CN104884470A - Acceptor framework for CDR grafting - Google Patents
Acceptor framework for CDR grafting Download PDFInfo
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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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
As therapeutical agent and diagnostic reagent, monoclonal antibody, their conjugate and derivative are commercially of crucial importance.Usually, after the injection of single low dosage, non-human antibody causes strong immunization reaction (Schroff, 1985 Cancer Res 45:879-85 in patients; Shawler.JImmunol 1985 135:1530-5; Dillman, Cancer Biother 1994 9:17-28).Therefore, develop for reducing muroid with immunogenic some methods of other rodent animal antibody and in order to use such as transgenic mice or phage display to prepare the technology of fully human antibodies.Chimeric antibody is engineered antibody, its combination rodent variable region and human constant region (such as Boulianne Nature 1984 312:643-6), Immunogenicity is made significantly to reduce (such as LoBuglio, Proc Natl Acad Sci 1989 86:4220-4; Clark, Immunol Today2000 21:397-402).Humanized antibody is also engineered antibody, wherein rodent variable region sequences self is come as far as possible close to human sequence by through engineering approaches, retain at least original CDR simultaneously, or wherein the CDR from rodent animal antibody to be transplanted in the framework of people's antibody (such as Riechmann, Nature 1988 332:323-7; US5,693,761).Rabbit polyclonal antibody is widely used in biological assay, as ELISA or western blotting.The avidity of multi-clone rabbit antibody often due to them is usually much higher and more favourable than polyclone rodent animal antibody.In addition, rabbit often can cause bad for immunogenicity in mouse and/or not produce the good antibody response of antigen of good combination agent when being used for phage display.Know advantage because rabbit antibody has these, they will be used for finding and exploitation therapeutic antibodies ideally.Usually the reason of this measure is not carried out mainly owing to producing the technological challenge of monoclonal rabbit antibodies.Because myelomatosis sample tumour is unknown in rabbit, the conventional hybridoma technology in order to produce monoclonal antibody is not useable for rabbit antibody.The development work of the fused cell system companion of rabbit antibody expressing cells is provided to carry out (Spieker-Polet etc. by Knight and colleague, PNAS 1995,92:9348-52), and a kind of fusion partner cells system that improves described (see such as U.S. Patent number 7429487) by Pytela etc. in 2005.But this technology is not extensively promoted, because corresponding tricks of the trade are controlled by single study group substantially.Relate to and being described in document from the alternative method for generation of monoclonal antibody of selected antibody expressing cells clonal antibody by RT-PCR, but never reported success is carried out to rabbit antibody.
Similar mouse antibodies, if expection rabbit antibody is for people's therapy, so can cause strong immunization reaction, therefore, rabbit antibody can need by humanization before Clinical practice at them.But owing between rabbit and mouse, and correspondingly, the textural difference between rabbit antibody and people's antibody, the method for the preparation of humanization rodent animal antibody can not easily be extrapolated for rabbit antibody.For example, light chain CDR3 (CDRL3) is normal more much longer than the previously known CDRL3 from people or mouse antibodies.
There is the humanized rabbit antibodies method that is described in a little in prior art, but it not the classical implantation method that the CDR of wherein non-human donor is implanted on people's receptor antibody.WO04/016740 describes a kind of so-called " surface is reinvented " strategy." surface is reinvented " strategy target be the inhuman framework of refigure solvent can and residue become more class people to make them.The similar humanization technologies for rabbit antibody as described in WO04/016740 is well known in the art.Both WO08/144757 and WO05/016950 are all open for making the humanized method of rabbit monoclonal antibodies, and it relates to the aminoacid sequence of aminoacid sequence and the similar people's antibody comparing parent rabbit antibody.Subsequently, the aminoacid sequence of parent rabbit antibody is changed with the equivalent framework district making its framework region more be similar to similar people's antibody in sequence.For obtaining good combination ability, needing individually to carry out effort exploitation for each immunoconjugator and attempting.
Potential problems of aforesaid method are not end user's frameworks but make rabbit frame engineering seem more class people to make it.Described method carries the risk that the amino acid segment be embedded in the core of protein still may comprise immmunogenic T-cell epitopes.
Up to now, applicant not yet identifies by application prior art state migration method in addition humanized rabbit antibody.This may explain by the fact that rabbit CDR extremely can be different from people or rodent CDR.As known in the art, many rabbit VH chains have extra halfcystine in pairs relative to muroid and people's counterpart.Except the conserved disulfide bridges formed between cys22 and cys92, also there is cys21-cys79 bridge, and S-S bridge between the CDR formed between the end of a CDRH1 residue and the first residue of CDR H2 in some rabbit chains.In addition, multipair cysteine residues is common in CDR-L3.In addition, many rabbit antibody CDR do not belong to any previously known typical structure.Specifically, CDR-L3 is often much longer than the CDR-L3 of people or muroid counterpart.
Therefore, being transplanted in people's framework by inhuman CDR antibody is main protein engineering duty.Must antigen binding loops be carried out to be transferred to from the framework of natural evolution people's framework of different artificial selection under making native annulus conformation be retained to reach antigen to combine.Usually after ring is transplanted, antigen-binding affinity is substantially reduced or eliminated.Use people's framework of careful selection can make the possibility retaining binding affinity in humanized molecule maximum (Roguzka etc. 1996) when transplantation antigen coupling collar.Although many transplantation experiments that can obtain in the literature are provided for the roughly guidance that CDR transplants, a certain pattern can not be summarized.Typical problem is to lose specificity, stability or can be generation after ring at transplanting CDR.
Therefore, to for reliably and make humanized rabbit antibodies there is emergency to be used as therapeutical agent and improving one's methods of diagnostic reagent fast.In addition, to for reliably making humanized rabbit antibodies, thus people's receptor framework of functional antibodies and/or the antibody fragment with medicine sample biophysical properties is provided to there are needs.
Summary of the invention
Shockingly find to measure (as in WO 0148017 and Auf der Maur etc. (2001) by quality control (QC), FEBS Lett 508, disclosed in 407-412 page) highly soluble identified and stable people's antibody framework be particularly suitable for receiving 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 " a58 " VH Frame sequence) of particular person antibody, it is especially suitable as the Multiple Antibodies from having different binding specificity, particularly from the receptor of the CDR of rabbit antibody, and whether be present in CDR irrelevant with disulphide bridges.
By rabbit CDR being transplanted to this humanization immunoconjugator that can produce in the variable chains framework of highly compatible all the time and the reliable spatial orientation retaining the rabbit antibody that donor CDR comes from.Therefore, the structure relevant position of donor immunity bonding agent is without the need to being introduced in receptor framework.Owing to these advantages, can reach do not optimize or under ability of optimizing integration a little to the high-throughput humanization of rabbit antibody.
Therefore, on the other hand, the invention provides for rabbit and other inhuman CDR being transplanted in solubility disclosed herein and stable light chain and/or heavy chain people antibody framework sequence, producing the method with the humanized antibody of superior biophysical properties thus.Specifically, the immunoconjugator produced by the inventive method represents superior functionality character, as solubility and stability.
Accompanying drawing is sketched
The CDR H1 that Fig. 1 is depicted in herein for being transplanted in highly soluble and stable people's antibody framework from rabbit monoclonal antibodies by antigen binding site defines.
Fig. 2: to CDR3 3 amino acid longer than its muroid counterpart usually of the analysis confirmation variable heavy chain of the rabbit antibody sequence extracted from Kabat database.
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 is defined in during entire chapter describes in detail and sets forth.
Term " antibody " refers to complete antibody and any Fab.Term " antigen-binding polypeptides " and " immunoconjugator " use in this article simultaneously." antibody " refers to and optionally glycosylatedly comprises protein by interconnective at least two weights (H) chain of disulfide linkage and two light (L) chains or its antigen-binding portion thereof.Each heavy chain comprises variable region of heavy chain and (is abbreviated as V in this article
h) and CH.CH comprises three structural domains, i.e. CH1, CH2 and CH3.Each light chain comprises variable region of light chain and (is abbreviated as V in this article
l) and constant region of light chain.Constant region of light chain comprises a domain C L.V
hdistrict and V
ldistrict can be divided into again be further called complementary determining region (CDR) there is denatured region, be scattered with the more conservative region being called framework region (FR) therebetween.Each V
hand V
lthree CDR and four the FR compositions primarily of arranging in the following order from N-terminal to C-terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.The binding domains with AI is contained in the variable region of heavy chain and light chain.The constant region of antibody can mediated immunity sphaeroprotein in conjunction with host tissue or the factor, comprise 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 antibody fragment of the ability of one or more reservation specific binding antigen (such as TNF).The antigen combined function having shown antibody can be performed by the fragment of full length antibody.The example being encompassed in the binding fragment in " antigen-binding portion thereof " of term antibody comprises (i) Fab fragment, namely by V
l, V
h, CL and CH1 structural domain composition monovalent fragment; (ii) F (ab') 2 fragment, namely comprises two bivalent fragment by the Fab fragment of the disulfide bridge connects of hinge area; (iii) by V
hwith the Fd fragment of CH1 structural domain composition; (iv) by the V of the single armed of antibody
land V
hthe Fv fragment of structural domain composition; V () single domain or dAb fragment (Ward etc., (1989) Nature341:544-546), it is by V
hstructural domain forms; And the combination of the CDR of the complementary determining region that (vi) is separated (CDR) or (vii) two or more separation that can optionally be engaged by synthetic linker.In addition, although two of Fv fragment structural domain V
land V
hbe by independent genes encoding, but they can use recombination method can be prepared into wherein V by making them
ldistrict and V
hdistrict (is called scFv (scFv) to form monovalent molecule in pairs; See (1988) Science 242:423-426 such as such as Bird; And (1988) Proc.Natl.Acad.Sci.USA85:5879-5883 such as Huston) the synthetic linker of single protein chain engaged.Described single-chain antibody is also intended to be encompassed in " antigen-binding portion thereof " of term antibody.These antibody fragments use routine techniques known to those skilled in the art to obtain, and screen the fragment of playing effectiveness in the mode identical with complete antibody.By recombinant DNA technology, or produce antigen-binding portion thereof by enzymatic or chemical cracking intact immunoglobulins.Antibody can have different isotype, 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, such as all or part of heavy chain and/or light variable domains, to make the molecule of immunoconjugator specific recognition target antigen.The limiting examples of immunoconjugator comprises total length immunoglobulin molecules and scFv and antibody fragment, includes but not limited to (i) Fab fragment, namely by V
l, V
h, C
land C
hthe monovalent fragment of 1 structural domain composition; (ii) F (ab')
2fragment, namely comprises two bivalent fragment by the Fab fragment of the disulfide bridge connects of hinge area; (iii) Fab ' fragment, it is the Fab (see FUNDAMENTALIMMUNOLOGY (Paul compiles, the 3rd edition 1993)) with a part of hinge area substantially; (iv) by V
hand C
hthe Fd fragment of 1 structural domain composition; V () is by the V of the single armed of antibody
land V
hthe Fv fragment of structural domain composition; (vi) single domain antibody, as by V
hor V
ldab fragment (Ward etc., (1989) Nature of structural domain composition
341: 544-546), Camelidae antibodies is (see Hamers-Casterman, etc., Nature 363:446-448 (1993); And Dumoulin, etc., Protein Science11:500-515 (2002)) or shark antibody (such as shark Ig-NAR
); And (vii) nano antibody, namely contain the variable region of heavy chain of single variable domains and two constant domain.
Term " single-chain antibody ", " scFv " or " scFv " refer to heavy chain of antibody variable domains (or the region comprising and connected by joint; V
h) and light chain of antibody variable domains (or region; V
l) molecule.Described scFv molecule can have general structure: NH
2-V
l-joint-V
h-COOH or NH
2-V
h-joint-V
l-COOH.Be applicable to prior art state joint by repeating GGGGS aminoacid sequence or its variant forms.In a preferred embodiment of the invention, use (GGGGS) 4 joint with the aminoacid sequence of setting forth in SEQ ID NO:8, but the variant with 1-3 tumor-necrosis factor glycoproteins is also possible (Holliger etc. (1993), Proc.Natl.Acad.Sci.USA 90:6444-6448).Other joint used in the present invention by (1995) such as Alfthan, Protein Eng.8:725-731; Choi etc. (2001), Eur.J.Immunol.31:94-106; Hu etc. (1996), Cancer Res.56:3055-3061; Kipriyanov etc. (1999), J.Mol.Biol.293:41-56 and Roovers etc. (2001), CancerImmunol describes.
As used herein, term " functional property " is such as in order to improve manufacture character or the therapeutic efficiency of polypeptide, and it improves (such as relative to conventional polypeptide) is cater to the need and/or favourable polypeptide (such as immunoconjugator) character to those skilled in the art.In one embodiment, functional property is stability (such as thermostability).In another embodiment, functional property is solubility (such as under cell condition).In another embodiment, functional property is aggregation properties.In another embodiment, functional property is protein expression (such as in prokaryotic cell prokaryocyte).In another embodiment, functional property is the refolding characteristic in the fabrication process after solubilization of inclusion bodies.In certain embodiments, functional property is not that antigen-binding affinity improves.In another preferred embodiment of the present, the binding affinity of improvement to immunoconjugator of one or more functional propertys does not have substantial effect.
Term " CDR " refers to one in six hypervariable regions mainly facilitating antigen to combine in the variable domains of antibody.A kind of definition being generally used for six CDR is most by Kabat E.A. etc., (1991) Sequences of proteins of immunological interest.NIH Publication91-3242) provide.As used herein, the Kabat definition of CDR is only for CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of light variable domains, and CDR2 and CDR3 of heavy-chain variable domains (CDR H2, CDR H3 or H2, H3).But the CDR1 (CDR H1 or H1) of heavy-chain variable domains as used herein is by initial with position 26, and resi-dues (Kabat numbering) definition stopped before position 36.This definition is the fusion (for diagram, also see Fig. 1) as the CDR H1 by Kabat and Chotia different definition substantially.
" antibody framework " refers to a part of variable domains VL or VH as the term is employed herein, and it serves as the skeleton of the antigen binding loops (CDR) of this variable domains.In fact, it is the variable domains without CDR.
Term " epi-position " or " antigenic determinant " refer to the site (specific site on such as TNF molecule) for immunoglobulin (Ig) or antibodies specific combination on antigen.Epi-position generally includes at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 continuous or non-contiguous amino acids in unique spatial conformation.See such as Epitope Mapping Protocols inMethods in Molecular Biology, the 66th volume, G.E.Morris compiles (1996).
Term " specific binding ", " selective binding ", " selectively combine " and " specifically combination " refer to the epi-position on antibodies predetermined antigens.Usually, the avidity (K of antibodies
d) be about less than 10
-7m, is less than 10 according to appointment
-8m, 10
-9m or 10
-10m or even lower.Term " K
d" or " Kd " refer to the Dissociation equilibrium constant of specific antibodies-AI.Usually, the Dissociation equilibrium constant (K of antibodies TNF of the present invention
d) be less than about 10
-7m, is less than 10 according to appointment
-8m, 10
-9m or 10
-10m or even lower, such as use in BIACORE instrument surface plasma body resonant vibration (SPR) technology measure.
" 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.When nucleic acid be placed in become functional relationship with another nucleotide sequence time, it is " operably connected ".For example, if promotor or enhanser affect transcribing of sequence, so it is operably connected to encoding sequence.
Term " carrier " refers to the nucleic acid molecule can transporting another nucleic acid that it has been attached thereto.In one embodiment, carrier is " plasmid ", and it refers to the circular double stranded DNA ring that wherein can connect other region of DNA section.In another embodiment, carrier is virus vector, and wherein other region of DNA section can be connected in viral genome.Self-replicating (such as there is the bacteria carrier of bacterial origin of replication and free mammalian vector) in the host cell that carrier disclosed herein can be introduced at them, or can in introducing host cell after be integrated in the genome of host cell, and to copy (such as non-free mammalian vector) together with host genome thus.
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), subtilis (Bacillus subtilis); Yeast saccharomyces cerevisiae (Saccharomycescerevisiae), pichia pastoris phaff (Pichia pastoris), CHO (Chinese hamster ovary strain) or NS0 cell.
Term " Lagomorph " refers to the member of the Lagomorpha classification comprising rabbit section (such as hare and rabbit) and Ochotonidae (pika).In the most preferred embodiment, Lagomorph is rabbit." rabbit " refers to the animal belonging to rabbit section 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 comparative sequences is all occupied by identical base or amino acid monomer subunit (if a certain position in such as two polypeptide is occupied by Methionin separately), so corresponding molecule is identical in that position." identity per-cent " between two sequences, with under the consideration number in room and the length in each room, the number of the same position had by sequence and becoming, described room needs to be introduced into the optimum comparison of reaching two sequences.Usually, compare be two sequences be compared with give carry out with during maximum identity.Described comparison such as can use the method for Needleman and Wunsch (J.MoI.Biol. (48): 444-453 (the 1970)) algorithm in the GAP program be incorporated in GCG software package, utilizes Blossum 62 matrix or PAM250 matrix and gap weight 16,14,12,10,8,6 or 4 and Length Weight 1,2,3,4,5 or 6 to provide.
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with by those skilled in the art usually understand identical implication.Although can use when implementing or test is of the present invention with those methods as herein described and material type like or the method for equivalence and material, following appropriate methodology and material are described.In the case of a conflict, be as the criterion with this specification sheets comprising definition.In addition, material, method and example only have illustrative and are not intended to have restricted.
All respects of the present invention are described in further detail in following trifle.Should be appreciated that various embodiment, preferably thing and scope can arbitrary combination.In addition, depending on specific embodiments, selected definition, embodiment or scope may be inapplicable.
If do not stated in addition, so amino acid position indicates according to AHo numbering flow process.AHo numbering system is further described in Honegger, A. and Pluckthun, A. (2001) J.Mol.Biol.
309: 657-670) in.Or, can use as (Kabat such as Kabat, E.A., Deng (1991) Sequences of Proteins of Immunological Interest, 5th edition, U.S.Department of Health and Human Services, NIH publication number 91-3242) in further described Kabat numbering system.Conversion table for the identification of two kinds of different numbering systems of the amino acid residue position in heavy chain of antibody and variable region of light chain is provided in A.Honegger, in J.Mol.Biol.309 (2001) 657-670.
In first aspect, the invention provides a kind of for transplanting from Lagomorph species, such as, from people's receptor Frame sequence of the CDR of rabbit.Shockingly finder's strand VH framework a58 (SEQ ID NO:1) in fact can with the antigen binding site highly compatible of rabbit antibody.Therefore, a58VH representative is in order to build the applicable skeleton of the stable humanization scFv antibody fragment obtained by the transplanting of rabbit ring.
Therefore, in one aspect, the invention provides a kind of immunoconjugator receptor framework, it comprises the VH sequence with SEQ ID No.1 with at least 70% identity.
Described sequence can combine with other applicable variable light any.Preferred variable light is also disclosed in WO03/097697 and is appointed as the SEQ ID NO:2 of KI27, or disclosed in WO03/097697 other VL sequence any.
In preferred embodiments, variable heavy chain framework is connected to variable light framework by joint.Joint can be any applicable joint, such as, comprise 1 to 4 joint repeated of sequence GGGGS (SEQ ID NO:5), preferably (GGGGS)
4peptide (SEQ ID NO:4), or disclosed in (1995) the Protein Eng.8:725-731 such as Alfthan joint.
Therefore, the invention provides a kind of immunoconjugator receptor framework, it comprises
I () and SEQ ID No.1 have at least 70% identity, and preferably at least 75%, 80%, 85%, 90%, the more preferably variable heavy chain framework of at least 95% identity; And/or
(ii) with SEQ ID No.2, there is at least 70% identity, preferably at least 75%, 80%, 85%, 90%, the more preferably variable light framework of at least 95% identity.
In very preferably embodiment, the invention provides a kind of sequence and SEQ ID NO:3 has at least 60%, the more preferably immunoconjugator of at least 65%, 70%, 75%, 80%, 85%, 90%, 95% identity.
Framework can be compatible with in fact any rabbit CDR.Containing under different rabbit CDR, contrary with rabbit wild-type strand, it is given full expression to and is well produced, and still almost retains the avidity of original donor rabbit antibody completely.
Immunoconjugator receptor framework as described herein in heavy chain framework, preferably can comprise solubility and strengthens replacement at position 12,103 and 144 (AHo numbering) place.Preferably, hydrophobic amino acid is by the larger aminoacid replacement of wetting ability.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).More preferably, heavy chain framework comprises (a) Serine (S) at position 12 place; Serine (S) at position 144 place of (b) Serine (S) at position 103 place or Threonine (T) and/or (c) or Threonine (T).
In addition, stability strengthens one or more positions 1,3,4,10,47,57,91 and 103 (AHo numbering) place that amino acid can be present in variable light framework.More preferably, variable light framework is included in the L-glutamic acid (E) at position 1 place, the α-amino-isovaleric acid (V) at position 3 place, the leucine (L) at position 4 place, Serine (S) at position 10 place; 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) tends to deamination, so in another preferred embodiment of the present, VH comprises glycine (G) at position 141 place.This replacement can improve the standing storage of protein.
For example, receptor framework disclosed herein can be used for the people's antibody or the humanized antibody that produce the binding property retaining the non-human antibody that inhuman CDR comes from.Therefore, in preferred embodiments, a kind of immunoconjugator receptor framework is as disclosed herein contained in the present invention, it comprises from donor immunity bonding agent further, 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; And
(ii) at least 70% is had with SEQ ID NO.1, preferably at least 75%, 80%, 85%, 90%, 95%, and people's variable heavy chain framework of most preferably 100% identity.
Preferably, Lagomorph is rabbit.More preferably, immunoconjugator 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 extra halfcystine in pairs relative to muroid and people's counterpart.Except the conserved disulfide bridges formed between cys22 and cys92, also there is cys21-cys79 bridge, and S-S bridge between the CDR formed between the end of a CDRH1 residue and the first residue of CDRH2 in some rabbit chains.In addition, multipair cysteine residues is common in CDR-L3.In addition, many rabbit antibody CDR do not belong to any previously known typical structure.Specifically, CDR-L3 is often much longer than the CDR-L3 of people or muroid counterpart.
As stated previously, inhuman CDR is transplanted to produce wherein CDR on framework disclosed herein be the molecule shown with suitable conformation.If needed, the AI Framework residues so by transplanting non-human donor's immunoconjugator improves the avidity of immunoconjugator.These positions can such as be identified in the following manner
I () identifies corresponding reproductive tract ancestor sequences, or, when very high homology Frame sequence, by using consensus sequence;
(ii) donor variable domain sequence and the reproductive tract ancestor sequences of step (i) or the sequence alignment of consensus sequence is produced; And
(iii) different residue is identified.
In many cases, in vivo during avidity production process, the different residue mutations on the surface of molecule, assuming that the former avidity that creates antagonism.
On the other hand, the invention provides a kind of immunoconjugator comprising immunoconjugator receptor framework as herein described.Described immunoconjugator can be such as scFv antibody, total length immunoglobulin (Ig), Fab fragment, Dab or nano antibody.
In preferred embodiments, immunoconjugator is made to be connected to one or more molecules, such as therapeutical agent (as cytotoxic agent), cytokine, chemokine, somatomedin or other signal transduction molecule, preparation or the second protein (as transcriptional activator or DNA binding domains).
Immunoconjugator as disclosed herein can such as in diagnostic use, treatment use, target checking or gene therapy.
The present invention further provides a kind of nucleic acid of encode immunoconjugator receptor framework disclosed herein or the separation of immunoconjugator as disclosed herein.
In another embodiment, providing package is containing the carrier of nucleic acid disclosed herein.
Nucleic acid as disclosed herein or carrier can such as in gene therapies.
A kind of host cell comprising carrier disclosed herein and/or nucleic acid is contained in the present invention further.
In addition, provide a kind of composition, it comprises immunoconjugator receptor framework as disclosed herein, as disclosed herein immunoconjugator, the nucleic acid be separated as disclosed herein or carrier as disclosed herein.
Sequence disclosed herein is following sequence (X residue is CDR insertion point, and contains at least 3 and maximum 50 amino acid):
SEQ ID NO:1: variable heavy chain framework a58
EVQLVESGGGLVQPGGSLRLSCAAS(X)
n=3-
50WVRQAPGKGLEWVS(X)
n=3-
50RESVSRDNSKNTVYLQINSLRAEDTAVYYCAM(X)
n=3-50WGQGTLVTVSS
SEQ ID NO:2: variable light framework KI27
EIVMTQSPSTLSASVGDRVIITC(X)
n=3-50WYQQKPGKAPKLLIY(X)
n=3-50
GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)
n=3-50FGQGTKLTVLG
SEQ ID NO:3: Frame sequence
EIVMTQSPSTLSASVGDRVIITC(X)
n=3-50WYQQKPGKAPKLLIY(X)
n=3-50
GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)
n=3-50FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGS
EVQLVESGGGLVQPGGSLRLSCAAS(X)
n=3-50WVRQAPGKGLEWVS(X)
n=3- 50RESVSRDNSKNTVYLOINSLRAEDTAVYYCAM(X)
n=3-50WGOGTLVTVSS
SEQ ID NO:4: joint
GGGGGSGGGGSGGGGSGGGGS
On the other hand, the invention provides for making the humanized method of non-human antibody by being transplanted by the CDR of non-human donor antibody on stable and soluble antibody framework.In a particularly preferred embodiment, from the CDR trunk of rabbit antibody and framework be above-mentioned those.
For CDR is transplanted to general approach in people's receptor framework by Winter at U.S. Patent number 5,225, in 539, and open in WO9007861A1 by Queen etc., described patent accordingly by reference entirety be incorporated to herein.Generality strategy for being transplanted by the CDR from rabbit monoclonal antibodies on selected framework is relevant with the strategy of Queen etc. to Winter etc., but disagrees at some critical aspects.Specifically, the difference of the inventive method and typical Winter and Queen method as known in the art is that people's antibody framework as disclosed herein is particularly suitable as the receptor of people or non-human donor antibody.Therefore, be different from the general approach of Winter and Queen, the sequence that the Frame sequence for humanization approach of the present invention needs not to be inhuman (such as rabbit) antibody come from donor CDR represents the homophylic Frame sequence of maximal sequence.In addition, do not need to transplant Framework residues to support CDR conformation from donor sequences.At the most, the antigen that is arranged in framework can be introduced in conjunction with amino acid or other sudden change of occurring during somatic hypermutation.
Below describe in order to produce the specific detail with the implantation method of the humanization rabbit endogenous antibody of high dissolubility and stability.
In the exemplary of the inventive method, first identify the aminoacid sequence of CDR donor antibody, and use routine sequence comparison instrument (such as Needleman-Wunsch algorithm and Blossum matrix) aligned sequences.Conventional antibody numbering system can be used to carry out room introduce and resi-dues name.For example, the AHo numbering system in immunoglobulin variable domain territory can be used.Also Kabat numbering flow process can be applied, because it is the standard for the residue numbering in antagonist the most extensively adopted.Kabat numbering can such as use SUBIM program to be specified.This program analyzes the variable region of antibody sequence according to the system set up by Kabat and colleague, and to sequence numbering (Deret etc. 1995).Framework and defining of CDR district are normally followed based on sequence variability, and is that the most normally used Kabat definition is carried out.But, for CDR-H1, preferably Kabat definition (meaning that contact data are that contact between antibody by analyzing a subgroup 3D composite structure and antigen produces (MacCallum etc., 1996)) is specified to define the combination (also see Fig. 1) of (position in its structure based ring region) with Chotia.Conversion table for the identification of two kinds of different numbering systems of the amino acid residue position in heavy chain of antibody and variable region of light chain is provided in A.Honegger, in J.Mol.Biol.309 (2001) 657-670.Kabat numbering system is further described in the (Kabat such as Kabat, E.A., Deng (1991) Sequences of Proteins of ImmunologicalInterest, 5th edition, U.S.Department of Health and Human Services, NIH publication number 91-3242) in.AHo numbering system is further described in (Honegger, A. and Pluckthun, A. (2001) J.Mol.Biol.
309: 657-670) in.
The variable domains of rabbit monoclonal antibodies such as can use the EXCEL steering routine of such as sequence analysis algorithm and be categorized into corresponding human subgroup (Knappik etc., 2000, the J Mol Biol.2 month 11 based on the sorting technique of analyst's antibody pedigree; 296 (1): 57-86).
The CDR conformation of antigen of donor land can be specified, also can be accredited as the resi-dues maintained needed for different typical structure subsequently.Chothia (1989) definition is used to determine the CDR typical 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 property showing enhancing further, the solubility such as strengthened and/or stability.In certain embodiments, optimize antibody of the present invention according to " function has " disclosed in the PCT application sequence number PCT/EP2008/001958 being entitled as " Sequence Based Engineering and Optimization of Single ChainAntibodies " that on March 12nd, 2008 submits to method, described application is incorporated to herein by reference.
Being entitled as in the PCT application PCT/CH2008/000284 of " SequenceBased Engineering and Optimization of Single Chain Antibodies " of being described in that the PCT application PCT/CH2008/000285 and 2008 being entitled as " Methods of Modifying Antibodies, andModified Antibodies with Improved Functional Properties " that submits on June 25th, 2008 submits to 25, on June is replaced for the example frame resi-dues that replaces and example frame.
In other embodiments, immunoconjugator of the present invention comprises the U.S.Provisional Serial 61/075 being entitled as " Solubility Optimization of Immunobinders " submitted on June 25th, 2008, and the one or more stability described in 692 strengthen sudden change.In some preferred embodiment, immunoconjugator comprises solubility and strengthens sudden change at the amino acid position place being selected from the heavy chain amino acid position group be made up of 12,103 and 144 (AHo numbering conventions).In a preferred embodiment, immunoconjugator comprises and is one or morely selected from by the replacement of the following group formed: (a) is at the Serine (S) at heavy chain amino acid position 12 place; (b) Serine (S) at heavy chain amino acid position 103 place or Threonine (T); And (c) Serine (S) at heavy chain amino acid position 144 place or Threonine (T).In another embodiment, immunoconjugator comprises following replacement: (a) is at the Serine (S) at heavy chain amino acid position 12 place; (b) Serine (S) at heavy chain amino acid position 103 place or Threonine (T); And (c) Serine (S) at heavy chain amino acid position 144 place or Threonine (T).
In some preferred embodiment, immunoconjugator light chain receptor framework variable region of light chain according to the Framework residues place at least one in the position 1,3,4,10,47,57,91 and 103 of AHo numbering system comprise stability strengthen sudden change.In preferred embodiments, light chain receptor framework comprises and is one or morely selected from by the replacement of the following group formed: (a) is at the L-glutamic acid (E) at position 1 place, b (), at the α-amino-isovaleric acid (V) at position 3 place, (c) is at the leucine (L) at position 4 place; D () is at the Serine (S) at position 10 place; E () is at the arginine (R) at position 47 place; E () is at the Serine (S) at position 57 place; F () is at the phenylalanine (F) at position 91 place; And (g) is at the α-amino-isovaleric acid (V) at position 103 place.
Any one that can use in multiple methods availalbe produces the humanized antibody comprising sudden change as above.
Therefore, the invention provides a kind of according to method as herein described in addition humanized immunoconjugator.
In some preferred embodiment, the target antigen of described immunoconjugator is VEGF or TNF α.
Can such as use technology as known in the art to synthesize described in the present invention or by the polypeptide that the inventive method produces.Or, can the nucleic acid molecule of variable region needed for composite coding, and produce polypeptide by recombination method.
For example, once determine the sequence of humanization variable region, the technology namely by knowing in biology field has been prepared that variable region or has been comprised its polypeptide.More particularly, recombinant DNA technology can be used for by with nucleotide sequence (the such as required variable region of coding (heavy chain such as modified or light chain; Its variable domains or its other Fab) DNA sequence dna) transformed host cell produces the polypeptide of broad range.
In one embodiment, can prepare to comprise and be operably connected at least V that encodes
hor V
lthe expression vector of promotor of DNA sequence dna.Necessary or when needing, (namely wherein parental expression vector is encoded V can to prepare the second expression vector of the promotor comprising the DNA sequence dna being operably connected to complementary variable domains of encode
h, the second expression vector codes V
l, and vice versa).Then with one or both expression vector transformation cell lines (such as immortalized mammalian clone), and (the international patent application no PCT/GB85/00392 see such as Neuberger etc.) can be cultivated under the condition allowing chimeric variable domains or chimeric antibody to express.
In one embodiment, the variable region comprising donor CDR and receptor FR aminoacid sequence can be prepared, then change be introduced in nucleic acid molecule and replace to realize cdr amino acid.
Exemplary this area authorization method for the preparation of the nucleic acid molecule of the amino acid sequence variation of coded polypeptide includes but not limited to that the DNA of the coding said polypeptide comparatively early prepared by fixed point (or oligonucleotide mediated) mutagenesis, PCR mutagenesis and cassette mutagenesis is prepared.
Site-directed mutagenesis is a kind of preferred method for the preparation of replacing variant.This technology is (see the Nucleic Acids Res.13:4431-4443 (1985) and Kunkel etc. such as such as Carter, the Proc.Natl.Acad.Sci.USA 82:488 (1987)) that know in the art.Inventionbriefly, when carrying out the site-directed mutagenesis of DNA, parent DNA is by first making the oligonucleotide hybridization suddenlyd change needed for coding change in the strand of described parent DNA.After hybridization, archaeal dna polymerase for using the oligonucleotide of hybridization as primer, and uses the strand of parent DNA as template to synthesize whole second chain.Therefore, the oligonucleotide of the required sudden change of coding is merged in gained double-stranded DNA.
PCR mutagenesis is also suitable for the amino acid sequence variation preparing polypeptide.See Higuchi, PCRProtocols, 177-183 page (Academic Press, 1990); And Vallette etc., Nuc.Acids Res.17:723-733 (1989).Inventionbriefly, when a small amount of template DNA is used as the initial substance in PCR, the primer of the respective regions be slightly different from template DNA in sequence can be used for producing relatively a large amount of specific DNA fragments being only different from template sequence in the primer position be different from residing for template.
Other method cassette mutagenesis for the preparation of variant is the technology based on being described by Wells etc., Gene34:315-323 (1985).Initial substance is the plasmid (or other carrier) comprising DNA to be suddenlyd change.Identify the codon in parent DNA to be suddenlyd change.Unique limiting acid endo enzyme site must be there is in each side in the mutational site of qualification.If there is no described restriction site, so they can use above-mentioned oligonucleotide mediated mutafacient system to produce with the appropriate location of they being introduced in the DNA of coded polypeptide.In these site cutting plasmid DNA to make its linearizing.Use standard program composite coding DNA between restriction site, but the double chain oligonucleotide of sequence containing required sudden change, wherein two chains of oligonucleotide synthesize respectively, then uses standard technique hybridization together.This double chain oligonucleotide is called box.This box is designed to have can with the compatible ends of linearization plasmid with the 5' end and the 3' end that make it can be directly connected in plasmid.This plasmid is now containing mutant DNA sequences.
The variable region produced by the inventive method can be increased antigen-binding further by refigure and further change.Therefore, above-mentioned steps can preposition with or succeeded by other step, comprise such as affinity maturation.In addition, experience can be used for further optimization in conjunction with data.
Except aminoacid replacement, the present invention also expects that other is modified, such as, to the Fc region variants that the modification of Fc region amino acid sequence changes to produce effector function.One or more amino-acid residues that such as can lack Fc district are to reduce or to strengthen and the combination of FcR.In one embodiment, one or more Fc districts residue can be modified to produce described Fc region variants.Usually, according to this embodiment of the present invention, 1 to about 10 Fc district residue at the most will be lacked.This paper Fc district comprising one or more aminoacid deletion by preferably 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 producing described in the present invention by recombination method or produced by the inventive method, such as humanized Ig variable region and/or comprise the polypeptide of humanized Ig variable region.For example, can the insertion of the polynucleotide sequence of coded polypeptide be applicable in expression vector recombinant expressed to reach.When polypeptide is antibody, the polynucleotide of encode other light chain and variable region of heavy chain of being optionally connected to constant region can be inserted in identical or different expression vector.Affinity tag sequence (such as His (6) label) can optionally connect or be included in peptide sequence to contribute to downstream purification.The region of DNA section of encoding immunoglobulin chains is made to be operably connected to the control sequence guaranteeing the expression of immunoglobulin polypeptides in expression vector.Expression control sequenc includes but not limited to promotor (such as natural relevant or allogeneic promoter), signal sequence, enhancer element and transcription termination sequence.Preferably, expression control sequenc be can transform or transfect eukaryotic host cells carrier in eukaryotic promoter system.Once carrier has been incorporated in suitable host, namely under the condition being suitable for high level expression nucleotide sequence and collection and purified polypeptide, maintain host.
These expression vectors usually can be used as episome or copy in host organisms as the integrated part of host chromosome DNA.Usually, expression vector contains selective marker (such as ampicillin resistant, hygromycin resistance, tetracyclin resistance or neomycin resistance) to allow to detect those cells of transforming with required DNA sequence dna (see such as U.S. Patent number 4,704,362).
Intestinal bacteria are prokaryotic hosts that one is used in particular for cloning polynucleotide of the present invention (such as DNA sequence dna).Other microorganism host be suitable for comprises Bacillaceae (as subtilis) and other enterobacteriaceae (as Salmonella (Salmonella), Serratia (Serratia) and various Rhodopseudomonas (Pseudomonas) species).
If other microorganism of yeast is also for expressing.Yeast belong and Pichia are exemplary yeast hosts, and wherein as required, applicable carrier has expression control sequenc (such as promotor), replication orgin, terminator sequence etc.Typical case's promotor comprises glycerol 3-phosphate acid kinase and other glycolytic ferment.Inducibility Yeast promoter especially comprises the promotor of the enzyme from alcoholdehydrogenase, different cell pigment C and responsible methyl alcohol, maltose and galactose utilization.
Within the scope of the invention, intestinal bacteria and yeast saccharomyces cerevisiae are preferred host cells.
Except microorganism, mammalian tissue culture also can be used for expressing and producing polypeptide of the present invention (such as the polynucleotide of encoding immune sphaeroprotein or its fragment).See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).Eukaryotic cell is actually preferred, because the many applicable host cell system of secretion heterogenous protein (such as intact immunoglobulins) can be developed in the art, and comprise Chinese hamster ovary celI system, various Cos clone, HeLa cell, 293 cells, myeloma cell line, transformed B cells and hybridoma.Expression vector for these cells can comprise expression control sequenc, as replication orgin, promotor and enhanser (Queen etc., Immunol.Rev.89:49 (1986)); And required process information site, as ribosome bind site, RNA splice site, site of polyadenylation and transcription terminator sequences.Preferred expression control sequence is the promotor coming from immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus etc.See Co etc., J.Immunol.148:1149 (1992).
Carrier containing targeting polynucleotide sequence (such as heavy chain and light chain encoding sequences and expression control sequenc) is transferred in host cell by the well-known process changed by the type of visual cell host.For example, calcium chloride transfection is generally used for prokaryotic cell prokaryocyte, and calcium phosphate process, electroporation, liposome transfection, particle gun or viral base transfection can be used for other cell host.(generally see Sambrook etc., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Press, the 2nd edition, 1989).Other method for transformed mammalian cell comprises use polybrene, protoplast fusion, liposome, electroporation and microinjection (generally see Sambrook etc., above).For generation transgenic animal, transgenosis can microinjection in fertilized oocyte, maybe can be incorporated in the genome of embryonic stem cell, and the consideration convey of described cell is moved in enucleation oocyte.
Theme polypeptide also can be incorporated to introduce in the genome of transgenic animal in transgenosis, and such as expresses subsequently in the milk of transgenic animal (see such as Deboer etc. 5,741,957; Rosen 5,304,489; And Meade 5,849,992.Applicable transgenosis comprises the light chain and/or heavy chain-coding sequence that are operatively connected with promotor and the enhanser from mammary gland specific gene (as casein or beta lactoglobulin).
Single carrier or two vector expression polypeptide can be used.For example, heavy chain of antibody and light chain can be cloned in single expression carrier, and cotransfection is in cell.
In one embodiment, signal sequence can be used for the expression promoting polypeptide of the present invention.
Once express, can according to the standard program purified polypeptide of this area, described standard program comprises ammonium sulfate precipitation, affinity column (such as a-protein or protein G), column chromatography, HPLC purifying, gel electrophoresis etc. (generally see Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)).
Can by cultivated host cell or expression of cell lines humanized Ig variable region or the polypeptide comprising them.They also can in vivo at cells.The clone being converted (such as transfection) next mutagenic antibody can be immortalized mammalian clone, as having lymphatic origin those (such as myelomatosis, hybridoma, trioma or four source hybridoma cell lines).Clone also can comprise by transforming the normal lymphoid like cell of in addition immortalization with viral (such as Epstein-Barr virus), as B cell.
Although for generation of the clone normally mammal cell line of polypeptide, the clone from other source (as bacterium and yeast) also can be used.Specifically, Escherichia coli source bacterial isolates can be used, especially such as phage display.
Ig light chain or the heavy chain of separation can be secreted under some immortalization lymphoidocytes as myeloma cell line tie up to their standard state.The vector of the antibody that the expression prepared during if described clone is used in the inventive method changes, so need not carry out all the other steps of method, prerequisite is that normocrinic chain is complementary to by the variable domains of the Ig chain of the vector encoded previously prepared.
If immortalized cell line is not secreted or do not secreted complementary strand, the carrier of the suitable complementary strand of coding or its fragment so must be introduced in cell.
When immortalized cell line secretion complementary light chain or heavy chain, can, such as by being applicable to bacterial cell with vector, bacterial cell and immortalized cell line be then made to merge (such as passing through spheraplast fusion) to produce transformation cell lines.Or, by electroporation, DNA is directly introduced in immortalized cell line.
In one embodiment, can be present in the Fab of any antibody as described in the present invention or by the humanized Ig variable region that the inventive method produces.Fragment can be recombinated generation and through engineering approaches, synthesis or by producing with proteolytic enzyme digest antibody.For example, fragment can be Fab fragment; Can at the interchain of joint two heavy chains (i.e. V with papain digestion
h-V
h) region before disulfide linkage makes antibody rupture.This causes the V formed containing light chain and heavy chain
hand C
htwo same clip of 1 structural domain.Or fragment can be F (ab ')
2fragment.These fragments are by producing with pepsin digested antibody, and described stomach en-is cracking heavy chain after interchain disulfide bond, and produce the fragment containing two antigen binding sites.Another replacement scheme is to use " strand " antibody.ScFv (scFv) fragment can be built in many ways.For example, V
hc-terminal can be connected to V
ln-terminal.Usually, by joint (such as (GGGGS)
4; SEQ ID NO:4) be placed on V
hwith V
lbetween.But, reversible can connect the order that each chain is complied with, and (label as these labels can attach to any antibody of the present invention or antibody fragment can to comprise the label (such as Myc label, His label or FLAG label) contributing to detection or purifying; Their use is not limited to scFv).Therefore, and as follows indicated by, the antibody of mark is within the scope of the invention.In an alternative embodiment, described herein or can be heavy chain homodimer or light chain dimer by the antibody that method as herein described produces.Further, light chain of antibody or heavy chain or its part, such as single domain antibody (DAb) can be used.
In another embodiment, be present in single-chain antibody (ScFv) or miniantibody (see such as U.S. Patent number 5 as described in the present invention or by the humanized Ig variable region that the inventive method produces, 837,821 or WO 94/09817A1).The dimeric molecule that miniantibody is made up of two polypeptide chains of each self-contained ScFv molecule (namely comprises the single polypeptide of one or more antigen binding site, such as V
lstructural domain is connected to V by flexible joint
hstructural domain, described V
hstructural domain is blended in CH3 structural domain by connection peptides).ScFv molecule can V
h-joint-V
lorientation or V
l-joint-V
horientation builds.Connect and compose the V of antigen binding site
lstructural domain and V
hthe flexible hinge of structural domain preferably comprises about 10 to about 50 amino-acid residues.Exemplary connection peptides for this object be (Gly4Ser) 3 (Huston etc. (1988) .PNAS, 85:5879).Other connection peptides is well known in the art.
The method preparing single-chain antibody is known in the art, such as Ho etc. (1989), Gene, 77:51; Bird etc. (1988), Science 242:423; Pantoliano etc. (1991), Biochemistry 30:10117; Milenic etc. (1991), Cancer Research, 51:6363; Takkinen etc. (1991), Protein Engineering 4:837.ScFv component and connection peptides-CH is built by using the method described in this area
3component prepares miniantibody (see such as United States Patent (USP) 5,837,821 or WO 94/09817A1).These components can restricted fragment form be separated from independent plasmid, then connect and are cloned in suitable carrier again.By restriction digestion and DNA sequence analysis checking proper mating.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, tetravalent minibody can be built.Tetravalent minibody can the mode identical with miniantibody build, and exception part uses such as to have aminoacid sequence (G
4s)
4g
3the flexible joint of AS (SEQ ID NO:7) connects two ScFv molecules.
In another embodiment, can be present in double-chain antibody as described in the present invention or by the humanization variable region that the inventive method produces.Double-chain antibody is similar to scFv molecule, but usually has short (be less than 10, and preferably 1-5 being individual) amino acid residue linker of connection two variable domains, to make the V on same polypeptide chain
lstructural domain and V
hstructural domain can not interact.Replace, the V of a polypeptide chain
lstructural domain and V
hv on structural domain and the second polypeptide chain
hstructural domain and V
lstructural domain (difference) interacts (WO 02/02781).
In another embodiment, in the humanization variable region of the present invention immunoreactivity fragment being operably connected to FcR bound fraction that can be present in antibody (such as scFv molecule, miniantibody, tetravalent minibody or double-chain antibody) or part.In an exemplary embodiment, FcR bound fraction is complete Fc district.
Preferably, humanization approach as herein described produces wherein compared to donor antibody, to the Ig variable region that the avidity of antigen does not change in fact.
In one embodiment, the polypeptide of variable domains of the present invention is comprised to be greater than (or equaling) following association constant Ka: 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 conjugated antigen of (comprising the avidity of the centre being in these values).
Avidity, avidity and/or specificity can be measured in many ways.Usually, and regardless of defining or measure avidity exact way used, when the antibody that the inventive method produces it clinical application any in a certain antibody (or multiple antibody) all prepared from it than it superior time, they make affinity of antibody improve (for example, when a certain antibody (or multiple antibody) prepared from it compared to it of antibody modified can compared with low dosage or compared with under small frequency or when being used by more convenient route of administration, the inventive method is regarded as effectively or success).
More particularly, the avidity between the antigen combined by various mensuration measurement antibody and it, described mensuration comprises such as ELISA mensuration, BiaCore and measures or KinExA
tM3000 measure (can obtain from Sapidyne Instruments (Boise, ID)).Inventionbriefly, by covalently bound, (can be any target antigen (such as cancer antigen for the antigen in the inventive method with antigen; The protein of cell surface proteins or secretion; The antigen (such as bacterium or virus antigen (such as HIV antigen, influenza antigens or hepatitis antigen)) of pathogenic agent or anaphylactogen)) coating agarose beads.Prepare the diluent of antibody to be tested, and add each diluent in the designation hole on plate.Then add and detect in antibody (such as Goat anti human IgG – HRP conjugate) to each hole, add subsequently and produce look substrate (such as HRP).Then in elisa plate reader, under 450nM, read plate, and calculate EC50 value.(but, should be appreciated that method as herein described can generally be suitable for; They are not limited to produce the antibody of the antigen in conjunction with any specific antigen or any particular category.)
One of skill in the art will recognize that to measure avidity not simple as checked single width figure all the time.Because antibody has two arms, so their apparent avidity usually more much higher than the inherent avidity between variable region and antigen (it is believed that this is owing to avidity).Inherent avidity can use scFv or Fab fragment to measure.
On the other hand, the invention is characterized in that one is puted together in treatment part, as humanized rabbit antibody or its fragment of cytotoxin, medicine (such as immunosuppressor) or radiotoxin.Described conjugate is referred to herein as " immune conjugate ".
Antibody conjugates of the present invention can be used for regulating given biological respinse, and drug moiety should not be construed as and is limited to classical chemical therapeutic agent.For example, drug moiety can be and has required bioactive protein or polypeptide.Described protein can comprise such as enzyme activity toxin or its active fragments, as abrin (abrin), ricin A (ricin A), Pseudomonas exotoxin (pseudomonas exotoxin) or diphtheria toxin (diphtheria toxin); Protein, as tumour necrosis factor or interferon-γ; Or biological response modifier, such as, as lymphokine, il-1 (" IL-1 "), interleukin-2 (" IL-2 "), interleukin-6 (" IL-6 "), granular leukocyte macrophage colony stimulating factor (" GM-CSF "), G CFS (" G-CSF ") or other somatomedin.
For making described treatment moiety conjugation know in the technology of antibody, see such as Arnon etc., " Monoclonal Antibodies For Immunotargeting Of Drugs InCancer Therapy ",
monoclonal Antibodies And Cancer Therapy, Reisfeld etc. (volume), 243-56 page (Alan R.Liss, Inc.1985); Hellstrom etc., " Antibodies For Drug Delivery ",
controlled Drug Delivery(the 2nd edition), Robinson etc. (volume), 623-53 page (Marcel Dekker, Inc.1987); Thorpe, " Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review ",
monoclonal Antibodies'84:Biological And Clinical Applications, Pinchera etc. (volume), 475-506 page (1985); " Analysis, Results, And FutureProspective Of The Therapeutic Use Of Radiolabeled Antibody In CancerTherapy ",
monoclonal Antibodies For Cancer Detection And Therapy, Baldwin etc. (volume), 303-16 page (Academic Press 1985); And Thorpe etc., " The Preparation And Cytotoxic Properties Of Antibody-ToxinConjugates ", Immunol.Rev.,
62: 119-58 (1982).
In one aspect, the invention provides the pharmaceutical preparation comprising the humanized rabbit antibody being used for the treatment of disease.Term " pharmaceutical preparation " refers to following preparation: it is in allowing the clear and definite effective form of the biological activity of antibody or antibody derivatives, and containing to preparation by other poisonous for the experimenter that uses component." pharmaceutically acceptable " vehicle (vehicle, additive) be can reasonably to theme administration to provide those of the activeconstituents used of effective dose.
Equivalent
Numerous amendment of the present invention and alternate embodiment will show in view of previously describing and easily know for those skilled in the art.Therefore, this description should be interpreted as only having illustrative, and is for performing the object of optimal mode of the present invention for instruction those skilled in the art.The details of structure can change in fact without departing from the spirit of the present invention, and retains the exclusive use of being included into all modifications of enclosing in the scope of claim.Intention the present invention is only limitted to enclose claim and the degree required by applicable regulation.
The all documents quoted in the application and analogous material, comprise patent, patent application, article, books, monograph, paper, webpage, graphic and/or annex, regardless of the form of described document and analogous material, entirety is clearly incorporated to herein all by reference.Be incorporated to document or contradiction different from the application with analogous material if one or more, comprise definition term, term use, described technology etc., be so as the criterion with the application.
Claims (16)
1. people's heavy chain receptor framework, it comprises SEQ ID NO:1.
2. people's heavy chain receptor 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 receptor framework as claimed in claim 2, wherein said replacement is
A () is at the Serine (S) at position 12 place;
(b) Serine (S) at position 103 place or Threonine (T); And/or
(c) Serine (S) at position 144 place or Threonine (T).
4. the nucleic acid be separated, its receptor framework according to claim 1 of encoding.
5. a carrier, it comprises nucleic acid according to claim 4.
6. a host cell, it comprises carrier according to claim 5.
7. have a specific immunoconjugator to required antigen, it comprises:
A () comprises the light chain receptor framework of the variable light CDR of Lagomorph immunoconjugator; And
B () comprises people's heavy chain receptor framework according to claim 1 of the variable heavy chain CDR of Lagomorph immunoconjugator.
8. immunoconjugator as claimed in claim 7, wherein said light chain receptor framework and SEQ ID NO:2 have at least 85% identity.
9. immunoconjugator as claimed in claim 7, it comprises the joint sequence connecting described variable light framework and described heavy chain receptor framework further, and wherein said joint sequence is SEQ ID NO:4.
10. immunoconjugator as claimed in claim 7, it comprises donor framework residues involved in antigen combination further.
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 at least one heavy chain CDR of the group be made up of CDR H1, CDR H2 and the CDR H3 sequence from donor rabbits immunoconjugator is transplanted in people's heavy chain receptor framework according to claim 1 by (); And
B at least one light chain CDR of the group be made up of CDR L1, CDR L2 and the CDR L3 sequence from donor rabbits immunoconjugator to be transplanted in people's light chain receptor framework with SEQ ID NO:2 with at least 85% identity in light chain receptor framework by ().
13. methods as claimed in claim 12, its comprise further with the Framework residues of described donor rabbits immunoconjugator replace described people's heavy chain receptor framework and described people's light chain framework one or two in Framework residues.
14. methods as claimed in claim 12, the one or more places of wherein said heavy chain receptor framework in the amino position 12,103 and 144 (AHo numbering) of heavy chain have replacement.
15. methods as claimed in claim 14, the described replacement at the one or more places wherein in position 12,103 and 144 is selected from by the following group formed:
A () is at the Serine (S) at position 12 place;
B () is at the Threonine (T) at position 103 place; And
C () is at the Threonine (T) at position 144 place.
16. 1 kinds of immunoconjugators, its method humanization according to claim 12.
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|---|---|---|---|
| PCT/EP2013/053039 WO2014124677A1 (en) | 2013-02-15 | 2013-02-15 | Acceptor framework for cdr grafting |
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| CN (1) | CN104884470A (en) |
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| HK (1) | HK1217960A1 (en) |
| RU (1) | RU2015139095A (en) |
| WO (1) | WO2014124677A1 (en) |
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| WO2016168755A1 (en) | 2015-04-17 | 2016-10-20 | Distributed Bio, Inc. | Method for mass humanization of non-human antibodies |
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- 2013-02-15 CN CN201380065987.9A patent/CN104884470A/en active Pending
- 2013-02-15 EP EP13704438.4A patent/EP2956478A1/en not_active Withdrawn
- 2013-02-15 RU RU2015139095A patent/RU2015139095A/en not_active Application Discontinuation
- 2013-02-15 JP JP2015557333A patent/JP2016506974A/en active Pending
- 2013-02-15 CA CA2894918A patent/CA2894918A1/en not_active Abandoned
- 2013-02-15 WO PCT/EP2013/053039 patent/WO2014124677A1/en active Application Filing
- 2013-02-15 AU AU2013378628A patent/AU2013378628A1/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 |
|---|---|
| CA2894918A1 (en) | 2014-08-21 |
| EP2956478A1 (en) | 2015-12-23 |
| AU2013378628A1 (en) | 2015-06-11 |
| RU2015139095A (en) | 2017-03-21 |
| JP2016506974A (en) | 2016-03-07 |
| HK1217960A1 (en) | 2017-01-27 |
| WO2014124677A1 (en) | 2014-08-21 |
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