CN102558355A - Heterodimeric FC (fragment crystallizable) modification method based on charge network and preparation method of heterodimeric proteins - Google Patents
Heterodimeric FC (fragment crystallizable) modification method based on charge network and preparation method of heterodimeric proteins Download PDFInfo
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Abstract
The invention discloses a heterodimeric FC (fragment crystallizable) modification method based on a charge network and a preparation method of heterodimeric proteins. The modification method is characterized by establishing a charge interaction network and mutating one or more interfacial amino acids based on interaction of heavy-chain CH3 two-arm amino acids of the FC and modifying the amino acids in the CH3 domains through charge effect, thus weakening self interactions of the domains (which is beneficial to forming homodimers) and enhancing the interactions between the domains ( which is beneficial to forming heterodimers) and finally selecting sense mutation. The invention also relates to heterodimeric proteins prepared by the preparation method, independent polypeptides forming the heterodimeric FC and nucleotide sequences coding the polypeptides.
Description
Technical field
The present invention relates to a kind of relevant albumen of heterodimer antibody FC and method of polypeptide of preparing, also relate to heterodimer antibody FC albumen and polypeptide itself, comprise the independent polypeptide of forming heterodimer antibody FC; The invention still further relates to the nucleotide sequence of these polypeptide of coding, and the medicine components that comprises one or more heterogeneous FC albumen or polypeptide.
Technical background
Monoclonal antibody drug increased in nearly 15 years rapidly, became the growth point of pharmaceutical industry.From 1996, one has 30 left and right sides monoclonal antibody medicines went through to go on the market.Wherein there are nine monoclonal antibody medicine years to sell above 1,000,000,000 dollars.The total sale of monoclonal antibody medicine surpassed 30,000,000,000 dollars in 2010, and annual growth surpasses 10%.Because the target high specificity of monoclonal antibody can only suppress single target spot.And in multiple disease, comprising tumour, autoimmunity needs to suppress the multiple signal path and avoids compensatory effect.For virus infection, because the high mutation rate of virus often need suppress many antigen sites and prevent to escape.In addition; Bifunctional antibody, albumen are used to special human activin immunity system (Wolf, Hofmeister et al. " BiTEs:bispecific antibody constructs with unique anti-tumor activity. " Drug Discov Today 10 (18): 1237-1244.2005).The Fc district of antibody forms homodimer, and Fc plays a crucial role to the body internal stability of keeping antibody and Fc fusion rotein simultaneously.Making it to form heterodimer through transformation Fc is to produce multipurpose antibody, albumen, and keeps the effective ways of its body internal stability.
The example of a typically used of heterodimer is a bispecific antibody, and (Bispecific antiboy BsAbs) is the immunoglobulin molecules that contains two different ligands binding sites to bispecific antibody.Bispecific antibody at least can to two not synantigen have activity (Carter; P. " Bispecific human IgG by design. " J Immunol Methods 248 (1-2): 7-15.2001); It has replaced the classical identical sequence of antibody fab two arms; But with two different fab sequences, so Y type two arms can combine different antigens.The application of bispecific antibody in cancer therapy summarized by many pieces of documents that (Carter 2001; Chames and Baty. " Bispecific antibodies for cancer therapy. " Curr Opin Drug Discov Devel 12 (2): 276-283.2009; Chames and Baty. " Bispecific antibodies for cancer therapy:the light at the end of the tunnel? " MAbs 1 (6): 539-547.2009).The other arm of related antigen that the arm of BsAbs can connect tumor cell surface then can trigger the further killer cell of immune effector cell, comes the kill cancer tumour cell through immune system.The application of other bispecific antibodies can be checked USP U.S.Pat.Nos 5,731,168 and 7,183,076.
There is not bi-specific antibody under the state of nature, can only prepares through special methods.The preparation method of bispecific antibody had chemical crosslink technique in the past, heterozygosis F (ab ') 2 molecule process and murine hybridoma method etc.Chemical crosslink technique is produced the heterology of bispecific antibody, batch with batch between unstable, and antibodies specific is subject to some modification or improper connection and the characteristic that changes, the bispecific antibody that makes this method produce is inappropriate for use in the body.Two specific hybridization molecules of producing with sulfydryl crosslinking protein enzymic digestion segment F (ab '), though composition than homogeneous, is wasted time and energy, and output is very low.The bispecific antibody that the hybridoma method is produced, from the horse's mouth, but, make bispecific antibody production, purifying become very difficult by the multiple possibility antibody formation that light chain, heavy chain random pair produce.
As far back as the nineties; The partial amino-acid of humans such as Carter " handle-hole " (knob into hole) model engineered antibody heavy chain; Relatively more successful realization bispecific antibody (Ridgway, Presta et al. " ' Knobs-into-holes ' engineering of antibody CH3domains for heavy chain heterodimeriz ation. " Protein Eng 9 (7): 617-621.1996; Carter 2001)." handle-hole " model be at first by Crick propose with solve amino acid side chain folding problem between the adjacent alpha-helix (Crick, F. H. " Is alpha-keratin a coiled coil? " Nature 170 (4334): 882-883.1952).Thereby Carter etc. through having become the big amino acid of side chain to create " handle " (like a T366Y) a little amino acid mutation of side chain, and have become the little amino acid of side chain to create " hole " (Y407T etc.) some amino acid mutation on the CH3 on the second heavy chain on the CH3 zone of FC article one heavy chain.The principle of " handle-hole " model is that " handle-hole " interact is to support heterodimer to form, and " handle-handle " model and " hole-hole " model are the formation that hinders homodimer.They have further introduced the binding ability that disulfide linkage in the CH3 zone further consolidates heterodimer on the basis of " handle-hole " sudden change.Yet in their result of study, " hole-hole " model is still not enough to the ability of the formation of obstruction homodimer, and still having left over probably has 5% homodimer.The content of heterodimer improves in this study group through one step of methods such as random mutation-phage display afterwards.But the result does not solve root problem yet.
US 2010/286374A discloses the preparation method of a kind of FC heterodimer albumen or polypeptide; Related heterodimer albumen comprises polypeptide and second polypeptide that comprises the CH3 zone that first comprises the CH3 zone; They are in contact with one another and form the interface of impelling heterodimer to form; Promptly on the interface, comprise one or more charged amino acid at first polypeptide and second polypeptide that comprises the CH3 zone that comprises the CH3 zone; Electrostatic interaction between charged amino acid is unfavorable for the formation of homodimer but helps the formation of heterodimer; Specifically be the amino acid that charged amino acid in first or second polypeptide that comprises in the CH3 zone is replaced with opposite charges, utilize electrostatic interaction to promote the formation of heterodimer.There are two defectives in this inventive method; This technical scheme lack of complete property of the first, the effect between the amino acid of interface is not limited between a pair of amino acid, and interface amino acid replaces with opposite charges; Particularly under the situation of a plurality of amino acid mutations; Based on interactively complicated between the amino acid of interface, electrostatic interaction always can not suppress homodimer or help the formation of heterodimer, helps the formation of heterodimer really though comprise the concrete alternative of part in this patent; But lack strategy or method that this is estimated and screens in its technical scheme; Under the situation of interface amino acid effect complicacy or multipoint mutation, for obtaining that the justice sudden change is arranged, this patent can't instruct those skilled in the art; It two is that this technical scheme is confined to electric charge amino acid and does opposite electrical replacement; In fact; Form a charge effect network between the amino acid of interface; The electrical variation of any one amino acid (electric charge or non-electric charge amino acid) all can cause the change of electrostatic interaction between two polypeptied chains, is confined to that the opposite electrical replacement of electric charge amino acid do can be ignored a large amount of justice that has that possibly exist and suddenlys change.
Summary of the invention
The object of the present invention is to provide the remodeling method of a kind of heterodimer antibody FC.
Another object of the present invention is to provide a kind of transformation further to prepare the relevant albumen of heterodimer antibody FC and the method for polypeptide based on heterodimer antibody FC.
Another object of the present invention also is to provide the heterodimer antibody FC albumen and the polypeptide itself of described method preparation, comprises the independent polypeptide of forming heterodimer antibody FC.
Another object of the present invention also is to provide the nucleotide sequence of these polypeptide of coding.
The objective of the invention is based on the amino acid whose coulombic interaction network of FC heavy chain CH3 two arms, reduce between the CH3 zone self bonded ability (formation homodimer) thereby revise CH3 zone amino acid, thereby obtain heterodimer through the electrical charge rejection effect.In general; At the CH3-CH3 contact surface; Revising related amino acid is that charge residue will form the electrical charge rejection effect, and certain positively charged amino acid (Methionin, l-arginine) is negative electricity amino acid (aspartic acid on the contact surface that suddenlys change in some cases; L-glutamic acid) or opposite, just can form repulsive interaction.
The CH3 amino acid that the invention describes a modification FC comes the method for the interaction (helping forming heterodimer) between relief regions self-interaction (helping forming homodimer) and the enhancement region.Through setting up the amino acid interactive network of CH3-CH3 action face; Obtain the effect situation between the electric charge amino acid in the network; Selecting one or more amino acid arbitrarily, analyze the influence situation of selected amino acid to homodimer and heterodimer, is charged amino acid with selected amino acid mutation; Investigate the influence situation of sudden change back for homodimer and heterodimer; Compare after will suddenling change with before the sudden change,, will produce the effect that strengthens heterodimer and weaken homodimer formation so if it is suitable to suddenly change.Investigate combination of all amino acid and contiguous amino acid whose number (number is big more, and then sudden change causes the variation of former attribute big more) at last, select the reasonable amino acid sudden change, maximized enhancing heterodimer and weaken the effect of homodimer.
The present invention adopts following technical scheme:
A kind of heterodimer FC remodeling method based on the electric charge network; It is characterized in that; Comprise at first of FC on polypeptide and/or second polypeptide that comprises the CH3 zone in CH3 zone; One or more amino acid mutations are positive charge or negative charge amino acid, and they are in contact with one another and form the interface of impelling heterodimer to form, and sport the justice of selecting to obtain by following method that has and one of suddenly change:
1) one or more interfaces amino acid in the CH3 of random mutation antibody FC zone is the amino acid that has positive charge or negative charge;
2), calculate the influence value of sudden change for homodimer and heterodimer based on the electric charge role of network between the amino acid of interface:
Electric charge sum before sudden change influence value=sudden change back electric charge sum-sudden change
The amino acid whose sub-network of electric charge sum=suddenlyd change (comprise mutating acid and interactionally contact amino acid whose minimum amino acid network) electric charge sum with it
Wherein, it is 1 that positive positive charge interacts, and it is 1 that negative negative charge interacts, and positive and negative charge interacts and is-1, just/and non-electric charge or negative/non-charge effect is 0;
3) screening for the homodimer influence value more than or equal to 0 sudden change, or to the heterodimer influence value smaller or equal to 0 sudden change.
Among the FC heterodimer albumen of US 2010/286374A record or the preparation method of polypeptide; Part relates in the aforesaid method electric charge amino acid and does that electrical phase back mutation is resulting to have a justice sudden change; Even but electric charge amino acid is done electrical phase back mutation; US 2010/286374A also can't obtain and the identical result of the inventive method, and US 2010/286374A neither comprises whole justice sudden changes that have, and is unfavorable for the scheme that heterodimer forms after can not getting rid of sudden change.
Above-mentioned heterodimer FC remodeling method has comprised for the amino acid whose sudden change of non-electric charge on the polypeptide that comprises the CH3 zone, can greatly expand the scope that the justice sudden change is arranged, and has concrete an application of enriching achievement as aforesaid method, and the technical scheme of employing is:
A kind of heterodimer FC remodeling method based on the electric charge network; It is characterized in that; Comprise at first of FC on polypeptide and/or second polypeptide that comprises the CH3 zone in CH3 zone; One or more not charged amino acid mutations are positive charge or negative charge amino acid, and they are in contact with one another and form the interface of impelling heterodimer to form, and sport the justice of selecting to obtain by following method that has and one of suddenly change:
1) one or more not charged interface amino acid in the CH3 of random mutation antibody FC zone are the amino acid that has positive charge or negative charge;
2), calculate the influence value of sudden change for homodimer and heterodimer based on the electric charge role of network between the amino acid of interface:
Electric charge sum before sudden change influence value=sudden change back electric charge sum-sudden change
The amino acid whose sub-network positive and negative charge sum of electric charge sum=suddenlyd change
Wherein, it is 1 that positive positive charge interacts, and it is 1 that negative negative charge interacts, and positive and negative charge interacts and is-1, just/and non-electric charge or negative/non-charge effect is 0;
3) screening for the homodimer influence value more than or equal to 0 sudden change, or to the heterodimer influence value smaller or equal to 0 sudden change.
Described method is based on the electric charge role of network between the amino acid of interface, and FC transforms to heterodimer, and screening has the justice sudden change with formation that suppresses homodimer or the formation that helps heterodimer, and described method specifically may further comprise the steps:
1) CH3 of FC zone aminoacid sequence and structure obtain;
2) interface amino acid obtains;
3) make up the coulombic interaction network;
4) calculating of node (amino acid) degree in the interactive network;
5) one or more interfaces amino acid in random mutation CH3 zone, screening has the justice sudden change.
In the amino acid whose interactive network in CH3-CH3 interface; One or more interfaces amino acid on random mutation first chain and/or second chain (comprising simple point mutation, two point sudden change etc. at random at random); Calculating comprises the preceding and sudden change back electric charge summation of sub-network sudden change of institute's mutating acid, and calculates the influence value that suddenlys change for homodimer and heterodimer:
The amino acid whose sub-network positive and negative charge sum of electric charge sum=suddenlyd change
Wherein, it is 1 that positive positive charge interacts, and it is 1 that negative negative charge interacts, and positive and negative charge interacts and is-1, just/and non-electric charge or negative/non-charge effect is 0;
Sudden change is calculated by following method the influence value that is brought on the electric charge meaning that is formed on of homodimer and heterodimer:
Electric charge sum before sudden change influence value=sudden change back electric charge sum-sudden change
Screening influences the sudden change (influence value=＜0) smaller or equal to 0 for homodimer is influenced the sudden change (being influence value＞=0) more than or equal to 0 to heterodimer.
Described heterodimer FC comprises human normal immunoglobulin FC.
Preferred simple point mutation of described sudden change or two point sudden change.
Described sudden change back positive charge amino acid is Methionin or l-arginine, and sudden change back negative electricity amino acid is aspartic acid or L-glutamic acid.
The present invention also can improve the defective that " handle-hole " model suppresses homodimer through introducing electrocharge effect; On the basis of " hole " chain; Further heterodimer FC is transformed through described method; Introduce the electrical charge rejection effect, then the repulsive interaction of " hole-hole " chain will be strengthened, the formation of final complete inhibition " hole-hole " homodimer.
In some aspects, the present invention also provides one to obtain the proteic method of heterodimer.Heterodimer albumen has comprised the polypeptide in a CH3 zone and the polypeptide interactive surfaces that another one comprises the CH3 zone.First polypeptide and second polypeptide that comprises the CH3 zone that comprises CH3 zone is through heterodimer FC remodeling method of the present invention, and the charged amino acid that is designed to comprise the CH3-CH3 action face makes it to suppress homodimer and the formation of supporting heterodimer.
The proteic preparation method of a kind of heterodimer; Described heterodimer albumen comprises polypeptide and second polypeptide that comprises the CH3 zone that first comprises the CH3 zone; May further comprise the steps: 1) cultivate host cell; Host cell comprises coding, and first comprises nucleic acid and second nucleic acid that comprises CH3 zone polypeptide of CH3 zone polypeptide, and first and second of the host cell expressions of wherein cultivating comprise the polypeptide in CH3 zone; 2) from the host cell culture, extract heterodimer albumen; It is characterized in that:
Described polypeptide and/or described second polypeptide that comprises the CH3 zone that first comprises the CH3 zone comprise the amino acid that one or more sudden changes produce on the interface; They are in contact with one another and form the interface of impelling heterodimer to form; Sudden change is positive charge or negative charge amino acid by charged interface amino acid mutation not, and meets by what following method was selected to obtain one of justice sudden change arranged:
1) the one or more not charged interface amino acid on the random mutation CH3 zone are the amino acid that has positive charge or negative charge;
2), calculate the influence value of sudden change for homodimer and heterodimer based on the electric charge role of network between the amino acid of interface:
Electric charge sum before sudden change influence value=sudden change back electric charge sum-sudden change
The amino acid whose sub-network positive and negative charge sum of electric charge sum=suddenlyd change
Wherein, it is 1 that positive positive charge interacts, and it is 1 that negative negative charge interacts, and it is 1 that positive and negative charge interacts, just/and non-electric charge or negative/non-charge effect is 0;
3) screening for to the homodimer influence value more than or equal to 0 sudden change, or to the heterodimer influence value smaller or equal to 0 sudden change.
The heterodimer molecule can be used laboratory facilities purifying from host cell of standard.For example, when heterodimer albumen has comprised FC, albumen then can come purifying with albumin A.Purification process includes but not limited to chromatographic technique such as volume-exclusion, IX, affinity chromatography and ultrafiltration process.The separation purification method of heterodimer of the present invention also comprises the appropriate combination of above-mentioned the whole bag of tricks.
Described heterodimer albumen comprises FC, preferred human normal immunoglobulin FC.In the ordinary course of things, the CH3 zone polypeptide in human normal immunoglobulin FC zone derives from the human normal immunoglobulin FC zone of wild-type.The human normal immunoglobulin FC of wild-type refers to occur in the aminoacid sequence among the crowd, and the FC sequence has some subtle differences in individuality certainly.Human normal immunoglobulin FC comprises that also the amino acid discrete for wild-type human normal immunoglobulin FC sequence changes among the present invention; Such as; Local some the amino acid whose change in FC zone, as comprise some amino acid, the perhaps sudden change of other nonsenses in the glycosylation site sudden change; Also comprise indivedual amino acid whose change according to the sudden change of " handle-hole " model.
Term " human normal immunoglobulin FC " refers to the carboxyl terminal of human normal immunoglobulin chain constant region, particularly immunoglobulin heavy chain constant region or a part wherein.For example, immunoglobulin FC region can comprise two or more structural domains of heavy chain CH1, CH2, CH3, CH4 and the combination of immunoglobulin hinge region.According to the aminoacid sequence of CH, Tegeline can be divided into different kinds, mainly contains 5 immunoglobulin like protein: IgA, IgD; IgE, IgG and IgM, some of them also can further be divided into subclass (isotype), like IgG-1; IgG-2, IgG-3, IgG-4, IgA-1 and IgA-2.From specific immunoglobulin class and subclass, select specific immunoglobulin FC region within the scope that those skilled in the art grasped.
In embodiment, the used human normal immunoglobulin FC of the present invention comprises at least one Tegeline hinge region, and a CH2 structural domain and a CH3 structural domain are specially human IgG1 FC (Fig. 1).In an embodiment, the CH3 in said human normal immunoglobulin FC zone is then by the sequence encoding shown in the SEQ ID N0:1.
The mammalian host cell that the present invention relates to includes but not limited to CHO, 293, and the myeloma cell.Host cell also can be yeast or prokaryotic cell prokaryocyte such as E. Coli.
Heterodimer albumen of the present invention is not only antibody, also can be bi-specific antibody, single unit price type antibody, single domain antibody, polypeptide type antibody, (see figure 2)s such as dual specific polypeptide type antibody.
The invention still further relates to the heterodimer albumen that sudden change according to the method described above produces, and the independent polypeptide of forming heterodimer antibody FC.
The present invention also further relates to the nucleotide sequence of these polypeptide of encoding.
The present invention further relates to the pharmaceutical composition of the independent polypeptide of the composition heterodimer antibody FC that comprises the sudden change generation.
Reduce other unwanted products such as the dimeric method of homology when the invention provides an increase heterodimer content.The heterodimer albumen that is obtained is mainly used in medical field; In some cases; Heterodimer albumen can be directed at tumour cell with targets such as medicine, affinity tag, cytotoxic cell, T cells; Thereby more effectively bring into play lethal effect, aspect the immunodiagnosis of tumour and the immunotherapy new method and approach is being provided.
Description of drawings
Fig. 1 .IgG1 antibody structure and different zones synoptic diagram.
Fig. 2. comprise the different heterodimer protein combination phenomenons of FC strand.
Fig. 3. the interactive network figure of the CH3-CH3 effect interface upper amino acid of human IgG1 FC.
Fig. 4. prepare the SDS-PAGE analyzing and testing result of heterodimer based on electric charge network rebuilding FC.
Embodiment
The invention provides a kind of modification CH3 amino acid transforms FC; Come the interaction (heterodimer) between relief regions self-interaction (homodimer) and the enhancement region; And further obtain the method for heterodimer, the step of said method is described below:
1. sequence and structure obtain
(PDB www.pdb.org) obtains 48 antibody crystals structures that comprise the FC zone, altogether through structural similarity searching algorithm (Ye and Godzik 2004) from Protein Data Bank.FC homodimer structure is from 1DN2 (PDB numbering).Two screening strategies can be used to discern the amino acid contact between the CH3-CH3: (i) distance of amino acid effect (ii) solvent can reach regional analysis.Here screen according to amino acid operating distance.
2. interface amino acid obtains
According to amino acid contact rule, interface amino acid refers to that distance between any one amino acid whose heavy atom of a side chain heavy atom and an other chain is less than those amino acid of a threshold value.Here threshold selection is
In some literature can also choose
(Bahar? and? Jernigan? 1997).Table 1 is the interactional amino acid tabulation of the CH3 of antibody first chain and antibody second chain.From then on the result can find out, first chain and the amino acid whose interaction of the antibody second chain CH3 are not only man-to-man relation, but the relation of one-to-many or multi-to-multi, this conclusion also can be found out from amino acid effect network chart (Fig. 2) more intuitively.What table 1 was listed is through amino acid contact 34 interface amino acid that screening rule filtered out.
The amino acid tabulation of table 1CH3-CH3 interface
3. make up the amino acid interactive network
On amino acid whose basis, CH3-CH3 interface, the pairing of the interaction between the amino acid is made up amino acid interactive network (see figure 2).Interaction between that amino acid effect network is comprised by all CH3-CH3 interface amino acid and any one interface amino acid and other interface amino acid constitutes.In the network chart, respectively charged amino acid is carried out the electric charge attribute labeling, aspartic acid, L-glutamic acid be for negative, and l-arginine, Methionin are for just.
4. the calculating of node (amino acid) degree in the interactive network
Node degree is meant that a node (amino acid) has the number of adjacent node (interactional amino acid), based on the coulombic interaction network, calculates each node respectively and closes on amino acid whose number (seeing table 2).Any one interface amino acid (node) amino acid interactional with it (adjacent node) constitutes the amino acid whose sub-network in this interface.If in general, the amino acid that certain amino acid closes on is more, and this amino acid that then suddenlys change exerts an influence to protein stability more easily.
The amino acid node degree tabulation of table 2CH3-CH3 interface
5. random mutation and screening have the justice sudden change
In the amino acid whose interactive network figure in CH3-CH3 interface, the one or more amino acid on random mutation first chain and/or second chain comprise at random simple point mutation, two point sudden change and multipoint mutation at random.Calculating comprises the preceding electric charge summation of sudden change of the sub-network of institute's mutating acid, and sudden change back electric charge summation.
The amino acid whose sub-network positive and negative charge sum of electric charge summation=suddenlyd change
Wherein, it is 1 that positive positive charge interacts, and it is 1 that negative negative charge interacts, and positive and negative charge interacts and is-1, just/and non-electric charge or negative/non-charge effect is 0.
Calculate the be formed on influence that electric charge meaning on brought of sudden change to homodimer and heterodimer:
Electric charge sum before the sudden change influence=sudden change back electric charge sum-sudden change.
Screening influences the sudden change (influence value＜=0) smaller or equal to 0 for homodimer is influenced the sudden change (being influence value＞=0) more than or equal to 0 to heterodimer.
Sudden change is rationally selected in comprehensive amino acidity (amino acidity is more little good more) and favourable sudden change (causing favourable influence to reach the disadvantageous effect to homodimer caused to heterodimer).
Positive charge amino acid in this patent after the sudden change is Methionin and l-arginine, and the negative electricity amino acid after the sudden change is aspartic acid and L-glutamic acid.
Simple point mutation
On the CH3-CH3 contact interface, selecting amino acid mutation is positive charge, has the sudden change (influence value >=0) of negative impact to see table 3 to homodimer.
Table 3 simple point mutation is the influence that positive charge amino acid is caused
Sequence number 1-6 is disclosed by US 2010/286374A for the negative charge amino acid mutation is a positive charge amino acid.7-22 is a positive charge amino acid for not charged amino acid mutation.It is thus clear that; According to the method for the invention; Suppress homodimer through not charged amino acid simple point mutation; Arbitrary method below can selecting, promptly at polypeptide first chain that comprises the CH3 zone, the amino acid that selection sports positively charged is PHE405, SER364, TYR407, VAL397, SER400, GLN362, VAL363 or LEU398.
On the CH3-CH3 contact interface, selecting amino acid mutation is negative charge, has the sudden change (influence value >=0) of negative impact to see table 4 to homodimer.
Table 4 simple point mutation is the influence that negative charge amino acid is caused
Sequence number 1-4 is disclosed by US 2010/286374A for the positive charge amino acid mutation is a negative charge amino acid.5-10 is a negative charge amino acid for not charged amino acid mutation.It is thus clear that, according to the method for the invention, suppress homodimer through not charged amino acid simple point mutation, arbitrary method below can selecting, promptly at polypeptide first chain that comprises the CH3 zone, selecting to sport electronegative amino acid is VAL348, TYR349 or THR350.
The two point sudden change
Two amino acid of random mutation on the CH3-CH3 contact interface select amino acid mutation for the sudden change (influence value＞=0) that homodimer had negative impact and/or to heterodimer the sudden change (influence value＜=0) of favourable influence to be arranged, and see table 5.
The influence that table 5 two point mutating acid is caused
a: sport charged amino acid whose electric charge (positive charge is 1, and negative charge is-1)
Sequence number 1-11 comprises that the positive charge amino acid mutation is negative charge amino acid or does the phase back mutation; Wherein the charged amino acid mutation of part is disclosed (as 1,2,3,5 and 9) by US 2010/286374A, but its enforcement is only limited on Fc the amino acid that is adapted for opposite charges with the contacted charge residue of these amino acid.12-33 is that two not charged amino acid sport positive electricity or negative charge amino acid respectively.It is thus clear that, according to the method for the invention,, following two kinds of selections are arranged through not charged amino acid two point sudden change preparation heterodimer albumen:
1, IgG1FC comprises that polypeptide that first comprises CH3 zone and second sequence that comprises the polypeptide in CH3 zone are different from the human normal immunoglobulin sequence of wild-type, and it is replaced with by not charged amino acid in CH3 zone on first chain, and the not charged amino acid in CH3 zone replaces with positive charge on amino acid and second chain of negative charge IgG sequence formed.Specifically comprise one of following mode:
The polypeptide first chain mutation T YR349 comprising the CH3 zone is a negative charge, is positive charge at polypeptide second chain sudden change SER354 that comprises the CH3 zone;
The polypeptide first chain mutation T HR350 comprising the CH3 zone is a negative charge, is positive charge at polypeptide second chain sudden change SER354 that comprises the CH3 zone;
Polypeptide first chain sudden change SER408 comprising the CH3 zone is a negative charge, is positive charge at the polypeptide second chain mutation T YR407 that comprises the CH3 zone;
The polypeptide first chain mutation T HR366 comprising the CH3 zone is a negative charge, is positive charge at the polypeptide second chain mutation T YR407 that comprises the CH3 zone;
Polypeptide first chain sudden change ASN390 comprising the CH3 zone is a negative charge, is positive charge at polypeptide second chain sudden change SER400 that comprises the CH3 zone;
The polypeptide first chain mutation T HR399 comprising the CH3 zone is a negative charge, is positive charge at polypeptide second chain sudden change VAL397 that comprises the CH3 zone;
Polypeptide first chain sudden change PRO395 comprising the CH3 zone is a negative charge, is positive charge at polypeptide second chain sudden change VAL397 that comprises the CH3 zone;
The polypeptide first chain mutation T HR394 comprising the CH3 zone is a negative charge, is positive charge at the polypeptide second chain mutation T YR407 that comprises the CH3 zone;
Polypeptide first chain sudden change LEU365 comprising the CH3 zone is a negative charge, is positive charge at the polypeptide second chain mutation T YR407 that comprises the CH3 zone;
Polypeptide first chain sudden change LEU368 comprising the CH3 zone is a negative charge, is positive charge at polypeptide second chain sudden change SER364 that comprises the CH3 zone.
2, IgG1FC comprises that polypeptide that first comprises CH3 zone and second sequence that comprises the polypeptide in CH3 zone are different from the human normal immunoglobulin sequence of wild-type, and it is replaced with by not charged amino acid in CH3 zone on first chain, and the not charged amino acid in CH3 zone replaces with negative charge on amino acid and second chain of positive charge IgG sequence formed.Specifically comprise one of following mode:
Polypeptide first chain sudden change SER364 comprising the CH3 zone is a positive charge, is negative charge at polypeptide second chain sudden change LEU368 that comprises the CH3 zone;
Polypeptide first chain sudden change VAL397 comprising the CH3 zone is a positive charge, is negative charge at polypeptide second chain sudden change PRO395 that comprises the CH3 zone;
Polypeptide first chain sudden change VAL397 comprising the CH3 zone is a positive charge, is negative charge at polypeptide second chain sudden change PRO395 that comprises the CH3 zone;
The polypeptide first chain mutation T RY407 comprising the CH3 zone is a positive charge, is negative charge at polypeptide second chain sudden change SER408 that comprises the CH3 zone;
The polypeptide first chain mutation T RY407 comprising the CH3 zone is a positive charge, is negative charge at the polypeptide second chain mutation T HR366 that comprises the CH3 zone;
Polypeptide first chain sudden change SER400 comprising the CH3 zone is a positive charge, is negative charge at polypeptide second chain sudden change ASN390 that comprises the CH3 zone;
The polypeptide first chain mutation T YR407 comprising the CH3 zone is a positive charge, is negative charge at polypeptide second chain sudden change LEU365 that comprises the CH3 zone;
The polypeptide first chain mutation T YR407 comprising the CH3 zone is a positive charge, is negative charge at the polypeptide second chain mutation T HR394 that comprises the CH3 zone;
Polypeptide first chain sudden change VAL397 comprising the CH3 zone is a positive charge, is negative charge at the polypeptide second chain mutation T HR394 that comprises the CH3 zone;
Polypeptide first chain sudden change VAL397 comprising the CH3 zone is a positive charge, is negative charge at the polypeptide second chain mutation T HR393 that comprises the CH3 zone;
Polypeptide first chain sudden change SER354 comprising the CH3 zone is a positive charge, is negative charge at the polypeptide second chain mutation T YR349 that comprises the CH3 zone;
Polypeptide first chain sudden change SER354 comprising the CH3 zone is a positive charge, is negative charge at the polypeptide second chain mutation T HR350 that comprises the CH3 zone.
In addition; Under complicated situation more; Can be according to the method for the invention; To random mutation on the CH3-CH3 contact interface more than 3, comprise 1 not charged amino acid whose electric charge at least, selecting behind the amino acid mutation has the sudden change (influence value＞=0) of negative impact and/or heterodimer is had the sudden change (influence value＜=0) of favourable influence homodimer, with preparation heterodimer albumen.
According to the present invention program heterodimer FC is transformed; And prepare the method for heterodimer antibody, and being not limited to the sudden change of simple point mutation mentioned above and two point, those skilled in the art can be according to aim of the present invention; 3 are suddenlyd change with upper amino acid, to form heterodimer albumen.
This embodiment will prove through transform the CH3 structural domain based on the charge analysis of interactive network can suppress homodimer when forming heterodimer.
Fusion rotein IL-1R-Fc and Fc make up by following mode:
The expression of homodimer and heterodimer detects with SDS-PAGE (polyacrylamide gel electrophoresis).The principle that detects is that fusion rotein IL-1R-Fc has bigger molecular weight than Fc; So in IL-1R-Fc and Fc mixing process; Homodimer (IL-1R-Fc/IL-1R-Fc; Fc/Fc) and heterodimer ((IL-1R-Fc/Fc) then has the different stripes position at SDS-PAGE, just can detect the ratio situation of homodimer and heterodimer through this principle, and detected result is seen Fig. 4.
Fc fragment (hing-CH2-CH3) gene order according to the human IgG1 who searches in the gene pool; Artificial synthesis obtains people's Fc gene; Synthetic good Fc (SEQ ID NO:1) subclone is in mammalian cell expression vector pcDNA3.1, and sequence verification makes up the accuracy of plasmid; Take out test kit in the employing day root, obtaining recombinant plasmid dna to specifications is pcDNA3.1-Fc.
Human IgG1 Fc sequence (SEQ ID NO:1) as follows, the nucleotide sequence of this sequence of encoding are seen SEQ ID NO:2:DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
According to people's cell interleukin-2-receptor (IL-1R) gene order that searches in the gene pool; Artificial synthesis obtains people's IL-1R gene; Synthetic good IL-1R gene subclone is in recombinant expression vector pcDNA3.1-Fc; Obtain IL-1R-Fc fusion rotein (SEQ ID NO:2, underscore is partly represented Fc), sequence verification makes up the accuracy of plasmid; Take out test kit in the employing day root, obtaining recombinant plasmid dna to specifications is pcDNA3.1-IL-1R-Fc.
IL-1R-Fc sequence (SEQ ID NO:3 as follows; Underscore is partly represented Fc), the nucleotide sequence of this sequence of encoding is seen SEQ ID NO:4:DKCKEREEKIILVSSANEIDVRPCPLNPNEHKGTITWYKDDSKTPVSTEQA SRIHQHKEKLWFVPAKVEDSGHYYCVVRNSSYCLRIKISAKFVENEPNLCYNAQAI FKQKLPVAGDGGLVCPYMEFFKNENNELPKLQWYKDCKPLLLDNIHF SGVKDRLIVMNVAEKHRGNYTCHASYTYLGKQYPITRVIEFITLEENKPTRPVIVS PANETMEVDLGSQIQLICNVTGQLSDIAYWKWNGSVIDEDDPVLGEDYYSVENPAN KRRSTLITVLNISEIESRFYKHPFTCFAKNTHGIDAAYIQLIYPVTN
GSGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN K ALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK
The 293H cell of the common transfection of recombinant plasmid pcDNA3.1-Fc and pcDNA3.1-IL-1R-Fc to suspension culture is cultivated after 3-4 days the collecting cell supernatant.Supernatant and proteinA agarose resin are carried out immunoprecipitation, through SDS-PAGE electrophoresis detection homodimer under the non-reduced condition (IL-1R-Fc/IL-1R-Fc, Fc/Fc) and heterodimer (the formation situation of (IL-1R-Fc/Fc).
Weaken the formation of homodimer through the CH3 amino acid of analyzing coulombic interaction network amendment IL-1R-Fc or Fc, and strengthen the formation of heterodimer, table 6 is seen in concrete mutational site for details, and two mutants is that the method through overlapping PCR obtains.
The concrete mutational site of table 6. two mutants
IL-1R-FC and FC expression vector cotransfection; Finally homodimer (IL-1R-Fc/IL-1R-FC can appear simultaneously; FC/FC) and heterodimer (IL-1R-Fc/FC), under the situation of wild-type, be IL-1R-Fc/IL-1R-FC, the ratio of FC/FC and IL-1R-Fc/FC was near 1: 1: 1.
When on FC, introducing E356K, introduce the K439D mutational site on the IL-1R-Fc fusion rotein after, the ratio of IL-1R-FC/FC heterodimer has significantly and rises, and homodimer (IL-1R-Fc/IL-1R-FC, FC/FC) the ratio (see figure 4) that then descends significantly; When on FC, introducing two mutational site E356K/F405K, introduce K439D on the IL-1R-Fc fusion rotein, express back albumen and exist with the form of IL-1R-FC/FC heterodimer basically, prove that further charge effect is most important for the formation of heterodimer.
Claims (24)
1. heterodimer FC remodeling method based on the electric charge network; It is characterized in that; Comprise at first of FC on polypeptide and/or second polypeptide that comprises the CH3 zone in CH3 zone; One or more amino acid mutations are positive charge or negative charge amino acid, and they are in contact with one another and form the interface of impelling heterodimer to form, and sport the justice of selecting to obtain by following method that has and one of suddenly change:
1) one or more interfaces amino acid in the CH3 of random mutation antibody FC zone is the amino acid that has positive charge or negative charge;
2), calculate the influence value of sudden change for homodimer and heterodimer based on the electric charge role of network between the amino acid of interface:
Electric charge sum before sudden change influence value=sudden change back electric charge sum-sudden change
The amino acid whose sub-network electric charge sum of electric charge sum=suddenlyd change
Wherein, it is 1 that positive positive charge interacts, and it is 1 that negative negative charge interacts, and positive and negative charge interacts and is-1, just/and non-electric charge or negative/non-charge effect is 0;
3) screening for the homodimer influence value more than or equal to 0 sudden change, or to the heterodimer influence value smaller or equal to 0 sudden change.
2. heterodimer FC remodeling method according to claim 1; It is characterized in that; Comprise at first of FC on polypeptide and/or second polypeptide that comprises the CH3 zone in CH3 zone; One or more not charged amino acid mutations are positive charge or negative charge amino acid, and they are in contact with one another and form the interface of impelling heterodimer to form, and sport the justice of selecting to obtain by following method that has and one of suddenly change:
1) one or more not charged interface amino acid in the CH3 of random mutation antibody FC zone are the amino acid that has positive charge or negative charge;
2), calculate the influence value of sudden change for homodimer and heterodimer based on the electric charge role of network between the amino acid of interface:
Electric charge sum before sudden change influence value=sudden change back electric charge sum-sudden change
The amino acid whose sub-network electric charge sum of electric charge sum=suddenlyd change
Wherein, it is 1 that positive positive charge interacts, and it is 1 that negative negative charge interacts, and positive and negative charge interacts and is-1, just/and non-electric charge or negative/non-charge effect is 0;
3) screening for the homodimer influence value more than or equal to 0 sudden change, or to the heterodimer influence value smaller or equal to 0 sudden change.
3. heterodimer FC remodeling method according to claim 1 and 2 is characterized in that described method specifically may further comprise the steps:
1) CH3 of FC zone aminoacid sequence and structure obtain;
2) interface amino acid obtains;
3) make up the coulombic interaction network;
4) calculating of node degree in the interactive network;
5) one or more interfaces amino acid in random mutation CH3 zone, screening has the justice sudden change.
4. heterodimer FC remodeling method according to claim 1 and 2 is characterized in that described heterodimer FC comprises human normal immunoglobulin FC.
5. heterodimer FC remodeling method according to claim 4 is characterized in that described heterodimer FC comprises human normal immunoglobulin IgGFC.
6. heterodimer FC remodeling method according to claim 1 and 2 is characterized in that, describedly sports the sudden change of simple point mutation or two point.
7. heterodimer FC remodeling method according to claim 1 and 2 is characterized in that, described sudden change back positive charge amino acid is Methionin or l-arginine, and sudden change back negative electricity amino acid is aspartic acid or L-glutamic acid.
8. proteic preparation method of heterodimer; Described heterodimer albumen comprises polypeptide and second polypeptide that comprises the CH3 zone that first comprises the CH3 zone; May further comprise the steps: 1) cultivate host cell; Host cell comprises coding, and first comprises nucleic acid and second nucleic acid that comprises CH3 zone polypeptide of CH3 zone polypeptide, and first and second of the host cell expressions of wherein cultivating comprise the polypeptide in CH3 zone; 2) from the host cell culture, extract heterodimer albumen;
It is characterized in that: described polypeptide and/or described second polypeptide that comprises the CH3 zone that first comprises the CH3 zone comprise the amino acid that one or more sudden changes produce on the interface; They are in contact with one another and form the interface of impelling heterodimer to form; Sudden change is positive charge or negative charge amino acid by not charged amino acid mutation, and meets by what following method was selected to obtain one of justice sudden change arranged:
1) the one or more not charged interface amino acid on the random mutation CH3 zone are the amino acid that has positive charge or negative charge;
2), calculate the influence value of sudden change for homodimer and heterodimer based on the electric charge role of network between the amino acid of interface:
Electric charge sum before sudden change influence value=sudden change back electric charge sum-sudden change
The amino acid whose sub-network electric charge sum of electric charge sum=suddenlyd change
Wherein, it is 1 that positive positive charge interacts, and it is 1 that negative negative charge interacts, and positive and negative charge interacts and is-1, just/and non-electric charge or negative/non-charge effect is 0;
3) screening for the homodimer influence value more than or equal to 0 sudden change, or to the heterodimer influence value smaller or equal to 0 sudden change.
9. the proteic preparation method of heterodimer according to claim 8 is characterized in that described heterodimer albumen comprises human normal immunoglobulin IgA, IgD, IgE, IgG or IgM FC.
10. the proteic preparation method of heterodimer according to claim 9 is characterized in that described heterodimer albumen comprises human normal immunoglobulin IgG1FC.
11. the proteic preparation method of heterodimer according to claim 10; It is characterized in that; Said IgG1FC comprises that polypeptide that first comprises CH3 zone or second sequence that comprises the polypeptide in CH3 zone are different from the human normal immunoglobulin sequence of wild-type; It is that the amino acid whose IgG sequence of positive charge or negative charge is formed by one on the CH3 zone not charged amino acid mutation, and the amino acid of selecting to sport positively charged is PHE405, SER364, TYR407, VAL397, SER400, GLN362, VAL363, LEU398; Perhaps selecting to sport electronegative amino acid is VAL348, TYR349, THR350.
12. the proteic preparation method of heterodimer according to claim 10; It is characterized in that; Described IgG1FC comprises that polypeptide that first comprises CH3 zone and second sequence that comprises the polypeptide in CH3 zone are different from the human normal immunoglobulin sequence of wild-type, and it is made up of by the IgG sequence of positive charge not charged amino acid mutation in CH3 zone on the amino acid that is negative charge of not charged amino acid mutation in CH3 zone on first chain and second chain.
13. the proteic preparation method of heterodimer according to claim 10; It is characterized in that; Described IgG1FC comprises that polypeptide that first comprises CH3 zone and second sequence that comprises the polypeptide in CH3 zone are different from the human normal immunoglobulin sequence of wild-type, and it is made up of by the IgG sequence of negative charge not charged amino acid mutation in CH3 zone on the amino acid that is positive charge of not charged amino acid mutation in CH3 zone on first chain and second chain.
14. the proteic preparation method of heterodimer according to claim 8 is characterized in that, described heterodimer albumen is antibody, bi-specific antibody, single unit price type antibody, single domain antibody, polypeptide type antibody or dual specific polypeptide type antibody.
15. heterodimer albumen; Comprise polypeptide and second polypeptide that comprises the CH3 zone that first comprises the CH3 zone; It is characterized in that described polypeptide and/or described second polypeptide that comprises the CH3 zone that first comprises the CH3 zone comprise one or more mutating acids on the interface, they are in contact with one another and form the interface of impelling heterodimer to form; Sudden change is positive charge or negative charge amino acid by charged interface amino acid mutation not; And for the homodimer influence value more than or equal to 0, or to the heterodimer influence value smaller or equal to 0, influence value calculates by following method:
Electric charge sum before sudden change influence value=sudden change back electric charge sum-sudden change
The amino acid whose sub-network electric charge sum of electric charge sum=suddenlyd change
Wherein, it is 1 that positive positive charge interacts, and it is 1 that negative negative charge interacts, and positive and negative charge interacts and is-1, just/and non-electric charge or negative/non-charge effect is 0.
16. heterodimer albumen according to claim 15 is characterized in that, described heterodimer albumen comprises human normal immunoglobulin IgA, IgE, the FC zone of IgD or IgM.
17. heterodimer albumen according to claim 16 is characterized in that, described FC zone comprises human normal immunoglobulin IgG1FC.
18. heterodimer albumen according to claim 17; It is characterized in that; First comprises the polypeptide in CH3 zone or the human normal immunoglobulin sequence that second sequence that comprises the polypeptide in CH3 zone is different from wild-type described IgG1FC; It is replaced with by one on the CH3 zone not charged amino acid, and the amino acid whose IgG sequence of positive charge or negative charge formed, and the amino acid of selecting to sport positively charged is PHE405, SER364, TYR407, VAL397, SER400, GLN362, VAL363, LEU398; Perhaps selecting to sport electronegative amino acid is VAL348, TYR349, THR350.
19. heterodimer albumen according to claim 17; It is characterized in that; Described IgG1FC comprises that polypeptide that first comprises CH3 zone and second sequence that comprises the polypeptide in CH3 zone are different from the human normal immunoglobulin sequence of wild-type, and it is replaced with by not charged amino acid in CH3 zone on first chain, and the not charged amino acid in CH3 zone replaces with positive charge on amino acid and second chain of negative charge IgG sequence formed.
20. heterodimer albumen according to claim 17; It is characterized in that; Described IgG1FC comprises that polypeptide that first comprises CH3 zone and second sequence that comprises the polypeptide in CH3 zone are different from the human normal immunoglobulin sequence of wild-type, and it is replaced with by not charged amino acid in CH3 zone on first chain, and the not charged amino acid in CH3 zone replaces with negative charge on amino acid and second chain of positive charge IgG sequence formed.
21. heterodimer albumen according to claim 15 is characterized in that, described heterodimer albumen is antibody, bi-specific antibody, single unit price type antibody, single domain antibody, polypeptide type antibody or dual specific polypeptide type antibody.
A 22. peptide species; The CH3 zone that comprises antibody; It is characterized in that described CH3 zone comprises the peptide sequence that is different from wild-type CH3 zone, formed, suddenly change for the homodimer influence value more than or equal to 0 by the amino acid of the one or more uncharged amino acid mutation in the wild-type CH3 zone by positively charged amino acid or negative charge; Or to the heterodimer influence value smaller or equal to 0, influence value calculates by following method:
Electric charge sum before sudden change influence value=sudden change back electric charge sum-sudden change
The amino acid whose sub-network electric charge sum of electric charge sum=suddenlyd change
Wherein, it is 1 that positive positive charge interacts, and it is 1 that negative negative charge interacts, and positive and negative charge interacts and is-1, just/and non-electric charge or negative/non-charge effect is 0.
23. the nucleotide sequence of the said polypeptide of coding claim 22.
24. a pharmaceutical composition is characterized in that wherein comprising the described polypeptide of claim 22.
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| CN109280085A (en) * | 2017-07-21 | 2019-01-29 | 赵磊 | A kind of heterodimeric protein and preparation method thereof |
| WO2019014912A1 (en) * | 2017-07-21 | 2019-01-24 | 赵磊 | Heterodimeric protein and preparation method thereof |
| CN111094356A (en) * | 2017-09-25 | 2020-05-01 | 苏州丁孚靶点生物技术有限公司 | Protein heterodimer and application thereof |
| CN111094356B (en) * | 2017-09-25 | 2022-11-01 | 苏州丁孚靶点生物技术有限公司 | Protein heterodimer and application thereof |
| WO2019057180A1 (en) * | 2017-09-25 | 2019-03-28 | Dingfu Biotarget Co., Ltd. | Proteinaceous heterodimer and use thereof |
| US11987609B2 (en) | 2017-09-25 | 2024-05-21 | Dingfu Biotarget Co., Ltd. | Proteinaceous heterodimer and use thereof |
| WO2021000886A1 (en) | 2019-07-01 | 2021-01-07 | 苏州康宁杰瑞生物科技有限公司 | Pertussis toxin binding protein |
| WO2022002233A1 (en) | 2020-07-03 | 2022-01-06 | 苏州康宁杰瑞生物科技有限公司 | Coagulation factor xi (fxi) binding protein |
| WO2023280092A1 (en) | 2021-07-05 | 2023-01-12 | 江苏康宁杰瑞生物制药有限公司 | Antibody-drug conjugate and application thereof |
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| Publication number | Publication date |
|---|---|
| CN102558355B (en) | 2015-02-25 |
| WO2013097430A1 (en) | 2013-07-04 |
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