56 described chimeric polyeptides, wherein this HSA partly comprises with SEQ IDNO:2 and has at least 98% conforming aminoacid sequence.
59. such as each described chimeric polyeptides among the embodiment 1-58, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 2 of HSA Domain III.
60. such as each described chimeric polyeptides among embodiment 1-23,30-32,35,36,40-42,45-49 or the 52-59, wherein the described aminoacid replacement of all in the HSA Domain III is all in the ring 2 of HSA Domain III.
61. such as embodiment 59 or 60 described chimeric polyeptides, be included in one in the HSA Domain III to five amino acid and replace, wherein said one is substituted in the ring 2 of HSA Domain III to five amino acid.
62. such as each described chimeric polyeptides among the embodiment 1-59, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 3 of HSA Domain III.
63. such as each described chimeric polyeptides among embodiment 1-23,30-36, the 40-58 or 62, wherein the described aminoacid replacement of all in the HSA Domain III is all in the ring 3 of HSA Domain III.
64. such as embodiment 62 or 63 described chimeric polyeptides, be included in one in the HSA Domain III to five amino acid and replace, wherein said one is substituted in the ring 3 of HSA Domain III to five amino acid.
65. such as each described chimeric polyeptides in embodiment 1-59 or 62, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 6 of HSA Domain III.
66. such as each described chimeric polyeptides among embodiment 1-23,30-32,35,36-49, the 52-58 or 65, wherein the described aminoacid replacement of all in the HSA Domain III is all in the ring 6 of HSA Domain III.
67. such as embodiment 65 or 66 described chimeric polyeptides, it is included in one to 18 aminoacid replacement in the HSA Domain III, wherein said one to 18 aminoacid replacement is in the ring 6 of HSA Domain III.
68. such as each described chimeric polyeptides among the embodiment 1-59,62 or 65, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the spiral 7 of HSA Domain III.
69. such as each described chimeric polyeptides among embodiment 1-23,30-32,35,36-49, the 52-58 or 68, wherein the described aminoacid replacement of all in the HSA Domain III is all in the spiral 7 of HSA Domain III.
70. such as embodiment 71 or 72 described chimeric polyeptides, it is included in one to three aminoacid replacement in the HSA Domain III, wherein said one to six aminoacid replacement is in the spiral 7 of HSA Domain III.
71. such as each described chimeric polyeptides among the embodiment 1-59,62,65,68, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 7 of HSA Domain III.
72. such as each described chimeric polyeptides among embodiment 1-23,30-32,35,36-49, the 52-58 or 71, wherein the described aminoacid replacement of all in the HSA Domain III is all in the ring 7 of HSA Domain III.
73. such as embodiment 71 or 72 described chimeric polyeptides, it is included in one to three aminoacid replacement in the HSA Domain III, wherein said one to three aminoacid replacement is in the ring 7 of HSA Domain III.
74. such as each described chimeric polyeptides among the embodiment 1-59,62,65,68 or 71, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the spiral 8 of HSA Domain III.
75. such as each described chimeric polyeptides among embodiment 1-23,30-31,41,42,45-48, the 53-58 or 74, wherein the described aminoacid replacement of all in the HSA Domain III is all in the spiral 8 of HSA Domain III.
76. such as embodiment 74 or 75 described chimeric polyeptides, it is included in one in the HSA Domain III to the five amino acid replacement, wherein said one to 18 aminoacid replacement is in the spiral 8 of HSA Domain III.
77. such as each described chimeric polyeptides among the embodiment 1-59,62,65,68,71 or 74, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 8 of HSA Domain III.
78. such as each described chimeric polyeptides among embodiment 1-23,30-31,41,42,45-48, the 53-58 or 77, wherein the described aminoacid replacement of all in the HSA Domain III is in the ring 8 of HSA Domain III.
79. such as embodiment 77 or 78 described chimeric polyeptides, be included in one in the HSA Domain III to five amino acid and replace, wherein said one is substituted in the ring 8 of HSA Domain III to five amino acid.
80. such as each described chimeric polyeptides among the embodiment 1-59,62,65,68,71,74 or 77, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 9 of HSA Domain III.
81. such as each described chimeric polyeptides among embodiment 1-23,30-31,45-48, the 53-58 or 80, wherein the described aminoacid replacement of all in the HSA Domain III is all in the ring 9 of HSA Domain III.
82. such as embodiment 80 or 81 described chimeric polyeptides, it is included in one to four aminoacid replacement in the HSA Domain III, wherein said one to four aminoacid replacement is in the ring 9 of HSA Domain III.
83. such as each described chimeric polyeptides among the embodiment 1-82, wherein at least one described aminoacid replacement comprises an amino acid residue is replaced with alanine.
84. such as each described chimeric polyeptides among the embodiment 1-83, wherein at least one described aminoacid replacement comprises that conservative amino acid replaces.
85. such as each described chimeric polyeptides among the embodiment 1-84, wherein at least one described aminoacid replacement comprises a basic amino acid with the displacement of another basic amino acid.
86. such as each described chimeric polyeptides among the embodiment 1-85, wherein at least one described aminoacid replacement comprises an acidic amino acid with the displacement of another acidic amino acid.
87. such as each described chimeric polyeptides among the embodiment 1-87, wherein at least one described aminoacid replacement comprises a neutral amino acid with the displacement of another neutral amino acid.
88. such as each described chimeric polyeptides among the embodiment 1-87, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: lysine, arginine and histidine.
89. such as each described chimeric polyeptides among the embodiment 1-88, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: aspartic acid and glutamic acid.
90. such as each described chimeric polyeptides among the embodiment 1-89, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: agedoite, glutamine, serine, threonine and tyrosine.
91. such as each described chimeric polyeptides among the embodiment 1-90, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: alanine, valine, isoleucine, leucine, proline, phenylalanine, tryptophan, methionine, cysteine and glycine.
92. such as each described chimeric polyeptides among the embodiment 1-91, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: phenylalanine, tryptophan and tyrosine.
93. such as each described chimeric polyeptides among the embodiment 1-92, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: cysteine, serine and threonine.
94. such as each described chimeric polyeptides among the embodiment 1-93, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: agedoite, glutamine, serine, threonine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid.
95. such as each described chimeric polyeptides among the embodiment 1-94, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: glycine, serine, threonine, alanine, valine, leucine and isoleucine.
96. such as each described chimeric polyeptides among the embodiment 1-95, wherein at least one described aminoacid replacement comprises that non-conservation replaces.
97. such as embodiment 83 described chimeric polyeptides, wherein all described aminoacid replacement comprise an amino acid residue are replaced with alanine.
98. such as embodiment 84 described chimeric polyeptides, wherein all described aminoacid replacement comprise that the conservative amino acid at place, each position replaces independently.
99. such as embodiment 85 described chimeric polyeptides, wherein all described aminoacid replacement comprise independently at place, each position a basic amino acid are replaced with another basic amino acid.
100. such as embodiment 87 described chimeric polyeptides, wherein all described aminoacid replacement comprise independently at place, each position an acidic amino acid are replaced with another acidic amino acid.
101. such as embodiment 87 described chimeric polyeptides, wherein all described aminoacid replacement comprise independently at place, each position a neutral amino acid are replaced with another neutral amino acid.
102. such as embodiment 88 described chimeric polyeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: lysine, arginine and histidine.
103. such as embodiment 89 described chimeric polyeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: aspartic acid and glutamic acid.
104. such as embodiment 90 described chimeric polyeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: agedoite, glutamine, serine, threonine and tyrosine.
105. such as embodiment 91 described chimeric polyeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: alanine, valine, isoleucine, leucine, proline, phenylalanine, tryptophan, methionine, cysteine and glycine.
106. such as embodiment 92 described chimeric polyeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: phenylalanine, tryptophan and tyrosine.
107. such as embodiment 93 described chimeric polyeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: cysteine, serine and threonine.
108. such as embodiment 94 described chimeric polyeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: agedoite, glutamine, serine, threonine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid.
109. such as embodiment 95 described chimeric polyeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: glycine, serine, threonine, alanine, valine, leucine and isoleucine.
110. such as embodiment 96 described chimeric polyeptides, wherein all described aminoacid replacement comprise that the non-conservation at place, each position replaces independently.
111. such as each described chimeric polyeptides among the embodiment 1-110, wherein this heterologous protein comprises antibody or its Fab.
112. such as each described chimeric polyeptides among the embodiment 1-111, wherein this heterologous protein comprises therapeutic protein.
113. such as each described chimeric polyeptides among the embodiment 1-112, it further comprises the constant region of IgG immunoglobulin.
114. such as each described chimeric polyeptides among the embodiment 1-113, wherein this HSA part is bonded to heterologous protein with chemical mode.
115. such as each described chimeric polyeptides among the embodiment 1-113, wherein this HSA part is bonded to heterologous protein with recombination form.
116. such as embodiment 115 described chimeric polyeptides, wherein this chimeric polyeptides is to use the recombinant vector of this HSA part of coding and this heterologous protein to produce.
117. such as embodiment 115 or 116 described chimeric polyeptides, wherein this chimeric polyeptides produces in prokaryotic cell or eukaryotic cell.
118. such as embodiment 117 described chimeric polyeptides, wherein this eukaryotic cell is selected from yeast cells, avian cell, insect cell or mammalian cell.
119. such as each described chimeric polyeptides among the embodiment 114-118, wherein each other directly combination of this HSA part and this heterologous protein.
120. such as each described chimeric polyeptides among the embodiment 114-118, wherein this HSA part and this heterologous protein are via the joint combination.
121. such as embodiment 120 described chimeric polyeptides, wherein this joint comprises one or more Gly-Gly-Gly-Gly-Ser repetitions.
122. such as each described chimeric polyeptides among the embodiment 1-121, wherein this HSA partly is bonded to the N terminal amino acid of this heterologous protein.
123. such as each described chimeric polyeptides among the embodiment 1-121, wherein this HSA partly is bonded to the C terminal amino acid of this heterologous protein.
124. such as each described chimeric polyeptides among the embodiment 1-121, wherein this HSA partly is bonded to the internal amino acid of this heterologous protein.
125. such as each described chimeric polyeptides among the embodiment 1-124, wherein this HSA part further comprises at least a portion of HSA domain I; Or at least a portion of HSA domain II; Or at least a portion of at least a portion of HSA domain I and HSA domain II.
126. such as each described chimeric polyeptides among the embodiment 1-125, wherein said chimeric polyeptides is purification basically.
127. a compositions, it comprises such as each described chimeric polyeptides and pharmaceutically acceptable carrier among the embodiment 1-126.
128. such as embodiment 127 described compositionss, wherein said compositions is aseptic composite.
129. such as embodiment 127 or 128 described compositionss, wherein said compositions is non-pyrogen.
130. treat the method that it is had the experimenter who needs for one kind, it comprises to described experimenter and giving according to each described chimeric polyeptides among the embodiment 1-126 or according to each described compositions among the embodiment 127-129.
131. an increase has the method for the serum half-life of the protein among the experimenter who needs to it, it comprises to described experimenter and giving according to each described chimeric polyeptides among the embodiment 1-126.
132. such as embodiment 130 or 131 described methods, wherein comprise that to described experimenter whole body gives described chimeric polyeptides.
133. such as embodiment 130 or 131 described methods, wherein comprise by following approach to described experimenter and give described chimeric polyeptides, this approach is selected from lower group, and this group is comprised of the following: in the Intradermal, percutaneous, intramuscular, intraperitoneal, intravenous, blood vessel, interior, subcutaneous, the lung of pericardium, intranasal, ophthalmic, epidural, part or oral.
134. such as embodiment 130 or 131 described methods, wherein comprise by intravenous to described experimenter giving described chimeric polyeptides.
135. a nucleic acid construct, it comprises coding such as the nucleotide sequence of each described chimeric polyeptides among the embodiment 1-125.
A 136. human serum albumin (HSA) variant polypeptide, it comprises the HSA Domain III, or its neonatal Fc receptor (FcRn) binding fragment, wherein said variant polypeptide can be combined with FcRn, and wherein said HSA Domain III comprises one to 18 aminoacid replacement, thereby increases described variant polypeptide to the affinity of FcRn with respect to the contrast HSA polypeptide that lacks described replacement.
137. such as embodiment 136 described variant polypeptides, wherein this variant polypeptide is combined with FcRn with the affinity higher than described contrast polypeptide, and wherein said affinity is measured under acid pH.
138. such as embodiment 136 or 137 described variant polypeptides, wherein this acid pH is between 5.0 and 6.0.
139. such as embodiment 138 described variant polypeptides, wherein this acid pH is 5.5 ± 0.2.
140. such as each described variant polypeptide among the embodiment 136, wherein said variant polypeptide is combined with FcRn with the affinity higher than described contrast polypeptide under acid pH, but described variant polypeptide is not combined with FcRn with the affinity higher than described contrast polypeptide under neutral pH.
141. such as embodiment 140 described variant polypeptides, wherein this neutral pH is between 6.9 and 7.9.
142. such as embodiment 141 described variant polypeptides, wherein this neutral pH is 7.4 ± 0.2.
143. such as each described variant polypeptide among the embodiment 136, wherein this variant polypeptide has than the longer serum half-life of described contrast HSA polypeptide.
144. such as each described variant polypeptide among the embodiment 136-143, wherein this HSA Domain III comprises one to ten aminoacid replacement.
145. such as each described variant polypeptide among the embodiment 136-144, wherein the described aminoacid replacement of at least one in the HSA Domain III is to be conservative residue in a plurality of species.
146. such as embodiment 145 described variant polypeptides, wherein the described aminoacid replacement of all in the HSA Domain III is to be conservative residue in a plurality of species.
147. such as each described variant polypeptide among the embodiment 136-144, wherein at least one the described aminoacid replacement in the HSA Domain III is to be conservative residue in from the serum albumin protein of following species: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.
148. such as embodiment 147 described variant polypeptides, wherein the described aminoacid replacement of all in the HSA Domain III is to be conservative residue in from the serum albumin protein of following species: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.
149. such as each described variant polypeptide among the embodiment 136-147, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from those that list in table 5.
150. such as each described variant polypeptide among the embodiment 136-147, wherein the described aminoacid replacement of at least one in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 381, residue 383, residue 391, residue 401, residue 402, residue 407, residue 411, residue 413, residue 414, residue 415, residue 416, residue 424, residue 426, residue 434, residue 442, residue 445, residue 447, residue 450, residue 454, residue 455, residue 456, residue 457, residue 459, residue 463, residue 495, residue 506, residue 508, residue 509, residue 511, residue 512, residue 515, residue 516, residue 517, residue 519, residue 521, residue 523, residue 524, residue 525, residue 526, residue 527, residue 531, residue 535, residue 538, residue 539, residue 541, residue 557, residue 561, residue 566, residue 569.
151. such as each described variant polypeptide in embodiment 136-147 or 150, wherein this variant polypeptide is included in the aminoacid replacement in the HSA Domain III of a plurality of positions that are numbered with respect to the position among the ripe HSA of total length, these positions are selected from lower group, and this group is comprised of the following: (a) residue 383 and 413; (b) residue 401 and 523; (c) residue 407 and 447; (d) residue 407 and 447 and 539; (e) residue 407 and 509; (f) residue 407 and 526; (g) residue 411 and 535; (h) residue 414 and 456; (i) residue 415 and 569; (j) residue 426 and 526; (k) residue 442 and 450 and 459; (l) residue 463 and 508; (m) residue 508 and 519 and 525; (n) residue 509 and 527; (o) residue 523 and 538; (p) residue 526 and 557; (q) residue 541 and 561; (r) residue 463 and 523; (s) residue 508 and 523; (t) residue 508 and 524; (u) residue 463,508 and 523; (v) residue 463,508 and 524; (w) residue 508,523 and 524; (x) residue 463,508,523 and 524; (y) residue 463 and 524; (z) residue 523 and 524; And (aa) residue 463,523 and 524.
152. such as embodiment 151 described variant polypeptides, wherein this variant polypeptide is included in the aminoacid replacement in the HSA Domain III of a plurality of positions that are numbered with respect to the position among the ripe HSA of total length, these positions are selected from lower group, and this group is comprised of the following: (a) residue 463 and 508; (b) residue 463 and 523; (c) residue 508 and 523; (d) residue 508 and 524; (e) residue 463,508 and 523; (f) residue 463,508 and 524; (g) residue 508,523 and 524; (h) residue 463,508,523 and 524; (i) residue 463 and 524; (j) residue 523 and 524; (k) residue 463,523 and 524.
153. such as each described variant polypeptide among embodiment 136-148 or the 150-152, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, V381Q, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y401D, Y401E, K402A, K402G, K402I, K402L, K402V, L407F, 407H, 407M, L407N, L407Q, 407R, L407W, L407Y, Y411Q, Y411N, K413C, K413S, K413T, K414S, K414T, V415C, V415L, V415S, V415T, Q416H, Q416P, V424A, V424D, V424G, V424I, V424L, V424M, V424N, V424Q, V424W, V426D, V426E, V426F, V426H, V426L, V426N, V426P, V426Q, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, P447S, P447T, E450D, E450E, S454C, S454M, S454T, V455N, V455Q, V456N, V456Q, L457F, L457W, L457Y, Q459K, Q459R, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T506F, T506M, T506N, T506R, T506W, T506Y, T508C, T508E, T508I, T508K, T508R, T508S, F509C, F509D, F509G, F509I, F509L, F509M, F509P, F509V, F509W, F509Y, A511D, A511F, A511I, A511R, A511T, A511V, A511W, A511Y, D512F, D512M, D512Q, D512W, D512Y, T515C, T515D, T515E, T515E, T515G, T515H, T515L, T515N, T515P, T515Q, T515S, T515W, T515Y, L516C, L516F, L516S, L516T, L516W, L516Y, S517C, S517F, S517M, S517T, S517W, S517Y, K519A, K519G, K519I, K519L, K519V, R521F, R521H, R521M, R521Q, R521T, R521W, R521Y, I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W, I523Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526A, Q526C, Q526F, Q526H, Q526L, Q526M, Q526P, Q526S, Q526T, Q526V, Q526Y, T527F, T527W, T527Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535K, H535L, H535N, H535P, H535S, K538F, K538W, K538Y, A539I, A539L, A539V, K541F, K541W, K541Y, K557A, K557G, K557I, K557L, K557N, K557S, K557V, A561F, A561W, A561Y, T566F, T566W, T566Y, A569H, and A569P.
154. such as each described variant polypeptide among embodiment 136-148 or the 150-153, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, E383G, N391V, Y401E, K402A, L407N, L407Y, Y411Q, K414S, K413S, V415T, V415C, Q416P, V424I, V424Q, V426E, V426H, G434C, E442K, R445W, P447S, E450D, S454C, V455N, V456N, L457F, Q459R, L463C, L463F, L463H, L463M, L463N, L463Q, E495D, T506Y, T508C, T508E, T508I, T508R, T508S, F509I, F509M, F509W, A511F, D512Y, T515P, T515Q, T515S, L516T, L516W, S517C, S517W, K519I, R521W, I523C, I523D, I523E, I523F, I523Q, I523H, I523K, I523L, I523N, I523P, I523G, I523R, I523Y, K524A, K524F, K524I, K524L, K524M, K524T, K524V, K525V, Q526T, Q526M, Q526Y, T527Y, E531I, H535N, H535P, K538Y, A539I, K541F, K557G, A561F, T566W, and A569P.
155. such as each described variant polypeptide among embodiment 136-148 or the 150-153, wherein this variant polypeptide is included in an aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: V381N, E383G, N391V, Y401E, K402A, L407N, L407Y, Y411Q, K414S, K413S, V415T, V415C, Q416P, V424I, V424Q, V426E, V426H, G434C, E442K, R445W, P447S, E450D, S454C, V455N, V456N, L457F, Q459R, L463C, L463F, L463H, L463M, L463N, L463Q, E495D, T506Y, T508C, T508E, T508I, T508R, T508S, F509I, F509M, F509W, A511F, D512Y, T515P, T515Q, T515S, L516T, L516W, S517C, S517W, K519I, R521W, I523C, I523D, I523E, I523F, I523Q, I523H, I523K, I523L, I523N, I523P, I523G, I523R, I523Y, K524A, K524F, K524I, K524L, K524M, K524T, K524V, K525V, Q526T, Q526M, Q526Y, T527Y, E531I, H535N, H535P, K538Y, A539I, K541F, K557G, A561F, T566W, and A569P.
156. such as embodiment 154 described variant polypeptides, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: L407N, L407Y, V415T, V424I, V424Q, V426E, V426H, P447S, V455N, V456N, L463N, E495D, T506Y, T508R, F509M, F509W, A511F, D512Y, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T527Y, H535P and K557G.
157. such as embodiment 155 described variant polypeptides, wherein this variant polypeptide is included in an aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: L407N, L407Y, V415T, V424I, V424Q, V426E, V426H, P447S, V455N, V456N, L463N, E495D, T506Y, T508R, F509M, F509W, A511F, D512Y, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T527Y, H535P and K557G.
158. such as each described variant polypeptide among embodiment 136-147 or the 150-152, wherein this variant polypeptide is included in a plurality of aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: (a) E383G/K413S; (b) Y401E/I523G; (c) L407N/P447S; (d) L407N/P447S/A539I; (e) L407N/F509M; (f) L407Y/Q526T; (g) Y411Q/H535N; (h) K414S/V456N; (i) V415T/A569P; (j) V426H/Q526Y; (k) E442K/E450D/Q459R; (l) L463N/T508R; (m) T508R/K519I/K525V; (n) F509I/T527Y; (o) I523Q/K538Y; (p) Q526M/K557G; (q) K541F/A561F; (r) L463N/K524L; (s) T508R/I523G; (t) T508R/K524L; (u) L463N/T508R/I523G; (v) L463N/T508R/K524L; (w) T508R/I523G/K524L; (x) L463N/T508R/I523G/K524L; (y) L463N/I523G; (z) I523G/K524L; (aa) L463N//I523G/K524L.
159. such as each described variant polypeptide among the embodiment 158, wherein this variant polypeptide is included in a plurality of aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: (a) L407N/P447S; (b) L407N/P447S/A539I; (c) L407N/F509M; (d) Y411Q/H535N; (e) K414S/V456N; (f) V426H/Q526Y; (g) L463N/T508R; (h) F509I/T527Y; (i) I523Q/K538Y; (j) Q526M/K557G; (k) K541F/A561F; (l) L463N/K524L; (m) T508R/I523G; (n) T508R/K524L; (o) L463N/T508R/I523G; (p) L463N/T508R/K524L; (q) T508R/I523G/K524L; (r) L463N/T508R/I523G/K524L; (r) L463N/T508R/I523G/K524L; (s) L463N/I523G; (t) I523G/K524L and (u) L463N//I523G/K524L.
160. such as each described variant polypeptide among the embodiment 158, wherein this variant polypeptide is included in a plurality of aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: (a) L463N/T508R; (b) L463N/K524L; (c) T508R/I523G; (d) T508R/K524L; (e) L463N/T508R/I523G; (f) L463N/T508R/K524L; (g) T508R/I523G/K524L; (h) L463N/T508R/I523G/K524L.
161. such as each described variant polypeptide among the embodiment 158, wherein this variant polypeptide is included in a plurality of aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: (a) L463N/K508R; (b) T508R/I523G; (c) T508R/K524L; (d) L463N/T508R/I523G.
162. such as each described variant polypeptide among the embodiment 136-147, wherein the described aminoacid replacement of at least one in the HSA Domain III be in from the serum albumin protein of people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse for conservative but in the chicken serum albumin conservative residue.
163. such as embodiment 162 described variant polypeptides, wherein the described aminoacid replacement of all in the HSA Domain III be in from the serum albumin protein of people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse for conservative but in the chicken serum albumin conservative residue.
164. such as embodiment 147 described variant polypeptides, wherein described at least one aminoacid replacement in the HSA Domain III with respect to total length ripe HSA(SEQ ID NO:2) in any following position that is numbered, position: residue 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.
165. such as embodiment 164 described variant polypeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 383, residue 391, residue 434, residue 442, residue 445 and residue 450.
166. such as embodiment 165 described variant polypeptides, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, E450D and E450E.
167. such as embodiment 164 described variant polypeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 417, residue 442, residue 499 and residue 502.
168. such as embodiment 162 described variant polypeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 380, residue 381, residue 384, residue 387, residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 419, residue 421, residue 422, residue 424, residue 428, residue 430, residue 431, residue 433, residue 441, residue 457, residue 458, residue 463, residue 464, residue 466, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 489, residue 491, residue 495, residue 500, residue 508, residue 510, residue 515, residue 516, residue 524, residue 525, residue 526, residue 528, residue 531, residue 535, residue 539, residue 544, residue 547, with residue 576.
169. such as embodiment 168 described variant polypeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 381, residue 401, residue 424, residue 457, residue 463, residue 495, residue 508, residue 515, residue 516, residue 524, residue 525, residue 526, residue 531, residue 535 and residue 539.
170. such as embodiment 169 described variant polypeptides, wherein this HSA Domain III is included in the aminoacid replacement of a plurality of positions that are numbered with respect to the position among the ripe HSA of total length, these positions are selected from lower group, and this group is comprised of the following: residue 463 and 508; Residue 463 and 524; Residue 508 and 524; Residue 463,508 and 524.
171. such as embodiment 169 or 170 described variant polypeptides, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, V381Q, Y401D, Y401E, V424N, V424Q, L457F, L457W, L457Y, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T508C, T508E, T508I, T508K, T508R, T508S, T515C, T515H, T515N, T515P, T515Q, T515S, L516F, L516S, L516T, L516W, L516Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526C, Q526M, Q526S, Q526T, Q526Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535P, A539I, and A539L, A539V.
172. such as embodiment 164 described variant polypeptides, wherein all described aminoacid replacement are selected from lower group member, and this group is comprised of the following: residue 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.
173. such as embodiment 168 described variant polypeptides, wherein all described aminoacid replacement are selected from lower group member, and this group is comprised of the following: residue 380, residue 381, residue 384, residue 387, residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 419, residue 421, residue 422, residue 424, residue 428, residue 430, residue 431, residue 433, residue 441, residue 457, residue 458, residue 463, residue 464, residue 466, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 489, residue 491, residue 495, residue 500, residue 508, residue 510, residue 515, residue 516, residue 524, residue 525, residue 526, residue 528, residue 531, residue 535, residue 539, residue 544, residue 547, with residue 576.
174. such as embodiment 164 or 168 described variant polypeptides, wherein all described aminoacid replacement are selected from lower group member, during this group is comprised of the following: residue 380, residue 381, residue 384, residue 383, residue 387, residue 389, residue 391, residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 410, residue 417, residue 419, residue 421, residue 422, residue 424, residue 425, residue 428, residue 430, residue 431, residue 433, residue 441, residue 442, residue 457, residue 458, residue 463, residue 464, residue 465, residue 466, residue 467, residue 468, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 486, residue 489, residue 491, residue 495, residue 499, residue 500, residue 502, residue 508, residue 510, residue 515, residue 516, residue 520, residue 524, residue 525, residue 526, residue 528, residue 531, residue 532, residue 535, residue 536, residue 539, residue 543, residue 544, residue 547, residue 571, with residue 576.
175. such as embodiment 174 described variant polypeptides, wherein all described aminoacid replacement are selected from lower group member, and this group is comprised of the following: residue 381, residue 383, residue 391, residue 401, residue 424, residue 442, residue 463, residue 495, residue 508, residue 515, residue 516, residue 524, residue 525, residue 526, residue 531, residue 535 and residue 539.
176. such as embodiment 175 described variant polypeptides, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, V381Q, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y401D, Y401E, V424A, V424G, V424I, V424L, V424N, V424Q, E442K, E442R, L463C, L463F, L463G, L463H, L463M, L463N, L463Q, E495D, T506F, T506W, T506Y, T508C, T508E, T508I, T508K, T508R, T508S, T515C, T515H, T515N, T515P, T515Q, T515S, L516S, L516T, L516W, L516Y, K524A, K524F, K524G, K524I, K524L, K524M, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526C, Q526M, Q526S, Q526T, Q526Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535P, A539I, A539L, and A539V.
177. such as each described variant polypeptide among the embodiment 136-174, wherein the described aminoacid replacement of at least one in the HSA Domain III is that the surface can reach residue.
178. such as embodiment 177 described variant polypeptides, wherein the described aminoacid replacement of all in the HSA Domain III is that the surface can reach residue.
179. such as each described variant polypeptide among the embodiment 136-174, wherein the described aminoacid replacement of at least one in the HSA Domain III is that the surface can reach and is conservative residue in a plurality of species.
180. such as embodiment 179 described variant polypeptides, wherein the described aminoacid replacement of all in the HSA Domain III is that the surface can reach and is conservative residue in a plurality of species.
181. such as embodiment 179 or 180 described variant polypeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.
182. such as embodiment 181 described variant polypeptides, wherein described at least one aminoacid replacement in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 383, residue 391 and residue 442.
183. such as embodiment 182 described variant polypeptides, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V and E442K, E442R.
184. such as each described variant polypeptide among the embodiment 136-180, wherein this HSA Domain III comprises with SEQ ID NO:l and has at least 90% conforming aminoacid sequence.
185. such as embodiment 184 described variant polypeptides, wherein this HSA Domain III comprises with SEQID NO:l and has at least 95% conforming aminoacid sequence.
186. such as embodiment 177185 described variant polypeptides, wherein this HSA Domain III comprises with SEQ ID NO:l and has at least 98% conforming aminoacid sequence.
187. such as the described variant polypeptide of embodiment 136-186, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 2 of HSA Domain III.
188. such as each described variant polypeptide among embodiment 136-155,162-164,168,172-174,177-181, the 184-187, wherein the described aminoacid replacement of all in the HSA Domain III is all in the ring 2 of HSA Domain III.
189. such as embodiment 187 or 188 described variant polypeptides, it is included in one in the HSA Domain III to five amino acid and replaces, wherein said one is substituted in the ring 2 of HSA Domain III to five amino acid.
190. such as each described variant polypeptide among the embodiment 136-187, wherein at least one the described aminoacid replacement in the HSA Domain III is in the ring 3 of HSA Domain III.
191. such as each described variant polypeptide among embodiment 136-155,162-168, the 172-174-186 or 190, wherein the described aminoacid replacement of all in the HSA Domain III is all in the ring 3 of HSA Domain III.
192. such as embodiment 190 or 191 described variant polypeptides, it is included in one in the HSA Domain III to five amino acid and replaces, wherein said one is substituted in the ring 3 of HSA Domain III to five amino acid.
193. such as each described variant polypeptide in embodiment 136-173 or 190, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 6 of HSA Domain III.
194. such as each described variant polypeptide among embodiment 136-155,162-164,167-181, the 184-186 or 193, wherein the described aminoacid replacement of all in the HSA Domain III is in the ring 6 of HSA Domain III.
195. such as embodiment 193 or 194 described variant polypeptides, it is included in one in the HSA Domain III to five amino acid and replaces, wherein said one is substituted in the ring 6 of HSA Domain III to five amino acid.
196. such as each described variant polypeptide among the embodiment 136-173,190 or 193, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the spiral 7 of HSA Domain III.
197. such as each described variant polypeptide among embodiment 136-155,162-164,167-181, the 184-186 or 196, wherein the described aminoacid replacement of all in the HSA Domain III is in the spiral 7 of HSA Domain III.
198. such as embodiment 196 or 200 described variant polypeptides, it is included in one to three aminoacid replacement in the HSA Domain III, wherein said one to six aminoacid replacement is in the spiral 7 of HSA Domain III.
199. such as each described variant polypeptide among the embodiment 136-173,190,193 or 196, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 7 of HSA Domain III.
200. such as each described variant polypeptide among embodiment 136-155,162-164,167-181, the 184-186 or 199, wherein the described aminoacid replacement of all in the HSA Domain III is in the ring 7 of HSA Domain III.
201. such as embodiment 199 or 200 described variant polypeptides, it is included in one to three aminoacid replacement in the HSA Domain III, wherein said one to three aminoacid replacement is in the ring 7 of HSA Domain III.
202. such as each described variant polypeptide among the embodiment 136-173,190,193,196 or 199, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the spiral 8 of HSA Domain III.
203. such as each described variant polypeptide among embodiment 136-155,162-164,173-174,177-181, the 184-186 or 202, wherein the described aminoacid replacement of all in the HSA Domain III is in the spiral 8 of HSA Domain III.
204. such as embodiment 202 or 203 described variant polypeptides, it is included in one in the HSA Domain III to the five amino acid replacement, wherein said one to 18 aminoacid replacement is in the spiral 8 of HSA Domain III.
205. such as each described variant polypeptide among the embodiment 136-173,190,193,196,199 or 202, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 8 of HSA Domain III.
206. such as each described variant polypeptide among embodiment 136-155,162-164,173-174,177-181, the 184-186 or 205, wherein the described aminoacid replacement of all in the HSA Domain III is in the ring 8 of HSA Domain III.
207. such as embodiment 205 or 206 described variant polypeptides, it is included in one in the HSA Domain III to five amino acid and replaces, wherein said one is substituted in the ring 8 of HSA Domain III to five amino acid.
208. such as embodiment 136-173,190,193 or 205 described variant polypeptides, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 9 of HSA Domain III.
209. such as embodiment 136-155,162,163,177-180,184-186 or 208 described variant polypeptides, wherein the described aminoacid replacement of all in the HSA Domain III is in the ring 9 of HSA Domain III.
210. such as embodiment 208 or 209 described variant polypeptides, it is included in one to four aminoacid replacement in the HSA Domain III, wherein said one to four aminoacid replacement is in the ring 9 of HSA Domain III.
211. such as each described variant polypeptide among the embodiment 136-210, wherein at least one described aminoacid replacement comprises an amino acid residue is replaced with alanine.
212. such as each described variant polypeptide among the embodiment 136-211, wherein at least one described aminoacid replacement comprises that conservative amino acid replaces.
213. such as each described variant polypeptide among the embodiment 136-212, wherein at least one described aminoacid replacement comprises a basic amino acid with the displacement of another basic amino acid.
214. such as each described variant polypeptide among the embodiment 136-213, wherein at least one described aminoacid replacement comprises an acidic amino acid with the displacement of another acidic amino acid.
215. such as each described variant polypeptide among the embodiment 136-214, wherein at least one described aminoacid replacement comprises a neutral amino acid with the displacement of another neutral amino acid.
216. such as each described variant polypeptide among the embodiment 136-215, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: lysine, arginine and histidine.
217. such as each described variant polypeptide among the embodiment 136-216, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: aspartic acid and glutamic acid.
218. such as each described variant polypeptide among the embodiment 136-217, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: agedoite, glutamine, serine, threonine and tyrosine.
219. such as each described variant polypeptide among the embodiment 136-218, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: alanine, valine, isoleucine, leucine, proline, phenylalanine, tryptophan, methionine, cysteine and glycine.
220. such as each described variant polypeptide among the embodiment 136-219, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: phenylalanine, tryptophan and tyrosine.
221. such as each described variant polypeptide among the embodiment 136-220, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: cysteine, serine and threonine.
222. such as each described variant polypeptide among the embodiment 136-221, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: agedoite, glutamine, serine, threonine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid.
223. such as each described variant polypeptide among the embodiment 136-222, wherein at least one described aminoacid replacement comprises an aminoacid with another amino acid replacement in following group: glycine, serine, threonine, alanine, valine, leucine and isoleucine.
224. such as each described variant polypeptide among the embodiment 136-223, wherein at least one described aminoacid replacement comprises that non-conservation replaces.
225. such as embodiment 211 described variant polypeptides, wherein all described aminoacid replacement comprise an amino acid residue are replaced with alanine.
226. such as embodiment 212 described variant polypeptides, wherein all described aminoacid replacement comprise that the conservative amino acid at place, each position replaces independently.
227. such as embodiment 213 described variant polypeptides, wherein all described aminoacid replacement comprise independently at place, each position a basic amino acid are replaced with another basic amino acid.
228. such as embodiment 214 described variant polypeptides, wherein all described aminoacid replacement comprise independently at place, each position an acidic amino acid are replaced with another acidic amino acid.
229. such as embodiment 215 described variant polypeptides, wherein all described aminoacid replacement comprise independently at place, each position a neutral amino acid are replaced with another neutral amino acid.
230. such as embodiment 216 described variant polypeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: lysine, arginine and histidine.
231. such as embodiment 217 described variant polypeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: aspartic acid and glutamic acid.
232. such as embodiment 218 described variant polypeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: agedoite, glutamine, serine, threonine and tyrosine.
233. such as embodiment 219 described variant polypeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: alanine, valine, isoleucine, leucine, proline, phenylalanine, tryptophan, methionine, cysteine and glycine.
234. such as embodiment 220 described variant polypeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: phenylalanine, tryptophan and tyrosine.
235. such as embodiment 221 described variant polypeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: cysteine, serine and threonine.
236. such as embodiment 222 described variant polypeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: agedoite, glutamine, serine, threonine, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid.
237. such as embodiment 223 described variant polypeptides, wherein all described aminoacid replacement comprise independently at place, each position an aminoacid another amino acid replacement in following group: glycine, serine, threonine, alanine, valine, leucine and isoleucine.
238. such as embodiment 224 described variant polypeptides, wherein all described aminoacid replacement comprise that the non-conservation at place, each position replaces independently.
239. such as each described variant polypeptide among the embodiment 136-238, wherein said HSA part further comprises at least a portion of HSA domain I; Or at least a portion of HSA domain II; Or at least a portion of at least a portion of HSA domain I and HSA domain II.
240. such as each described variant polypeptide among the embodiment 136-239, wherein said variant polypeptide is purification basically.
241. such as each described variant polypeptide among the embodiment 136-240, wherein said variant polypeptide further comprises the binding site of the epi-position on the target.
242. such as embodiment 241 described variant polypeptides, the described target of this binding site antagonism wherein.
243. such as embodiment 241 described variant polypeptides, the exciting described target of this binding site wherein.
244. a compositions, it comprises such as each described variant polypeptide and pharmaceutically acceptable carrier among the embodiment 136-243.
245. such as embodiment 244 described compositionss, wherein said compositions is aseptic composite.
246. such as embodiment 244 or 245 described compositionss, wherein said compositions is non-pyrogen.
247. a method that increases the serum half-life of protein, it comprise with described protein with according to embodiment 136-243 in each described variant polypeptide be combined.
248. treat the method that it is had the experimenter who needs for one kind, it comprises to described experimenter and giving according to each described variant polypeptide among the embodiment 241-243.
249. a nucleic acid construct, it comprises coding such as the nucleotide sequence of each described variant polypeptide among the embodiment 136-239.
250. nucleic acid construct, it comprises the nucleotide sequence of (a) encoding human serum albumin (HSA) part, this HSA partly comprises HSA Domain III or its FcRn binding fragment, wherein this HSA Domain III comprises one to 18 aminoacid replacement, it may be operably coupled to the nucleotide sequence of (b) coding heterologous protein, wherein said nucleic acid construct coding chimeric polyeptides, described chimeric polyeptides has kept the functional activity of heterologous protein and can be combined with FcRn, and wherein said chimeric polyeptides does not comprise the contrast chimeric polyeptides of described aminoacid replacement and has the serum half-life of increase and/or the affinity to FcRn of increase with respect to this HSA part wherein.
251. such as embodiment 250 described nucleic acid constructs, wherein this chimeric polyeptides is combined with FcRn with the affinity higher than described contrast chimeric polyeptides.
252. such as embodiment 250 or 251 described nucleic acid constructs, wherein this chimeric polyeptides is combined with FcRn with the affinity higher than described contrast chimeric polyeptides, and wherein said affinity is measured under acid pH.
253. such as embodiment 252 described nucleic acid constructs, wherein acid pH is between 5.0 and 6.0.
254. such as embodiment 253 described nucleic acid constructs, wherein this acid pH is 5.5 ± 0.2.
255. such as each described nucleic acid construct among the embodiment 250-255, wherein this chimeric polyeptides is combined with FcRn with the affinity higher than described contrast chimeric polyeptides under acid pH, but this chimeric polyeptides is not combined with FcRn with the affinity higher than described contrast chimeric polyeptides under neutral pH.
256. such as embodiment 255 described nucleic acid constructs, wherein this neutral pH is between 6.9 and 7.9.
257. such as embodiment 256 described nucleic acid constructs, wherein this neutral pH is 7.4 ± 0.2.
258. such as each described nucleic acid construct among the embodiment 250, wherein (i) comprises the nucleotide sequence of encoding human serum albumin (HSA) part, this HSA partly comprises HSA Domain III or its FcRn binding fragment, and this HSA Domain III comprises one to ten aminoacid replacement.
259. such as each described nucleic acid construct among the embodiment 250-258, wherein the described aminoacid replacement of at least one in the HSA Domain III is to be conservative residue in a plurality of species.
260. such as embodiment 259 described nucleic acid constructs, wherein the described aminoacid replacement of all in the HSA Domain III is to be conservative residue in a plurality of species.
261. such as each described nucleic acid construct among the embodiment 250-258, wherein the described aminoacid replacement of at least one in the HSA Domain III is to be conservative residue in from the serum albumin protein of following species: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.
262. such as embodiment 261 described nucleic acid constructs, wherein the described aminoacid replacement of all in the HSA Domain III is to be conservative residue in from the serum albumin protein of following species: people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.
263. such as each described nucleic acid construct among the embodiment 250-262, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from those that list in table 5.
264. such as each described nucleic acid construct among the embodiment 250-262, wherein the described aminoacid replacement of at least one in the HSA Domain III is in any following position that is numbered with respect to the position among the total length maturation HSA: residue 381, residue 383, residue 391, residue 401, residue 402, residue 407, residue 411, residue 413, residue 414, residue 415, residue 416, residue 424, residue 426, residue 434, residue 442, residue 445, residue 447, residue 450, residue 454, residue 455, residue 456, residue 457, residue 459, residue 463, residue 495, residue 506, residue 508, residue 509, residue 511, residue 512, residue 515, residue 516, residue 517, residue 519, residue 521, residue 523, residue 524, residue 525, residue 526, residue 527, residue 531, residue 535, residue 538, residue 539, residue 541, residue 557, residue 561, residue 566, residue 569.
265. such as each described nucleic acid construct among the embodiment 250-262, wherein this HSA Domain III is included in the aminoacid replacement of a plurality of positions that are numbered with respect to the position among the ripe HSA of total length, these positions are selected from lower group, and this group is comprised of the following: (a) residue 383 and 413; (b) residue 401 and 523; (c) residue 407 and 447; (d) residue 407 and 447 and 539; (e) residue 407 and 509; (f) residue 407 and 526; (g) residue 411 and 535; (h) residue 414 and 456; (i) residue 415 and 569; (j) residue 426 and 526; (k) residue 442 and 450 and 459; (l) residue 463 and 508; (m) residue 508 and 519 and 525; (n) residue 509 and 527; (o) residue 523 and 538; (p) residue 526 and 557; (q) residue 541 and 561; (r) residue 463 and 523; (s) residue 508 and 523; (t) residue 508 and 524; (u) residue 463,508 and 523; (v) residue 463,508 and 524; (w) residue 508,523 and 524; (x) residue 463,508,523 and 524; (y) residue 463 and 524; (z) residue 523 and 524; And (aa) residue 463,523 and 524.
266. such as each described nucleic acid construct among the embodiment 250-262, wherein this HSA Domain III is included in the aminoacid replacement of a plurality of positions that are numbered with respect to the position among the ripe HSA of total length, these positions are selected from lower group, and this group is comprised of the following: (a) residue 463 and 508; (b) residue 463 and 523; (c) residue 508 and 523; (d) residue 508 and 524; (e) residue 463,508 and 523; (f) residue 463,508 and 524; (g) residue 508,523 and 524; (h) residue 463,508,523 and 524; (i) residue 463 and 524; (j) residue 523 and 524; And (k) residue 463,523 and 524.
267. such as each described nucleic acid construct among embodiment 250-262 or the 264-266, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, V381Q, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y401D, Y401E, K402A, K402G, K402I, K402L, K402V, L407F, 407H, 407M, L407N, L407Q, 407R, L407W, L407Y, Y411Q, Y411N, K413C, K413S, K413T, K414S, K414T, V415C, V415L, V415S, V415T, Q416H, Q416P, V424A, V424D, V424G, V424I, V424L, V424M, V424N, V424Q, V424W, V426D, V426E, V426F, V426H, V426L, V426N, V426P, V426Q, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, P447S, P447T, E450D, E450E, S454C, S454M, S454T, V455N, V455Q, V456N, V456Q, L457F, L457W, L457Y, Q459K, Q459R, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T506F, T506M, T506N, T506R, T506W, T506Y, T508C, T508E, T508I, T508K, T508R, T508S, F509C, F509D, F509G, F509I, F509L, F509M, F509P, F509V, F509W, F509Y, A511D, A511F, A511I, A511R, A511T, A511V, A511W, A511Y, D512F, D512M, D512Q, D512W, D512Y, T515C, T515D, T515E, T515E, T515G, T515H, T515L, T515N, T515P, T515Q, T515S, T515W, T515Y, L516C, L516F, L516S, L516T, L516W, L516Y, S517C, S517F, S517M, S517T, S517W, S517Y, K519A, K519G, K519I, K519L, K519V, R521F, R521H, R521M, R521Q, R521T, R521W, R521Y, I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W, I523Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526A, Q526C, Q526F, Q526H, Q526L, Q526M, Q526P, Q526S, Q526T, Q526V, Q526Y, T527F, T527W, T527Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535K, H535L, H535N, H535P, H535S, K538F, K538W, K538Y, A539I, A539L, A539V, K541F, K541W, K541Y, K557A, K557G, K557I, K557L, K557N, K557S, K557V, A561F, A561W, A561Y, T566F, T566W, T566Y, A569H, and A569P.
268. such as each described nucleic acid construct among embodiment 250-262 or the 264-266, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: V381N, E383G, N391V, Y401E, K402A, L407N, L407Y, Y411Q, K414S, K413S, V415T, V415C, Q416P, V424I, V424Q, V426E, V426H, G434C, E442K, R445W, P447S, E450D, S454C, V455N, V456N, L457F, Q459R, L463C, L463F, L463H, L463M, L463N, L463Q, E495D, T506Y, T508C, T508E, T508I, T508R, T508S, F509I, F509M, F509W, A511F, D512Y, T515P, T515Q, T515S, L516T, L516W, S517C, S517W, K519I, R521W, I523C, I523D, I523E, I523F, I523Q, I523H, I523K, I523L, I523N, I523P, I523G, I523R, I523Y, K524F, K524I, K524L, K524M, K524T, K524V, K525V, Q526T, Q526M, Q526Y, T527Y, E531I, H535N, H535P, K538Y, A539I, K541F, K557G, A561F, T566W, and A569P.
269. such as embodiment 268 described nucleic acid constructs, wherein the described aminoacid replacement of at least one in the HSA Domain III is selected from lower group, and this group is comprised of the following: L407N, L407Y, V415T, V424I, V424Q, V426E, V426H, P447S, V455N, V456N, L463N, E495D, T506Y, T508R, F509M, F509W, A511F, D512Y, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T527Y, H535P and K557G.
270. such as each described nucleic acid construct in embodiment 250-262 or 264269, wherein this chimeric polyeptides is included in the aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: (a) E383G/K413S; (b) Y401E/I523G; (c) L407N/P447S; (d) L407N/P447S/A539I; (e) L407N/F509M; (f) L407Y/Q526T; (g) Y411Q/H535N; (h) K414S/V456N; (i) V415T/A569P; (j) V426H/Q526Y; (k) E442K/E450D/Q459R; (l) L463N/T508R; (m) T508R/K519I/K525V; (n) F509I/T527Y; (o) I523Q/K538Y; (p) Q526M/K557G; (q) K541F/A561F; (r) L463N/K524L; (s) T508R/I523G; (t) T508R/K524L; (u) L463N/T508R/I523G; (v) L463N/T508R/K524L; (w) T508R/I523G/K524L; (x) L463N/T508R/I523G/K524L; (y) L463N/I523G; (z) I523G/K524L; (aa) L463N//I523G/K524L.
271. such as embodiment 270 described nucleic acid constructs, wherein this chimeric polyeptides is included in the aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: (a) L407N/P447S; (b) L407N/P447S/A539I; (c) L407N/F509M; (d) Y411Q/H535N; (e) K414S/V456N; (f) V426H/Q526Y; (g) L463N/T508R; (h) F509I/T527Y; (i) I523Q/K538Y; (j) Q526M/K557G; (k) K541F/A561F; (l) L463N/K524L; (m) T508R/I523G; (n) T508R/K524L; (o) L463N/T508R/I523G; (p) L463N/T508R/K524L; (q) T508R/I523G/K524L; (r) L463N/T508R/I523G/K524L; (s) L463N/I523G; (t) I523G/K524L and (u) L463N//I523G/K524L.
272. such as embodiment 270 described nucleic acid constructs, wherein this chimeric polyeptides is included in the aminoacid replacement in the HSA Domain III, described replacement is selected from lower group, and this group is comprised of the following: (a) L463N/T508R; (b) L463N/K524L; (c) T508R/I523G; (d) T508R/K524L; (e) L463N/T508R/I523G; (f) L463N/T508R/K524L; (g) T508R/I523G/K524L; (h) L463N/T508R/I523G/K524L.
273. such as embodiment 270 described nucleic acid constructs, wherein this chimeric polyeptides is included in the aminoacid replacement in the Domain III, described replacement is selected from lower group, and this group is comprised of the following: (a) L463N/K508R; (b) T508R/I523G; (c) T508R/K524L; (d) L463N/T508R/I523G.
274. such as each described nucleic acid construct among the embodiment 250-262, wherein the described aminoacid replacement of at least one in the HSA Domain III is that the surface can reach residue.
275. such as each described nucleic acid construct among the embodiment 274, wherein the described aminoacid replacement of all in the HSA Domain III is that the surface can reach residue.
276. such as each described nucleic acid construct among the embodiment 250-258, wherein the described aminoacid replacement of at least one in the HSA Domain III is that the surface can reach and is conservative residue in a plurality of species.
277. such as each described nucleic acid construct among the embodiment 276, wherein the described aminoacid replacement of all in the HSA Domain III is that the surface can reach and is conservative residue in a plurality of species.
278. such as each described nucleic acid construct among the embodiment 250-277, wherein (i) comprises coding and has the nucleotide sequence of at least 90% conforming HSA Domain III with SEQ ID NO:l.
279. such as embodiment 278 described nucleic acid constructs, wherein (i) comprises coding and has the nucleotide sequence of at least 95% conforming HSA Domain III with SEQ ID NO:l.
280. such as embodiment 279 described nucleic acid constructs, wherein (i) comprises coding and has the nucleotide sequence of at least 98% conforming HSA Domain III with SEQ IDNO:l.
281. such as each described nucleic acid construct among the embodiment 250-280, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 2 of HSA Domain III.
282. such as each described nucleic acid construct among the embodiment 250-281, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 3 of HSA Domain III.
283. such as each described nucleic acid construct among the embodiment 250-282, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 6 of HSA Domain III.
284. such as each described nucleic acid construct among the embodiment 250-272, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the spiral 7 of HSA Domain III.
285. such as each described nucleic acid construct among the embodiment 250-284, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 7 of HSA Domain III.
286. such as each described nucleic acid construct among the embodiment 250-285283, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the spiral 8 of HSA Domain III.
287. such as each described nucleic acid construct among the embodiment 250-286, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 8 of HSA Domain III.
288. such as each described nucleic acid construct among the embodiment 250-287, wherein the described aminoacid replacement of at least one in the HSA Domain III is in the ring 9 of HSA Domain III.
289. such as each described nucleic acid construct among the embodiment 250-288, wherein (ii) comprises the nucleotide sequence of the heterologous protein of encoding, this heterologous protein comprises antibody or its Fab.
290. such as each described nucleic acid construct among the embodiment 250-289, it further comprises the nucleotide sequence of the joint of encoding.
291. such as embodiment 290 described nucleic acid constructs, wherein this nucleotide sequence codedly comprises joint that one or more Gly-Gly-Gly-Gly-Ser repeat.
292. such as each described nucleic acid construct among the embodiment 250-291, wherein this HSA part further comprises at least a portion of HSA domain I; Or at least a portion of HSA domain II; Or at least a portion of at least a portion of HSA domain I and HSA domain II.
293. library that comprises a plurality of polypeptide, each of wherein said a plurality of polypeptide comprises HSA Domain III or its FcRn binding fragment, and each of wherein said a plurality of polypeptide is included at least one aminoacid replacement of the residue in the described HSA Domain III independently, and described residue is guarded in the serum albumin protein from people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.
294. library that comprises a plurality of polypeptide, each of wherein said a plurality of polypeptide comprises HSA Domain III or its FcRn binding fragment, and each of wherein said a plurality of polypeptide is included at least one aminoacid replacement of the residue in the described HSA Domain III independently, described residue is guarded in the serum albumin protein from people, pig, rat, mice, Canis familiaris L., rabbit, cattle, donkey, mongolian gerbil, sheep, cat and horse, and does not guard in the serum albumin from chicken.
295. library that comprises a plurality of polypeptide, each of wherein said a plurality of polypeptide comprises HSA Domain III or its FcRn binding fragment, and each of wherein said a plurality of polypeptide is included at least one aminoacid replacement of the residue in the described HSA Domain III independently, and described residue is that the surface can reach residue.
296. such as embodiment 295 described libraries, wherein said surface can reach residue in the ring 2 of HSA Domain III.
297. such as embodiment 295 or 296 described libraries, wherein said surface can reach residue in the ring 3 of HSA Domain III.
298. such as each described library among the embodiment 295-297, wherein said surface can reach residue in the ring 6 of HSA Domain III.
299. such as each described library among the embodiment 295-273, wherein said surface can reach residue in the ring 7 of HSA Domain III.
300. such as each described library among the embodiment 295-299, wherein said surface can reach residue in the ring 8 of HSA Domain III.
301. such as each described library among the embodiment 295-300, wherein said surface can reach residue in the ring 9 of HSA Domain III.
302. library that comprises a plurality of polypeptide, each of wherein said a plurality of polypeptide comprises HSA Domain III or its FcRn binding fragment, and each of wherein said a plurality of polypeptide is included at least one aminoacid replacement of the residue in the described HSA Domain III independently, and described residue can reach residue for (i) surface and (ii) guard in the serum albumin protein from people, pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.
303. library that comprises a plurality of polypeptide, each of wherein said a plurality of polypeptide comprises the HSA Domain III, or its FcRn binding fragment, and each of wherein said a plurality of polypeptide is included at least one aminoacid replacement of the residue in the described HSA Domain III independently, it is the aminoacid of guarding that described residue is substituted by in the serum albumin protein of two or more species of group under from being selected from except the people, and this group is comprised of the following: pig, rat, mice, Canis familiaris L., rabbit, cattle, chicken, donkey, mongolian gerbil, sheep, cat and horse.
304. such as embodiment 302 or 303 described libraries, wherein said at least one aminoacid replacement is in the ring 2 of HSA Domain III.
305. such as each described library among the embodiment 302-304, wherein said at least one aminoacid replacement is in the ring 3 of HSA Domain III.
306. such as each described library among the embodiment 302-305, wherein said at least one aminoacid replacement is in the ring 6 of HSA Domain III.
307. such as arbitrary described library among the embodiment 302-306, wherein said at least one aminoacid replacement is in the spiral 7 of HSA Domain III.
308. such as each described library among the embodiment 302-307, wherein said at least one aminoacid replacement is in the ring 7 of HSA Domain III.
309. such as each described library among the embodiment 302-308, wherein said at least one aminoacid replacement is in the spiral 8 of HSA Domain III.
310. such as each described library among the embodiment 302-309, wherein said at least one aminoacid replacement is in the ring 8 of HSA Domain III.
311. such as each described library among the embodiment 302-310, wherein said at least one aminoacid replacement is in the ring 9 of HSA Domain III.
312. such as each described library among the embodiment 293-311, each of wherein said a plurality of polypeptide further comprises at least a portion of HSA domain I; Or at least a portion of HSA domain II; Or at least a portion of at least a portion of HSA domain I and HSA domain II.
313. such as each described library among the embodiment 293-312, wherein said library is display libraries.
314. such as embodiment 313 described libraries, wherein the type of display libraries is selected from lower group, this group is comprised of the following: yeast, phage and mammal.
315. the method in each described library among a screening such as the embodiment 293-314, described method comprises: a plurality of polypeptide of (a) selecting to be used for screening; (b) screening has the polypeptide to the serum half-life of the binding affinity of FcRn or increase of increase; And (c) determine to have the sequence to the polypeptide of the serum half-life of the binding affinity of FcRn or increase of increase.
8 illustrate
Can be more readily understood now general described the present invention with reference to following examples, these included embodiment only for the purpose of explanation some aspect of the present invention and embodiment, are not intended to limit the present invention.For example, particular build body disclosed here and experimental design represent the tool and method of the exemplary suitable function of checking.It is evident that equally the particular build body of any disclosure and planning of experiment can be substituted in the category scope of this disclosure.In addition, should be understood that unless clearly so explanation, the concrete inventory of employed concrete equipment and reagent, size, manufacturer etc. or description should not be considered to limitation of the present invention.Should be understood that further that other equipment and the reagent that play a role similarly can easily be substituted.
8.1 example 1: kinetics and affinity analysis that people FcRn is combined with human serum albumin's (HSA) Domain III
This example use surface plasma body resonant vibration (SPR) measure the Domain III of HSA to the association of people FcRn, dissociate and the balance affinity constant.Domain III (also being abbreviated as " DIII ") is the fragment of crossing over human serum albumin's protein of amino acid residue 381-585.The aminoacid sequence of Domain III is listed in SEQ ID NO:l.The aminoacid sequence of the ripe HSA of total length is listed in SEQ ID NO:2.Express and purification structure territory III from Pichia sp. (Pichia Patoris), in order in these experiments, use.
8.1.1 recombinant protein expression and purification
From German Geneart AG, Regensburg, the recombiant plasmid of acquisition coding structure territory III gene.The carrier that uses restricted enzyme EcoRI and Notl to provide from supplier excises the Domain III gene and is cloned into (hero company, catalog number (Cat.No.) V195-20) pPICZ-α-A yeast expression vector.Express recombination structure territory III albumen in the mode of describing in manufacturer's explanation.Make recombination structure territory III protein excretion in medium, and make it by hydrophobic interaction chromatography purification (catalog number (Cat.No.) 17519701) on from the Hi Trap butyl-agarose rapid flow post of GE Healthcare.In brief, at first the salinity with culture medium is adjusted into 1.5M ammonium sulfate and 50mM sodium phosphate, and pH is adjusted into 7.0.Filter culture medium and make it pass through the butyl-agarose post, then use the Domain III of the less salt sodium phosphate buffer elution of bound of pH7.0.Use remains on 50 ℃ cyclic water jacket, with the protein part of purification by through Hydroxyaloxypsopyl Dextran(Sigma-Aldrich company, catalog number (Cat.No.) H6258) the post degrease.Visual by the 4%-12%SDS PAGE of coomassie brilliant blue staining as passing through, the purity of degrease and non-degrease form all is 99%(Figure 1A).Measure protein concentration by A280.By closely-UV-CD and far away-UV-CD measure the suitably folding of the Domain III of confirming purification, described measurement and previous measurement of announcing closely related (referring to, such as people such as Giancola, International Journal of Biological Macromolecules(" international bio macromole magazine ") 20(1997) 193-204).
8.1.2 kinetic measurement
Under 25 ℃, upward measure balance, associate and the speed constant of dissociating at BIAcore Tl00 instrument (Sweden, Uppsala), and use BIAcore Tl00 assessment software, version 1.1(BIAcore company, Sweden, Uppsala) analytical data.By standard amine coupling (BIAcore handbook, 2002), with the non-degrease of Domain III and degrease form with coupling density be respectively 1016 and the 1184RU covalency be fixed on CM4(catalog number (Cat.No.) BR-1005-39) or CM5 chip (catalog number (Cat.No.) BR-1000-14) on.Use does not have identical fixed solution simulation one of them flow cell of coupling of protein so that as blank.All injections are all carried out in the 50mM phosphate solution under pH5.5 and the 150mM NaCl buffer, and make chip surface regeneration between the injection with the phosphate buffered saline (PBS) (PBS) of pH7.4.In order in single experiment, to measure association constant (k On), dissociation constant (k Off) and equilibrium dissociation constant (K D), on the III protein of fixed structure territory with the people FcRn(39nM-40 μ M of 50 μ L/min injection progressive concentration) (Figure 1B, left figure).Allow in conjunction with reaching balance, and by simultaneously the association phase (4min) of curve being obtained kinetic constant k with Lang Gemiuer (langmuir) model that dissociate mutually (1min) fits to 1:l OnAnd k OffBy using nonlinear regression analysis that the association reaction (Req) when the balance is obtained K to the curve fitting of analyte concentration as stable state affinity model D(Figure 1B, right figure).The degrease of Domain III and non-degrease form show similar in conjunction with sensing figure and Req to the ligand concentration curve chart.
The interaction of FcRn and Domain III shows the (k that associates fast On≈ 7e 3) and (k that dissociates Off≈ 4e - 2) kinetics (table 1).Domain III is to the K of FcRn DBetween 5-8 μ M, it is than the K of total length HSA to FcRn DLarge approximately 7 times (for example, larger dissociation constant) (table 1).K DThis species diversity mainly owing to the faster k of Domain III with respect to HSA Off, and the k of two kinds of molecules OnQuite.Be derived from k OnAnd k OffK DWith the value that obtains by experiment near consistent, thereby verified kinetic measurement.And the two kinds of different sensor chips (CM4 and CM5) with variable carboxy moiety have similar affinity.Kinetic constant and equilibrium constant are suitable between the degrease of Domain III and non-degrease form, show that lipid molecular does not mediate or promote the combination of FcRn and Domain III.
Table 1. this table provide the kinetics that is derived from SPR of the people FcRn of being combined with human serum albumin's (HSA) Domain III and equilibrium constant and with the comparison (figure below) of the kinetic constant of disclosed total length HSA.
Figure BDA0000365918090001101
8.2 example 2: merge separately the enhancing human IgG to the affinity of FcRn with total length HSA or Domain III
This experiment showed, that the fusion of the Domain III of HSA and therapeutic protein or antibody or its variant has strengthened therapeutic protein/antibody affinity to FcRn under acid pH, and can not affect synergistic pH dependency.Under pH5.5, the affinity increase of FcRn is converted into the serum half-life (for example, in the body or the life-span of improving in the suitable model system) of increase.Can in the people FcRn gene of for example the expressing single copy transgenic mice of (but lacking Mus FcRn), comprise the pharmacokinetics half-life measurement that merges to the HSA part of the Domain III of therapeutic protein, so that the fusion of assessment and the part that comprises wild type or variant structure territory III is on the impact of half-life.
8.2.1 construct design
As representative protein, end user IgG1 come comparison independent IgG and and the IgG that merges of HSA or Domain III between the FcRn affinity.Via the joint that comprises 4 Gly-Gly-Gly-Gly-Ser repetitives HSA or Domain III are merged C-terminal (Fig. 2 A) to human IgG1's heavy chain.Based on the length of coming designed joint in the IgG binding site on the FcRn and the distance between the HSA binding site, to allow two parts simultaneously in conjunction with its corresponding binding site.The HSA and the Domain III that produce equally in a similar manner the high-affinity variant of previous described IgG merge form (IgG-YTE, referring to people such as Dall ' Acqua, 2002, J Immunol.(" IMMUNOLOGY KEY WORDS INDEX), 169:5171-5180), described IgG variant demonstrates with respect to natural IgG and improves 10 times the affinity to FcRn.Therefore, preparation and the following construct of use:
IgG
IgG(YTE)
IgG-(G 4S) 4-HSA
IgG-(G 4S) 4-Domain III
IgG(YTE)-(G 4S) 4-HSA
IgG (YTE)-(G 4S) 4-Domain III
8.2.2 purification and sign
In brief, all 6 constructs are cloned in the expression vector, and pass through transient expression in 293F cell (GIBCO catalog number (Cat.No.) R79007) and protein purification.Use is from the HiTrap of GEHealthcare TMPurifying secreted IgG1 and the fusion rotein to culture medium of protein A affinity column (catalog number (Cat.No.) 17-0403-03).Under reduction and non-reducing condition, resolve the protein of purification by SDS PAGE, and as visual by coomassie dyeing, observe 99% purity (Fig. 2 B).
IgG-(G 4S) 4The estimation molecular weight of-HSA fusion rotein is 284 kilodaltons (KDa), and IgG-(G 4S) 4The estimation molecular weight of-Domain III is 196KDa.The molecular weight of observing and these estimated values are closely related.Whether assemble because of the physicochemical characteristics of its large-size or change in order to assess these fusion rotein forms, use AgilentTechnologies1200 series SEC to analyze these fusion rotein (Fig. 2 C) by size exclusion chromatography (SEC) (SEC).IgG-(G 4S) 4-HSA and IgG-(G 4S) 4-Domain III A280 to retention time (minute) on all demonstrate unimodal corresponding to monomer, show that fusion rotein do not assemble (in any measurable degree).SEC spectrum and the IgG-(G of the fusion rotein of IgG-YTE variant 4S) 4-HSA or IgG-(G 4S) 4The SEC spectrum of-HSA chimeric protein is undistinguishable.
8.2.3 equilibrium association constant (K D) measurement
At the affinity (Kd) of the upper measurement of BIAcore T100 instrument (Sweden, Uppsala) people FCRn to fused protein.In brief, use the described standard amine coupling of apparatus manufacturer chemical method, with human IgG, IgG-(G 4S) 4-Domain III, IgG-(G 4S) 4-HSA, IgG-YTE, IgG-YTE-(G 4S) 4-Domain III and IgG-YTE-(G 4S) 4-HSA is fixed on the flow cell of the separation on two Series5 sensor chips (GE health care companies) with high density.Final surperficial IgG density is respectively 5116,5258,6097,5256,5561 and 5531RU.Use identical fixed solution, also at each the sensor chip preparation reference flow cell without any protein.(scope is from 5.86nM to 3000nM, at 50mM PO with the twice serial dilution of people FcRn 4, in the 150mM NaCl buffer, under pH5.5) with the flow velocity of 5 μ L/min be expelled to the cell surface of albumen coupling and with reference to cell surface on.Collection continues 50 minutes in conjunction with data, carries out repeatedly injection in 60 seconds by the phosphate buffered saline (PBS) with the pH7.4 that contains 0.05% polysorbas20 subsequently and regenerates.Use BIAcore T100 assessment software 1.1 versions (BIAcore company, Sweden, the Uppsala), the association reaction (Req) of per injection when the balance drawn for concentration and it is fitted to stable state affinity model (Fig. 3), thereby obtain equilibrium association constant K DIllustration represents the combination of FcRn and the fixed ligands of a series of concentration.Also obtain in an identical manner the K of IgG-YTE variant and corresponding fusion rotein D(data do not show).
1.51 μ M K with independent IgG DCompare IgG-(G 4S) 4The KD of-HSA is 183nM and IgG-(G 4S) 4The K of-Domain III DBe 305nM, prove that fusion HSA or Domain III have improved FcRn adhesion (table 2) with 10 times and 5 times respectively to IgG.
Equally also observe the similar of YTE variant but more inapparent trend, wherein, with respect to IgG-YTE, IgG-YTE-(G 4S) 4-HSA shows that affinity improves 3.8 times (42.5nM), and IgG-YTE-(G 4S) 4-Domain III shows that affinity improves 2.5 times (65.1nM) (table 2).
This table of table 2. provide under pH5.5 with and the IgG that merges of HSA or and the equilibrium constant that is derived from SPR of the IgG that merges of Domain III and YTE variant analog thereof the people FcRn of being combined.
Construct K D(nM)
IgG 1510
IgG-(G4S)4-HSA 183
IgG-(G4S)4-DIII 305
IgG-YTE 161
IgG-YTE-(G4S)4-HSA 42.5
IgG-YTE-(G4S)4-DIII 65.1
Yet the raising of affinity does not affect the FcRn combination under neutral pH under pH5.5.This tests by inject 1 μ M FcRn at identical fixed surface under pH7.2.Than IgG/IgG-YTE contrast, with the FcRn of any fusion rotein coupling surface combination in do not detect measurable difference (data do not show).
Under acid pH (about 5.5-6.0), the HSA fusions is combined with FcRn, and make in the release of neutral pH (about 7.4) relevant with effect in the body because such characteristic simulation Binding in vivo.Therefore, preferred HSA variant has the variant of the affinity of raising and the variant of (ii) observing the affinity of raising under acid pH for (i) with respect to natural HSA or the conjugate that comprises natural HSA.And the variant that under neutral pH FcRn is had a binding affinity of increase may damage effect and reduce the advantageous effect of the affinity that increases under acid pH.
8.3 example 3: strengthen the serum persistency of human IgG with the fusion of total length HSA
This experiment showed, that the fusion of HSA and antibody has increased the serum half-life of antibody.As shown in FIG. 7, the serum persistency that merges at the IgG-HSA described in the example 1 has increased than independent IgG.The increase of serum half-life be suitable from IgG-YTE variant finding.Yet, in this research, add HSA to IgG-YTE variant and seem and can not cause remarkable enhancing than independent YTE.
Use the people FcRnC57BL/6 transgenic mice (JAX laboratory) at 4-5 monthly age to carry out PK research, described transgenic mice has the mice neonatal Fc receptor (mFcRn) with people FcRn (huFcRn) replacement of single copy.Via the tail vein to the suitable protein in the phosphate buffered saline (PBS) that is diluted in pH7.2 of injected in mice 15mg/kg dosage.All animals are collected (75 μ l) serum rear 1 hour of injection from vena orbitalis posterior clump (retro-orbital plexus) blood-letting be injected at actual amount the circulation with mensuration.Then after injection, collected blood serum samples and be stored in-80 ℃ in 24,72,168 and 240 hours.Amount by the indicator protein that keeps in the elisa assay serum.In brief, catch various IgG fusion constructs with the plate that anti-_ HSA is coated, and use anti--Kappa to detect the described plate of antibody test.For IgG and YTE construct, catch IgG and use anti-heavy chain to detect the described plate of antibody test with antigen coated plate.It is the amount (1 hour sample) of injecting and the mark of time that the % of the protein that keeps in the serum is drawn.
8.4 example 4: the epi-position for FcRn on HSA is conformation
As by visual with the immunoblotting of anti-beta-2 microglobulin antibody, observe people FcRn and be combined with natural HSA hole in the concentration dependent mode.Yet, use the degeneration HSA that under similar experiment condition, tests not observe similar result.
Will be from the human serum albumin (HSA of Sigma-Aldrich with 10mg protein/ml agarose (Sepharose); Catalog number (Cat.No.) A-8763), human IgG (hIgG; Catalog number (Cat.No.) I-4506), the Tris buffer is fixed on the sepharose 4B (GE health care companies) of CNBr activation.By with 0.1M Trizma alkali, 0.5M NaCl(pH8) reactive group of the sepharose 4B of blocking-up CNBr activation prepares agarose-Tris.In the presence of SDS, the agarose gel pearl (20 μ l pearls are equivalent to the protein that about 180 μ g connect) that is connected to HSA, hIgG or Tris was boiled 10 minutes, described SDS contains under reduction (1%2-mercaptoethanol) or non-reduced condition or untreated sample buffer (60mM Tris(pH6.8), 2.3%SDS, 10% glycerol, 0.01% bromophenol blue).Under pH5.5 with the 50mM sodium phosphate, contain 0.1% fish glue (BIOFX Laboratories company, catalog number (Cat.No.) PFGP-1000-01) protein that the washing of 150mM NaCl buffer is so processed or the pearl of Tris coupling, and then make itself and the people FcRn(0-20 μ g of 200 μ l variable concentrations in the pH5.5 buffer) at room temperature hatched 2 hours.Use the buffer of pH5.5 to wash unconjugated protein.By in connection with protein boil its eluting with the sample buffer that contains SDS that contains the 1%2-mercaptoethanol, and analyze at sds page, then carry out immunoblotting with anti-β2-microglobulin antibody (Abeam catalog number (Cat.No.) Ab6608).
For whole FcRn concentration range, be maximum for the people FcRn of natural HSA and the combination of agarose-HSA.Yet when making the HSA degeneration under reduction and non-reduced condition, in conjunction with significantly reducing, the epi-position most probable that shows the FcRn on HSA is comformational epitope (Fig. 4).As expected, agarose-IgG is in conjunction with people FcRn, and the pearl of Tris blocking-up under any condition not in conjunction with FcRn.
8.5 example 5:HSA and Domain III can be illustrated on the yeast cell surface and the protein of showing has kept the FcRn binding ability
Evaluate such as the improvement flow cytometry that carries out under at acid pH, this examples prove can be on yeast cell surface successful expression HSA and Domain III, and the protein of these displayings in pH dependency mode in conjunction with FcRn.Therefore, provide a kind of screening (for example to contain in Domain III vicissitudinous construct in the expression on the yeast cells, the HSA of independent Domain III, total length HSA, truncate or comprise at least chimeric polyeptides of Domain III) method, thereby evaluate such construct (i) in conjunction with the ability of FcRn, and (ii) with respect to the affinity of for example increase of the non-variant construct ability in conjunction with FcRn.
8.5.1 yeast cell surface is showed
Be cloned into HSA, Domain III or Single-Chain Fv Fragment of Murine (scfv) in the pYD1 yeast display carrier (the catalog number (Cat.No.) V835-01 of hero company) and be transformed in the saccharomyces cerevisiae, be used for presenting in yeast cell surface.Under the control of galactose inducible promoter, pYD1 is shown as interested protein the C end fusions with saccharomyces cerevisiae a-protein ga2p.Described in supplier's handbook, carry out all test procedures.Use auxotroph selected marker uracil and tryptophan to select the cell that transforms, and it is cultivated in suitable selection culture medium (Teknova company, catalog number (Cat.No.) C8140).Culture medium induced with galactose reach 48 hours allow to express the Aga2p fusion rotein.At 0,24 and 48 hour cell is taken a sample.With cell sample washing and with the PBS(pH7.2 that contains 0.1% fish glue) blocking-up, dye with the anti-HSA antibody of rabbit polyclonal (Abeam company, catalog number (Cat.No.) AB34669) of FITC combination, and pass through flow cytometry.
In the time of 0 hour, do not observe the cell surface expression of HSA or Domain III, and in the time of 24 hours, observe this two kinds of protein expression on yeast cells.Such expression kept after inducing 48 hours.Making to express dyes visual by positive FITC.Fig. 5 A show can be on yeast cell surface successful expression HSA and Domain III.As expected, the cell of scfv conversion is not with anti-HSA dyeing (for example, negative).Therefore, the cell that scfv is transformed is as negative control.
8.5.2 the FcRn binding ability of the HSA of surface expression or Domain III
The yeast cells of expressing HSA, Domain III or scfv and induced with galactose 48 hours was blocked 1 hour with the 50mM sodium phosphate of pH5.5, the 150mM NaCl buffer (being also referred to as the FACS buffer) that contains 0.1% fish glue.Then with cell and the biotinylated FcRn(70 μ M in the pH5.5 phosphate buffer) hatch, and use Streptavidin phycoerythrin (PE) (Invitrogen company) to make the FcRn of combination visual.50mM sodium phosphate, 150mM NaCl buffer with pH5.5 replace the conventional PBS that uses by the cell of flow cytometry dyeing like this.
Such as the transformation from rectangular histogram as seen, than scfv(Fig. 5 B), HSA and Domain III express cell are to the PE dyeing that all is positive, and negative control scfv express cell is not, proves that the HSA that expresses in yeast cell surface and Domain III have kept the FcRn binding ability and is functional therefore.In independent experiment, processing cell with the similar mode of the low pH flow cytometry of being combined for FcRn, and use the high flux sampling technique, System (IntelliCyt company) measures, and has obtained similar result.
8.6 example 6: comprise the adenovirus mammalian cell surface display carrier of OriP for generation of having highly multifarious library
Use exists for glycosyl-phosphatidyl inositol (GPI) anchor of surface display scFv-Fc albumen
Figure BDA0000365918090001162
Make up the mammal display library in the entry vector, described entry vector is engineered to the scFv-Fc expression cassette (referring to Fig. 8 A) that contains called after pENDisplay.The library of different scFv sequences is easily inserted in the Sfi/NotI site.According to the explanation of manufacturer, with the library in the pENDisplay carrier and pAd/PL_DEST TMCarrier (hero company, catalog number (Cat.No.) V494-20) combination to produce the gland virus expression library, obtains altogether about 5x10 6Individual colony forming unit (cfu).In order to produce adenovirus, with gland virus expression library transfection 293A cell (hero company, catalog number (Cat.No.) R70507), described cell contains the stable integration copy of E1 gene, it provides the required E1 albumen of trans generation recombinant adenovirus (E1a and E1b), and this gland virus expression library is linearized to expose left side and right side oppositely terminal repetition (ITR).Find that by facs analysis at least 50% the 293A cell with linearisation adenovirus library direct transfection is showed antibody in its surface.Yet, when linearizing adenovirus library being transfected in the 293A cell for generation of adenovirus, every 110mm(diameter) and culture dish obtains to be less than 50 plaques.Separate at the 10th day results adenovirus and with viral DNA, use pcr amplification again to clone the scFv coding region that is back in the pENDisplay carrier, select 96 colonies and it is checked order.From 96 clones that analyze, only identify the VH sequence of 14 uniquenesses.The poor efficiency that plaque reclaims may and cause the remarkable reduction of adenovirus library complexity owing to the degraded of linearisation adenovirus vector.
Epstein-Barr nuclear antigen 1(EBNA-1) contains nuclear localization signal (NLS) and be combined with the OriP that contains nucleic acid (such as plasmid).EBNA-1 albumen (referring to Fig. 9 A) can help to make the OriP transposition that contains nucleic acid to nucleus and strengthen episome and keep via NLS.Although not thinking that adenovirus is rescued needs episome to keep, but still after the polyadenylic acid sequence of scFv-Fc box, between the attL1 of pENDisplay carrier and attL2 sequence, introduce OriP sequence (referring to Fig. 9 C).The new support of called after pENDisplay-OriP is depicted among Fig. 8 B.With the library among the pENDisplay-OriP and pAd/PL-DEST TMCarrier (hero company, catalog number (Cat.No.) V494-20) combination also has about 5 * 10 to produce 6The second gland virus expression library of cfu.To and be transfected into 293E cell (hero company from library linearisation that pENDisplay-OriP produces, catalog number (Cat.No.) R620-07) in, described 293E cytotostatic is expressed Epstein-Barr virus nuclear antigen (EBNA-1) and adenovirus E 1 a albumen, causing every 110mm(diameter) culture dish substantially exceeds 10,000 plaques.Also analyze as mentioned above 96 clones in the 7th day results virus.Compare with the uniqueness clone of isolated low quantity in first library in OriP site never, 96 VH sequences of all that analyze all are unique.These results prove that together interpolation OriP sequence has strengthened from the cell of expressing EBNA-1 greatly to the gland virus expression carrier library rescues the ability of adenovirus and the multiformity in adenovirus library.
Add OriP sequence (for example, Fig. 9 C) to adenovirus vector and strengthened the efficient that in the presence of EBNA-1 protein, produces the recombinant adenovirus granule from host cell.When making up the gland virus expression library, clone's quantity that the viral generation efficiency of enhancing is lost by minimizing keeps the multiformity/complexity in library.Hereinafter example 7 has described structure and the screening of the mammal display library of expressing HSA Domain III variant in detail, and described variant is attached to the OriP sequence in the substantially aforesaid adenovirus expression carrier.
Figure 10 provides the sketch map of the representative general adenovirus expression carrier of expressing interested one or more protein.In this example, sudden change adenoviral gene group is provided, wherein lacked E1 and/or E3 part.Rescue to be used for virus by the trans viral gene of disappearance that provides of host cell.These disappearances prevent that adenovirus from copying for the host cell of expressing interested one or more protein.Interested DNA will comprise all component that protein expression is required, includes but not limited to, and coded sequence, promoter sequence, termination signal, polyA sequence, etc.Interested protein can be soluble maybe can comprising with the sequence of protein grappling to the cell surface, such as membrane spaning domain or GPI grappling signal.As illustrational at this, adenovirus vector can be engineered to the library of expressing misfolded proteins.The adenovirus expression carrier of describing in Figure 10 provides the position of att recombination site, and described site is derived from uses the Gateway that is used for structure TMCross the threshold and the purpose plasmid.Also described the possible position that the EBNA-1DNA sequence may be positioned.Other positions and the orientation of carrier component have been expected.Those skilled in the art should be understood that the orientation in carrier zone and/or relative position can change.
8.7 example 7: be used for the use of other display platforms of HSA and Domain III
Phage display and mammalian cell surface display technique have been assessed equally as the potential display platform of HSA and Domain III.The phage display platform is not expressed any one in HSA or the Domain III on the bacterial cell surface, infer that this is owing to a large amount of disulfide bond (data do not show) in these molecules.Yet, the mammalian cell display systems of the temporary transient surface expression of use in the 293-F cell has successfully been showed HSA and Domain III, described 293-F cell mediates via glycosyl-phosphatidyl inositol (GPI) the grappling signal from decay accelerating factor (decay accelerating factor) (for example, the sudden change DAF described in the US2007/0111260).The protein of showing has kept FcRn binding ability (data do not show).
The wherein interpolation that has produced called after pEN-HSA-GPI (for example is used for amphophilic epitope tag, the Flag label) other mammalian cell expression construct, and add joint increasing the flexible of fusion rotein and to promote HSA to be combined with FcRn (Figure 11 A) at 5 ' and 3 ' of HSA, this construct is directly used in transient transfection or for generation of called after pAd-HSA-GPI and also in conjunction with the adenovirus expression carrier of OriP sequence.In transient transfection is measured (data do not show) and use the gland virus expression system, expressive function HSA on from the surface of the mammalian cell (for example, 293F cell) of this construct.Figure 11 has shown with anti-HSA(Figure 11 B) or the FcRn(of 25 μ g/ml and 5 μ l/ml be respectively Figure 11 C and Figure 11 D) transformation of cell gained in rectangular histogram of dyeing.Therefore, exist for the some potential system of expressing HSA and Domain III variant, and be used for this type of variant that screening meets the following conditions: (i) be combined with FcRn and (ii) have an ability of being combined with FcRn with respect to the affinity of for example increase of non-variant construct.
Transfection 293F cell is showed HSA at cell surface:The plasmid of 1.5 μ g and the 293fectin of 2.25 μ l are added in the Optimem culture medium (hero company) of 100 μ l in independent pipe, at room temperature hatch 5 minutes, and then these two components are merged together.After at room temperature hatching other 20 minutes, the density that mixture is added in 24 deep-well plates is 1 * 10 6In the 2ml293F cell of cell/ml.At 8%CO 2Make transfectional cell growth 24 hours with the 250rpm rotating speed under existing.
The generation of adenovirus expression carrier and useUse
Figure BDA0000365918090001181
Technology (hero company) recombinates in the Invitrogen purpose carrier HSA in the entry vector (pEN-HSA-GPI) to produce adenovirus expression carrier.In brief, with the pEN-HSA-GPI carrier of 150ng, the pAd/PL-DEST(hero company of 300ng), the LR Clonase II(hero company of 2 μ l) and the TE buffer be added in the reactant mixture of 10 μ l altogether.After 25 ℃ of lower overnight incubation, follow the scheme of manufacturer, the reactant mixture of 2 μ l is used for transforming One-shot Top10 competent cell (hero company).With the TOP10 cell plating that transforms to the plate of ampicillin and 37 ℃ of lower overnight incubation.Single colony is selected to the LB culture medium with the preparation plasmid.The HSA gene that will contain adenovirus expression carrier used Pac I linearisation to produce adenovirus before transfection.Come transfection 293E cell to produce adenovirus with the linearisation adenovirus vector of 2 μ g and the lipofecatine-2000 of 6 μ l.After through 7 days transfection, by alternately freezing (under-80 ℃) and thawing (under 37 ℃) discharge adenovirus 2 to 3 times from the cell of transfection.Removed cell debris in 10 minutes by the 3000rpm centrifugalize, and the adenovirus that will contain supernatant is distributed in the new test tube and-80 ℃ of lower preservations.According to the operation instruction of manufacturer, use the quick titration test kit of Adeno-XTM (Clontech:PT3651-2) to determine virus titer.Be to use adenovirus to express the HSA construct 1 time at MOI.
8.8 example 8: the surface on DIII exposes the alanine scanning mutagenesis of ring to identify that FcRn is in conjunction with epi-position
This example has been described on the surface on the Domain III and has been exposed ring in the effect of adjusting FcRn in HSA is combined.
Domain III is comprised of 205 amino acid residues and encodes via 9 ring connections and by 6 people such as 10 spirals (Sugio(China fir tail) that disulfide bond is stable, 1999, Protein Eng.(" protein engineering ") 12:439-46 and PDB:1BM0).The below lists the position of the amino acid residue that comprises these rings and the length of each ring.Aminoacid numbering is with respect to the position of these rings in the ripe HSA albumen of total length (SEQID NO:2):
Ring 1: a residue 398-400=3 aminoacid
Ring 2: a residue 415-419=5 aminoacid
Ring 3: a residue 439-443=5 aminoacid
Ring 4: a residue 468-470=3 aminoacid
Ring 5: a residue 480-482=3 aminoacid
Ring 6: a residue 492-509=18 aminoacid
Ring 7: a residue 516-517=2 aminoacid
Ring 8: a residue 537-541=5 aminoacid
Ring 9: a residue 561-564=4 aminoacid
Be numbered 2,3,6,8 and 9 ring and be solvent can and and be exposed on the molecular surface people such as (, ibid, and PDB:1BM0) China fir tails.
In some instances, the amino acid mutation that replaces in each independent surface can and be encircled is become alanine (except the proline and cysteine that are not suddenlyd change), the residue of sudden change odd-numbered in one group wherein, and in another group the residue of sudden change even-numbered.Except encircling 9, each ring produces two such sudden change groups, and its medium ring 9 only needs a construct to satisfy experimental design.Produce in carrier altogether that 9 such constructs carry out cell surface display (for example, pYD1 yeast display carrier), and for assessment of the FcRn binding ability.Can use the described standard external test of the application (for example flow cytometry) assessment variant.Identify and show the one or more variants to the FcRn affinity that improve.Can also screen each variant with determine to improve whether the FcRn affinity is only occurred at acid pH, and under neutral pH, not occur.For (i) independent variant structure territory III construct; (ii) the variant structure territory III construct that in total length HSA situation, exists; Or (iii) at truncate HSA or comprise in the situation of the chimeric polyeptides of Domain III at least, can test that the affinity to FcRn increases under acid pH, and under neutral pH, do not increase.With aforementioned circumstances and wild type Domain III, wild type full-length HSA or the chimeric polyeptides that do not contain sudden change relatively.
In some instances, the information that obtains from aforementioned screening identified such surface can and ring residue, it is easy to change simultaneously and keeps (or even improving) FcRn binding ability.Made up a series of variant, wherein the position of such evaluation sported other 20 seed amino acids each, and screened such variant.Then make up and screen other variants that comprise sudden change in more than one position.
In some instances, produce and assessed the variant library.
8.9 example 9: the alanine scanning mutagenesis of residue conservative, that the surface exposes
By the aminoacid sequence identify can reach amino acid residue for conservative surface in the Domain III in the different animal species of l3.Such conservative residue is mutated into separately alanine to determine its effect in the FcRn combination.
With the Domain III aminoacid sequence of HSA with from the serum albumin Domain III sequence of 12 different plant species relatively, these 12 species comprise rat, mice, cattle, Canis familiaris L., rabbit, pig, chicken, donkey, mongolian gerbil, sheep, cat and horse, and have identified the residue (Fig. 6 A-D) for guarding in all these species.Because chicken HSA is obviously different from mammal HSA albumen, only therefore provides and compared (Fig. 6 E-H) for second of mammalian species.By ELISA, immunoblotting and SPR, show and be combined (data do not show) with people FcRn from the serum albumin of pig, rat, mice, Canis familiaris L., sheep, rabbit and cattle.In independent analysis, use can be in the Internet (http://curie.utmb.edu/getarea.html) GET ARE A of obtaining β software l.0, identified the residue that the surface in Domain III exposes.Accessible surface area and the gradient thereof of the atom that this computed in software is independent, and the probability of the surperficial accessibility of each amino acid residue given a mark are expressed as " i " or " o ", represent respectively unreachable and can and (table 3).As calculating by software and confirming by the manual detection to the HSA crystal structure, identified in all these different plant species to be aminoacid conservative and that expose for the surface.(in table 3, adding frame)
All 18 seed amino acid residues of so identifying are mutated into separately alanine and use cell surface display (for example, pYD1 yeast display systems) assessment on the impact of FcRn combination.Introducing Domain III sudden change, and in following one or more situations, it being screened: the HSA of independent Domain III, total length HSA albumen, truncate or comprise at least chimeric polyeptides of Domain III.Cysteine and the proline guarded, the surface exposes are not included in this analysis.
In another example, all 18 seed amino acid residues (can not allow replacement if the alanine experiment shows ad-hoc location, then be less than all 18 kinds) are mutated into individually each of other 19 seed amino acid residues and use pYD1 yeast display systems (or another kind of display systems) assessment on the impact of FcRn combination.
In another example, made up and assessed the variant of the combination that comprises sudden change.Can use the standard external test described in the application (for example flow cytometry) assessment variant.The one or more variants to the affinity of FcRn that show raising have been identified.Can also screen each variant and whether only occur at acid pH with the affinity to FcRn of determining to improve, and under neutral pH, not occur.For following one or more situations: (i) independent variant structure territory III construct; (ii) the variant structure territory III construct in total length HSA situation, or (iii) at truncate HSA or comprise under the situation of the chimeric polyeptides of Domain III has at least been tested at acid pH rather than the affinity to FcRn that improves under neutral pH.Aforementioned circumstances and wild type Domain III, wild type full-length HSA or the chimeric polyeptides that do not contain sudden change are compared.
Table 3. this table has been described all the amino acid whose solvent accessibility parameters in Domain III.Be that conservative these residues (the ripe total length HSA that provides in respect to SEQ ID NO:2 is numbered) show with runic and labelling (##) in all species of in Fig. 6 A-D, comparing, and be that the surface can reach and conservative residue adds frame in the species of all comparisons. http://curie.utmb.edu/getarea.html。And that compares in Fig. 6 E-H is labeled () for conservative residue in all species except chicken.With the residue note be (i) and (o) and surface unreachable with presentation surface respectively can and.
The nonpolar main chain side chain ratio of residue sum (%) can and/unreachable
Figure BDA0000365918090001221
Figure BDA0000365918090001241
8.10 example 10: each residue on the Domain III sport all possible aminoacid to produce the library of single amino acids mutant
Except cysteine and proline, each aminoacid in the Domain III is sported all 20 seed amino acids (namely, wild-type amino acid and all 19 kinds of non-wild-type amino acids) to produce mutant library, so that each single mutant only has a single mutation a position.In library construction, contain 205 amino acid whose whole length; Therefore the 184 kinds of residues that suddenly change seriatim cause total library multiformity of 3496.Introducing Domain III sudden change, and in following one or more situations, it being screened: independent Domain III, total length HSA albumen, truncate HSA or comprise at least chimeric protein of Domain III.Can produce the Domain III mutant library by the Application standard method of mutagenesis.Randomly or alternately, the library by commercial facility (for example Geneart AG company, Germany) preparation Domain III mutant.Mutant library is cloned in the display carrier, and use in this application that described standard external test (for example flow cytometry) screens its FcRn binding ability, described display carrier is above-mentioned pYD1 yeast display carrier or mammal display carrier pEN-HSA-GPI for example, its comprise the OriP sequence with the generation that strengthens recombinant adenovirus (referring to, comprise the sketch map of the general entry vector of OriP among Figure 10).The one or more variants to the affinity of FcRn that identify to show improve.Also can screen each variant and whether only under acid pH, occur with the affinity to FcRn of determining raising, and under neutral pH, not occur.For (i) independent variant structure territory III construct; (ii) be present in variant structure territory III construct in the total length HSA situation; Or (iii) in the situation of chimeric polyeptides, test improves under acid pH the FcRn affinity, and improves under neutral pH.With aforementioned circumstances and wild type Domain III, wild type full-length HSA or the chimeric polyeptides that do not contain sudden change relatively.Experimental design allows to identify in conjunction with epi-position, and raising is to the sudden change of the affinity of FcRn.
Produced as mentioned above composite structure territory III(DIII) library, it has 6 * 10e4 independently transformant.Although the library is designed so that each independent mutant only has single mutation a position, but still has produced a plurality of dual or even triple mutants.Synthetic DIII library is assembled to form total length HSA library by overlapping PCR by pcr amplification and with DI and DII.With Sfi I and EcoR I digestion and be cloned among the carrier pEN-HSA-GPI of similar digestion, its mammal for the enhancing that comprises the OriP sequence as indicated above is showed Gateway with PCR TMEntry vector (referring to, Fig. 8 B and 10 for example).The primer that is used for amplification DIII library has six (6) wild-type amino acid residues at N and C-terminal, has therefore eliminated these 12 the amino acid whose multiformity at Domain III.In the pEN-HSA-GPI carrier, produce two libraries and except using 12 μ gPAC I linearisation adenovirus expression carriers for the generation of library, produce substantially as mentioned above corresponding adenovirus library.The size in each library is shown in (table 4).Check the multiformity in pEN-HSA-GPI library, in library 1: about 50% clone is wild type, and in library 2: about 30% clone is wild type.
Table 4:DIII library size
? pEN-HSA pAd-HSA
Library
1 1.3×10 6 5.4×10 6
Library 2 6.4×10 6 1.7×10 7
Basic as described in the example 5, the cell that infects gland virus expression wild type HSA, HSA-DIII literary composition village, often used in village names l or HSA-DIII library 2 is dyeed with anti--HSA-FITC antibody and FcRN-biotin (detecting with SA-PE), and as described below by facs analysis/screening.The expression in wild type HSA and two libraries is (Figure 12 A) quite.When dyeing with 10 μ g/ml FcRn, the cell mass of only expressing the HSA-DIII library has shown the transformation (Figure 12 B) in rectangular histogram.Figure 13 has shown corresponding amphophilic FACS scattergram (FACS profile).As described below, the cell mass of enrichment is from minute choosing recoverys, increase, and stands second by sorting and take turns enrichment or for separating of independent clone.Rectangular histogram from Figure 14 A as seen, the expression in wild type HSA, initial library and sorting library is suitable.Yet, showing that with FcRn dyeing the 1st takes turns the cell of expressing the HSA-DIII mutant with the 2nd enrichment of library of taking turns sorting, described mutant can be combined (Figure 14 B and C) with the low concentration that l μ g/ml even 0.1 μ g/ml present with FcRn.
Basic as described in the example 2, except experiment is carrying out under the pH7.2, separated a plurality of independent clones (as described below) and for pH dependency FcRn in conjunction with screening.Figure 15 has shown the A at pH5.5(figure) and pH7.2(figure B) lower control cells, wild type HSA and several representative representative rectangular histogram of cloning.Expression between these HSA mutants and wild type HSA is (Figure 15 C) quite.As shown in Figure 16, keep the pH dependency in conjunction with (namely to being accredited as, preferential combination at low pH) 16 clones check order and can use some FcRn concentration (for example, the FcRn-biotin of 0.1 μ g/ml, l μ g/ml and 10 μ g/ml) to make these clones stand other facs analysis to analyze these sudden changes to the relative affinity of FcRn together with wild type HSA and control cells.Before carrying out any other sign, other about 1100 clones from enriched populations are checked order.Table 5 provides gathering at the aminoacid replacement that identifies from the clone of library separation and/or order-checking.The position of the positional representation of overstriking between about 1%-5% clone found to replace and can be called as " preferred point ".Overstriking and the underlined positional representation position in surpassing 5% clone is found to replace and can be called as " focus ".Only in the situation of dual/tri sudden change, identify the aminoacid replacement (the AA hurdle of replacement) of listing with italic.The aminoacid replacement (the AA hurdle of replacement) of finding in 5 or more clone represents with runic.The compositions of finding in three or more clones shows with runic that also clone's quantity of wherein identifying is presented at parenthetic.The aminoacid numbering is with regard to the ripe total length HSA in being provided at SEQ ID NO:2.Separated to contain in the same amino acid at same position place and replaced and/or a plurality of clones (referring to table 5) that the different aminoacids of same position replaces, show that these positions can represent mutantional hotspot.
The positional representation of the some preferred and/or focus on the HSA analytic structure is on Figure 17.Except aminoacid 407,415 and 463, most of focuses and preferred point are found in the ring 6 and the 7(that iris out among Figure 16 and contain respectively residue 492-509 and 516-518) and spiral 7(contain respectively residue 510-515 and 519-536) in.
Gathering of the DIII sudden change that table 5 is identified
Figure BDA0000365918090001271
Figure BDA0000365918090001281
Figure BDA0000365918090001291
Figure BDA0000365918090001301
Figure BDA0000365918090001311
Figure BDA0000365918090001321
Figure BDA0000365918090001331
* with respect to ripe total length HSA(SEQ ID NO:2) be numbered; The number of times that the amino acid residue heel that replaces is found with a numeral residue; Usually list these combinations in being expert at for each position of finding in the combination
Ω for 2 among 3 clones in the position 407 uncertain sequences
At first be accredited as clone 12 L463N
Euro at first be accredited as clone 45 L463N/T508R
$ is the 3rd replacement of w/a (for example, Y411L, V433T, E495D, A504G, E531G, 571K) occasionally
These clones can also have a disappearance at 523 places
Figure BDA0000365918090001343
At first be accredited as clone 46 I523Y
By direct mutagenesis, produced the mutant of some evaluations like this, be soluble protein, and as described below being purified being used for analyzed further.As described below by ProteOn() and/or BIAcore(is substantially as mentioned above) determine that binding affinity is (at the K of pH5.5 D) and pH dependency (7.2vs.5.5).Table 6 provides gathering of binding and has comprised protein density for these researchs.Find that a plurality of mutants have the combination of enhancing under pH5.5, comprise L407Y/Q526T; L463N/T508R; I523G; V424Q; L83N/128R/I143G; And K144, be presented in the table 6 with runic.The mutant that all these tests are presented at pH5.5 enhancing combination also is found to keep simultaneously the pH dependency in conjunction with (referring to the last string of table 6).Some mutants of find measuring have unaltered under pH5.5 or even the K that increases D(being unaltered or worse combination).Why identify such clone and have some possible reasons, for example, these sudden changes can strengthen protein expression or the stability in display systems.Also possiblely be, be used for the GPI anchor showed in conjunction with making some difference, these mutants can compensate the impact that can not repeat when mutant HSA is expressed as soluble molecule.In addition, the optimization of design of screening technique is to catch the stable mutant of dissociation rate, and these mutants may or may not change into global affinity and improve.What also might reflect is that these sudden changes provide enhancing when making up with one or more other sudden change.In combination, find a plurality of sudden changes (referring to table 5).
Table 6 binding gathers
Figure BDA0000365918090001342
Figure BDA0000365918090001351
Not test of blank expression; Nb is illustrated in not combination under the employed condition
* estimate-weak affinity+high concentration causes Req only to have edge curvature * * assessment-strong affinity-low concentration curve to be combined and not reach " real " stable state with the isothermal of concentration under 10 μ M
Cell dyeing and facs analysis and sorting:With 1 * 10 63,000 ten thousand 293F cells the under/ml density infect under MOI=1 with HSA adenovirus library.Washed with cold FACS buffer by centrifugal cell harvesting in 16 hours after transduction, and with about 1 * 10 7Individual cell/ml carries out resuspending.Divide in the first round and to choose with 10 μ glml(212nM) add biotinylation FcRn.After cell hatched 60 minutes under 4 ℃, with FACS buffer washed twice and with the dilution factor of 1:500 it is suspended among Streptavidin-PE again.After under 4 ℃, hatching 30 minutes, with FACS buffer washing once and for FcRn in conjunction with carrying out sorting, this so that under pH5.5 in conjunction with these cell enrichments of FcRn.The cell of amplification sorting is to carry out other screening.Take turns sorting for second, the cell mass of sorting enrichment as mentioned above basically is except with l μ g/ml(21.2nM) use biotinylation FcRn with the further enrichment high-affinity HSA mutant.Equally with 0.l μ g/ml(2.12nM) enriched library is carried out other analysis, referring to (for example) Figure 14.In some screenings, take turns in the screening in conjunction with " cancellation is selected " step the 1st or the 2nd, wherein sorting the enrichment of cell group to remove under neutral pH (pH7.4) cell in conjunction with FnRc.For identifying independent clone, be cloned into the mammalian expression vector with transient transfection 293F cell from the cell mass extraction viral DNA of enrichment and with DIII HSA variant.Basically as mentioned above, by flow cytometry, screen the independent pH dependency combination that is cloned under pH5.5 and the pH7.4.
The generation of HSA-DIII mutant and expression:The Application standard scheme (
Figure BDA0000365918090001361
II XL direct mutagenesis test kit, Agilent catalog number (Cat.No.) 200521) make wild type HSA sudden change, thus in the mammalian expression vector that uses specific mutagenic primer, produce some DIII mutants.According to the scheme of manufacturer, use 293fectin TMAt 293F cells mutant, and the Application standard program is at the upper purified mutant body of ANTI-FLAG M2 affinity gel (Sigma-Aldrich, catalogue #A2220) by transient transfection for transfection reagent (hero company, catalogue #Sku12347-019).All mutants of purification like this are evaluated purity and gathering by SDS PAGE and size exclusion chromatography (SEC) analysis.All mutants are judged as about 99% purity, without significantly assembling (surpassing 95% monomer) (data do not show).
ProteOn analyzes:Measure people FcRn to the affinity (KD) of HSA variant at ProteOn XPR36 protein interaction array system (BioRad).In brief, as described in manufacturer, use ProteOn amine coupling reagent box, with high density the HSA variant is fixed on the separated flow surface on the ProteOnGLM sensor chip.Final surperficial HSA density is between 3740-3950RU.Use identical fixed solution, also prepared the reference flow surface at the chip without any protein.(scope is from 5.86nM to 10000nM, at 50mM P0 with the twice serial dilution of people FcRn 4, in the 150mM NaCl buffer, under pH5.5) with the flow velocity of 25 μ L/min be expelled to the albumen coupling cell surface and with reference to cell surface on.Collection continues 8 minutes in conjunction with data, regenerates by repeatedly injecting in 60 seconds with the pH7.4 phosphate buffered saline (PBS) that contains 0.05% polysorbas20 subsequently.Use ProteOn Manager software, the association reaction (Req) when the balance of per injection is drawn for concentration and it is fitted to stable state affinity model, thereby obtain equilibrium association constant KD.
Analyze based on this, the combinatorial mutagenesis of identifying is further analyzed to evaluate the activity of each independent sudden change.Whether the different components (construct that for example, is not had sudden change in screening by an above position of Direct Identification) that in addition, further screens to identify sudden change provides the affinity to FcRn that improves.
8.11 example 11: the combinatorial mutagenesis of the residue of selecting at Domain III
In order to check at example 10(above) in the combination of the highest frequency sudden change identified, produced a synthetic Domain III library, so that residue 407; 415; 463; 495; 508; 509; 511; 512; 515; 516; 517; 521; 523; 524; 526; 527; With 557 residues that sport at table 7 indicating, thereby produce so that each independent mutant has the mutant library of 2-4 sudden change.Alternately, the library can be produced so that each residue of listing is mutated into all 20 seed amino acids (that is, wild-type amino acid and all 19 kinds of non-wild-type amino acids) to check widely combination series.
Introduce the Domain III combinatorial mutagenesis, and in following one or more situations, it is screened: independent Domain III, total length HSA albumen, truncate HSA or comprise at least chimeric protein of Domain III.Can produce the Domain III mutant library by the Application standard method of mutagenesis.Randomly or alternately, the library by commercial facility (for example Geneart AG company, Germany) preparation Domain III mutant.
With the library clone of combination mutant in display carrier (for example above-mentioned pYD1 yeast display carrier or mammal display carrier pEN-HSA-GPI), and the standard external test described in use the application (for example, flow cytometry) screens its FcRn binding ability.Can adopt the positive and/or negative system of selection, as at described in the example 10 those.Identified and showed the combinatory variants to the affinity of FcRn that improves.Can also screen each combinatory variants and whether only under acid pH, occur with definite affinity that FcRn is improved, and under neutral pH, not occur.For (i) independent variant structure territory III construct; (ii) be present in variant structure territory III construct in the total length HSA situation; Or (iii) in the chimeric polyeptides situation, test improves under acid pH the affinity of FcRn, and improves under neutral pH.Can be with aforementioned circumstances and wild Domain III, wild type full-length HSA or the chimeric polyeptides that does not contain sudden change relatively.Alternately, or randomly, can be seriatim combinatorial mutagenesis and Domain III, total length HSA or the chimeric polyeptides that comprises each sudden change be compared, whether further increased affinity and/or the serum half-life to FcRn to determine this combination.Experimental design allows to identify and improves the affinity of FcRn and/or the combinatorial mutagenesis of serum half-life.
The sudden change of table 7 combinatorial library
The position Sudden change The position Sudden change The position Sudden change
407 N,Y 511 F 523 D,E,F,G,K,R
415 T 512 M,Y 524 L
463 F,N 515 Q 526 A,M,Y
495 D 516 T,W 527 Y
508 R,S 517 W 557 G
509 I,M,W 521 W ? ?
8.12 example 12: the FcRn affinity that the mutation of the residue on Domain III improves in order to screening to produce the single amino acid mutant
Conserved amino acid from Domain III is selected 18 kinds of single amino acids and is used in this application described standard method that it is mutated into alanine seriatim, so that each variant only has single mutation a position.At total length HSA albumen or alternately at truncate HSA or comprise in the chimeric protein situation of Domain III at least, introduce 18 kinds of variants and it is screened.The FcRn binding ability of using described standard external test in this application to screen 18 kinds of variants.Evaluation shows the variant to the affinity of FcRn of raising.Also screened every kind of variant and whether only under acid pH, occurred with the affinity to FcRn of determining raising, and under neutral pH, do not occurred.For (i) independent variant structure territory III construct; (ii) be present in variant structure territory III construct in the total length HSA situation; Or in the situation of affine polypeptide, test increases under acid pH the affinity of FcRn, and does not increase under neutral pH.Aforementioned circumstances and wild type Domain III, wild type full-length HSA or the chimeric polyeptides that do not contain sudden change are compared.
Analyze based on this, screen further to identify whether the various combination (construct that for example, has sudden change an above position) of sudden change provides the affinity to FcRn that improves.
8.13 other " focus " variant and combinatory variants
Position 463,508,523 and 524 is sported respectively all 19 kinds of non-wild-type amino acids and the group of 76 independent point mutation is basically cloned as mentioned above and expressed (referring to above, name is called the chapters and sections of " generation of HSA-DIII mutant and expression ").Use be coupled on the chip with from cell conditioned medium liquid, catch the HSA variant anti--HSA(is non--the Domain III conjugate) carry out BIAcore and analyze.Then with people FcRn by this chip allowing combination under pH5.5, and measured under pH5.5 association with dissociate.In this form, dissociation constant (kd (1/s)) will be more accurate than association constant.Binding constant for single mutant is provided in table 8, and this single mutant has than wild type HSA slowly at least about 2.5 times dissociation rate.As can be seen from Table 8, the mass mutation of these positions causes that the slower dissociation rate of comparing with wild type HSA and the FcRn of raising be combined.
The binding constant of table 8. " focus " single mutant
Sudden change ka(1/Ms) kd(1/s) KD(M) Rmax(RU)
K524L 3.39E+04 1.14E-03 3.36E-08 37.2
L463C 1.72E+04 1.34E-03 7.77E-08 155
I523D 3.94E+04 1.37E-03 3.48E-08 224
I523N 4.09E+04 1.58E-03 3.86E-08 109
K254I 2.07E+04 1.71E-03 8.22E-08 176
I523H 3.51E+04 1.85E-03 5.27E-08 302
K524V 1.92E+04 1.85E-03 9.65E-08 373
I523C 2.95E+04 1.99E-03 6.74E-08 262
I523E 3.05E+04 2.03E-03 6.66E-08 224
L463G 3.16E+04 2.14E-03 6.78E-08 58.4
T508E 3.52E+04 2.17E-03 6.17E-08 160
K524M 2.76E+04 2.18E-03 7.88E-08 134
I523G 3.29E+04 2.23E-03 6.77E-08 92.1
K524T 2.04E+04 2.27E-03 1.11E-07 155
I523P 2.59E+04 2.39E-03 9.24E-08 188
L463Q 2.93E+04 2.42E-03 8.27E-08 428
I523Q 2.47E+04 2.44E-03 9.88E-08 272
I523L 2.68E+04 2.45E-03 9.14E-08 403
T508R 3.40E+04 2.53E-03 7.44E-08 259
L463H 3.19E+04 2.71E-03 8.50E-08 451
K524F 2.88E+04 2.95E-03 1.02E-07 25.3
L463M 3.31E+04 2.97E-03 8.96E-08 18.4
I523R 3.25E+04 3.01E-03 9.27E-08 150
T508C 2.70E+04 3.04E-03 1.12E-07 369
T508I 3.12E+04 3.05E-03 9.76E-08 34.6
I523K 2.75E+04 3.06E-03 1.11E-07 398
L463N 2.48E+04 3.13E-03 1.26E-07 360
L463F 2.60E+04 3.17E-03 1.22E-07 54
K524N 2.07E+04 3.27E-03 1.58E-07 242
I523F 3.42E+04 3.31E-03 9.69E-08 141
Be combined in example 10(above) in each " focus " sudden change of identifying produce one group of combination HSA double, triple and quadruple and suddenly change.Basically as indicated above, " focus " combination mutant cloned and expresses and definite equilibrium association constant and pH dependency FcRn in conjunction with (people and machin) (referring to above, name is called the chapters and sections of " generation of HSA-DIII mutant and expression " and " kinetic measurement ").By pH5.5 and 7.4 times, inject the pH dependency that 3 μ M FcRn calculate the FcRn combination with the flow velocity of 5 μ l/min, and obtain through the balancing response speed of pH5.5 to pH7.4.In at table 9, sum up, show that each " focus " combinatorial mutagenesis with quadruple sudden change of at least 2-3 times of affinity raising has shown maximum raising (20-75 doubly).In addition, some mutants have been kept about wild type level (L463N/T128R of pH dependency FcRn combination; L463N/K524L; T508R/I523G; And L463N/T508R/I523G).Some " focus " combination HSA kinetics that is derived from SPR and the equilibrium constant of variant are provided in table 10.In addition, the equilibrium constant that is derived from SPR for some " focus " combinatory variants of machin FcRn is shown as similar to people FcRn being seen those (tables 9) with pH dependency (table 10).
Table 9 is for the equilibrium constant that is derived from SPR and the pH dependency of people FcRn
Figure BDA0000365918090001401
In the balancing response speed that arrives pH7.4 through pH5.5 * 100
Table 10 is derived from kinetics and the equilibrium constant of SPR
Figure BDA0000365918090001403
Table 11 is for the equilibrium constant that is derived from SPR and the pH dependency of machin FcRn
Figure BDA0000365918090001404
Figure BDA0000365918090001405
In the balancing response speed that arrives pH7.4 through pH5.5 * 100.
8.14 half-life measurement
Can be used at present unique mouse model of test concept (FcRn of raising will have the half-life of prolongation in conjunction with the HSA mutant) checking is huFcRn tgn mice (isozygoty KO and the human gene's of little musculus cdna heterozygosis the KI) (people such as Petkova S.B, (2006) International Immunology(" international immunology "), 18 volumes, 12 phases, the 1759-1769 page or leaf).From measuring the viewpoint of HSA half-life, this model has a large amount of restricted; (600 μ M) has the affinity higher 10 times than Wt HSA in conjunction with the albuminous high circulation composition of the mice serum of people FcRn.Yet, if tested HSA variant can successfully be competed with muroid serum albumin (MSA), can use this model.
External Biacore competitive assay is designed to prediction: the mutant of the affinity that show to improve whether can be successfully for huFcRn in conjunction with competing with muroid serum albumin (MSA).By premixing 1 μ MFcRn and the MSA(41nM-100 of a series of concentration μ M) and be expelled on the mutant HSA surface with test MSA and finish these experiments with respect to the HSA variant competition ability that FcRn is combined of winning.These data fittings are become single type competitive binding site model and calculate IC 50
Three mutant L463N/K524L have been tested for the first time; T508R/I523G; And L463N/T508R/I523G, each of these mutants has shown the affinity to FcRn that improves, and keeps pH dependency (referring to table 8).Find that T508R/I523G and L463N/K524L have the IC that 10-15 doubly is higher than Wt HSA or MSA 50Value (table 11).These IC 50Value is consistent with the dissociation rate measurement, shows that T508R/I523G and L463N/K524L and FcRn are compound more stable.
Then in huFcRn tgn Mice Body, determine each half-life (PK that vide infra analysis) of these three mutants.As shown in the table 11, in this model system, mutant L463N/K524L has shown 5 hours half-life raising, and mutant T508R/I523G has shown 3 hours half-life raising.Yet in this model, the removing of mutant L463N/T508R/I523G is faster than Wt.Because this mouse model of above pointing out is restricted, faster L463N/T508R/I523G removing may be false picture (artifact).Alternately, the sudden change stability that can affect the body internal protein maybe can affect its bio distribution.The elimination half life values of L463N/K524L and T508R/I523G is consistent with the dissociation rate scattergram of these mutants.Similarly, these data also have the IC that the competitive assay of MSA is carried out with use 50Measure consistent.
Table 11
HSA t1/2(hr) IC50(μM)
L463N/K524L 30.2 7.41
L463N/T508R/I523G 17.7 0.93
T508R/I523G 28.7 4.42
Wild type 24.7 0.72
PK analyzes: produced wild type HSA(Wt HSA) or L463N/T508R/I523G, T508R/I523G, L463N/K524L variant carry N end His10 label.As previously mentioned at all protein of 293F cells, and according to the explanation of manufacturer, use the HisTrap HP post (catalog number (Cat.No.) 17-5248-02) from GE health care companies to carry out purification.Use 5 mices/time point/group, via the tail vein to the Wt HSA of huFcRn tgn mouse vein infusion 15 mg/Kg or L463N/T508R/I523G, T508R/I523G, L463N/K524L variant, and 1,16,24,48,72,96 and the time point of 120hr collect blood serum sample.By the blood serum sample of elisa assay from these mices.In brief, elisa plate is produced with anti-His mAb(inside) coated, then hatch with suitable dilution blood serum sample, and with the anti-HSA polyclonal antibody of rabbit (AbCam catalog number (Cat.No.) AB60191) and the anti-rabbit igg HRP(Jackson of donkey Immunoresearch catalog number (Cat.No.) 711-035-152) detect the amount that is retained in the HSA/ variant in the serum.All zooscopies are all ratified through institutional review board (Institutional Review Board).
Use WinNonlin Professional(version 5.2, Pharsight company) carry out non-compartment analysis, to determine the pharmacokinetic parameter from average serum protein concentration-time graph.Use the logarithm trapezoidal method, by estimating from C P, last/ λ zLast measured value (C P, last) area (AUC), wherein λ under in addition the regional calculated curve ZIt is the slope of straight line of eliminating the least square fitting of phase by terminal.Calculate removing (CL) value of these protein according to CL=dosage/AUC, wherein dosage is the cpm of infusion.According to ln 2/ λ zCalculate half-life (t 1/2).
9 sequences
SEQ ID NO:1 – people HSA DIII protein sequence
VEEPQNLIKQNCELFEQLGFYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
SEQ ID NO:2 – people total length HSA protein sequence
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYELARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLLTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCLAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
SEQ?ID?NO:?18
VEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCV(X 1)HEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETF(X 2)FHADICTLSEKERQ(X 3)(X 4)KQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
Fig. 6 provides the comparison from the Domain III of the serum albumin protein of different plant species.
The wild-type amino acid sequence of the Domain III of rat serum albumin protein is listed as SEQ IDNO:3 in Fig. 6.The wild-type amino acid sequence of the Domain III of mice serum albuminous protein employed is shown in Figure 6 for as SEQ ID NO:4 and lists.The wild-type amino acid sequence of the Domain III of bovine serum albumin is listed as SEQ ID NO:5 in Fig. 6.The wild-type amino acid sequence of human serum albumin's Domain III is listed as SEQ ID NO:2 in Fig. 6.The wild-type amino acid sequence of the Domain III of dog serum albumin is listed as SEQ ID NO:6 in Fig. 6.The wild-type amino acid sequence of the Domain III of albumin rabbit serum is listed as SEQ ID NO:7 in Fig. 6.The wild-type amino acid sequence of the Domain III of porcine hemoglobin is listed as SEQ ID NO:8 in Fig. 6.The wild-type amino acid sequence of the albuminous Domain III of chicken serum is listed as SEQ ID NO:9 in Fig. 6.The wild-type amino acid sequence of the sero-abluminous Domain III of donkey is listed as SEQ ID NO:10 in Fig. 6.The wild-type amino acid sequence of the sero-abluminous Domain III of mongolian gerbil is listed as SEQ ID NO:11 in Fig. 6.The wild-type amino acid sequence of the albuminous Domain III of sheep serum is listed as SEQ ID NO:12 in Fig. 6.The wild-type amino acid sequence of the sero-abluminous Domain III of cat is listed as SEQ ID NO:13 in Fig. 6.The wild-type amino acid sequence of the albuminous Domain III of horse serum is listed as SEQ ID NO:14 in Fig. 6.
All publications and patent are hereby with it in full by reference and combination clearly and is individually shown by reference and combination as each independent publication or patent referred in this.In addition, for all purposes with U.S. Provisional Patent Application number: submitted on February 16th, 2010 61/304,954; And on July 15th, 2010 submit to 61/364,503 by reference with its in full combination.
Although specific embodiment of the present invention has been discussed, above-mentioned explanation is illustrative and nonrestrictive.After this description of summary and following claims, many modifications of the present invention it will be apparent to those of skill in the art.Should change to determine gamut of the present invention together with this type of by gamut and the description of reference claims and equivalent thereof.
Figure IDA0000365918150000021
Figure IDA0000365918150000031
Figure IDA0000365918150000061
Figure IDA0000365918150000081
Figure IDA0000365918150000091
Figure IDA0000365918150000101
Figure IDA0000365918150000111
Figure IDA0000365918150000121
Figure IDA0000365918150000131
Figure IDA0000365918150000141
Figure IDA0000365918150000151
Figure IDA0000365918150000161
Figure IDA0000365918150000171
Figure IDA0000365918150000181
Figure IDA0000365918150000191

Claims (32)

1. one kind comprises human serum albumin (HSA) polypeptide partly, this HSA partly comprises HSA Domain III or its neonatal Fc receptor (FcRn) binding fragment, wherein this HSA Domain III or its neonatal Fc receptor (FcRn) binding fragment are included in the aminoacid replacement of a plurality of positions that are numbered with respect to the position among the ripe HSA of total length, these positions are selected from lower group, and this group is comprised of the following:
(a) residue 463 and 508;
(b) residue 463 and 523;
(c) residue 463 and 524;
(d) residue 508 and 523;
(e) residue 508 and 524;
(f) residue 523 and 524;
(g) residue 463,508 and 523;
(h) residue 463,508 and 524;
(i) residue 463,523 and 524;
(j) residue 508,523 and 524; And
(k) residue 463,508,523 and 524,
Wherein these replace with respect to this HSA part wherein and do not comprise the contrast polypeptide of described aminoacid replacement and increase the one or both to affinity and the serum half-life of FcRn of this polypeptide.
2. a polypeptide that comprises SEQ ID NO:18, wherein X 1Not leucine and/or X 2Not threonine and/or X 3Not isoleucine and/or X 4Be not lysine, wherein this polypeptide is with respect to X wherein 1Leucine, X 2Threonine, X 3Isoleucine and X 4It is the one or both to the serum half-life of the affinity of FcRn and increase that the contrast polypeptide of lysine has increase.
3. polypeptide as claimed in claim 2, wherein X 1Cysteine, phenylalanine, glycine, histidine, isoleucine, agedoite, serine or glutamine; X 2Cysteine, glutamic acid, isoleucine, lysine, arginine or serine; X 3Alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, agedoite, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine; And X 4Alanine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, glutamine, threonine or valine.
4. polypeptide as claimed in claim 2, wherein X 1Agedoite and/or X 2Arginine and or X 3Glycine and/or X 4It is leucine.
5. such as each described polypeptide among the claim l-4, wherein this polypeptide is combined with FcRn with the affinity higher than described contrast polypeptide.
6. such as each described polypeptide among the claim l to 5, wherein this polypeptide is combined with FcRn with the affinity higher than described contrast polypeptide, and wherein said affinity is measured under acid pH.
7. such as each described polypeptide among claim l or the 5-6, wherein the replacement at residue 463 places is selected from L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q; Replacement at residue 508 places is selected from T508C, T508E, T508I, T508K, T508R, T508S; Replacement at residue 523 places is selected from I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W, I523Y; And the replacement at residue 524 places is selected from K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V.
8. polypeptide as claimed in claim 7, wherein the replacement at residue 463 places is selected from L463F, L463N; Replacement at residue 508 places is selected from T508R, T508S; Replacement at residue 523 places is selected from I523D, I523E, I523F, I523G, I523K, I523R; And the replacement at residue 524 places is K524L.
9. such as claim 7 or 8 described polypeptide, wherein the aminoacid replacement in the HSA Domain III is selected from lower group, and this group is comprised of the following:
(a)L463N/T508R；
(b)L463N//I523G；
(c)L463N/K524L；
(d)T508R/I523G；
(e)T508R/K524L；
(f)I523G/K524L；
(g)L463N/T508R/I523G；
(h)L463N/T508R/K524L；
(i)L463N//I523G/K524L；
(j) T508R/I523G/K524L; With
(k)L463N/T508R/I523G/K524L。
10. polypeptide as claimed in claim 9, wherein the aminoacid replacement in the HSA Domain III is L463N/T508R.
11. polypeptide as claimed in claim 9, wherein the aminoacid replacement in the HSA Domain III is L463N//I523G.
12. polypeptide as claimed in claim 9, wherein the aminoacid replacement in the HSA Domain III is L463N/K524L.
13. polypeptide as claimed in claim 9, wherein the aminoacid replacement in the HSA Domain III is T508R/I523G.
14. polypeptide as claimed in claim 9, wherein the aminoacid replacement in the HSA Domain III is T508R/K524L.
15. polypeptide as claimed in claim 9, wherein the aminoacid replacement in the HSA Domain III is L463N/T508R/I523G.
16. polypeptide as claimed in claim 9, wherein the aminoacid replacement in the HSA Domain III is L463N/T508R/K524L.
17. polypeptide as claimed in claim 9, wherein the aminoacid replacement in the HSA Domain III is L463N//I523G/K524L.
18. polypeptide as claimed in claim 9, wherein the aminoacid replacement in the HSA Domain III is T508R/I523G/K524L.
19. polypeptide as claimed in claim 9, wherein the aminoacid replacement in the HSA Domain III is L463N/T508R/I523G/K524L.
20. a peptide species comprises SEQ ID NO: aminoacid sequence.
21. such as each described polypeptide among the claim 1-15, comprise heterologous protein, wherein this polypeptide has kept the functional activity of this heterologous protein and can be combined with FcRn.
22. polypeptide as claimed in claim 21, wherein this heterologous protein comprises antibody or its Fab.
23. such as claim 21 or 22 described polypeptide, wherein this heterologous protein comprises therapeutic protein.
24. such as each described polypeptide among the claim 21-23, wherein this heterologous protein is combined with this HSA part with chemical mode or recombination form.
25. such as each described polypeptide among the claim 1-15, it is combined with non-proteinaceous matter, wherein this polypeptide has kept the functional activity of this allos non-proteinaceous matter and can be combined with FcRn.
26. such as each described polypeptide among the claim 21-25, wherein this heterologous protein or this non-proteinaceous matter are combined with the N-terminal aminoacid of this HSA part, the C-terminal aminoacid of this HSA part or the internal amino acid of this HSA part.
27. such as each described polypeptide among the claim 1-26, wherein this HSA part further comprises at least a portion of HSA domain I; Or at least a portion of HSA domain II; Or at least a portion of at least a portion of HSA domain I and HSA domain II.
28. a compositions comprises such as each described polypeptide among the claim 1-27 and a kind of pharmaceutically acceptable carrier.
29. treat the method that it is had the experimenter who needs, comprise to described experimenter giving according to claim 1 each described polypeptide or compositions according to claim 28 in-22 for one kind.
30. a nucleic acid construct comprises coding such as the nucleotide sequence of polypeptide as described in each among the claim 1-27.
31. one kind increases protein to the method for the one or both of the affinity of FcRn and its serum half-life, comprise make this protein with such as claim 1-15 or 27 in each described polypeptide be combined.
32. one kind increases non-proteinaceous matter to the method for the one or both of the affinity of FcRn and its serum half-life, comprise make this non-proteinaceous matter with such as claim 1-15 or 27 in each described polypeptide be combined.
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