CN104884471A - Acceptor framework for CDR grafting - Google Patents
Acceptor framework for CDR grafting Download PDFInfo
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- CN104884471A CN104884471A CN201380069162.4A CN201380069162A CN104884471A CN 104884471 A CN104884471 A CN 104884471A CN 201380069162 A CN201380069162 A CN 201380069162A CN 104884471 A CN104884471 A CN 104884471A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/567—Framework region [FR]
Abstract
The present invention relates to an antibody acceptor framework and to methods for grafting non-human antibodies, e.g., rabbit antibodies, using a particularly well suited antibody acceptor framework. Antibodies generated by the methods of the invention are useful in a variety of diagnostic and therapeutic applications.
Description
[background of invention]
Monoclonal antibody, its conjugate and derivative are commercially in large quantities as important therapeutical agent and diagnostic reagent.Non-human antibody causes strong immune response (Schroff, 1985Cancer Res 45:879-85, the Shawler.J Immunol 1985135:1530-5 of patient usually after the injection of single low dosage; Dillman, Cancer Biother 19949:17-28).Therefore, have developed several immunogenic method for reducing muroid and other rodent animal antibody and use such as transgenic mice or phage display to manufacture the technology of human antibody.Chimeric antibody (the such as Boulianne Nature 1984312:643-6) certain degree that rodent variable region and human constant region merge by engineering design decreases Immunogenicity (such as LoBuglio, Proc NatlAcad Sci 198986:4220-4; Clark, Immunol Today 200021:397-402).Also engineering design humanized antibody, wherein the rodent sequences of variable region itself is as far as possible close to human sequence by engineering design, at least retain initial CDR simultaneously, or wherein the CDR from rodent animal antibody is transplanted to (such as Riechmann, Nature1988332:323-7 in the framework of people's antibody; US5,693,761).Rabbit polyclonal antibody is widely used in the biological analysis of such as ELISA or western blotting.Multi-clone rabbit antibody is often better than polyclone rodent animal antibody because of its usual higher avidity.In addition, rabbit often can to having bad immunogenicity and/or cause good antibody response at the antigen for not producing good combination agent during phage display in mouse.Due to these advantages known of rabbit antibody, they will be used for finding and research and development therapeutic antibodies ideally.This reason of usually not carrying out is mainly owing to producing the technological challenge in monoclonal rabbit antibodies.Because myelomatoid tumour is unknown in rabbit body, therefore the conventional hybridoma technology for generation of monoclonal antibody is not suitable for rabbit antibody.Knight and colleagues' cell carried out as expressing rabbit antibody provide the original work (people such as Spieker-Polet of fused cell system mating partner, PNAS 1995,92:9348-52) and the people such as Pytela described a kind of fusion partner clone (see, for example U.S. Patent No. 7429487) of improvement in 2005.But this technology not wide-scale distribution, because corresponding know-how is controlled by single research team substantially.There is the alternative method described for generation of monoclonal antibody in document, relate to by RT-PCR clonal antibody from selected antibody expressing cells, but have no precedent reported success for rabbit antibody.
Rabbit antibody is desirably in for causing strong immune response during human therapy as mouse antibodies, and therefore rabbit antibody can need humanization before Clinical practice.But due to textural difference respective between rabbit and mouse and between rabbit and people's antibody, the method therefore for the manufacture of humanization rodent animal antibody cannot easily be extrapolated for rabbit antibody.For example, light chain CDR3 (CDRL3) usually than people or the previously known CDRL3 of mouse antibodies longer.
Few humanization approach describing rabbit antibody in prior art, but these methods non-classical implantation method, wherein transplant the CDR of non-human donor on people's receptor antibody.WO04/016740 describes a kind of so-called " surface is reinvented " strategy.The target of " surface is reinvented " strategy be to reinvent inhuman framework solvent can and residue to make its similar people.In this area known as described in WO 04/016740 for the similar humanization technologies of rabbit antibody.WO08/144757 and WO05/016950 is open for making the humanized method of rabbit monoclonal antibodies, and it relates to and comparing with the aminoacid sequence of similar people's antibody the aminoacid sequence of parent rabbit antibody.Then the aminoacid sequence of parent rabbit antibody is changed to make the sequence of its framework region more similar to the equivalent framework district of similar people's antibody.In order to obtain good binding ability, often kind of immunoconjugator is needed individually to carry out difficult development efforts.
The potential problems of aforesaid method are not end user's framework, but carry out engineering design to rabbit framework and seem similar people to make it.There is risk in this method, the amino acid be namely embedded in protein core extends still may comprise immunogenic t cell epitope.
Up to now, applicant not yet confirms that one carries out humanized rabbit antibody by the state-of-the-art implantation method of application.This may can be explained with people or the rodent CDR fact far from each other by rabbit CDR.As known in the art, many rabbit VH chains have the halfcystine of additional pairs for muroid and mankind's counterpart.Except the conserved disulfide bridges formed between cys22 and cys92, in some rabbit chain, between last residue and first residue of CDR H2 of CDRH1, be also formed with the S-S bridge between cys21-cys79 bridge and CDR.In addition, common cysteine residues pair in CDR-L3.In addition, many rabbit antibody CDR do not belong to any previously known norm structure (canonical structure).Especially CDR-L3 is often longer than the CDR-L3 of the mankind or muroid counterpart.
Therefore, being migrated in people's framework by inhuman CDR antibody is a great protein engineering design objective.Antigen binding loops must be carried out and be transferred to people's framework through different artificial selection from the framework of natural evolution, be configured to antigen combine to retain natural ring.Antigen-binding affinity is normal after ring is transplanted significantly to be reduced or eliminates.In transplantation antigen coupling collar, use people's framework of careful selection to maximize possibility people such as (, 1996) Roguzka retaining binding affinity in humanized molecule.Although many transplantation experiments available in document provide rough guide for CDR transplants, a pattern can not be summarized in.Typical problem is that specificity, stability or producibility are loosened after CDR ring is transplanted.
Therefore, the method being badly in need of improving is come reliably and is made humanized rabbit antibodies to be used as therapeutical agent and diagnostic reagent rapidly.In addition, need people's acceptor framework reliably to make humanized rabbit antibodies, function antibody and/or the antibody fragment of the biophysical properties with similar medicine are provided.
[summary of the invention]
Surprisingly find, by the highly soluble of quality control (QC) analysis confirmation and stability people antibody framework (as people (2001) such as WO 0148017 and Auf der Maur, FEBS Lett508, disclosed in 407 to 412 page) be especially applicable to adapting to the CDR from other non-human animal's species, such as rabbit CDR.Therefore, in first aspect, the invention provides the variable region of heavy chain (so-called " a72 " VH Frame sequence) of particular person antibody, it is especially suitable as the various antibody from having different binding specificity, especially from the acceptor of the CDR of rabbit antibody, and with CDR in whether exist disulphide bridges have nothing to do.
By rabbit CDR being transplanted to the humanization immunoconjugator that produces in the variable chains framework of this high compatibility consistently and the spatial orientation of the rabbit antibody reliably keeping donor CDR to be derived from.Therefore, the structure relevant position without donor immunity bonding agent needs to introduce in acceptor framework.Due to these advantages, the high-throughput humanization to rabbit antibody can be realized, and do not optimize or ability of optimizing integration hardly.
Therefore, on the other hand, the invention provides the method for rabbit and other inhuman CDR being migrated in solubility disclosed herein and stability light chain and/or heavy chain people antibody framework sequence, producing the humanized antibody with excellent biophysical properties thus.Specifically, the immunoconjugator produced by method of the present invention represents good functional property, such as solvability and stability.
[accompanying drawing explanation]
Fig. 1 describes the CDR H1 for being transplanted to people's antibody framework of highly soluble and stability from rabbit monoclonal antibodies by antigen binding site used herein and defines.
Fig. 2: CDR3 three amino acid longer than its muroid counterpart usually analysis of the rabbit antibody sequence extracted from Kabat database being confirmed to variable heavy chain.
[detailed Description Of The Invention]
[definition]
Can be easier to for making the present invention understand, will as some term of giving a definition.Other definition is listed in a specific embodiment.
Term " antibody " refers to complete antibody and any Fab.Term " antigen-binding polypeptides " and " immunoconjugator " use in this article simultaneously." antibody " refers to optionally past glycosylated albumen, and it comprises at least two heavy chains (H) and two light chains (L) or its antigen-binding portion thereof that are interconnected by disulfide linkage.Each heavy chain comprises variable region of heavy chain and (is abbreviated as V herein
h) and CH.CH comprises three domain Cs H1, CH2 and CH3.Each light chain comprises variable region of light chain and (is abbreviated as V herein
l) and constant region of light chain.Constant region of light chain comprises a domain C L.V
hand V
ldistrict can be divided into hypervariable region further again, is called complementary determining region (CDR), is interspersed with more conservative district therebetween, is called framework region (FR).Each V
hand V
lbe made up of, from N-terminal to C-terminal with following sequential arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 three CDR and four FR.The binding domains with AI is contained in the variable region of heavy chain and light chain.The constant region immunity-regulating sphaeroprotein of antibody is combined with host tissue or the factor, comprises first component (Clq) of immune various cell (such as, effector cell) and classical complement system.
" antigen-binding portion thereof " (or referred to as " antibody moiety ") of term antibody refers to the one or more antibody fragments kept with the ability of antigen (such as, TNF) specific binding.Confirm that the antigen combined function of antibody can be performed by the fragment of full length antibody.The example of the binding fragment contained in " antigen-binding portion thereof " of term antibody comprises (i) Fab fragment, a kind of by V
l, V
h, CL and CH1 structural domain composition monovalent fragment; (ii) F (ab')
2fragment, is a kind ofly included in the bivalent fragment of hinge area by two Fab fragments of disulfide bridge connects; (iii) by V
hwith the Fd fragment of CH1 structural domain composition; (iv) by the V of antibody single armed
land V
hthe Fv fragment of structural domain composition, (v) is by V
hthe single structure territory of structural domain composition or dAb fragment people such as (, (1989) Nature 341:544-546) Ward; (vi) combination of two or more CDR be separated that the complementary determining region (CDR) be separated or (vii) are optionally connected by the joint synthesized.In addition, although two of Fv fragment structural domain V
land V
hby independently genes encoding, but they can use recombination method to be connected by synthetic linker, can form V
land V
hdistrict matches wherein and forms the albumen strand of monovalent molecule and (be called scFv (scFv); See, for example the people such as Bird (1988) Science 242:423-426; With people (1988) Proc.Natl.Acad.Sci.USA85:5879-5883 such as Huston).This kind of single-chain antibody also intends to be encompassed in " antigen-binding portion thereof " of term antibody.These antibody fragments use routine techniques well known by persons skilled in the art to obtain, and screen fragment for use in the mode identical with complete antibody.Antigen-binding portion thereof can be produced by recombinant DNA technology, or is produced by enzymatic lysis or the complete immunoglobulin (Ig) of chemical cracking.Antibody can have different isotypes, such as IgG (such as IgG1, IgG2, IgG3 or IgG4 hypotype), IgA1, IgA2, IgD, IgE or IgM antibody.
Term " immunoconjugator " refers to all or part of antigen binding site containing antibody, and such as all or part of heavy chain and/or the molecule of light variable domains, identify target antigen specifically to make immunoconjugator.The limiting examples of immunoconjugator comprises total length immunoglobulin molecules and scFv, and antibody fragment, includes, but is not limited to (i) Fab fragment, a kind of by V
l, V
h, C
land C
hthe monovalent fragment of 1 structural domain composition; (ii) F (ab')
2fragment, is a kind ofly included in the bivalent fragment of hinge area by two Fab fragments of disulfide bridge connects; (iii) Fab ' fragment, it is Fab (the reference FUNDAMENTALIMMUNOLOGY (Paul compiles, the 3rd supplementary issue version 1993) with part hinge district substantially; (iv) by V
hand C
hthe Fd fragment of 1 structural domain composition; V () is by the V of antibody single armed
land V
hthe Fv fragment of structural domain composition, (vi) is by V
hor V
lthe single structure domain antibodies of structural domain composition, such as Dab fragment (people such as Ward, (1989) Nature
341: 544-546), a kind of camellid is (with reference to people such as Hamers-Casterman, the people such as Nature 363:446-448 (1993) and Dumoulin, Protein Science 11:500-515 (2002)) or shark antibody (such as, shark Ig-NARs
); (vii) nano antibody, a kind of variable region of heavy chain containing single variable domains and two constant domain.
Term " single-chain antibody ", " scFv " or " scFv " refer to heavy chain of antibody variable domains (or the district comprising and connected by joint; V
h) and light chain of antibody variable domains (or district; V
l) molecule.This kind of scFv molecule can have universal architecture: NH
2-V
l-joint-V
h-COOH or NH
2-V
h-joint-V
l-COOH.The joint of suitable prior art is made up of the GGGGS aminoacid sequence repeated or its varient.In a preferred embodiment of the invention, use (GGGGS) of aminoacid sequence listed in SEQ ID NO:8
4joint, but also may be 1 to 3 varient people (1993) such as (, Proc.Natl.Acad.Sci.USA90:6444-6448) Holliger repeated.The people such as Alfthan (1995), the people (2001) such as Protein Eng.8:725-731, Choi, the people such as Eur.J.Immunol.31:94-106, Hu (1996), the people (1999) such as Cancer Res.56:3055-3061, Kipriyanov, the people such as J.Mol.Biol.293:41-56 and Roovers (2001), Cancer Immunol describes other joint used in the present invention.
As used herein, term " functional performance " be polypeptide (such as, immunoconjugator) a kind of characteristic, it improves (such as, for conventional polypeptide) be favourable and/or useful to those skilled in the art, such as, in order to improve manufacturing characteristics or the therapeutic efficiency of polypeptide.In one embodiment, functional performance is stability (such as, thermostability).In another embodiment, functional performance is solvability (such as, under cell condition).In still another embodiment, functional performance is Assembling Behavior.In still another embodiment, functional performance is protein expression (such as in prokaryotic cell prokaryocyte).In still another embodiment, functional performance is the refolding behavior after occlusion body dissolves in the fabrication process.In certain embodiments, functional performance is not the improvement of antigen-binding affinity.In another preferred embodiment, the essentially no impact of the binding affinity of improvement on immunoconjugator of one or more functional performances.
Term " CDR " refers to one of six hypervariable regions mainly contained in the variable domains helping the antibody that antigen combines.One of definition the most frequently used for six CDR by people such as Kabat E.A., (1991) Sequences of proteins of immunological interest.NIHPublication 91-3242) provide.As used herein, the Kabat definition of CDR is only applicable to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDRL3 or L1, L2, L3) of light variable domains and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of heavy-chain variable domains.But the CDR1 (CDR H1 or H1) of heavy-chain variable domains as used herein is defined by the resi-dues (Kabat numbering) terminated from position 26 and before position 36.This definition is as distinguished the CDR H1 syzygy (being also described with reference to figure 1) defined by Kabat and Chotia substantially.
" antibody framework " refers to the variable domains VL of support or the part of VH that serve as the antigen binding loops (CDR) of this variable domains as the term is employed herein.Substantially it is the variable domains without CDR.
Term " epi-position " or " antigenic determinant " refer to the site (specific position such as, on TNF molecule) on the antigen that immunoglobulin (Ig) or antibodies specific combine.Epi-position generally includes at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 continuous or discrete amino acid in unique spatial conformation.See, for example Epitope Mapping Protocols in Methods in Molecular Biology, the 66th volume, G.E.Morris compiles (1996).
Term " specific binding ", " selective binding ", " optionally combine " and " specifically combination " refer to the epi-position on antibodies predetermined antigens.Antibody is usually to be approximately less than 10
-7m, is such as approximately less than 10
-8m, 10
-9m or 10
-10avidity (the K of M or even lower
d) combine.
Term " K
d" or " Kd " refer to the Dissociation equilibrium constant of specific antibodies-AI.As use in BIACORE instrument surface plasma body resonant vibration (SPR) technology measure, antibody of the present invention is usually such as to be less than about 10
-7m, is such as less than about 10
-8m, 10
-9m or 10
-10dissociation equilibrium constant (the K of M or even lower
d) in conjunction with TNF.
" nucleic acid molecule " refers to DNA molecular and RNA molecule as the term is employed herein.Nucleic acid molecule can be strand or double-strand, but is preferably double-stranded DNA.Nucleic acid is " operability is connected " when being placed in the functional relationship with another nucleotide sequence.For example, if promotor or enhanser affect transcribing of sequence, then it is connected with encoding sequence operability.
Term " carrier " refers to the nucleic acid molecule that can transport another nucleic acid connected.In one embodiment, carrier is " plasmid ", refers to the circular double stranded DNA ring that wherein can engage other region of DNA section.In another embodiment, carrier is virus vector, and wherein other region of DNA section can be bonded in viral genome.Carrier disclosed herein in its host cell introduced can self-replicating (such as, there is bacteria carrier and episomal mammalian vectors that bacterium copies source) or can be incorporated in the genome of host cell when introducing host cell, and therefore copy together with host genome (such as, non-free type mammalian vector).
Term " host cell " refers to the cell wherein having introduced expression vector.Host cell comprises bacterium, microorganism, plant or zooblast, preferably intestinal bacteria (Escherichia coli), Bacillus subtilus (Bacillus subtilis); Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), pichia spp (Pichia pastoris), CHO (Chinese hamster ovary line, Chinese HamsterOvary line) or NS0 cell.
Term " Lagomorph (1agomorph) " refers to the member of taxonomy Lagomorpha, comprises rabbit section (Leporidae) family (such as, hare and rabbit) and Ochotonidae (pika).In the most preferred embodiment, Lagomorph is rabbit." rabbit " refers to the animal belonging to Tu Ke family as the term is employed herein.
As used herein, " identity " refers to the sequences match between two polypeptide, molecule or between two nucleic acid.When a certain position in two both comparative sequences is occupied by identical base or amino acid monomer subunit (such as, if a certain position in two polypeptide each is all occupied by Methionin), so respective molecule is identical in this position." identity per-cent " between two sequences be sequence the function of same position quantity shared, consider the quantity of breach and the length of each breach, these need to introduce the best comparison for two sequences.Usually compare to provide maximum identity when two sequence alignments.The method of Needleman and Wunsch (J.MoI.Biol. (48): 444-453 (1970)) algorithm such as can be used to provide this comparison, this algorithm has been incorporated in the GAP program in the GCG software package using Blossum 62 matrix or PAM250 matrix, and Gap Weight (gap weight) is 16,14,12,10,8,6 or 4 and Length Weight (length weight) is 1,2,3,4,5 or 6.
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have usual the understood identical meanings of persons skilled in the art belonging to the present invention.Although can use and those similar or equivalent method and materials described herein in practice or test the present invention, suitable method and material are hereafter described.In the case of a conflict, will be as the criterion with this specification sheets (comprising definition).In addition, material, method and example are illustrative and are not intended to have restricted.
Hereafter in sub-chapters and sections, all respects of the present invention are described in further detail.Should be appreciated that, various embodiment, preferable and scope can arbitrary combination.In addition, according to specific embodiments, selected definition, embodiment or scope may be inapplicable.
If do not specified in addition, then according to AHo numbering plan instruction amino acid position.AHo numbering system at Honegger, A. and Pluckthun, A. (2001) J.Mol.Biol.
309: 657-670) in further describe.Or, can use as at the people such as Kabat (Kabat, E.A., Deng people (1991) Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Service (U.S.Department of Health and HumanServices), NIH announces No.91-3242) in the Kabat numbering system that further describes.The conversion table of two kinds of different numbering systems of the amino acid residue position identified in heavy chain of antibody and variable region of light chain is provided in A.Honegger, J.Mol.Biol.309 (2001) 657-670.
In first aspect, the invention provides a kind of for transplanting from Lagomorph species, people's acceptor framework sequence of the CDR of such as rabbit.Surprisingly finder's strand VH framework a72 (SEQID NO:1) in essence with the antigen binding site highly compatible of rabbit antibody.Therefore, a72VH represents a kind of for constructing the suitable holder being derived from the stable humanization scFv antibody fragment transplanting rabbit ring.
Therefore, in one aspect, the invention provides a kind of immunoconjugator acceptor framework, it comprises the VH sequence with SEQ ID No.1 with at least 70% identity.
Described sequence can merge with other suitable variable light any.A kind of preferred variable light be also open in WO03/097697 and be expressed as KI27 SEQ ID NO:2 or as other VL sequence any disclosed in WO03/097697.
In a preferred embodiment, variable heavy chain framework is connected with variable light framework by joint.Joint can be any suitable joint, and the joint repeated for 1 to 4 time that such as comprises sequence GGGGS (SEQ IDNO:5), is preferably (GGGGS)
4peptide (SEQ ID NO:4) or disclosed in the people such as Alfthan (1995) Protein Eng.8:725-731 joint.
Therefore, the invention provides a kind of immunoconjugator acceptor framework, it comprises
I () and SEQ ID NO:1 have the variable heavy chain framework of at least 70% identity, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% identity; And/or
(ii) there is with SEQ ID NO:2 the variable light framework of at least 70% identity, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% identity.
In a preferred embodiment, the invention provides a kind of immunoconjugator, it has the sequence with SEQ ID NO:3 with at least 60%, more preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 95% identity.
Framework is in fact all compatible with any rabbit CDR.Containing different rabbit CDR, obtain on the contrary giving full expression to rabbit wild-type strand and well produce, and almost still retaining the avidity of initial donor rabbits antibody completely.
Immunoconjugator acceptor framework as described herein, in heavy chain framework, preferably can comprise solubleness and strengthen replacement at position 12,103 and 144 (AHo numbering) place.Hydrophobic amino acid is preferably had more hydrophilic aminoacid replacement.Hydrophilic amino acid is such as arginine (R), l-asparagine (N), aspartic acid (D), glutamine (Q), glycine (G), Histidine (H), Methionin (K), Serine (S) and Threonine (T).Heavy chain framework more preferably comprises the Serine (S) at (a) position 12 place; The Serine (S) at (b) position 103 place or the Serine (S) at Threonine (T) and/or (c) position 144 place or Threonine (T).
In addition, can in 1 of variable light framework, 3,4,10,47,57, one or more positions (AHo numbering) existence and stability of 91 and 103 strengthens amino acid.Variable light framework more preferably comprises L-glutamic acid (E), the α-amino-isovaleric acid (V) at position 3 place, the leucine (L) at position 4 place, the Serine (S) at position 10 place at position 1 place; The arginine (R) at position 47 place, the Serine (S) at position 57 place, the phenylalanine (F) at position 91 place and/or the α-amino-isovaleric acid (V) at position 103 place.
Because glutamine (Q) is easy to deamination, therefore in another preferred embodiment, VH comprises glycine (G) at position 141 place.This replacement can improve the standing storage of albumen.
For example, acceptor framework disclosed herein can be used for the people or the humanized antibody that produce the binding characteristic retaining the non-human antibody that inhuman CDR is derived from.Therefore, in a preferred embodiment, immunoconjugator acceptor framework as disclosed herein is contained in the present invention, its comprise in addition from donor immunity bonding agent, preferably from mammalian immune bonding agent, more preferably from Lagomorph immunoconjugator and most preferably from heavy chain CDR1, CDR2 and CDR3 and/or light chain CDR1, CDR2 and CDR3 of rabbit.Therefore, in one embodiment, the invention provides one and have specific immunoconjugator to required antigen, it comprises
The variable light CDR of (i) Lagomorph; With
(ii) there is with SEQ ID NO:1 people's variable heavy chain framework of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% and most preferably 100% identity.
Lagomorph is preferably rabbit.Immunoconjugator more preferably comprises heavy chain CDR1, CDR2 and CDR3 from donor immunity bonding agent and light chain CDR1, CDR2 and CDR3.
As known in the art, many rabbit VH chains have the halfcystine of additional pairs for muroid and mankind's counterpart.Except the conserved disulfide bridges formed between cys22 and cys92, in some rabbit chain, between last residue and first residue of CDR H2 of CDRH1, also form the S-S bridge between cys21-cys79 bridge and CDR.In addition, common cysteine residues pair in CDR-L3.In addition, many rabbit antibody CDR do not belong to any previously known norm structure.Especially CDR-L3 is often longer than the CDR-L3 of the mankind or muroid counterpart.
As mentioned before, be transplanted to by inhuman CDR on framework disclosed herein and produce a kind of molecule, wherein CDR presents with suitable conformation.If needed, the avidity of immunoconjugator can be improved by the AI Framework residues transplanting non-human donor's immunoconjugator.These positions are such as identified by following methods
(i) identify respective germline progenitor cell sequence or, or when very high homology Frame sequence by use consensus sequence;
(ii) donor variable domain sequence and the germline progenitor cell sequence of step (i) or the sequence alignment of consensus sequence is produced; With
(iii) different residues is identified.
Different residues on molecular surface suddenly change, assuming that the former avidity that creates antagonism during avidity production process in many cases in vivo.
On the other hand, the invention provides a kind of immunoconjugator comprising immunoconjugator acceptor framework as herein described.Described immunoconjugator can be such as scFv antibody, total length immunoglobulin (Ig), Fab fragment, Dab or nano antibody.
In a preferred embodiment, immunoconjugator and one or more points of sub-connections, such as therapeutical agent (such as cytotoxic agent, cytokine, chemokine, somatomedin or other signal transducers), developer or the second albumen (such as transcriptional activation agent or DNA binding domains).
Immunoconjugator as disclosed herein such as can be used for diagnostic use, treatment use, target validation or gene therapy.
The present invention also provides a kind of nucleic acid of separation, its encode immunoconjugator acceptor framework disclosed herein or immunoconjugator as disclosed herein.
In another embodiment, a kind of carrier comprising nucleic acid disclosed herein is provided.
Nucleic acid as disclosed herein or carrier such as can be used in gene therapy.
A kind of host cell comprising carrier disclosed herein and/or nucleic acid is also contained in the present invention.
In addition, provide a kind of composition, it comprises immunoconjugator acceptor framework as disclosed herein, as disclosed herein immunoconjugator, the nucleic acid be separated as disclosed herein or carrier as disclosed herein.
Sequence disclosed herein following (X residue be CDR insertion point and containing at least 3 and at the most 50 amino acid):
SEQ ID NO.1: variable heavy chain framework a72
EVQLVESGPGLVKPSQTLSLTCGVS(X)
n＝3-50WIRQHPVKGLEWIG(X)
n=3-50RLTISVDTSKTQVSLNLRSVTAADTAVYYCAR(X)
n=3-50WGQGTTVTVSS
SEQ ID NO.2: variable light framework KI27
EIVMTQSPSTLSASVGDRVIITC(X)
n＝3-50WYQQKPGKAPKLLIY(X)
n＝3-50GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)
n=3-50FGQGTKKTVLG
SEQ ID NO.3: Frame sequence
EIVMTQSPSTLSASVGDRVIITC(X)
n＝3-50WYQQKPGKAPKLLIY(X)
n＝3-50GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)
n＝3- 50FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGPGLVKPSQTLSLTCGVS(X)
n=3-50WIRQHPVKGLEWIG(X)
n＝3-50RLTISVDTSKTQVSLNLRSVTAADTAVYYCAR(X)
n＝3-50WGQGTTVTVSS
SEQ ID NO:4: joint
GGGGSGGGGSGGGGSGGGGS
On the other hand, the invention provides by the CDR of non-human donor antibody being transplanted to stable and the antibody framework of solubility making the humanized method of non-human antibody.In an especially preferred embodiment, being derived from the CDR of rabbit antibody and framework is mentioned above those.
CDR is transplanted to universal method in people's acceptor framework by Winter in U.S. Patent No. 5,225, by people such as Queen open in WO9007861A1 in 539, the mode that they are quoted in full is incorporated herein.Strategy for the CDR from rabbit monoclonal antibodies being transplanted to the people such as people and Queen such as general policies on selected frame and Winter is relevant, but different in some importance.Specifically, the difference of method of the present invention and typical Winter and Queen method as known in the art is that people's antibody framework as disclosed herein is especially suitable as the acceptor of people or non-human donor antibody.Therefore, different from the universal method of Winter and Queen, the Frame sequence for humanization approach of the present invention needs not to be the Frame sequence of the maximum sequence similarities of the sequence shows of inhuman (such as, the rabbit) antibody be derived from donor CDR.In addition, do not need the Framework residues transplanted from donor sequences to support CDR conformation.The antigen that is arranged in framework can be introduced at most in conjunction with amino acid or other sudden change of occurring during somatic hypermutation.
Hereafter describe in order to produce the concrete details being derived from the implantation method of the antibody of humanization rabbit with high solubility and stability.
In the exemplary of the inventive method, first identify the aminoacid sequence of CDR donor antibody and use conventional sequence alignment tools (such as, Needleman-Wunsch algorithm and Blossum matrix) aligned sequences.The introducing of breach and the name of resi-dues can use conventional antibody numbering system to carry out.For example, the AHo numbering system for immunoglobulin variable domain territory can be used.Also Kabat numbering plan can be applied, because it is the most extensive accepted standard be numbered for the residue in antagonist.Kabat numbering such as can use SUBIM program to specify.The variable region of this programanalysis antibody sequence the system set up according to Kabat and co-worker are numbered people such as (, 1995) Deret to sequence.The definition in framework and CDR district is normally carried out according to the Kabat definition based on sequence variability and the most frequently used.But, for CDR-H1, title is preferably the combination (also with reference to figure 1) that Kabat definition (the mean exposure data produced by the contact analyzed between the antibody of 3D composite structure subset and antigen) people such as (, 1996) MacCallum and Chotia define (position in structure based ring district).The conversion table of two kinds of different numbering systems of the amino acid residue position identified in heavy chain of antibody and variable region of light chain is provided in A.Honegger, J.Mol.Biol.309 (2001) 657-670.Kabat numbering system is at the people such as Kabat (Kabat, E.A. people is waited, (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S. Department of Health and Human Service, NIH announces No.91-3242) in further describe.AHo numbering system at Honegger, A. and Pluckthun, A. (2001) J.Mol.Biol.
309: 657-670) in further describe.
The variable domains of rabbit monoclonal antibodies such as can use such as EXCEL implementation sequence analytical algorithm and be categorized as corresponding human sub-population (people such as Knappik, 2000, the J Mol Biol.2 months 11 based on the sorting technique analyzing a complete set of people's antibody library; 296 (1): 57-86).
CDR conformation can be appointed as antigen of donor land, then also can identify the resi-dues needing to keep different specification structure.Chothia (1989) definition is used to determine the CDR norm structure of five (L1, L2, L3, H1 and H2) in six antibody hypervariable regions of rabbit antibody.
Antibody of the present invention can optimize the functional performance showing enhancing further, the solvability such as strengthened and/or stability.In certain embodiments, be that " function has " method disclosed in the PCT application sequence No.PCT/EP2008/001958 (its be incorporated to by reference herein) of " Sequence Based Engineering and Optimization of SingleChain Antibodies " optimizes antibody of the present invention according to the title submitted on March 12nd, 2008.
The title submitted to 25, on June at the title submitted on June 25th, the 2008 PCT application No.PCT/CH2008/000285 and 2008 that is " Methods of ModifyingAntibodies, and Modified Antibodies with Improved FunctionalProperties " is describe exemplary Framework residues the position of substitution and exemplary framework substitution in the PCT application No.PCT/CH2008/000284 of " Sequence Based Engineering and Optimization ofSingle Chain Antibodies ".
In other embodiments, immunoconjugator of the present invention is included in the U.S. Provisional Application sequence No.61/075 that the title submitted on June 25th, 2008 is " Solubility Optimization of Immunobinders ", and one or more stability described in 692 strengthens sudden change.In certain preferred aspects, immunoconjugator comprises solubleness enhancing sudden change at the amino acid position place being selected from the heavy chain amino acid position be made up of 12,103 and 144 (AHo numbering conventions).In a preferred embodiment, immunoconjugator comprises one or more and is selected from following replacement: the Serine (S) at (a) heavy chain amino acid position 12 place; The Serine (S) at (b) heavy chain amino acid position 103 place or Threonine (T); (c) Serine (S) at heavy chain amino acid position 144 place or Threonine (T).In another embodiment, immunoconjugator comprises following replacement: the Serine (S) at (a) heavy chain amino acid position 12 place; The Serine (S) at (b) heavy chain amino acid position 103 place or Threonine (T); (c) Serine (S) at heavy chain amino acid position 144 place or Threonine (T).
In certain preferred aspects, in at least one of immunoconjugator in the position 1,3,4,10,47,57,91 and 103 of the variable region of light chain according to AHo numbering system, the stability comprising the Framework residues place being positioned at light chain acceptor framework strengthens sudden change.In a preferred embodiment, light chain acceptor framework comprises one or more and is selected from following replacement: the leucine (L) at the L-glutamic acid (E) at (a) position 1 place, the α-amino-isovaleric acid (V) at (b) position 3 place, (c) position 4 place; The Serine (S) at (d) position 10 place; The arginine (R) at (e) position 47 place; The Serine (S) at (e) position 57 place; The phenylalanine (F) at (f) position 91 place; (g) α-amino-isovaleric acid (V) at position 103 place.
Any one that can use various methods availalbe produces the humanized antibody of the sudden change comprised as described above.
Therefore, the invention provides one according to the humanized immunoconjugator of method as herein described.
In certain preferred aspects, the target antigen of described immunoconjugator is VEGF or TNF α.
Of the present invention or such as can use technology as known in the art to synthesize by the polypeptide that method of the present invention produces.Or, can variable region needed for composite coding nucleic acid molecule and produce polypeptide by recombination method.
For example, once determine the sequence of humanization variable region, just can be manufactured variable region by the technology known in biology field or comprise its polypeptide.More particularly, recombinant DNA technology can be used, by (such as, the DNA sequence dna of the required variable region of coding is (such as, through heavy chain or the light chain of modification with nucleotide sequence; Its variable domains or its other Fab)) transformed host cell produces each peptide species.
In one embodiment, can prepare expression vector, it comprises and the V that at least encodes
hor V
ldNA sequence dna operability connect promotor.If necessary or desired, can prepare the second expression vector, it comprises the promotor be connected with the DNA sequence dna operability of the complementary variable domains of coding, and (that is, wherein parental expression vector is encoded V
h, the second expression vector codes V
l, vice versa).(the international patent application No.PCT/GB85/00392 see, for example people such as Neuberger) is cultivated under can expressing the condition being fitted together to variable domains or chimeric antibody with one or both expression vector transformation cell lines (such as, immortalized mammalian clone) and in permission subsequently.
In one embodiment, the variable region comprising donor CDR and acceptor FR aminoacid sequence can be manufactured and replace to affect cdr amino acid subsequently to introducing change in nucleic acid molecule.
The illustrative methods approved in this area for the manufacture of the nucleic acid molecule of the amino acid sequence variants of coded polypeptide includes, but is not limited to fixed point (or oligonucleotide mediated) mutagenesis of the DNA by the coded polypeptide previously prepared, PCR mutagenesis and cassette mutagenesis and prepares.
Site-directed mutagenesis is the preferred method for the preparation of replacing varient.This technology (see, for example people such as Carter, the people such as Nucleic Acids Res.13:4431-4443 (1985) and Kunkel, Proc.Natl.Acad.Sci.USA 82:488 (1987)) well known in the art.In brief, in the site-directed mutagenesis carrying out DNA, first parent DNA is made to become this parent DNA of strand by the oligonucleotide of the required sudden change of hybridization coding.After hybridization, use the oligonucleotide of hybridization as primer and use the parent DNA of strand as template, using archaeal dna polymerase to synthesize whole Article 2 chain.Therefore, the oligonucleotide that suddenlys change needed for coding is incorporated in the double-stranded DNA obtained.
PCR mutagenesis is also suitable for the amino acid sequence variants manufacturing polypeptide.With reference to Higuchi, at PCR Protocols, in the 177 to 183 page (Academic Press, 1990); With people such as Vallette, Nuc.Acids Res.17:723-733 (1989).In brief, when using small amounts of template DNA as parent material in PCR, the primer that sequence can be used slightly different from the respective area in template DNA produces relatively a large amount of DNA fragment specific, and this DNA fragment specific is only different from template sequence in the position that primer is different from template.
The method cassette mutagenesis that another kind prepares varient is based on people such as Wells, the technology described in Gene34:315-323 (1985).Parent material is the plasmid (or other carrier) comprising DNA to be suddenlyd change.Identify the codon in parent DNA to be suddenlyd change.The restriction endonuclease site that one unique must be had in every side in identified mutational site.If there is no this restriction site, can use above-mentioned oligonucleotide mediated mutafacient system to produce to be introduced into the appropriate location in the DNA of coded polypeptide.In these sites cutting plasmid DNA to make its linearizing.Use the DNA sequence dna between standard program composite coding restriction site and the double chain oligonucleotide containing required sudden change, wherein two chains of oligonucleotide synthesize separately and use standard technique hybridization subsequently together.This double chain oligonucleotide is called box.5' and the 3' end had with the compatible ends of linearization plasmid is appointed as by this box, so that it can directly engage with plasmid.The DNA sequence dna of this plasmid now containing sudden change.
The variable region produced by method of the present invention can be reinvented and change further and be combined to increase antigen further.Therefore, above-mentioned steps prior to other step, or then can carry out other step, such as, comprise affinity maturation.In addition, experimentally can be used for further optimization in conjunction with data.
Except aminoacid replacement, the present invention expects that other is modified, the Fc district varient that such as Fc region amino acid sequence changes to produce effector function.The one or more amino-acid residues such as can deleting Fc district are to reduce or to strengthen and the combination of FcR.In one embodiment, one or more Fc districts residue can carry out modifying to produce this Fc district varient.According to this embodiment of the present invention, usually deletion is no more than one to about ten Fc district residue.The Fc district comprising one or more aminoacid deletion herein preferably by retain initial Fc district or native sequences people Fc district at least about 80% and preferably at least about 90%, and most preferably at least about 95%.
In one embodiment, the polypeptide produced described in the present invention or by method of the present invention, such as humanized Ig variable region and/or comprise the polypeptide of humanized Ig variable region, can be produced by recombination method.For example, the polynucleotide sequence of coded polypeptide can insert in suitable expression vector for recombinant expressed.When polypeptide is antibody, the polynucleotide of other light chain of the coding be optionally connected with constant region and variable region of heavy chain can be inserted in identical or different expression vector.In peptide sequence, optionally connect or comprise avidity sequence label (such as, His (6) label) so that downstream purification.The region of DNA section of encoding immunoglobulin chains with guarantee in expression vector that the control sequence operability that immunoglobulin polypeptides is expressed is connected.Expression control sequenc includes, but is not limited to promotor (such as, natural relevant or heterogeneous promotor), signal sequence, enhancer element and transcription termination sequence.Can transform or transfect eukaryotic host cells carrier in, expression control sequenc is preferably eukaryotic promoter system.Once carrier is incorporated in suitable host, under just host being remained on the condition of applicable high level expression nucleotide sequence and collection and purified polypeptide.
These expression vectors are usually reproducible in host body is episome (episome) or the integral part of host chromosome DNA.Expression vector usually containing selective marker (such as amicillin resistance, hygromycin resistance, tetracyclin resistance or neomycin resistance) to allow to detect those cells of being transformed by required DNA sequence dna (see, for example U.S. Patent No. 4,704,362).
Intestinal bacteria (E.coli) are the prokaryotic hosts that one is particularly useful for cloning polynucleotide of the present invention (such as, DNA sequence dna).Other microorganism host be suitable for comprises bacillus, such as Bacillus subtilus (Bacillus subtilus), with other enterobacteria, such as Salmonellas (Salmonella), serratia (Serratia) and various pseudomonas (Pseudomonas) species.
Other microorganism (such as yeast) is also applicable to express.Yeast belong (Saccharomyces) and Pichia (Pichia) are exemplary yeast hosts, and wherein suitable carrier optionally has expression control sequenc (such as promotor), replication orgin, terminator sequence etc.Typical promotor comprises 3-phosphoglycerate kinases and other glycolytic ferment.Induction type Yeast promoter especially comprises the promotor of the enzyme from alcoholdehydrogenase, different cell pigment C (isocytochrome C) and responsible methyl alcohol, maltose and galactose utilization.
In category of the present invention, intestinal bacteria and yeast saccharomyces cerevisiae (S.cerevisiae) are preferred host cells.
Except microorganism, mammalian tissue culture also may be used for expressing and producing polypeptide of the present invention (such as, the polynucleotide of encoding immune sphaeroprotein or its fragment).With reference to Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).Eukaryotic cell is in fact preferred, because developed in the art multiple can heterologous protein secretion (such as, complete immunoglobulin (Ig)) suitable host cell lines, and comprise Chinese hamster ovary celI system, various Cos clone, HeLa cell, 293 cells, myeloma cell line, the B cell of conversion and hybridoma.Expression vector for these cells can comprise expression control sequenc, such as replication orgin, promotor and the enhanser (people such as Queen, Immunol.Rev.89:49 (1986)) and necessary machining information site, such as ribosome bind site, RNA splice site, site of polyadenylation and transcription terminator sequences.Preferred expression control sequenc is the promotor being derived from immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus etc.With reference to people such as Co, J.Immunol.148:1149 (1992).
Carrier containing the polynucleotide sequence paid close attention to some extent (such as, heavy chain and light chain encoding sequences and expression control sequenc) is transferred in host cell by well-known process, and the method changes with the type of cell host.For example, calcium chloride transfection is usually used in prokaryotic cell prokaryocyte, and calcium phosphate process, electroporation, lipofection, particle gun or the transfection based on virus can be used for other cell host.(usually with reference to people such as Sambrook, Molecular Cloning:A LaboratoryManual (Cold Spring Harbor Press, the 2nd edition, 1989).Other method for transformed mammalian cell comprises use polybrene, protoplast fusion, liposome, electroporation and microinjection (usually with reference to people such as Sambrook, above).For producing transgenic animal, by transgenosis microinjection in zygote, maybe can be incorporated to the genome of embryonic stem cell, and the core of these cells can be transferred in seedless ovocyte.
Subject matter polypeptide also can be incorporated in transgenosis for introduce transgenic animal genome in and then such as in the milk of transgenic animal express (see, for example the people such as Deboer 5,741,957; Rosen 5,304,489; With Meade 5,849,992).Suitable transgenosis comprises and the encoding sequence for light chain and/or heavy chain be operatively connected from the promotor of mammary gland specific genes and enhanser, such as casein or beta lactoglobulin.
Polypeptide can use single carrier or two carriers to express.For example, heavy chain of antibody and light chain can be cloned and be jointly transfected in cell on independent expression vector.
In one embodiment, signal sequence can be used for the expression promoting polypeptide of the present invention.
Once expression, just can carry out purified polypeptide according to the standard program of this area, comprise ammonium sulfate precipitation, affinity column (such as, albumin A or Protein G), column chromatography, HPLC purifying, gel electrophoresis etc. is (usually with reference to Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)).
Humanized Ig variable region or the polypeptide comprising them can be expressed in culture by host cell or clone.They also can cells in vivo.Through transforming (such as, transfection) clone of mutagenic antibody can be the mammal cell line of immortalization, those clones (such as, myelomatosis, hybridoma, trioma (trioma) or four source hybridoma (quadroma) clones) of such as lymphatic origin.Clone also can comprise normal lymphocyte, such as by transforming and the B-cell of immortalization through virus (such as, epstein-Barr virus, Epstein-Barr virus).
Although for generation of the clone normally mammal cell line of polypeptide, the clone from other source (such as, bacterium and yeast) also can be used.Specifically, can use and be derived from colibacillary bacterial isolates, especially such as phage display.
Some immortalization lymphocyte series (such as myeloma cell line) secrete Ig light chain or the heavy chain of separation in normal state.If this clone passes through the vector of the antibody of the expression change prepared during process of the present invention, by the unnecessary remaining step carrying out this process, prerequisite is that normocrinic chain is complementary with the variable domains of the Ig chain coded by the carrier prepared in the early time.
If the clone of immortalization is not secreted or do not secreted complementary strand, the carrier introducing the suitable complementary strand of coding or its fragment in cell will be necessary.
When immortalized cell line secretion complementary light chain or heavy chain, such as by making the clone of bacterial cell and immortalization merge (such as, passing through spheraplast fusion) to produce the clone of conversion subsequently with the suitable bacterial cell of vector.Or, by electroporation, DNA is directly introduced in the clone of immortalization.
In one embodiment, as described in the present invention or the humanized Ig variable region produced by method of the present invention can be present in the Fab of any antibody.Fragment can recombinate produce and by carrying out engineering design, synthesis or generation with proteolytic enzyme digest antibody.For example, fragment can be Fab fragment; Make antibody at interchain (that is, V with papain digestion
h-V
h) connect the region place fracture of two heavy chains before disulfide linkage.This causes the V formed containing light chain and heavy chain
hand C
hthe fragment that two of 1 structural domain are identical.Or fragment can be F (ab ')
2fragment.These fragments by producing with pepsin digested antibody, this cracking heavy chain and producing containing the fragment of two antigen binding sites after interchain disulfide bond.Another alternative method uses " strand " antibody.ScFv (scFv) fragment builds by various ways.For example, V
hc-terminal can with V
ln-terminal connect.Joint (such as, (GGGGS)
4; SEQID NO:4) be usually located at V
hand V
lbetween.But the order of connection chain can be put upside down, and the label (such as, Myc-, His-or FLAG-label) that can comprise promotion detection or purifying (such as can be attached to the label of any antibody of the present invention or antibody fragment; Their use is not limited to scFv).Therefore and as mentioned below, through the antibody of mark in category of the present invention.In an alternative embodiment, antibody described herein or that produced by method as herein described can be heavy chain homodimer or light chain dimer.Light chain of antibody or heavy chain or its part, such as single domain antibody (DAb) can also be used in addition.
In another embodiment, the humanized Ig variable region produced as described in the present invention or by method of the present invention is present in single-chain antibody (ScFv) or miniantibody (see, for example U.S. Patent No. 5,837,821 or WO 94/09817A1).The dimer molecule that miniantibody is made up of two polypeptide chains, every bar polypeptide chain all comprises a ScFv molecule and (comprises the single polypeptide of one or more antigen binding site, such as, by flexible joint and V
hthe V that structural domain connects
lstructural domain, V
hstructural domain is by connection peptides and CH3 domain fusion).ScFv molecule can be built into V
h-joint-V
lorientation or V
l-joint-V
horientation.Connect and compose the V of antigen binding site
land V
hthe flexible hinge of structural domain preferably comprises about 10 to about 50 amino-acid residues.Exemplary connection peptides for this object is (Gly4Ser) 3 (people (1988) .PNAS, the 85:5879 such as Huston).Other connection peptides known in this area.
The method of manufacture single-chain antibody well known in the art, the people (1989) such as such as Ho, Gene, 77:51; The people such as Bird (1988), Science 242:423; The people such as Pantoliano (1991), Biochemistry 30:10117; The people such as Milenic (1991), Cancer Research, 51:6363; The people such as Takkinen (1991), Protein Engineering 4:837.ScFv component is built and connection peptides-CH by using the method described in this area
3component manufactures miniantibody (see, for example United States Patent (USP) 5,837,821 or WO 94/09817A1).These components can be used as restriction fragment and are separated from independent plasmid, and engage subsequently and be again cloned in suitable carrier.Suitable assembling can be confirmed by restrictive diges-tion and DNA sequence analysis.In one embodiment, miniantibody of the present invention comprises connection peptides.In one embodiment, connection peptides comprises Gly/Ser joint, such as GGGSSGGGSGG (SEQ ID NO:6).
In another embodiment, tetravalence miniantibody can be built.Tetravalence miniantibody can be built by the mode identical with miniantibody, such as has aminoacid sequence (G except using
4s)
4g
3the flexible joint of AS (SEQ ID NO:7) connects beyond two ScFv molecules.
In another embodiment, as described in the present invention or the humanization variable region produced by method of the present invention can be present in bivalent antibody.Bivalent antibody and scFv molecular mimicry, but there is short (be less than 10 and be preferably 1 to 5) amino acid residue linker usually connect two variable domains, so that the V on same polypeptide chain
land V
hstructural domain can not interact.On the contrary, the V on a polypeptide chain
land V
hv on structural domain and the second polypeptide chain
hand V
lstructural domain (difference) interacts (WO 02/02781).
In another embodiment, in the humanization variable region of the present invention immunoreactivity fragment that can be present in the antibody be connected with FcR bound fraction operability or part (such as, scFv molecule, miniantibody, tetravalence miniantibody or bivalent antibody).In an exemplary embodiment, FcR bound fraction is whole Fc district.
Humanization approach as herein described preferably produces Ig variable region, wherein to avidity essentially no change compared with donor antibody of antigen.
In one embodiment, the polypeptide comprising variable domains of the present invention is to be greater than (or equaling) about 10
5m
-1, 10
6m
-1, 10
7m
-1, 10
8m
-1, 10
9m
-1, 10
10m
-1, 10
11m
-1or 10
12m
-1the binding affinity (comprising the avidity intermediate value of these values) of dissociation constant Ka be combined with antigen.
Avidity, avidity and/or specificity are measured by various ways.Usually and no matter definition or measure avidity butt formula really, method of the present invention all improves the avidity of antibody when producing antibody, this antibody its clinical application any in be all better than by its obtained antibody (or Multiple Antibodies) (for example, when through modification antibody can be lower dosage less frequency is used or by than by its obtain antibody (or Multiple Antibodies) route of administration is used more easily time, just think that method of the present invention is effective or successful).
More particularly, measure the avidity between antibody and its antigen that combines by various analysis, comprise such as elisa assay, BiaCore and analyze or KinExA
tM3000 analyze (can obtain from Sapidyne Instruments (Boise, ID)).In simple terms, (antigen used in method of the present invention can be paid close attention to any antigen (such as, cancer antigen to be coated with agarose beads by covalently bound antigen; The albumen of cell surface protein or secretion; The antigen (such as, bacterium or virus antigen (such as, HIV antigen, influenza antigens or hepatitis antigen)) of pathogenic agent or anaphylactogen).Prepare antibody diluent to be tested and every part of diluent joined in the designation hole on flat board.Detecting antibody (such as, Goat anti human IgG-HRP conjugate) subsequently to adding in each hole, then adding and producing look substrate (such as, HRP).Read dull and stereotyped subsequently under 450nM in ELISA plate reader, and calculate EC50 value.(but, should be appreciated that method described herein is usually all applicable; They are not limited to the antibody produced in conjunction with any specific antigen or antigen type.)
Persons skilled in the art will be recognized, determine that avidity is not always simple as observed as single features.Because antibody has two arms, therefore their apparent avidity usually more much higher than the inherent avidity between variable region and antigen (believing that this is due to avidity).Inherent avidity can use scFv or Fab fragment to measure.
On the other hand, feature of the present invention and treatment part (such as cytotoxin, medicine (such as, immunosuppressor) or the radiotoxin) humanized rabbit antibody that puts together or its fragment.This kind of conjugate is referred to herein as " immunoconjugates ".
Antibody conjugates of the present invention can be used for changing set biological respinse, and drug moiety should not be construed as the chemotherapeutic being confined to classics.For example, drug moiety can be possess required bioactive albumen or polypeptide.This proteinoid such as can comprise enzymatic activity toxin or its active fragments, such as toxalbumin, ricin A, Pseudomonas exotoxin or diphtheria toxin; Albumen, such as tumour necrosis factor or interferon-γ; Or biological response modifier, such as lymphokine, il-1 (" IL-1 "), interleukin-2 (" IL-2 "), interleukin-6 (" IL-6 "), rHuGM-CSF (" GM-CSF "), granulocyte colony-stimulating factor (" G-CSF ") or other somatomedin.
Know the technology for making described treatment part and antibody conjugate, such as, with reference to people such as Arnon, " Monoclonal Antibodies For Immunotargeting Of Drugs InCancer Therapy ",
monoclonal Antibodies And Cancer Therapy, the people such as Reisfeld (volume), in 243-56 page (Alan R.Liss, Inc.1985); The people such as Hellstrom, " Antibodies For Drug Delivery ",
controlled Drug Delivery(the 2nd edition), the people such as Robinson (volume), in 623-53 page (Marcel Dekker, Inc.1987); Thorpe, " Antibody Carriers Of Cytotoxic Agents In CancerTherapy:A Review ",
monoclonal Antibodies ' 84:Biological And clinical Applications, the people such as Pinchera (volume), in 475-506 page (1985); " Analysis, Results, And Future Prospective Of The Therapeutic UseOf Radiolabeled Antibody In Cancer Therapy ",
monoclonal antibodies For Cancer Detection And Therapythe people such as Baldwin (volume), the people such as (Academic Press 1985) and Thorpe in 303-16 page, " The PreparationAnd Cytotoxic Properties Of Antibody-Toxin Conjugates ", Immunol.Rev.
62: 119-58 (1982).
In one aspect, the invention provides the pharmaceutical preparation comprising humanized rabbit antibody being used for the treatment of disease.Term " pharmaceutical preparation " refers to can allow the clear and definite effective form of the biological activity of antibody or antibody derivatives and the formulation do not contained experimenter's tool other component virose of administered formulation." pharmaceutically acceptable " vehicle (vehicle, additive) be can reasonably be applied to targeting mammalian with provide effective dose those of employing activeconstituents.
[equivalent]
In view of above description, many amendments of the present invention and alternate embodiment will be apparent for those skilled in the art institute.Therefore, this description is interpreted as being only illustrative and for instructing those skilled in the art to carry out optimal mode of the present invention object.Do not depart under spirit of the present invention can the details of change structure substantially, and retain the specificity of all modifications belonged in following claims category.The present invention's expection is only limitted to enclose claim and the applicable degree needed for statutory rules.
The all documents quoted in the application and analogous material, comprise patent, patent application, file, book, paper, special topic, webpage, figure and/or annex, and regardless of the form of these documents and analogous material, the mode all quoted in full is clearly incorporated to.Different from the application with analogous material or when conflicting at one or more document be incorporated to, comprise the technology etc. of the term of definition, term usage, description, be as the criterion with the application.
Claims (16)
1. people's heavy chain acceptor framework, it comprises SEQ ID NO:1.
2. people's heavy chain acceptor framework as claimed in claim 1, it comprises aminoacid replacement at position 12,103 and/or 144 (Aho numbering) place.
3. people's heavy chain acceptor framework as claimed in claim 2, wherein said replacement is
The Serine (S) at (a) position 12 place;
The Serine (S) at (b) position 103 place or Threonine (T); And/or
The Serine (S) at (c) position 144 place or Threonine (T).
4. the nucleic acid be separated, its acceptor framework as claimed in claim 1 of encoding.
5. a carrier, it comprises nucleic acid as claimed in claim 4.
6. a host cell, it comprises carrier as claimed in claim 5.
7. have a specific immunoconjugator to required antigen, it comprises:
A () comprises the light chain acceptor framework of the variable light CDR of Lagomorph immunoconjugator; With
B () comprises people's heavy chain acceptor framework as claimed in claim 1 of the variable heavy chain CDR of Lagomorph immunoconjugator.
8. immunoconjugator as claimed in claim 7, wherein said light chain acceptor framework and SEQ ID NO:2 have at least 85% identity.
9. immunoconjugator as claimed in claim 7, it also comprises the joint sequence connecting described variable light framework and described heavy chain acceptor framework, and wherein said joint sequence is SEQ IDNO:4.
10. immunoconjugator as claimed in claim 7, it is also included in donor framework residues involved in antigen combination.
11. immunoconjugators as claimed in claim 7, wherein said immunoconjugator is scFv antibody, total length immunoglobulin (Ig) or Fab fragment.
12. 1 kinds make the humanized method of rabbit immunoconjugator, and described method comprises:
A () will be transplanted in people's heavy chain acceptor framework as claimed in claim 1 from least one heavy chain CDR in the group be made up of CDR H1, CDR H2 and CDR H3 sequence of donor rabbits immunoconjugator; With
B () will be transplanted in people's light chain acceptor framework from least one light chain CDR in the group be made up of CDR L1, CDR L2 and CDR L3 sequence of donor rabbits immunoconjugator, described light chain acceptor framework and SEQ ID NO:2 have at least 85% identity.
13. methods as claimed in claim 12, it also comprises the Framework residues replaced with the Framework residues of described donor rabbits immunoconjugator in the one or both of described people's heavy chain acceptor framework and described people's light chain framework.
14. methods as claimed in claim 12, wherein said heavy chain acceptor framework has replacement at a place of the amino position 12,103 and 144 (AHo numbering) of heavy chain or many places.
15. methods as claimed in claim 14, are wherein selected from a place of position 12,103 and 144 or the described replacement of many places:
The Serine (S) at (a) position 12 place;
The Threonine (T) at (b) position 103 place; With
The Threonine (T) at (c) position 144 place.
16. 1 kinds of immunoconjugators, it is according to method humanization as claimed in claim 12.
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|---|---|---|---|
| PCT/EP2013/053329 WO2014127811A1 (en) | 2013-02-20 | 2013-02-20 | Acceptor framework for cdr grafting |
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| JP (1) | JP2016506752A (en) |
| CN (1) | CN104884471A (en) |
| AU (1) | AU2013379444A1 (en) |
| CA (1) | CA2896174A1 (en) |
| HK (1) | HK1219485A1 (en) |
| RU (1) | RU2015139890A (en) |
| WO (1) | WO2014127811A1 (en) |
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| WO2016173719A1 (en) * | 2015-04-30 | 2016-11-03 | Abcheck S.R.O. | Method for mass humanization of rabbit antibodies |
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| WO2008144753A2 (en) * | 2007-05-21 | 2008-11-27 | Alder Biopharmaceuticals, Inc. | Antibodies to tnf alpha and use thereof |
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- 2013-02-20 AU AU2013379444A patent/AU2013379444A1/en not_active Abandoned
- 2013-02-20 CN CN201380069162.4A patent/CN104884471A/en active Pending
- 2013-02-20 EP EP13705169.4A patent/EP2958939A1/en not_active Withdrawn
- 2013-02-20 RU RU2015139890A patent/RU2015139890A/en not_active Application Discontinuation
- 2013-02-20 CA CA2896174A patent/CA2896174A1/en not_active Abandoned
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2016
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| WO2003097697A2 (en) * | 2002-05-22 | 2003-11-27 | Esbatech Ag | Immunoglobulin frameworks which demonstrate enhanced stability in the intracellular environment and methods of identifying same |
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Also Published As
| Publication number | Publication date |
|---|---|
| JP2016506752A (en) | 2016-03-07 |
| RU2015139890A (en) | 2017-03-27 |
| HK1219485A1 (en) | 2017-04-07 |
| CA2896174A1 (en) | 2014-08-28 |
| WO2014127811A1 (en) | 2014-08-28 |
| AU2013379444A1 (en) | 2015-06-11 |
| EP2958939A1 (en) | 2015-12-30 |
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