CN1291995A - Enhancing circulating half-life of antibody-based fusion proteins - Google Patents

Enhancing circulating half-life of antibody-based fusion proteins Download PDF

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CN1291995A
CN1291995A CN99803277.8A CN99803277A CN1291995A CN 1291995 A CN1291995 A CN 1291995A CN 99803277 A CN99803277 A CN 99803277A CN 1291995 A CN1291995 A CN 1291995A
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S·D·吉利斯
劳健明
兰燕
J·维索洛夫斯基
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EMD Serono Research Center Inc
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Abstract

Disclosed are methods for the genetic construction and expression of antibody-based fusion proteins with enhanced circulating half-lives. The fusion proteins of the present invention lack the ability to bind to immunoglobulin Fc receptors, either as a consequence of the antibody isotype used for fusion protein construction, or through directed mutagenesis of antibody isotypes that normally bind Fc receptors. The fusion proteins of the present invention may also contain a functional domain capable of binding an immunoglobulin protection receptor.

Description

Raising is based on the method for the circulating half-life of the fusion rotein of antibody
The cross-reference of related application
This paper has quoted the U.S. Provisional Patent Application sequence number (SN) of submitting on February 25th, 1,998 60/075,887 as a reference and has required its right of priority and interests.
Invention field
The present invention relates generally to fusion rotein.More particularly, the present invention relates to improve method based on the circulating half-life of the fusion rotein of antibody.
Background of invention
The purposes that antibody is used for the treatment of human body diseases is that determine very much and closely related with the introducing of genetic engineering.Having developed several technology increases the purposes of antibody.They comprise: (1) is created " hybridoma " or is generated monoclonal antibody by heavy (H) chain of molecular cloning antibody from the cell that produces antibody and light (L) chain by cytogamy; (2) other molecule is engaged with antibody so that they are transported to position in the preferred body, described molecule is radio isotope, drug toxicity, proteotoxin and cytokine for example; (3) operation antibody mediated effect device function is so that increase or reduce biological activity; (4) with other protein such as endotoxin and cytokine class on hereditary level with antibodies so that produce fusion rotein based on antibody; (5) with one or more groups antibodies district on hereditary level in conjunction with so that produce bi-specific antibody.
When protein mutually combined by chemistry or genetic manipulation, which kind of characteristic common end product difficult to calculate had kept from parent molecule.Along with chemical bond, the different sites that cohesive process can be on molecule takes place, and generally causes molecule to have in various degree the change that can influence one or both proteinic functions.On the other hand, the heredity application of merging makes that connection procedure is more harmonious and causes keeping the generation of the stable end product of two kinds of composition protein functions.Referring to (PROC.NATL.ACAD.SCI.USA) 89:1428-1432 (1992) and United States Patent (USP) 5,650,150 such as for example Gillies " NAS's journal ".
Yet the application based on the fusion rotein of antibody that reorganization produces can be subjected to its restriction of removing fast from circulation in vivo.For example confirmed antibody-cytokine fusion protein in vivo specific ionization antibody have significantly lower circulating half-life.When detecting various antibody-cytokine fusion protein, α phase (distribution phase) transformation period that all examined fusion roteins of reports such as Gillies have all is lower than 1.5 hours.In fact, by by 2 hours the time, most of fusion roteins based on antibody all are eliminated the free antibodies serum-concentration to 10%.Referring to (BIOCOJ.CHEM.) 4:230-235 (1993) such as Gillies " biological joiner chemistry ".Therefore, exist in this area the demand of raising based on the method for the fusion rotein circulating half-life in vivo of antibody.
Invention is summarized
Had been found that the novel method of a kind of raising at present based on the fusion rotein circulating half-life in vivo of antibody.Especially, the invention provides to be used to produce the Fc acceptor is had the immunoglobulin (Ig) of binding affinity of reduction and the method for the fusion rotein between second kind of NIg protein.The fusion rotein based on antibody that the Fc acceptor is had the binding affinity of reduction has significantly longer circulating half-life in vivo than the second kind of NIg protein that does not connect.
It is that Fc γ RI, Fc γ RII and Fc γ RIII interact that IgG molecule and three classes have specific Fc acceptor (FcR) to the IgG antibody-like.In preferred embodiments, the immunoglobulin (Ig) of fusion rotein (Ig) composition has to the constant region of small part IgG, and this district is at least a binding affinity with reduction among Fc γ RI, Fc γ RII and the Fc γ RIII.
In one aspect of the invention, by using the heavy chain isotype to reduce the binding affinity of fusion rotein to the Fc acceptor as the fusion partner that Fc acceptor on the pair cell has the binding affinity of reduction.For example, human IgG1 and IgG3 combine with the Fc γ RI with high-affinity according to reports, and IgG4 and its 10 times below in conjunction with well, and IgG2 does not combine with it fully.Important sequence with IgG and Fc receptors bind is arranged in the CH2 territory according to reports.Therefore, in a preferred embodiment, by will be at least CH2 territory among IgG2 or the IgG4 be connected the fusion rotein based on antibody of the circulating half-life that obtains to have in vivo raising with second kind of NIg protein.
In another aspect of the present invention, reduce the binding affinity of fusion rotein to the Fc acceptor by introduce one or more amino acid whose genetic modifications in the constant region of IgG1 or IgG3 heavy chain, described constant region can reduce the binding affinity of these isotypes to the Fc acceptor.This class is modified and is comprised and change contact Fc acceptor or change the necessary residue of the other factors that contacts that influences between other heavy chain residue and the Fc acceptor by the inductive conformational change.Therefore, in a preferred embodiment, obtain to have in vivo the fusion rotein based on antibody of the circulating half-life of raising through the following steps: at first introduce sudden change, disappearance on the one or more amino acid in the IgG1 constant region or insert, described amino acid is selected from Leu 234, Leu 235, Gly 236, Gly 237, Asn 297And Pro 331And immunoglobulin (Ig) or its part with gained is connected with second kind of NIg protein then.In another preferred embodiment, introduce sudden change, disappearance or insertion on one or more amino acid of IgG3 constant region, described amino acid is selected from Leu 281, Leu 282, Gly 283, Gly 284, Asn 344And Pro 378And immunoglobulin (Ig) or its part of gained be connected with second kind of NIg protein.The fusion rotein based on antibody of gained has longer circulating half-life than the second kind of NIg protein that does not connect in vivo.
In a preferred embodiment, second of fusion rotein kind of NIg composition is cytokine.Term " cytokine " used herein " be used for describing protein, its analogue and the fragment thereof that produces justacrine by cell, and they cause the intracellular specific reaction that has this cytokine receptor.Preferred situation is that cytokine comprises: interleukin-such as interleukin II (IL-2); Hemopoietic factor such as granulocyte-macrophage colony stimutaing factor (GM-CSF); Tumour necrosis factor (TNF) such as TNF α and lymphokine are such as lymphotoxin.Preferred situation is that the fusion rotein based on antibody of the present invention demonstrates the biological activity of cytokine.
In another preferred embodiment, second of fusion rotein kind of NIg composition is biologically active part is conjugated protein.For example, this class part conjugated protein can: (1) is in cell surface blocking-up receptor-ligand binding; Or (2) offset the biological activity of molecule (for example cytokine) in the liquid phase of blood, prevents that thus it from reaching its cell-targeting thing.Preferred situation is conjugated protein CD4, CTLA-4, TNF acceptor or interleukin-1 receptor such as IL-1 and the IL-4 acceptor of comprising of part.Preferred situation is that the fusion rotein based on antibody of the present invention demonstrates the protein-bonded biological activity of part.
In another selectable preferred embodiment, the NIg composition of second kind of described fusion rotein is a kind of proteotoxin.Preferred situation is that antibody of the present invention-toxin fusion rotein demonstrates the toxicity activity of proteotoxin.
In a preferred embodiment, the fusion rotein based on antibody comprises that target antigen is had specific variable region and the constant region that is connected by the peptide with second kind of NIg protein bound.Described constant region can be that the constant region of usually being correlated with the variable region or a kind of different district are for example from different types of variable region and constant region.Heavy chain can comprise CH1, CH2 and/or CH3 territory.The implication of term " fusion rotein " also comprises the construct that has in conjunction with the territory, described in conjunction with the territory comprise from such as by Winter etc. at GB2, disclosed different types of framework region and variable region (being complementarity-determining region) in 188,638.The fusion rotein based on antibody that comprises the variable region demonstrates antigen-binding specificity better.In another preferred embodiment, the fusion rotein based on antibody further comprises a kind of light chain.The present invention provides fusion rotein that effective biological activity combination of the activity of antigen-binding specificity and antibody and second kind of NIg protein such as a kind of cytokine is got up thus.Fusion rotein of the present invention can be used for optionally second kind of NIg protein transduction being transported to target cell so that second kind of NIg protein can be brought into play localized biological action in vivo.
In a selectable preferred embodiment, comprise the CH that is connected by peptide and do not comprise variable region of heavy chain with second kind of NIg protein bound based on the fusion rotein of antibody.The present invention further provides the fusion rotein that keeps second kind of effective biological activity of NIg protein and lack antigen-binding specificity and antibody activity thus.
In preferred embodiments, the fusion rotein based on antibody of the present invention comprises that in conjunction with the necessary sequence of Fc protection acceptor (FcRp) described acceptor is such as the introduction stage intestines transhipment acceptor (FcRn) that contains beta-2 microglobulin.
In preferred embodiments, describedly comprise that two kinds contain to the chimeric chain of small part heavy chain and are connected by disulfide linkage with second kind of proteic fusion rotein of non--Ig.
Feature of the present invention also is the DNA construct of the above-mentioned fusion rotein of codified and is myelomatosis for example with these construct cells transfected.
These and other objects of the present invention disclosed herein and advantage and feature can be observed from following description, accompanying drawing and claim more significantly.
Brief description of drawings
If read in conjunction with the accompanying drawings, from the description of following preferred embodiment, can more completely understand above and other objects of the present invention so, feature and advantage and the present invention itself, wherein:
Accompanying drawing 1 is the homologous sequence comparison diagram of the aminoacid sequence of C γ 1 and C γ 3 constant regions, and the sequence of carrying out contrast reaches to greatest extent amino acid whose homogeny, and wherein non-conserved amino acid is with box indicating;
Accompanying drawing 2 is that the homology sequence comparison diagram of the aminoacid sequence of C γ 1, C γ 2 and C γ 4 constant regions carries out sequence contrast amino acid whose homogeny is reached to greatest extent, and wherein non-conserved amino acid is with box indicating;
Accompanying drawing 3 is to demonstrate the synoptic diagram of the coding in relevant limit site based on the collection of illustrative plates of the genetic constructs of the fusion rotein of antibody;
Accompanying drawing 4 is to describe antibody hu-KS-1/4 and (solid bars) arranged or do not having under the situation that (bar of band point) excessive mouse IgG exists on mouse J774 cell clavate synoptic diagram with the Fc receptors bind based on the fusion rotein hu-KS γ 1-IL-2 of antibody and hu-KS γ 4-IL-2;
Accompanying drawing 5 is to describe the linear synoptic diagram of the plasma concentration of total antibody (free antibodies and fusion rotein) of hu-KS γ 1-IL-2 (solid diamond) and hu-KS γ 4-IL-2 (black triangle) and hu-KS γ 1-IL-2 (open diamonds) and the complete fusion rotein of hu-KS γ 4-IL-2 (hollow triangle) in the body as the function of time;
Accompanying drawing 6 is to be used to make up have the graphic representation based on the technical scheme of the fusion rotein of antibody that reduces with the sudden change of Fc receptor binding affinity;
Accompanying drawing 7 be the hu-KS γ 1-IL-2 that describes hu-KS γ 1-IL-2 (◇) in the body, sudden change (
Figure A9980327700091
) and the plasma concentration of the complete fusion rotein of hu-KS γ 4-IL-2 (△) as the linear synoptic diagram of the function of time.
Detailed Description Of The Invention
Have been found that at present second kind of protein such as cytokine and immunoglobulin (Ig) merged to change antibody structure, thereby cause binding affinity to one or more cell bonded Fc acceptor to increase and cause fusion rotein removing fast from circulation based on antibody.The invention describes the circulating half-life that has raising in vivo based on the fusion rotein of antibody and comprise the method that one or more Fc acceptors is had the binding affinity of reduction by recombinant DNA technology production based on the fusion rotein of antibody.
Can obtain to have in vivo the fusion rotein based on antibody of the circulating half-life of raising at first, through the following steps: use to have the isotype construction of fusion protein and avoiding that the Fc acceptor is had a binding affinity of reduction and use from sequence in conjunction with the antibody isotype of Fc acceptor.For example, in 4 kinds of known IgG isotypes, known IgG1 (C γ 1) and IgG3 (C γ 3) can be in conjunction with the FcR γ I with high-affinity, then low 10 times of the binding affinities of IgG4 (C γ 4), and IgG2 (C γ 2) debond FcR γ I.Therefore, have the fusion rotein in C γ 2 constant regions (Fc district) or C γ 4 Fc districts and avoid using construct can obtain the Fc acceptor is had the fusion rotein based on antibody of the binding affinity of reduction by structure with C γ 1 Fc district or C γ 3 Fc districts.
The second, by being modified at the Fc acceptor is had in the isotype of binding affinity the fusion rotein based on antibody that can obtain to have in vivo the circulating half-life of raising in conjunction with the necessary sequence of Fc acceptor, thereby reduce or eliminated keying action.As mentioned above, IgG molecule and three class Fc acceptors (FcR) are that Fc γ RI, Fc γ RII and Fc γ RIII interact.C γ 1 and C γ 3 be in conjunction with the Fc γ RI with high-affinity, and C γ 4 and 2 couples of Fc γ of C γ RI have a reduction or do not have binding affinity.To comparison shows that of C γ 1 and C γ 3: the twisting section that extends in C γ 3, the amino acid sequence homology between these two kinds of isotypes is very high.Even take place in those verified and complement Clq fragments and various Fc γ R class that this also is correct in the interactional district.Accompanying drawing 1 provides the sequence comparison diagram of G γ 1 and C γ 3 aminoacid sequences.Two kinds of isotypes of other of human IgG (C γ 2 and C γ 4) have and the relevant sequence difference of Fc γ R combination.Accompanying drawing 2 provides the sequence comparison diagram of the aminoacid sequence of C γ 1, C γ 2 and C γ 4.Be used for the important sequence of Fc γ R bonded and be being arranged in Leu-Leu-Gly-Gly (C γ 1 residue 234 to 237) with the CH2 territory of hinge adjacency.Canfield and Morrison " experiment medicine magazine " be 173:1483-1491 (1991) (J.EXP.MED.).The motif of these sequences is guarded in C γ 1 and C γ 3, and is consistent with its similar biological nature; And may be relevant when they are used to make up the IL-2 fusion rotein with the similarity of pharmacokinetic properties.Carried out many mutation analysises prove specific mutant to FcR in conjunction with, comprise residue 234-237 and the crooked residue Pro of twisting near-end that is replaced by Ser among the IgG4 331In those effect.Another kind be used for effective FcR in conjunction with necessary important structure composition be exist with covalent and Asn 297The carbohydrate chain that bonded N-connects.Enzymatic is removed this structure of Asn residue or sudden change can be eliminated effectively or reduce at least significantly and all types of combination of Fc γ R.
Supposition such as Brumbell exist the Fc by binding antibody that the katabolism speed of circulating antibody is slowed down and it can be redirected the protection acceptor (FcRp) that returns in the circulation after its pinocytosis are gone into cell.Brumbell etc. " nature " are 203:1352-135 (1964) (NATURE).The introduction stage intestines transhipment acceptors (FcRn) that will contain beta-2 microglobulin recently are accredited as FcRp.Referring to (PROC.NATL.ACAD.SCI.USA) 93:5512-5516 (1996) such as Junghans " NAS's journal ".In conjunction with the necessary sequence of this acceptor in all four class human IgGs be guard and they are on the interface between CH2 and the CH3 territory.Referring to " IMMUNOLOGY KEY WORDS INDEX (J.IMMUBOL.) 158:2211-2217 (1997) such as Medesan.Having reported these sequences is important for antibody circulating half-life in vivo.Referring to international pct application WO 97/34631.Therefore, the present invention preferably has the necessary sequence in conjunction with FcRp based on the fusion rotein of antibody.
Described the present invention synthetic with embodiment method and be used to detect the active detection method of its pharmacokinetics, comprise the interior animal model of the body that uses external and preclinical phase.The preferred gene construct of chimeric chain of encoding comprises the dna fragmentation that is encoding to the small part immunoglobulin (Ig) and second kind of NIg protein DNA of coding of 5 '-3 ' direction.Another kind of preferred gene construct comprises second kind of NIg protein DNA of coding fragment of 5 '-3 ' direction and is encoding to the DNA of small part immunoglobulin (Ig).With fusion gene assembling and insert the expression vector that is used for the suitable recipient cell of transfection, it is expressed therein.
The present invention further explains by following non-limiting examples:
Embodiment 1 by carry out from C γ 1 to C γ 4 IgG constant regions classification conversion increase antibody-
IL2 fusion rotein circulating half-life in vivo
According to the present invention, by using from reducing with the Fc receptor binding affinity or not having the sequence construct of the antibody isotype of binding affinity can obtain to have the fusion rotein of the body-internal-circulation transformation period of raising based on the fusion rotein of antibody with it based on antibody.
In order to assess by using from reducing with the Fc receptor binding affinity or not having the sequence of the antibody isotype of binding affinity whether can improve body-internal-circulation transformation period based on the fusion rotein of antibody with it, the antibody-IL2 fusion rotein that will have the antibody-IL2 fusion rotein of people C γ 1 constant region (Fc district) and have people C γ 4 Fc districts compares.1.1 have the structure of the antibody-IL2 fusion rotein of C γ 4 IgG constant regions
The structure of the antibody-IL2 fusion rotein with C γ 1 constant region has been described in the prior art.Referring to for example, Gillies etc. " NAS's journal " are 89:1428-1432 (1992) and United States Patent (USP) 5,650,150 (PROC.NATL.ACAD.SCI.USA), and the document is incorporated herein by reference.
In order to make up antibody-IL2 fusion rotein with C γ 4 constant regions, by removing C γ 1 gene fragment and use corresponding sequence from people C γ 4 genes to replace it and modify a kind of plasmid vector, this plasmid vector can be expressed the humanized antibody-IL2 fusion rotein with people C γ 1 heavy chain that merges to the full cancer antigen of people (KSA) specific variable (V) district with the human IL-2.The collection of illustrative plates in the insertion site of some relevant limit site and C γ 4 gene fragments is provided in accompanying drawing 3.These plasmid construction bodies contain cytomegalovirus (CMV) early promoter that is useful on transcript mRNA, and described mRNA codified derives from light (L) and heavy (H) chain variable (V) district of mouse antibodies KS-1/4.Make mouse V district's humanization and its coded DNA sequence is synthesized with chemical mode by standard method.Add functional donor splicing site the end of each V district to so that it can be used for containing the carrier of H and L chain constant region gene.People C γ light chain gene is inserted into the downstream of the cloning site that is used for the VL gene and is its endogenous 3 ' non-translational region and polyadenylation site after it.After this transcription unit the second kind of independent transcription unit that is used for heavy chain-IL2 fusion rotein.It also starts by the CMV promotor.The VH encoding sequence is inserted into the upstream of the DNA of the specific C gamma heavy chain gene that merges with human IL-2's encoding sequence of coding.This class C γ gene contains the acceptor splicing site that is useful on first kind of heavy chain exon (CH1), the downstream of the common unique Hind III of everyone C γ gene that does for oneself just.The end that will be inserted into the IL-2 encoding sequence from the 3 ' untranslated and the polyadenylation site of SV40 virus.The residuum of carrier contains in the intestinal bacteria the necessary bacteria plasmid DNA of breeding and is used to select the selected marker of mammalian cell transfectant (Tetrahydrofolate dehydrogenase-dhfr).
By containing the plasmid DNA of original C γ 1 and finish C γ 1 and C γ 4 segmental exchanges by the big 7.8kb fragment of agarose gel electrophoresis purifying with Hind III and Xho I digestion.Digest second kind of plasmid DNA containing C γ 4 genes and the fragment of purifying 1.75kb with HindIII and Nsi I.Digest the fragment that contains the little 470bp of the third plasmid that C-terminal human IL-2 cDNA and SV40 poly A site and people C γ 1 gene merges and purifying with Xho I and Nsi I.All three kinds of fragments are connected to each other with about equimolar amount and the intestinal bacteria that product is used for transformed competence colibacillus will be connect.This connection product is used for the transformed competence colibacillus intestinal bacteria and by selecting the clone containing on the flat board of penbritin growth.Identify that by the plasmid DNA prepared product from isolating transformant being carried out restriction analysis the recombinant plasmid of correct assembling also will use the digest of Fsp I to be used to distinguish the gene insertion fragment of C γ 1 (no Fsp I) and C γ 4 (site).The final carrier that will contain the replacement of C γ 4-IL2 heavy chain is introduced murine myeloma cell and is selected transformant by growth in the substratum that contains methotrexate (0.1 μ M).To express the cell clone amplification of high-level antibody-IL2 fusion rotein and use a-protein Sepharose chromatography from the culture supernatants purified fusion protein.Measure the purity and the integrity of C γ 4 fusion roteins by the SDS-polyacrylamide gel electrophoresis.Detect in T-cell proliferation that to measure I1-2 in the test active and find its active identical with C γ 1 construct.1.2 by antibody and have the antibody-IL2 fusion rotein of C γ 1 and C γ 4 IgG constant regions in conjunction with the Fc acceptor
Various mouse and human cell line can express one or more Fc acceptors.For example, the expression of mouse J774 macrophage system can be in conjunction with the mouse of suitable subclass or the FcR γ I of human IgG.Equally, people K562 erythroleukemia cell system expresses FcR γ II and does not express FcR γ I.In order to assess the potential contribution based on the fusion rotein of antibody of Fc receptors bind to removing from circulation, comparison antibody, C γ 1-IL2 fusion rotein and C γ 4-IL2 fusion rotein are to the binding affinity of FcR γ I in mouse J774 clone.
Is 2 * 10 of 0.2ml containing 0.1% bovine serum albumin(BSA) (BSA) with final volume with two kinds of antibody described in the embodiment 1-IL2 fusion rotein hu-KS γ 1-IL2 and hu-KS γ 4-IL2 5Be diluted to 2 μ g/ml among the PBS of J774 cell.Cultivate the anti-human IgG Fc antibody (Fab that adds the FITC joint after 20 minutes on ice 2) and will cultivate and continue again to carry out 30 minutes.By removing unconjugated antibody with the PBS-BSA washed twice and coming analysis of cells with fluorescence-activated cell sorter (FACS).Contain the second antibody that is mixed with the FITC mark just in the control reaction thing or be mixed with the same cell of humanization KS γ 1 antibody (not conforming to IL-2).
Just as expected, C γ 4-IL2 fusion rotein and combining of J774 cell are starkly lower than combining of C γ 1-IL2 fusion rotein and J774 cell.Referring to accompanying drawing 4.Yet surprisingly C γ 1-IL2 and C γ 4-IL2 fusion rotein and J774 cell combines apparently higher than KS γ 1 antibody (not containing IL-2).This prompting can change the structure of antibody with second kind of protein such as a kind of cytokine and immunoglobulin (Ig) fusion, thereby causes the binding affinity of one or more cell bonded Fc acceptor is increased, and causes the quick removing from circulation thus.
In order determine to use whether the viewed stronger keying action of IL-2 fusion rotein is because the IL-2 acceptor or the FcR γ I acceptor that exist on the cell cause, excessive mouse IgG (mIgG) is used to compete combination on the Fc acceptor.As attached illustrated in fig. 4, under the situation that the excessive mIgG that 50 times of moles are arranged exists, use antibody and two kinds of antibody-IL2 fusion roteins can observe the combination of background level.The signal combination of the increase of this prompting antibody-IL2 fusion rotein is owing to cause with the increase of Fc receptors bind.
The clone of expressing the Fc acceptor is used to detect the binding affinity of candidate's fusion rotein and Fc acceptor so that identify the fusion rotein based on antibody of transformation period in the body with raising.Can detect candidate's fusion rotein based on antibody by aforesaid method.To be accredited as the fusion rotein of transformation period in the body with raising to the material standed for that the Fc acceptor has greatly the binding affinity that reduces based on the fusion rotein of antibody based on antibody.1.3 measure the circulating half-life of antibody-IL2 fusion rotein with C γ 1 and C γ 4 IgG constant regions
Whether can improve intravital circulating half-life in order to assess use has the affinity of reduction to the Fc acceptor the Fc district of IgG isotype, the fusion rotein (being hu-KS γ 1-IL2) that will contain C γ 1 isotype heavy chain and the fusion rotein that contains C γ 4 isotype heavy chains (are hu-KS γ-IL2) compare.
Going into phosphate-buffered saline (PBS) by diafiltration comes humanization KS-1/4-IL2 fusion rotein to the purifying that contains C γ 1 or C γ 4 isotype heavy chains to carry out buffer exchange and further be diluted to～concentration of 100 μ g/ml.Use the method slowly inject the fusion rotein based on antibody (0.2ml) of about 20 μ g to be injected the tail vein of the Balb/c mouse in 6-8 all ages.4 mouse of every group of injection.At different time point places, the blood sample that takes a morsel by bloodletting behind the eye socket of anesthetized animal is also collected the pipe that fills citrate buffer it in case hemostasis liquid grumeleuse.By removing cell in centrifugal 5 minutes with the Eppendorf high speed tabletop centrifuge.Take out blood plasma and freezing down with micropipet at-70 ℃.Measure the concentration of the people's paratope in the mouse blood by ELISA.To have specific capture antibodies to people H and L antibody chain and be used to catch fusion rotein from the plasma sample of dilution.In 96 hole flat boards of antibody sandwich, cultivate after 2 hours, by removing unconjugated material three times with ELISA damping fluid (the PBS solution of 0.01%Tween 80) washing.Second kind of culturing step uses anti-people Fc antibody (being used to detect antibody and complete fusion rotein) or anti-human IL-2's antibody (only being used to detect complete fusion rotein).Two kinds of antibody are connected with horseradish peroxidase (HRP).After cultivating 1 hour, remove unconjugated detection antibody and by with substrate cultivation with determine the amount of bonded HPR with spectrophotometric determination by washing with the ELISA damping fluid.
As what described in the accompanying drawing 5, the α phase transformation period of hu-KS γ 4-IL2 fusion rotein significantly is lower than the α phase transformation period of hu-KS γ 1-IL2 fusion rotein.The transformation period that increases serve as best typical with the concentration (60ng/ml) that the concentration (3.3 μ g/ml) of the hu-KS γ 4-IL2 fusion rotein found in mouse after 24 hours is significantly higher than hu-KS γ 1-IL2 fusion rotein.
As in early days chimeric 14,18-IL2 fusion rotein being reported, hu-KS γ 1-IL2 albumen has quick distribution (α) phase, is slow katabolism (β) phase thereafter.Referring to (BIOCONJ.CHEM.) 4:230-235 (1993) such as Gillies " biological joiner chemistry ".In the research of Gillies etc., only measured paratope, so whether not clear clearance rate has represented the antibody component of the fusion rotein of the complete fusion rotein removed or removing.In the present embodiment, use (1) antibodies specific ELISA and (2) fusion rotein specific ELISA (i.e. the antibody that need be connected with physics mode and the ELISA of IL-2 composition) to come test sample.。As attached illustrated in fig. 5, in the animal of using the injection of hu-KS γ 1-IL2 fusion rotein, the amount of round-robin fusion rotein is lower than the total amount of circulating antibody, and is particularly all the more so at 24 hours time point place.This prompting fusion rotein continues circulation with the antibody of cracking of proteolysis mode and release in vivo.Surprisingly, in the animal of using the injection of hu-KS γ 4-IL2 fusion rotein, between the total amount of the amount of round-robin fusion rotein and round-robin antibody, there is not significant difference.This prompting in 24 hours that are measured time limit in these animal bodies hu-KS γ 4-IL2 fusion rotein not with proteoclastic mode cracking.
As mentioned above, 3 pairs of Fc acceptors of C γ 1 and C γ have binding affinity, and binding affinity and 2 pairs of Fc acceptors of C γ that this moment, C γ 4 had reduction do not have binding affinity.Present embodiment has been described and has been used the production of C γ 4 Fc districts with the method for the complete fusion rotein of antibody (a kind of IgG isotype that the Fc acceptor is had the affinity of reduction) and determined that this class has the fusion rotein based on antibody of the body-internal-circulation transformation period of raising.Therefore, those skilled in the art can use these methods to produce to have with C γ 2 Fc districts substitute C γ 4 Fc districts based on the fusion rotein of antibody so that the circulating half-life of raising fusion rotein.The aforesaid method that uses identical restricted fragment to replace and be used for C γ 4-IL2 fusion rotein can make up the hu-KS-IL2 fusion rotein in C γ 2 districts that should choose and use method as herein described to detect so that prove the circulating half-life of increase.What have C γ 2 Fc districts compares the circulating half-life that the fusion rotein based on antibody that the Fc acceptor has binding affinity has raising in vivo based on the fusion rotein of antibody or any other Fc district that the Fc acceptor is had binding affinity or lacks binding affinity.
Embodiment 2 makes people C γ 1 or C γ 3 in the fusion protein construct based on antibody
Gene is undergone mutation to increase its circulating half-life in vivo
Several molecules in IgG molecule and the circulation comprise that the member's (for example Clq fragment) and the three class FcR of proteinic complement system interact.Being used for the important residue of Clq bonded is the residue Glu that is positioned at people's heavy chain CH2 territory 318, Lys 320And Lys 322Tao etc. " experiment medicine magazine " are 178:661-667 (1993) (J.EXP.MED.).Remove mechanism fast for the conduct of the difference between distinguishing FcR and Clq combining, we are replaced to C γ 1 heavy chain with the C γ 2 twisting proximal segment of more thorough change.Estimate this sudden change can influence FcR in conjunction with and do not influence the complement combination.
By cloning and adopt kind of the hinge and the sub-district between the initial part that are C γ 1 gene C H2 exon, using overlapping polymerase chain reaction (PCR) to finish sudden change.The PCR design of primers is become crossing over the new sequence (referring to accompanying drawing 6) of segmental two the segmental contact of the adjacent PCR places replacement of Psi I to Drd I.In the first step, use two the independent PCR reactants of C γ 1 gene as template preparation use primer 1 and 2 (being respectively SEQ ID NOS:5 and 6) or primer 3 and 4 (being respectively SEQ IDNOS:7 and 8).The cycling condition that is used for one-level PCR is 35 circulations of following condition: 94 ℃, 45 seconds; Annealed 45 seconds down at 48 ℃; With 72 ℃ of following primer extensions 45 seconds.The product of each PCR reaction is used as the template of secondary ligation step./ 10th of each first order reaction is mixed with each other and merges so that the merging product of two the initial p CR products that only increase with primer 1 and 4.The condition that is used for secondary PCR is: 94 ℃, 1 minute; Annealed 1 minute down at 51 ℃; With 72 ℃ of following primer extensions 1 minute.After sex change and annealing, ligation as and two independent segments of the terminal paired of another fragment between the result that overlaps take place.Shown in accompanying drawing 6, form by the fragment of the crossbred of Taq polymeric enzymatic amplification and by optionally the increase product of full mutation of the guiding of outer primer.Final PCR product and its sequence of clone identified by dna sequence analysis in plasmid vector.
In a plurality of steps, carry out the assembling of mutator gene.In the first step, contain the cloning vector of people C γ 1 gene so that remove the not twisting-CH2-CH3 encoding sequence of sudden change with Psi I and XhoI digestion.As mentioned above, by Drd I to the Xho I fragment of human IL-2's encoding sequence of C γ 1-IL2 preparing carriers encoding part CH2, all CH3 and fusion.By preparing the 3rd fragment by PCR product with Psi I and DrdI digestion subclone.Come whole three fragments of purifying and in a kind of single reaction mixture, they are connected to each other by agarose gel electrophoresis.To connect that product is used for the transformed competence colibacillus intestinal bacteria and by selecting the clone containing on the flat board of penbritin growth.By the gene that the plasmid DNA prepared product from isolating transformant is carried out restriction analysis is identified the recombinant plasmid of correct assembling and confirm by dna sequence analysis to suddenly change.To be used to ressemble complete hu-KS antibody-IL2 fusion protein expression vector from Hind III to the Xho I fragment of sudden change C γ 1-IL2.
For the raising situation of assessing the inductive body-internal-circulation transformation period by being used for FcR bonded important amino acid sudden change and in FcR and the mechanism of distinguishing between Clq combine as quick removing, the plasma concentration of the hu-KS γ 1-IL2 with the plasma concentration of the hu-KS γ 1-IL2 of vivo mutations during with different specified time compares.As attached illustrated in fig. 7, clearance rate significantly is lower than the clearance rate of hu-KS γ 1-IL2 in the body of the hu-KS γ 1-IL2 of sudden change and hu-KS γ 4-IL2.These results suggest are by modifying the fusion rotein based on antibody that can obtain to have the body-internal-circulation transformation period of raising in conjunction with the necessary sequence of Fc acceptor in the isotype that the Fc acceptor is had binding affinity.In addition, the quick mechanism of removing of these results suggest comprises the FcR combination but not the Clq combination.
From instruction of the present invention, it will be appreciated by those skilled in the art that can introduce several other sudden changes of C γ 1 or C γ 3 genes in case reduce with FcR combine and raising based on body-internal-circulation transformation period of the fusion rotein of antibody.In addition, sudden change can also be introduced C γ 4 genes so that further reduce combining of C γ 4 fusion roteins and FcR.For example, possible in addition sudden change comprises the sudden change in the twisting near-end amino-acid residue: make Pro 331Undergo mutation or undergo mutation by the glycosylation site that N-is connected.The latter is positioned the Asn as the part canonical sequence 287: Asn-X-Thr/Ser wherein can be to be Thr or Ser on amino acid (may except that Pro) and the 3rd arbitrarily on second.For example, almost to not influence of protein, but can prevent to connect any carbohydrate side chain to the conservative sudden change of amino acid Gln.The strategy that this residue is undergone mutation can adopt the aforesaid general program that is used for the twisting proximal end region.Fully set up the method and the commercially available reagent box that are used in cloned dna sequence generation point mutation in this area and can obtain being used for this purpose there from some sellers.
Embodiment 3 increases the circulating half-life that acceptor-antibody is the fusion rotein on basis
Several reference have been reported the Fc part of human IgG can be conjugated protein as the part of many biologically actives or the useful carrier of acceptor.The N-of the Fc of some in conjugated protein such as CD4, CTLA-4 and TNF acceptor and Ig part is terminal with these parts merges.Referring to for example, Capon etc. " nature " are 337:525-531 (1989) (NATURE); Linsley etc. " experiment medicine magazine " are 174:561-569 (1991) (J.EXP.MED.); " IMMUNOLOGY KEY WORDS INDEX (J.IMMUNOL.) 151:6602-6607 (1993) such as Wooley.Increase acceptor-antibody and be main fusion rotein circulating half-life can so that the conjugated protein mating partner of part (i.e. second kind of non-ig protein) more effectively: (1) blocks the receptor-ligand binding of cell surface; Or (2) offset the biological activity of the molecule (for example cytokine) of blood flow in mutually, prevents that thus it from arriving its cell-targeting thing.Whether can improve its intravital circulating half-life for the fusion rotein on basis in conjunction with the ability of IgG acceptor in order to assess reduction acceptor-antibody, the acceptor-antibody that will have people C γ 1 a Fc district compares for the fusion rotein on basis and the fusion rotein based on antibody with people C γ 4 Fc districts.
In order to make up the fusion rotein of CD4-antibody, use the clone people extracellular domain of cd4 cell surface receptor of PCR from human peripheral blood mononuclear cell (PBMC) for the basis.Described clone's CD4 acceptor comprises consistency restriction site and the donor splicing site described in the embodiment 1.Expression vector contains the unique Xba I cloning site downstream of CMV early promoter and the people C γ 1 or the C γ 4 gene downstreams in endogenous HindIII site thereof.The residuum of plasmid contains be useful on bacterium genetic information and the dhfr selected marker who breeds in intestinal bacteria.The DNAs that connects is used for the transformed competence colibacillus bacterium also according to identifying recombinant plasmid from the restriction analysis of each bacterial clone.Two kinds of plasmid DNA construct: CD4-C γ 1 and CD4-C γ 4 have been obtained.
By electroporation expression plasmid is used for the transfection rat bone marrow tumour cell and selects transformant by growing at the substratum that contains methotrexate (0.1 μ M).By elisa assay identify the transfectant of expressed fusion protein and in substratum amplification so that generate be used for by in conjunction with and come the fusion rotein of purifying from a-protein Sepharose wash-out.To go into PBS and be diluted to the final concentration of 100 μ g/ml with the protein diafiltration of chromatography eluant damping fluid purifying.CD4-C γ 1 or CD4-C γ 4 fusion roteins with 0.2ml (20 μ g) are given the Balb/c injected in mice and carry out pharmacokinetics test described in embodiment 1.3.CD4-C γ 4 fusion roteins have the transformation period that is significantly higher than CD4-C γ 1 fusion rotein.
Equivalents
The present invention can be included in other particular form can not break away from its essence or principal character.Above-mentioned embodiment all is counted as indicative thus in all respects but not the present invention as herein described is played the qualification effect.Therefore, scope of the present invention is by appended claim but not shown by foregoing description, and the institute that is included in the implication of the equivalents in the claim scope and scope changes and includes therein.<110>?GILLIES，Stephen?D
LO，Kin-Ming
LAN，Yan
WESOLOWSKI; John＜120〉improve method take antibody as the circulation half-life of the fusion on basis＜130〉LEX-003PC＜140〉＜141＜150〉US 60/075,887＜151〉1998-02-25＜160〉8＜170〉PatentIn Ver.2.0＜210〉1＜211〉447＜212〉PRT＜213〉people＜220〉＜223〉IGG-1 chain C districts＜400〉1Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa, 15 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
35 40 45Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
50 55 60Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?65 70 75 80Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
85 90 95Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
100 105 110Xaa?Xaa?Xaa?Xaa?Xaa?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu
115 120 125Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys
130 135 140Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser145 150 155 160Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser
165 170 175Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser
180 185 190Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn
195 200 205Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His
210 215 220Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val225 230 235 240Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr
245 250 255Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu
260 265 270Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys
275 280 285Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser
290 295 300Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys305 310 315 320Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile
325 330 335Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro
340 345 350Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu
355 360 365Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn
370 375 380Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser385 390 395 400Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg
405 410 415Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu
420 425 430His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys
435 440 445＜210〉chain C district＜400〉2Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa, 15 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 2＜211〉443＜212〉PRT＜213〉people＜220〉＜223〉IGG-2
20 25 30Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
35 40 45Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
50 55 60Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?65 70 75 80Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
85 90 95Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
100 105 110Xaa?Xaa?Xaa?Xaa?Xaa?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu
115 120 125Ala?Pro?Cys?Ser?Arg?Ser?Thr?Ser?Glu?Ser?Thr?Ala?Ala?Leu?Gly?Cys
130 135 140Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser145 150 155 160Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser
165 170 175Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Asn
180 185 190Phe?Gly?Thr?Gln?Thr?Tyr?Thr?Cys?Asn?Val?Asp?His?Lys?Pro?Ser?Asn
195 200 205Thr?Lys?Val?Asp?Lys?Thr?Val?Glu?Arg?Lys?Cys?Cys?Val?Glu?Cys?Pro
210 215 220Pro?Cys?Pro?Ala?Pro?Pro?Val?Ala?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro225 230 235 240Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr
245 250 255Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Gln?Phe?Asn
260 265 270Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg
275 280 285Glu?Glu?Gln?Phe?Asn?Ser?Thr?Phe?Arg?Val?Val?Ser?Val?Leu?Thr?Val
290 295 300Val?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser305 310 315 320Asn?Lys?Gly?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Thr?Lys
325 330 335Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Glu
340 345 350Glu?Met?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe
355 360 365Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu
370 375 380 Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Met?Leu?Asp?Ser?Asp?Gly?Ser?Phe385 390 395 400Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly
405 410 415Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr
420 425 430Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys
435 440＜210〉chain C district＜400〉3Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa, 15 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 3＜211〉494＜212〉PRT＜213〉people＜220〉＜223〉IGG-3
20 25 30Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
35 40 45Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
50 55 60Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?65 70 75 80Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
85 90 95Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
100 105 110Xaa?Xaa?Xaa?Xaa?Xaa?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu
115 120 125Ala?Pro?Cys?Ser?Arg?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys
130 135 140Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser145 150 155 160Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser
165 170 175Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser
180 185 190Leu?Gly?Thr?Gln?Thr?Tyr?Thr?Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn
195 200 205Thr?Lys?Val?Asp?Lys?Arg?Val?Glu?Leu?Lys?Thr?Pro?Leu?Gly?Asp?Thr
210 215 220Thr?His?Thr?Cys?Pro?Arg?Cys?Pro?Glu?Pro?Lys?Ser?Cys?Asp?Thr?Pro225 230 235 240Pro?Pro?Cys?Pro?Arg?Cys?Pro?Glu?Pro?Lys?Ser?Cys?Asp?Thr?Pro?Pro
245 250 255Pro?Cys?Pro?Arg?Cys?Pro?Glu?Pro?Lys?Ser?Cys?Asp?Thr?Pro?Pro?Pro
260 265 270Cys?Pro?Arg?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe
275 280 285Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro
290 295 300Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val305 310 315 320Gln?Phe?Lys?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr
325 330 335Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Phe?Arg?Val?Val?Ser?Val
340 345 350Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys
355 360 365Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser
370 375 380Lys?Thr?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro385 390 395 400Ser?Arg?Glu?Glu?Met?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val
405 410 415Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Ser?Gly
420 425 430Gln?Pro?Glu?Asn?Asn?Tyr?Asn?Thr?Thr?Pro?Pro?Met?Leu?Asp?Ser?Asp
435 440 445 Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp
450 455 460Gln?Gln?Gly?Asn?Ile?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His465 470 475 480Asn?Arg?Phe?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys
485 490＜210〉chain C district＜400〉4Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa, 15 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 4＜211〉444＜212〉PRT＜213〉people＜220〉＜223〉IGG-4
20 25 30Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
35 40 45Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
50 55 60Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?65 70 75 80Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
85 90 95Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa
100 105 110Xaa?Xaa?Xaa?Xaa?Xaa?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu
115 120 125Ala?Pro?Cys?Ser?Arg?Ser?Thr?Ser?Glu?Ser?Thr?Ala?Ala?Leu?Gly?Cys
130 135 140Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser145 150 155 160Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser
165 170 175Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser
180 185 190Leu?Gly?Thr?Lys?Thr?Tyr?Thr?Cys?Asn?Val?Asp?His?Lys?Pro?Ser?Asn
195 200 205Thr?Lys?Val?Asp?Lys?Arg?Val?Glu?Ser?Lys?Tyr?Gly?Pro?Pro?Cys?Pro
210 215 220Ser?Cys?Pro?Ala?Pro?Glu?Phe?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe225 230 235 240Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val
245 250 255Thr?Cys?Val?Val?Val?Asp?Val?Ser?Gln?Glu?Asp?Pro?Glu?Val?Gln?Phe
260 265 270Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro
275 280 285Arg?Glu?Glu?Gln?Phe?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr
290 295 300Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val305 310 315 320Ser?Asn?Lys?Gly?Leu?Pro?Ser?Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala
325 330 335Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Gln
340 345 350Glu?Glu?Met?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly
355 360 365Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro
370 375 380Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser385 390 395 400Phe?Phe?Leu?Tyr?Ser?Arg?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Glu
405 410 415Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His
420 425 430Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Leu?Gly?Lys
435 440＜210〉5＜211〉17＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer 1＜400〉5catcggtctt ccccctg 17＜210〉6＜211〉35＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer 2＜400〉6cggtcctgcg acgggaggtg ctgaggaaga gatgg 35＜210〉7＜211〉45＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer 3＜400〉7tcttcctcag cacctcccgt cgcaggaccg tcagtcttcc tcttc 45＜210〉8＜211〉17＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: primer 4＜400〉8gaggcgtggt cttgtag 17

Claims (26)

1. fusion rotein with circulating half-life of raising based on antibody, comprise the sphaeroprotein of partial immunity at least (Ig) heavy chain that the Fc acceptor is had the binding affinity that greatly reduces, be connected with second kind of non--Ig albumen on the described part heavy chain, described fusion rotein based on antibody has the second kind of non--longer circulating half-life of Ig albumen that connects than not in vivo.
2. the fusion rotein based on antibody of claim 1, wherein said part heavy chain comprises the CH2 territory of IgG2 or IgG4 constant region at least.
3. the fusion rotein based on antibody of claim 1, wherein said part heavy chain comprise have on one or more amino acid, undergo mutation or lack to small part IgG1 constant region, described amino acid is selected from Leu 234, Leu 235, Gly 236, Gly 237, Asn 297And Pro 331The group of forming.
4. the fusion rotein based on antibody of claim 1, wherein said part heavy chain comprise have on one or more amino acid, undergo mutation or lack to small part IgG3 constant region, described amino acid is selected from Leu 281, Leu 282, Gly 283, Gly 284, Asn 344And Pro 378The group of forming.
5. the fusion rotein based on antibody of claim 1, wherein said part heavy chain further have binding affinity to immunoglobulin (Ig) protection acceptor.
6. the fusion rotein based on antibody of claim 1, wherein said part heavy chain has the binding affinity that greatly reduces to the Fc acceptor, and described Fc acceptor is selected from the group that Fc γ RI, Fc γ RII and Fc γ RIII form.
7. the fusion rotein based on antibody of claim 1, the group that wherein said second kind of non--Ig albumen is selected from cytokine, part is conjugated protein and proteotoxin is formed.
8. the fusion rotein based on antibody of claim 1, wherein said cytokine are selected from the group that tumour necrosis factor, interleukin-and lymphokine are formed.
9. the fusion rotein based on antibody of claim 8, wherein said tumour necrosis factor is a α-Zhong Liuhuaisiyinzi.
10. the fusion rotein based on antibody of claim 8, wherein said interleukin-is an interleukin II.
11. the fusion rotein based on antibody of claim 8, wherein said lymphokine is lymphotoxin or G CFS.
12. the fusion rotein based on antibody of claim 11, wherein said G CFS is a rHuGM-CSF.
13. the fusion rotein based on antibody of claim 1, the conjugated protein group that is selected from CD4, CTLA-4, TNF acceptor and interleukin-1 receptor composition of wherein said part.
14. an increase is based on the method for the circulating half-life of the fusion rotein of antibody, this method comprise the following steps: near small part Ig heavy chain non-with second kind-Ig albumen is connected, described part heavy chain has the binding affinity that greatly reduces to the Fc acceptor, forms thus to have than the fusion rotein based on antibody that does not connect second kind of non--Ig albumen longer body-internal-circulation transformation period.
15. the method for claim 14, wherein said part heavy chain comprises the CH2 territory of IgG2 or IgG4 constant region at least.
16. an increase is based on the method for the circulating half-life of the fusion rotein of antibody, this method comprises the following steps:
(a) the one or more amino acid place in the IgG1 constant region introduces sudden change or disappearance, and described amino acid is selected from Leu 234, Leu 235, Gly 236, Gly 237, Asn 297And Pro 331The group of forming produces the Ig heavy chain that the Fc acceptor is had the binding affinity that greatly reduces thus; With
(b) with step (a) to the small part heavy chain non-with second kind-Ig albumen is connected, and forms the fusion rotein based on antibody that has than the second kind of non--Ig albumen longer body-internal-circulation transformation period that does not connect thus.
17. an increase is based on the method for the circulating half-life of the fusion rotein of antibody, this method comprises the following steps:
(a) the one or more amino acid place in the IgG3 constant region introduces sudden change or disappearance, and described amino acid is selected from Leu 281, Leu 282, Gly 283, Gly 284, Asn 344And Pro 378The group of forming produces the Ig heavy chain that the Fc acceptor is had the binding affinity that greatly reduces thus; With
(b) with step (a) to small part Ig heavy chain non-with second kind-Ig albumen is connected, form thus to have than the fusion rotein that does not connect second kind of non--Ig albumen longer body-internal-circulation transformation period based on antibody.
18. claim 14,16 or 17 method, wherein said part heavy chain further have binding affinity to immunoglobulin (Ig) protection acceptor.
19. claim 14,16 or 17 method, wherein said part heavy chain has significantly reduced binding affinity to the Fc acceptor, and described Fc acceptor is selected from the group that Fc γ RI, Fc γ RII and Fc γ RIII form.
20. claim 14,16 or 17 method, the group that wherein said second kind of non--Ig albumen is selected from cytokine, part is conjugated protein and proteotoxin is formed.
21. claim 14,16 or 17 method, wherein said cytokine are selected from the group that tumour necrosis factor, interleukin-and lymphokine are formed.
22. the method for claim 21, wherein said tumour necrosis factor is a α-Zhong Liuhuaisiyinzi.
23. the method for claim 21, wherein said interleukin-is an interleukin II.
24. the method for claim 21, wherein said lymphokine are lymphotoxin or G CFS.
25. the fusion rotein based on antibody of claim 24, wherein said G CFS is a rHuGM-CSF.
26. claim 14,16 or 17 method, conjugated protein CD4, CTLA-4, TNF acceptor and the interleukin-1 receptor composition group of being selected from of wherein said part.
CN99803277.8A 1998-02-25 1999-02-24 Enhancing circulating half-life of antibody-based fusion proteins Expired - Fee Related CN1204147C (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104507504A (en) * 2012-08-10 2015-04-08 罗切格利卡特公司 Interleukin-2 fusion proteins and uses thereof
CN110914296A (en) * 2019-02-22 2020-03-24 武汉友芝友生物制药有限公司 Engineered Fc fragments, antibodies comprising same and uses thereof
US11254728B2 (en) 2012-08-02 2022-02-22 Hoffmann-La Roche Inc. Method for producing monomeric and multimeric molecules and uses thereof

Families Citing this family (127)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7749498B2 (en) 1997-03-10 2010-07-06 Genentech, Inc. Antibodies for inhibiting blood coagulation and methods of use thereof
US20030109680A1 (en) 2001-11-21 2003-06-12 Sunol Molecular Corporation Antibodies for inhibiting blood coagulation and methods of use thereof
US20060235209A9 (en) 1997-03-10 2006-10-19 Jin-An Jiao Use of anti-tissue factor antibodies for treating thromboses
US5986065A (en) 1997-03-10 1999-11-16 Sunol Molecular Corporation Antibodies for inhibiting blood coagulation and methods of use thereof
US6528624B1 (en) 1998-04-02 2003-03-04 Genentech, Inc. Polypeptide variants
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
US6242195B1 (en) 1998-04-02 2001-06-05 Genentech, Inc. Methods for determining binding of an analyte to a receptor
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US7183387B1 (en) 1999-01-15 2007-02-27 Genentech, Inc. Polypeptide variants with altered effector function
PL209786B1 (en) 1999-01-15 2011-10-31 Genentech Inc Variant of mother polypeptide containing Fc region, polypeptide containing variant of Fc region with altered affinity of Fc gamma receptor binding (Fc R), polypeptide containing variant of Fc region with altered affinity of Fc gamma neonatal receptor binding (Fc Rn), composition, isolated nucleic acid, vector, host cell, method for obtaining polypeptide variant, the use thereof and method for obtaining region Fc variant
GB9926084D0 (en) * 1999-11-03 2000-01-12 King S College London Recombinant fusion molecules
ES2269366T3 (en) * 2000-02-11 2007-04-01 Merck Patent Gmbh IMPROVEMENT OF AVERAGE LIFE IN CIRCULATION OF FUSION PROTEINS BASED ON ANTIBODIES.
EP1272526A4 (en) 2000-04-13 2004-10-13 Univ Rockefeller Enhancement of antibody-mediated immune responses
DE10045591A1 (en) * 2000-09-15 2002-04-04 Klaus Pfizenmaier Site-specific, antibody-mediated activation of proapoptotic cytokines - AMAIZe (Antibody-Mediated Apoptosis Inducing Cytokines)
EP2357187A1 (en) * 2000-12-12 2011-08-17 MedImmune, LLC Molecules with extended half-lives, compositions and uses thereof
AU2011253690C1 (en) * 2000-12-12 2018-01-18 Board Of Regents, The University Of Texas System Molecules with extended half-lives, compositions and uses thereof
US7658921B2 (en) 2000-12-12 2010-02-09 Medimmune, Llc Molecules with extended half-lives, compositions and uses thereof
US20030133939A1 (en) 2001-01-17 2003-07-17 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
US7754208B2 (en) 2001-01-17 2010-07-13 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
DK1366067T3 (en) * 2001-03-07 2012-10-22 Merck Patent Gmbh EXPRESSION TECHNOLOGY FOR PROTEINS CONTAINING A HYBRID ISOTYPE ANTIBODY UNIT
WO2002079415A2 (en) 2001-03-30 2002-10-10 Lexigen Pharmaceuticals Corp. Reducing the immunogenicity of fusion proteins
TWI338009B (en) * 2001-10-29 2011-03-01 Genentech Inc Antibodies for inhibiting blood coagulation and methods of use thereof
EP1495055B1 (en) * 2002-04-18 2013-08-14 Genencor International, Inc. Production of functional antibodies in filamentous fungi
US7132100B2 (en) 2002-06-14 2006-11-07 Medimmune, Inc. Stabilized liquid anti-RSV antibody formulations
US8968730B2 (en) 2002-08-14 2015-03-03 Macrogenics Inc. FcγRIIB specific antibodies and methods of use thereof
US8946387B2 (en) 2002-08-14 2015-02-03 Macrogenics, Inc. FcγRIIB specific antibodies and methods of use thereof
PT1562972E (en) * 2002-10-15 2010-11-10 Facet Biotech Corp Alteration of fcrn binding affinities or serum half-lives of antibodies by mutagenesis
US7217797B2 (en) * 2002-10-15 2007-05-15 Pdl Biopharma, Inc. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
US7960512B2 (en) 2003-01-09 2011-06-14 Macrogenics, Inc. Identification and engineering of antibodies with variant Fc regions and methods of using same
US7355008B2 (en) 2003-01-09 2008-04-08 Macrogenics, Inc. Identification and engineering of antibodies with variant Fc regions and methods of using same
EP2298347B1 (en) 2003-05-06 2015-09-30 Biogen Hemophilia Inc. Clotting factor chimeric proteins for treatment of a hemostatic disorder
TWI353991B (en) 2003-05-06 2011-12-11 Syntonix Pharmaceuticals Inc Immunoglobulin chimeric monomer-dimer hybrids
JP2005073528A (en) * 2003-08-28 2005-03-24 Genetics Inst Llc Method for inhibiting adhesion of blood to biological tissue in biological system, and composition for being used for the method
WO2005037867A1 (en) 2003-10-15 2005-04-28 Pdl Biopharma, Inc. ALTERATION OF Fc-FUSION PROTEIN SERUM HALF-LIVES BY MUTAGENESIS OF POSITIONS 250, 314 AND/OR 428 OF THE HEAVY CHAIN CONSTANT REGION OF IG
US8420087B2 (en) 2004-01-05 2013-04-16 Antisoma Research Limited Interleukin-12 targeted to oncofoetal fibronectin
EP1810035A4 (en) 2004-11-10 2010-03-17 Macrogenics Inc Engineering fc antibody regions to confer effector function
US9284375B2 (en) 2005-04-15 2016-03-15 Macrogenics, Inc. Covalent diabodies and uses thereof
US11254748B2 (en) 2005-04-15 2022-02-22 Macrogenics, Inc. Covalent diabodies and uses thereof
US9963510B2 (en) 2005-04-15 2018-05-08 Macrogenics, Inc. Covalent diabodies and uses thereof
US9296816B2 (en) 2005-04-15 2016-03-29 Macrogenics, Inc. Covalent diabodies and uses thereof
EP2298815B1 (en) 2005-07-25 2015-03-11 Emergent Product Development Seattle, LLC B-cell reduction using CD37-specific and CD20-specific binding molecules
DK1919503T3 (en) 2005-08-10 2014-12-15 Macrogenics Inc Identification and preparation of antibodies with variant fc regions and methods of use thereof
WO2007146968A2 (en) 2006-06-12 2007-12-21 Trubion Pharmaceuticals, Inc. Single-chain multivalent binding proteins with effector function
HUE030269T2 (en) 2006-06-26 2017-04-28 Macrogenics Inc Fc riib-specific antibodies and methods of use thereof
EP2032159B1 (en) 2006-06-26 2015-01-07 MacroGenics, Inc. Combination of fcgammariib antibodies and cd20-specific antibodies and methods of use thereof
ATE480568T1 (en) * 2006-06-30 2010-09-15 Conaris Res Inst Ag IMPROVED SGP 130FC DIMERE
US8652466B2 (en) 2006-12-08 2014-02-18 Macrogenics, Inc. Methods for the treatment of disease using immunoglobulins having Fc regions with altered affinities for FcγRactivating and FcγRinhibiting
TW200907056A (en) * 2007-03-28 2009-02-16 Astrazeneca Ab New method
PE20120259A1 (en) 2007-08-09 2012-04-04 Boehringer Ingelheim Int ANTI-CD37 ANTIBODIES
JP5809415B2 (en) 2007-11-09 2015-11-10 ペレグリン ファーマシューティカルズ，インコーポレーテッド Compositions and methods of anti-VEGF antibodies
WO2009117030A2 (en) 2007-12-19 2009-09-24 Macrogenics, Inc. Improved compositions for the prevention and treatment of smallpox
CA2711736A1 (en) 2008-01-18 2009-07-23 Medimmune, Llc Cysteine engineered antibodies for site-specific conjugation
KR101614494B1 (en) 2008-04-02 2016-04-22 마크로제닉스, 인크. Bcr-complex-specific antibodies and methods of using same
JP5555223B2 (en) 2008-04-02 2014-07-23 マクロジェニクス，インコーポレーテッド HER2 / neu specific antibody and method of use thereof
NZ603059A (en) 2008-04-11 2014-07-25 Emergent Product Dev Seattle Cd37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof
TWI440469B (en) 2008-09-26 2014-06-11 Chugai Pharmaceutical Co Ltd Improved antibody molecules
EP2373689A1 (en) 2008-12-12 2011-10-12 MedImmune, LLC Crystals and structure of a human igg fc variant with enhanced fcrn binding
AU2010225951B2 (en) * 2009-03-19 2014-03-13 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical formulation containing improved antibody molecules
EP2233500A1 (en) 2009-03-20 2010-09-29 LFB Biotechnologies Optimized Fc variants
EP2421896A1 (en) 2009-04-22 2012-02-29 Merck Patent GmbH Antibody fusion proteins with modified fcrn binding sites
US20120134984A1 (en) 2009-06-01 2012-05-31 Olga Lubman Molecules with extended half-lives and uses thereof
CA2766405A1 (en) 2009-06-22 2011-01-13 Medimmune, Llc Engineered fc regions for site-specific conjugation
RU2583298C2 (en) 2009-10-07 2016-05-10 Макродженикс, Инк. POLYPEPTIDES CONTAINING Fc-SECTION THAT DISPLAY INCREASED EFFECTOR FUNCTION OWING TO VARYING FUCOSYLATION LEVEL AND THEIR APPLICATIONS
CA2782320A1 (en) 2009-12-02 2011-06-09 Acceleron Pharma Inc. Compositions and methods for increasing serum half-life of fc fusion proteins
TWI505838B (en) 2010-01-20 2015-11-01 Chugai Pharmaceutical Co Ltd Stabilized antibody
AU2011223782B2 (en) 2010-03-04 2014-09-18 Macrogenics, Inc. Antibodies reactive with B7-H3, immunologically active fragments thereof and uses thereof
US8802091B2 (en) 2010-03-04 2014-08-12 Macrogenics, Inc. Antibodies reactive with B7-H3 and uses thereof
CA2807127C (en) 2010-08-02 2019-02-12 Leslie S. Johnson Covalent diabodies and uses thereof
UA112062C2 (en) 2010-10-04 2016-07-25 Бьорінгер Інгельхайм Інтернаціональ Гмбх CD33-Binding Agent
TR201810773T4 (en) 2010-11-23 2018-08-27 Glaxo Group Ltd Oncostatin (osm) antigen binding proteins.
CN103459422A (en) 2010-11-24 2013-12-18 葛兰素集团有限公司 Multispecific antigen binding proteins targeting HGF
EP2654790B1 (en) 2010-12-22 2019-02-06 Teva Pharmaceuticals Australia Pty Ltd Modified antibody with improved half-life
PT2691417T (en) 2011-03-29 2018-10-31 Roche Glycart Ag Antibody fc variants
EA201892619A1 (en) 2011-04-29 2019-04-30 Роше Гликарт Аг IMMUNOCONJUGATES CONTAINING INTERLEUKIN-2 MUTANT POLYPETIPS
UA111612C2 (en) 2011-05-21 2016-05-25 Макродженікс, Інк. DOMAINS WHICH ARE CONNECTED TO DEIMMUNIZED SERUM
WO2012170938A1 (en) 2011-06-08 2012-12-13 Acceleron Pharma Inc. Compositions and methods for increasing serum half-life
CA2842099A1 (en) 2011-07-27 2013-01-31 Glaxo Group Limited Antigen binding constructs
BR112014008804A2 (en) 2011-10-11 2017-06-13 Medimmune Llc cd40l-specific tn3-derived carriers and methods of use
CA2859755C (en) 2011-12-23 2021-04-20 Pfizer Inc. Engineered antibody constant regions for site-specific conjugation and methods and uses therefor
HUE056217T2 (en) 2012-07-13 2022-02-28 Roche Glycart Ag Bispecific anti-vegf/anti-ang-2 antibodies and their use in the treatment of ocular vascular diseases
WO2014029752A1 (en) 2012-08-22 2014-02-27 Glaxo Group Limited Anti lrp6 antibodies
TWI682941B (en) 2013-02-01 2020-01-21 美商再生元醫藥公司 Antibodies comprising chimeric constant domains
US9487587B2 (en) 2013-03-05 2016-11-08 Macrogenics, Inc. Bispecific molecules that are immunoreactive with immune effector cells of a companion animal that express an activating receptor and cells that express B7-H3 and uses thereof
DK2968520T3 (en) 2013-03-14 2021-08-09 Macrogenics Inc BISPECIFIC MOLECULES THAT ARE IMMUNORE ACTIVE WITH IMMUNE EFFECTOR CELLS EXPRESSING AN ACTIVATING RECEPTOR
UA118750C2 (en) 2013-03-15 2019-03-11 Ґлаксосмітклайн Інтеллектуал Проперті Дивелопмент Лімітед Anti-lag-3 binding proteins
US11384149B2 (en) 2013-08-09 2022-07-12 Macrogenics, Inc. Bi-specific monovalent Fc diabodies that are capable of binding CD32B and CD79b and uses thereof
UA116479C2 (en) 2013-08-09 2018-03-26 Макродженікс, Інк. Bi-specific monovalent fc diabodies that are capable of binding cd32b and cd79b and uses thereof
EP2839842A1 (en) 2013-08-23 2015-02-25 MacroGenics, Inc. Bi-specific monovalent diabodies that are capable of binding CD123 and CD3 and uses thereof
EP2840091A1 (en) 2013-08-23 2015-02-25 MacroGenics, Inc. Bi-specific diabodies that are capable of binding gpA33 and CD3 and uses thereof
NZ719654A (en) 2014-02-06 2020-06-26 Hoffmann La Roche Interleukin-2 fusion proteins and uses thereof
GB201403775D0 (en) 2014-03-04 2014-04-16 Kymab Ltd Antibodies, uses & methods
TWI754319B (en) 2014-03-19 2022-02-01 美商再生元醫藥公司 Methods and antibody compositions for tumor treatment
ES2869459T3 (en) 2014-05-16 2021-10-25 Medimmune Llc Molecules with altered neonate fc receptor binding that have enhanced therapeutic and diagnostic properties
US10562946B2 (en) 2014-06-20 2020-02-18 Genentech, Inc. Chagasin-based scaffold compositions, methods, and uses
KR20170035910A (en) 2014-08-11 2017-03-31 데리니아, 인크. Modified il-2 variants that selectively activate regulatory t cells for the treatment of autoimmune diseases
WO2016054101A1 (en) 2014-09-29 2016-04-07 Duke University Bispecific molecules comprising an hiv-1 envelope targeting arm
PL3221359T3 (en) 2014-11-17 2020-11-16 Regeneron Pharmaceuticals, Inc. Methods for tumor treatment using cd3xcd20 bispecific antibody
KR101892883B1 (en) 2015-02-27 2018-10-05 추가이 세이야쿠 가부시키가이샤 Composition for treating il-6-related diseases
EP3277725B1 (en) 2015-03-30 2020-11-25 Regeneron Pharmaceuticals, Inc. Heavy chain constant regions with reduced binding to fc gamma receptors
GB201506389D0 (en) * 2015-04-15 2015-05-27 Berkel Patricius H C Van And Howard Philip W Site-specific antibody-drug conjugates
GB201506393D0 (en) * 2015-04-15 2015-05-27 Berkel Patricius H C Van And Howard Philip W Site-specific antibody-drug conjugates
WO2017019729A1 (en) 2015-07-27 2017-02-02 The General Hospital Corporation Antibody derivatives with conditionally enabled effector function
EP3798234A1 (en) 2015-09-02 2021-03-31 Immutep S.A.S. Anti-lag-3 agonistic antibodies
TWI799366B (en) 2015-09-15 2023-04-21 美商建南德克公司 Cystine knot scaffold platform
SG10202002577XA (en) 2015-09-21 2020-04-29 Aptevo Res & Development Llc Cd3 binding polypeptides
WO2017093947A1 (en) 2015-12-04 2017-06-08 Novartis Ag Antibody cytokine engrafted compositions and methods of use for immunoregulation
US20170204154A1 (en) 2016-01-20 2017-07-20 Delinia, Inc. Molecules that selectively activate regulatory t cells for the treatment of autoimmune diseases
TWI781098B (en) 2016-04-15 2022-10-21 美商宏觀基因股份有限公司 Novel b7-h3-binding molecules, antibody drug conjugates thereof and methods of use thereof
US9567399B1 (en) 2016-06-20 2017-02-14 Kymab Limited Antibodies and immunocytokines
WO2018049275A1 (en) 2016-09-09 2018-03-15 Genentech, Inc. Selective peptide inhibitors of frizzled
WO2018073365A1 (en) * 2016-10-19 2018-04-26 F. Hoffmann-La Roche Ag Method for producing an immunoconjugate
WO2018083248A1 (en) 2016-11-03 2018-05-11 Kymab Limited Antibodies, combinations comprising antibodies, biomarkers, uses & methods
EP3538548A4 (en) 2016-11-08 2020-08-19 Delinia, Inc. Il-2 variants for the treatment of autoimmune diseases
WO2018203545A1 (en) 2017-05-02 2018-11-08 国立研究開発法人国立精神・神経医療研究センター Method for predicting and evaluating therapeutic effect in diseases related to il-6 and neutrophils
JOP20190271A1 (en) 2017-05-24 2019-11-21 Novartis Ag Antibody-cytokine engrafted proteins and methods of use for immune related disorders
EP3694885A1 (en) 2017-10-14 2020-08-19 CytomX Therapeutics, Inc. Antibodies, activatable antibodies, bispecific antibodies, and bispecific activatable antibodies and methods of use thereof
MX2020006322A (en) 2017-12-19 2020-09-18 Xencor Inc Engineered il-2 fc fusion proteins.
KR20210054528A (en) 2018-08-31 2021-05-13 리제너론 파아마슈티컬스, 인크. Dosing strategies to alleviate cytokine release syndrome against CD3/C20 bispecific antibodies
CN114502593A (en) 2019-08-06 2022-05-13 葛兰素史密斯克莱知识产权发展有限公司 Biopharmaceutical compositions and related methods
EP4347656A1 (en) 2021-05-28 2024-04-10 GlaxoSmithKline Intellectual Property Development Limited Combination therapies for treating cancer
WO2023012669A2 (en) 2021-08-03 2023-02-09 Glaxosmithkline Intellectual Property Development Limited Biopharmaceutical compositions and stable isotope labeling peptide mapping method
CA3233953A1 (en) 2021-10-05 2023-04-13 Matthew Bruce Combination therapies for treating cancer
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WO2023212304A1 (en) 2022-04-29 2023-11-02 23Andme, Inc. Antigen binding proteins
WO2024042112A1 (en) 2022-08-25 2024-02-29 Glaxosmithkline Intellectual Property Development Limited Antigen binding proteins and uses thereof
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US20240166728A1 (en) 2022-11-02 2024-05-23 VIIV Healthcare UK (No.5) Limited Antigen binding proteins

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9511935D0 (en) * 1995-06-13 1995-08-09 Smithkline Beecham Plc Novel compound
AU2527397A (en) * 1996-03-13 1997-10-01 Protein Design Labs, Inc. Fas ligand fusion proteins and their uses

Cited By (6)

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Publication number Priority date Publication date Assignee Title
US11254728B2 (en) 2012-08-02 2022-02-22 Hoffmann-La Roche Inc. Method for producing monomeric and multimeric molecules and uses thereof
CN104507504A (en) * 2012-08-10 2015-04-08 罗切格利卡特公司 Interleukin-2 fusion proteins and uses thereof
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CN110914296A (en) * 2019-02-22 2020-03-24 武汉友芝友生物制药有限公司 Engineered Fc fragments, antibodies comprising same and uses thereof
CN112142847A (en) * 2019-02-22 2020-12-29 石家庄石友生物技术有限公司 Engineered Fc fragments, antibodies comprising same and uses thereof
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