CN1327388A - Modified antibodies of induced anti-idiotype reaction enhancement - Google Patents

Modified antibodies of induced anti-idiotype reaction enhancement Download PDF

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CN1327388A
CN1327388A CN98813119A CN98813119A CN1327388A CN 1327388 A CN1327388 A CN 1327388A CN 98813119 A CN98813119 A CN 98813119A CN 98813119 A CN98813119 A CN 98813119A CN 1327388 A CN1327388 A CN 1327388A
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virus
receptor
antigen
immunoglobulin
variable region
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R·M·布尔克
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Euro Celtique SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
    • C07K16/4266—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig against anti-tumor receptor Ig
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/18—Kallidins; Bradykinins; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/10—Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to modified immunoglobulin molecules in which one or more variable region residues that form intrachain disulfide bonds are substituted with amino acid residues that do not contain sulfydryl groups, such that the intrachain disulfide bond does not form. Such immunoglobulin molecules have an enhanced ability to elicit an anti-idiotype response. The invention further provides for the methods of prevention and treatment of cancer and/or infectious diseases using the modified immunoglobulins of the invention.

Description

The enhanced modified antibodies of induced anti-idiotype reaction
The cross reference of related application:
It number is that the provisional application submitted on April 10th, 60/065,716 and 1998 number is the right of two parts of applications of 60/081,403 that the application requires the provisional application submitted on November 14th, 1997, and this paper is incorporated by reference in its entirety.
1. invention field:
Immunoglobulin that the present invention relates to modify and vaccine combination thereof wherein with the one or more cysteine residues that form intrachain disulfide bond in the amino acid replacement variable region that does not contain mercapto groups, do not form disulfide bond thus.The invention still further relates to and use vaccine combination treatment of the present invention or prevention some diseases and imbalance, especially cancer and infectious disease.
2. background of invention
2.1 immunoglobulin structure
The ultimate unit of immunoglobulin structure is complex-two the same low-molecular-weight " gently " chain that four polypeptide chains constitute, and two the same high molecular " weight " chains constitute together by non-covalent bond and disulfide bond.Amino terminal at the weight chain of antibody molecule has a variable region, at carboxyl terminal a constant region (see figure 1) is arranged.The variable region of each antibody all is unique, contains antigen binding site.And each variable region comprises four conservative relatively framework region and the called after complementary determining region of three alterable heights or regional (see figure 2)s of CDRs.In most cases, CDRs forms the antigen binding site of antibody and gives its antigenicity.Constant region is that conservative is strong, only exists because the different slight variation of haplotype with respect to the variable region.
According to the difference of aminoacid sequence, light chain is divided into κ and λ type again.CH constitutes (CH1, CH2, CH3 by a plurality of domains ... CHx), concrete domain number depends on the kind of specific antibody.Have hinge region separately between CH1 and the CH2, hinge region is given antibody molecule and is stretched tropism.The variable region of light chain and the variable region of heavy chain are arranged in together, and first constant region of the constant region of light chain and heavy chain is arranged in together.The CH2-CHx domain of heavy chain has constituted the Fc district, this district is relevant with the effector function of immunoglobulin molecules, is the Fc receptors bind on cell, killer cell, mastocyte and other immunocyte as the complement combination with being expressed in lymphocyte, granulocyte, monokaryon.
As shown in Figure 3, the light chain of IgG molecule and heavy chain have constituted variable region and constant region domain.Each domain constitutes (referring to Roitt et al., immunology, the third edition, London by single disulfide bond again by two extends parallel, about 100 structures that aminoacid constitutes; Mosby, 1993, p4.4). each in the albumen lamella of two extends parallel of domain comprises two antiparallel adjacent chains again, each adjacent chain contains β sheet conformation structure (Stryer, 1975, a biochemistry again, WH Freeman and Co., p.950).After the immunoglobulin folding, each domain all has a similar three dimensional structure.
2.2 immunization therapy and anti-idiotype antibody
In modern medicine, immunization therapy or vaccine have in fact been eradicated such as poliomyelitis, tetanus, tuberculosis, chickenpox, measles, hepatitis etc.Immune system used verified by the eqpidemic disease vaccine can the infection prevention disease.
Immunization therapy equally also is applied to tumor treatment.Tumour immunity starts from the experiment that PREHN and MAIN do epoch, they studies show that, with the inductive sarcoma antigen of methyl cholanthrene (MCA) is tumour-specific, transplant to detect and in the normal structure of mice, can not find this antigen (Prehn et al., 1957, J.Natl.Cancer Inst.18:79-778).The experiment that further confirms this point is: the resistance of the tumour-specific of anti-MCA is also induced generation (Klein et al., 1990, cancer research 20:151-1572) intrinsic (autochthonous) host of tumor source Mus.
Tumour patient uses the immunization therapy reason abundant.At first, if patient's immunoreation is suppressed in the operation, as using anesthetis and using chemotherapy subsequently, immunosuppressant may further worsen, and carries out immunization therapy before operation, and possible epidemic prevention suppresses or make it to reverse.This will reduce infection and impel wound healing.Secondly, behind operation and the chemicotherapy, the tumor cell quantitative change is little, and use immunization therapy this moment will be more effective.At last, in operation process, tumor cell might enter blood circulation, and this moment, effectively immunization therapy then can be removed these cells.
Immunization therapy is divided into two kinds, i.e. " active immunity " and " passive immunity "." active immunity " is as vaccine, to the protective immunological reaction of inducing patient after patient's medication with antigen." passive immunity " is to use antibody to the patient, do not induce the immunoreation of following.When the antibody that comes from certain animal is injected to second kind of suitable animal as immunogen, host's immunoreation will be induced.Usually Antybody therapy is the feature of passive immunity, does not produce because antibody is patient oneself.But a passive speech tends to make the people to misunderstand, and patient can produce the two anti-of antiidiotype in fact, can challenge and with Proantigen generation cross reaction.After using antibody, the immune participation of patient, sustainable and the cell effect that contains specific antigen, so, produce two anti-patients' immunization therapy better effects if.
In anti-idiotype reaction, the antibody of antibody that begins to produce or introducing body has the inconsistent new epi-position of body itself, therefore induce and produced second antibody (called after " Ab2 "), wherein partial antibody is the idiotype (being antigen binding site) at initial antibodies (called after " Abl "), the antibody that promptly first antibody that produces of initial antibodies or external source are introduced.These two antibody anti-or two anti-samples also have its idiotype site, will induce the 3rd production of antibodies to be called " Ab3 ", and the antigen binding site of some antibody recognition Ab2 is wherein arranged, and so analogize.Famous network theory that Here it is.Therefore the similar initial antigen of binding site that part two resists will reproduce initially antigenic " interior image ".Three of the antigen binding site that identification two resists resists, and discerns initial antigen equally.(see figure 4)
Therefore, the binding site of anti-idiotype antibody is similar to antigen aspect conformation and electric charge, can induce the reaction the same or stronger with tumor antigen.Therefore use the ectogenic antibody that to induce strong anti-idiotype antibody, can be used as a kind of effective vaccine by keeping constant immunoreation.
Because anti-idiotype reaction is the part of typical human antimouse antibody reaction (HAMA), till in face of arriving, the antiidiotype vaccine comprises murine antibody.But behind Abl destructurized, give antibody stronger antigenicity, can see strong anti-idiotype reaction chain (Madlyalakanet a1., 1995, hybridoma 14:199-203).Had directly use ectogenic anti-idiotype antibody to the patient after, produced the idiotype antibody (U.S.PulicationNo.4,918,14) of anti-tumour antibody.After the medication, the anti-antibody that body produces is not only discerned the antibody of antiidiotype, but also discerns former tumor antigen, and therefore direct activating complement reaction and other external immune system response, attacks the tumor cell of expressing tumor epitope.
Though the antiidiotype vaccine is ideal target, and the example of success is arranged, yet can repeat to cause ability normally impossible (Foonet al., 1995, the J.Clin.Invest.9:334-342 of such anti-idiotype reaction behind the use antibody; (Madlyalakan etal., 1995, Hybridoma 14:199-203).One of reason of failure is, is ectogenic though produce the Ab1 of anti-idiotype reaction, because antibody molecule is all closely similar, thus very similar to self molecule, and body is limited to the tendency type that self molecule produces anti-idiotype reaction.Therefore, this area needs reliable method, produces the anti-idiotype antibody at specific antibodies.
3. summary of the invention
The present invention is based on inventor's practice, it is the amino acid replacement that one or more cysteine of forming one or more disulfide bond in the antibody molecule variable region is not contained sulfydryl, the antibody disulfide bond can not form like this, but the complete stronger anti-idiotype reaction of antibody molecule of disulfide bond in this antibody induction ratio variable region.
Correspondingly, the invention provides the immunoglobulin molecules or antibody (the functional activity fragment of modification, derivant and analog) and comprise the vaccine combination of these immunoglobulin molecules, the variable region of modified immunoglobulin molecule wherein, as disconnect in the chain or interchain one or more disulfide bond, the conformation compactness is tended to reduce, but is not limited to these methods.The invention provides the immunoglobulin of modification, comprise a variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin molecules, the wherein said second immunoglobulin molecules immunologic opsonin conjugated antigen (is to determine in this area that the confirmable immunoglobulin of the bonded method of antigen-antibody combines with its antigenic specificity, wherein do not comprise and other antigenic non-specific but non-essential cross reactivity), said one or more amino acid whose replacement refers to replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with the aminoacid that does not contain sulfydryl.The second immunoglobulin molecules immunologic opsonin in preferred version in conjunction with tumor antigen; In other preferred version, the second immunoglobulin molecules immunologic opsonin in conjunction with the infectious disease pathogen antigen or in conjunction with the cell receptor of this cause of disease.
The present invention also provides, and uses the method for modified immunoglobulin induced anti-idiotype reaction of the present invention the experimenter.In specific scheme, immunoglobulin of the present invention can be used for treatment or prophylaxis of tumours, especially use derive from immunologic opsonin in conjunction with the modified immunoglobulin of tumor antigen (promptly according to method of modifying of the present invention, with the one or more cysteine that form disulfide bond in the amino acid replacement variable region that does not contain sulfydryl), said tumor antigen is the tumor antigen with the tumor correlated expression of particular type.In addition, in other scheme, immunoglobulin molecules of the present invention can be used for treatment or keeps off infection, and especially uses to derive from immunologic opsonin in conjunction with the infectious disease pathogen antigen or in conjunction with the modified immunoglobulin molecule of the cell receptor of the infectious disease cause of disease that produces communicable diseases.
The production method that the present invention also provides modified immunoglobulin molecule production method of the present invention and comprises the vaccine combination of modified immunoglobulin molecule of the present invention.
4. accompanying drawing summary
Fig. 1. the weight chain structure sketch map of immunoglobulin molecules.Every chain is by amino terminal (H 2N-) (constant region COOH) is formed for variable region and carboxyl terminal.
Fig. 2 .IgG molecule weight chain variable region (is designated as V accordingly LAnd V H) in four framework regions (FR1, FR2, FR3, FR4) and three complementary determining regions (CDR1, CDR2, CDR3) sketch maps.Constant region of light chain is designated as C L, three domains of CH are designated as CH1, CH2 and CH3.F AbBe to comprise weight chain variable region and C LAnd C H1The antibody fragment of part.F cIt is the constant region fragment that comprises CH2 and CH3 domain.
Fig. 3. the antibody structure sketch map.The same with Fig. 2, but emphasize (correspondingly to be designated as V by determined each the structural domain of the disulfide bond of keeping three dimensional structure (shown in the black line) L, V H, C L, CH1, CH2 and CH3) (Roitt et al., immunology, second edition London:GowerMedical Publishing, 1989, p5.3).
Fig. 4. in the anti-idiotype reaction chain, the anti-idiotype antibody (Ab2) that causes by the idiotype antibody (Ab1) of anti-aglucon and the interior mapping generating process sketch map of anti-anti-idiotype antibody (Ab3).
Fig. 5. substitute the cysteine that forms disulfide bond in the variable region with alanine, the immune globulin variable region that disconnects the variable region disulfide bond is modified sketch map.CH1, CH2, CH3 are constant regions, V HBe variable region of heavy chain, V LIt is variable region of light chain.
The structure of Fig. 6 A-C. (A) expression vector pMRR010.1 wherein comprises human kappa light chain constant region sequence.(B) structure of expression vector pGammal, wherein comprise human IgG1's CH (CH1, CH2, CH3) and hinge region.(C) structure of expression vector pNEPuDGV wherein comprises the sequence of coding light chain κ constant region and the sequence of CH and hinge region.Three carriers are referring to Bebbington et al., 1991, Enzymology method 2:136-145.
The consensus sequence of Fig. 7 A and B. (A) variable region of light chain ConVL1 comprises aminoacid and the corresponding nucleic acids sequence of leading peptide.(B) aminoacid of the consensus sequence of variable region of heavy chain ConVH1 and corresponding nucleic acids sequence are comprising leading peptide sequence.
Aminoacid and the corresponding nucleic acids sequence of Fig. 8 A-B. (A) 2CAVLCOL1, wherein the variable region of light chain of antibody comes from mAb31.1, and wherein 23 and 88 s' cysteine is substituted by alanine, lives with the square frame frame.(B) aminoacid of 2CAVHCOL1 and corresponding nucleic acids sequence, wherein the variable region of heavy chain of antibody comes from mAb31.1, and wherein 23 and 88 s' cysteine is substituted by alanine, lives with the square frame frame.
The oligonucleotide of Fig. 9 A-D. (A) assembling 2CAVHCOL1 is the heavy chain variable region gene at the human colon carcinoma tumor antigen.(B) oligonucleotide of assembling 2CAVLCOL1 is the chain variable region gene at the human colon carcinoma tumor antigen.(C) oligonucleotide of assembling light chain consensus is with reference to ConVL1.(D) oligonucleotide of assembling heavy chain consensus is with reference to ConVL1.
Figure 10. the installation step sketch map of the synthetic antigenic modified antibodies gene of specific recognition human colon carcinoma.
Figure 11. the immunoblotting result.Compared and be derived from mAb31.1 and cysteine of no use substitutes the same antibody of the antibody of alanine and labelling biotin and antigenic competitive combination of LS-174T cell preparation.The unmarked antibody concentration of nM, the no antigen of " blk " band expression.
Figure 12 A-D. (A) is competitive in conjunction with testing result: the antibody and the serum of using the empty carrier immune mouse that comprise biotin labeled resistive connection colon-cancer cell, discern colon cancer cell but not modified control antibodies, and CDR1, CDR2, CDR3, CDR4, CDR5, these contain the competition result of polypeptide of the CDR region sequence of bradykinin receptor binding site CDR6, and wherein these polypeptide are the percentage ratio contrasts that are used to represent in conjunction with the LS-174T cell.(B) competitive in conjunction with testing result: the antibody and the serum of using the empty carrier immune mouse that comprise biotin labeled resistive connection colon-cancer cell, identification colon cancer cell but not modified control antibodies and with the competition result of 2CAVHCOL1 and 2CAVLCOL1 mice immunized serum.(C) sketch map that combines of biotin labeling (representing) antibody (inverted Y) and antigen (solid triangle) with b.(D) anti-idiotype antibody (filled arrows) suppresses the sketch map that combines of biotin labeling (representing with b) antibody (inverted Y) and antigen (solid triangle).
Figure 13. comprise the nucleotide sequence of HMFG-1 binding sequence among the variable region of light chain CDR.
5. detailed Description Of The Invention:
The invention provides with modified immunoglobulin (especially antibody and active function fragment, derivative and analog etc.) and induce stronger immune response, especially stronger anti-idiotype reaction than corresponding not modified immunoglobulin (Ig). Particularly, after but the immunoglobulin molecules of modifying front immunologic opsonin conjugated antigen is modified, immunoglobulin molecules variable region conformation compactness reduces, has an amino acid replacement that is not contained sulfydryl at least in the cysteine that the participation intrachain disulfide bond forms in the preferred variable region, thereby can not form disulfide bond, so variable region conformation compactness reduces (Fig. 5). In the preferred embodiments of the invention, modified immunoglobulin molecular source self energy immunologic opsonin is in conjunction with the immunoglobulin (Ig) of tumour antigen. And in other preferred embodiment, modified immunoglobulin be derived from can immunologic opsonin in conjunction with the infectious disease pathogen antigen or in conjunction with the immunoglobulin (Ig) of the cell receptor of this antigen.
The present invention also provides the vaccine combination that contains immunoglobulin molecules of the present invention. In addition, the present invention also provides and has used modified immunoglobulin of the present invention, induces the method that produces anti-idiotype reaction a certain experimenter.
In specific embodiments, the present invention also provides the method for using modified immunoglobulin molecule prevention of the present invention and treatment tumour, before wherein said immunoglobulin (Ig) is modified, can with the specific combination of expression antigen immune of certain Tumor-assaciated. Use this modified immunoglobulin to induce the experimenter to produce anti-idiotype reaction in the host, the antibody of inducing antitumor antigen produces. And in another embodiment, before said immunoglobulin (Ig) is modified, can immune immunologic opsonin in conjunction with the infectious disease pathogen antigen or in conjunction with the cell receptor of this antigen. This immunoglobulin (Ig) also can be used for the communicable disease for the treatment of or preventing to be caused by the infectious disease cause of disease.
For making narration more detailed, detailed Description Of The Invention is divided into following components, but is not restriction the present invention.
5.1 the antibody of modifying
Modified immunoglobulin of the present invention, especially antibody, conjugated antigen that can immune immunologic opsonin before modification at least, and the ability of modifying its induced anti-idiotype reaction of back is enhanced.This modification can reduce the compactness of immune globulin variable region conformation, method of modifying as: remove or the reduction chain in or interchain disulfide bond, any other known method in chemical modification or this area.Especially the invention provides first immunoglobulin, in the variable region one or more aminoacid replaced, all the other aminoacid are consistent with second immunoglobulin molecules, but this second immunoglobulin immunological characteristic conjugated antigen, described substituting is the cysteine of using on the relevant position that forms intrachain disulfide bond in one or more amino acid replacements second immunoglobulin molecules that does not contain sulfydryl.The present invention also provides the nucleotide sequence that comprises code book invention modified immunoglobulin molecule at interior nucleotide sequence.
Determining to form in the antibody specific molecule cysteine method of disulfide bond, can be any method as known in the art.Such as: well-known, the cysteine that forms intrachain disulfide bond at antibody all types of and plant between high conservative, therefore just can determine cysteine position in the antibody molecule of the unknown with the antibody molecule aminoacid sequence comparison of the cysteine position of known formation intrachain disulfide bond.
Table 1 has been listed the position that forms the cysteine of intrachain disulfide bond in a lot of antibody molecules.
Table 1 (from Katal et al, the sequence of immune destination protein, the 5th edition U.S.Department of Health and Human Services, Bethesda, Marryland)
Kind variable region subgroup forms the cysteine (position) of disulfide bond
Human kappa light chain I 23,88
Human kappa light chain II 23,88
Human kappa light chain III 23,88
Human kappa light chain IV 23,88
People's lambda light chain I 23,88
People's lambda light chain II 23,88
People's lambda light chain III 23,88
People's lambda light chain IV 23,88
People's lambda light chain V 23,88
People's lambda light chain VI 23,88
Mice κ light chain I 23,88
Mice κ light chain II 23,88
Mice κ light chain III 23,88
Mice κ light chain IV 23,88
Mice κ light chain V 23,88
Mice κ light chain VI 23,88
Mice κ light chain VII 23,88
Mice lambda light chain heterozygosis 23,88
Mice lambda light chain 23,88
Chimpanzee lambda light chain 23,88
Rat κ light chain 23,88
Rat lambda light chain 23,88
Rabbit κ light chain 23,88
Rabbit lambda light chain 23,88
Canis familiaris L. κ light chain 23,88
Pig κ light chain 23, (88)
Pig lambda light chain 23,88
Cavia porcellus lambda light chain 23, (88)
Sheep lambda light chain 23,88
Chicken lambda light chain 23,88
Turkey lambda light chain 23, (88)
Silver shark lambda light chain 23, (88)
Shark κ light chain 23,88
People's heavy chain I 22,92
People's heavy chain II 22,92
People's heavy chain III 22,92
Murine heavy chain I (A) 22,92
Murine heavy chain I (B) 22,92
Murine heavy chain II (A) 22,92
Murine heavy chain II (B) 22,92
Murine heavy chain II (C) 22,92
Murine heavy chain III (A) 22,92
Murine heavy chain III (B) 22,92
Murine heavy chain III (C) 22,92
Murine heavy chain III (D) 22,92
Murine heavy chain V (A) 22,92
Murine heavy chain V (B) 22,92
Murine heavy chain heterozygosis 22,92
Rat heavy chain 22,92
Rabbit heavy chain 22,92
Cavia porcellus heavy chain 22,92
Cat heavy chain 22, (92)
Canis familiaris L. heavy chain 22,92
Pig heavy chain 22, (92)
Weasel heavy chain 22, (92)
Sea lion heavy chain 22, (92)
Sea dog heavy chain 22, (92)
Turkey heavy chain 22,92
Duck heavy chain 22, (92)
Goose heavy chain 22, (92)
Penguin heavy chain 22, (92)
Turkey heavy chain 22, (92)
Alligator heavy chain 22,92
Pawl frog heavy chain 22,92
Elops heavy chain 22,92
Carassius auratus heavy chain 22,92
Silver shark heavy chain 22, (92)
Shark heavy chain 22,92
Location number in () draws by relatively inferring with known array, is not this albumen position of mensuration.
It should be noted that the cysteine that forms intrachain disulfide bond in the listed antibody molecule of table 1 all is positioned at 23 or 88 of variable region of light chain, is positioned at 22 or 92 of variable region of heavy chain.Related residue location number corresponding to Katal etc. (from Katal et al, the sequence of immunity destination protein the 5th edition, U.S.Department of Health and HumanServices, Bethesda, Marryland) the variable region sequences of weight chain among fixed consensus sequence residue or Fig. 7 A and the B.(the corresponding meaning is the variable region sequences by weight chain among the consensus sequence in the arrangement appointment antibody molecule or Fig. 7 A or the B.)
Correspondingly, the immunoglobulin of modifying in the embodiment of the present invention is a kind of antibody, and wherein 23 of light chains and/or 88 amino acids residues are not contained the amino acid replacement of sulfydryl, and perhaps 22 of heavy chains and/or 92 amino acids residues are not contained the amino acid replacement of sulfydryl.
In modified immunoglobulin of the present invention, the aminoacid that substitutes the cysteine that forms disulfide bond is the arbitrary amino acid that does not contain sulfydryl, as: alanine, arginine, agedoite, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline serine threonine, tyrosine, tryptophan, or valine.In preferred embodiments, what substitute cysteine residues is glycine, serine, and threonine, tyrosine, agedoite, glutamine residue is alanine residue in the most preferred embodiment.
In addition, the cysteine that forms disulfide bond can substitute (such as with conventional albumen synthetic method, but being not limited to these methods) with atypia aminoacid that does not contain sulfydryl or chemical amino acid analogue.Atypia aminoacid comprises: typical amino acid whose dextrorotation aminoacid, α aminobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx ,-aminocaproic acid, Aib, the 2-aminoisobutyric acid, the amino and propanoic acid of 3-, ornithine, nor-leucine, norvaline, hydroxyproline, sarcosine, citrulline, tert-butyl group glycine, tert-butyl group alanine, phenylglycine, Cyclohexylalanine, Beta-alanine, fluoro-aminoacid is such as the Beta-methyl aminoacid of design, C Alpha-Methyl aminoacid, N Alpha-Methyl aminoacid, amino acid analogue.And aminoacid can be dextrorotation or left-handed.In alternative embodiment, the amino acid residue that forms disulfide bond can be deleted.
In specific embodiments, the amino acid residue of the formation disulfide bond in two chain variable regions of heavy chain or light chain or weight is replaced.In other embodiments, one of residue that forms concrete disulfide bond replaced (or deletion) perhaps forms two residues replaced (or deletion) of disulfide bond.
In other embodiments, the invention provides immunoglobulin molecules, wherein in this immunoglobulin molecules one or more amino acid replacements are arranged, comprising with the amino acid replacement that does not contain sulfydryl corresponding to the aminoacid that forms disulfide bond in the second antibody molecule variable region, also have one or more amino acid whose substituting (that is: with the amino acid replacement that does not contain sulfydryl be not two residues that form disulfide bond) in addition.
Especially, the invention provides first immunoglobulin molecules, this molecule comprises a variable region, and this variable region sequences is except that one or more amino acid replacements, and is consistent with second immunoglobulin sequences.But the conjugated antigen of this second immunoglobulin immunologic opsonin, wherein in first immunoglobulin corresponding in the one or more cysteine that form disulfide bond in the second immunoglobulin molecules variable region, have one or more amino acid replacements that do not contained sulfydryl at least.
In preferred embodiments, using the amino acid replacement that does not contain sulfydryl to form the variation of the cysteine of disulfide bond, is unsettled amino acid replacement.Stable variation is meant that those aminoacid variations cause the enhanced variation of antibody molecule stability.These stable aminoacid change: in the antibody specific molecule on the particular location, uncommon aminoacid is with (Katal etc. are in the sequence of immune destination protein on the assigned address, the 5th edition, U.S.Department of Health andHuman Services, Bethesda, in Marryland one book fixed antibody consensus sequence number) common amino acids substitutes, as using in the consensus sequence of antibody molecule, this locational amino acid replacement is (referring to PCT Publication WO96/02574,1996,2,1, Steipe etal).
The amino acid replacement of other type also is that to induce the ability of anti--anti--idiotype reaction be prerequisite not change modified immunoglobulin, such as, the example described in known 5.5.This alternative comprising, such as, with the close amino acid replacement of function; The one or more aminoacid of another amino acid replacement identical with polarity, that function is close.Amino acid whose alternative other member that can be selected from its affiliated amino acids in the sequence, such as: nonpolar (hydrophobic) aminoacid comprises: alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; Polar neutral amino acid comprises: glycine, serine, threonine, cysteine, tyrosine, agedoite, glutamine.Positively charged (alkalescence) aminoacid comprises: arginine, lysine, histidine; Electronegative (acidity) aminoacid comprises: aspartic acid, glutamic acid.
But modified antibodies of the present invention is derived from immunologic opsonin in conjunction with any antigenic antibody.In preferred embodiments, but modified antibodies is derived from immunologic opsonin knot cancer antigen, the especially antibody of tumor antigen.In specific embodiments, modified antibodies derives from and can comprise in conjunction with following antigenic antibody: polymorphic epidermis mucin antigen, human colon carcinoma associated protein antigen, the carbohydrate antigen that human colon carcinoma is relevant, human milk fat bead antigen, perhaps mammary gland, ovary, uterus, prostate, bladder, lung, skin, colon, pancreas, gastrointestinal tract, B or T lymphocyte or other tumor-marker antigen expressed are as those antigens of being discussed in 5.21.In preferred embodiments, modified antibodies is derived from Mab31.1 and (is derived from American type culture collection 10801, Uinversity Boulevard, Manassas, Virginia 20110-2201 preserving number No.12314), Mab33.28 or (preserving number No.12315 Mab HMFG-1 (seeing PCT Publication W090/0514 and PCT Publication W092/04380).
In another embodiment, but modified antibodies is derived from immunologic opsonin in conjunction with the infectious disease pathogen antigen or in conjunction with the antibody of this cell antigen receptor.In preferred embodiments, antigen is bacterial antigens, viral antigen, or parasite antigen, and perhaps other infectious disease pathogen antigen is as those infectious disease pathogen antigens of being discussed in 5.22.
Modified antibodies of the present invention can be the immunoglobulin molecules of any classification, type or hypotype.In the preferred embodiment, employing be antibody molecule, be preferably selected from: IgG, IgE, IgM, IgD and IgA are more preferably the IgG molecule.In the alternative embodiment, immunoglobulin molecules is T cell or B-cell receptor, the adhesion molecule of cell surface, and such as CD4, cofactors such as CD8 or CD19, or the constant domain in the MHC molecule.
Modified antibodies can be derived from natural antibody, preferred monoclonal antibody, synthetic antibody or genetic engineering antibody.On the one hand, modified immunoglobulin of the present invention is derived from certain antibody, wherein in conjunction with binding site to binding site or antigen part, insert or substitute all or part of variable region CDR, as common unsettled Application No. _ _ _ _ described in, exercise question is " immunoglobulin molecules that comprises a synthetic variable region and modified specificity ", provide application by Burch, the applying date is that December in 1998 (attorney docket No.6750-016) this paper on the 13rd quotes in full at this.
Especially, synthetic antibody can immunologic opsonin ground in conjunction with first component in the pairing coalition, wherein have a CDR district first component binding site in the CDR district of antibody at least, this binding site comes from and matches the aminoacid sequence of another component of coalition.Among the present invention, the binding site aminoacid sequence is not found under natural surroundings in the CDR district on the one hand.In addition, have at least a district to comprise antigen part in the CDR district, especially the epi-position part.
The aminoacid sequence of binding site, any known method is determined in available this area.Under some situation, clearly known in conjunction with the concrete sequence of a component of centering and participated in directly and the combining of other component.In this case, this sequence just can be used for making up the CDR district of synthetic antibody, and this antibody specific recognition is in conjunction with the second right component.If in conjunction with a certain component of centering and the bonded aminoacid sequence the unknown of second component, can determine by other any known method in this area, as determining with branch submodule construction method or experimental technique, as: by detecting and the bonded composition of another component (as: peptide), perhaps determine binding site by sudden change and the bonded mutant of searching blocking-up.
In conjunction with to being any interactional two molecules, comprise albumen, nucleic acid, carbohydrate or lipid, but the combination that preferably can therefrom find out binding site is to being interactional albumen.In preferred embodiments, the immunoglobulin of modification comprises tumor antigen, the communicate illness pathogen antigen, and the pathogen cells receptor perhaps participates in receptor-aglucon interaction receptor or aglucon binding sequence.
In specific embodiments, in conjunction with right to being interactional albumen, they can be that homotype interacts (that is: the interaction between the albumen of the same race), or special-shaped interact (that is: the interaction between the different albumen).
In specific embodiments,, after wherein preferred aglucon-receptors bind, can induce the physiological reaction, as signal conduction in the cell in conjunction with to first component being a kind of in aglucon-receptor.By the example explanation, aglucon or receptor can be hormones, autacoid, somatomedin, cytokine or nuclear mediator, or hormone, autacoid, somatomedin, the receptor of cytokine or neurotransmitter, (summary of signal transduction path is seen Campbell perhaps to participate in the aglucon of signal conduction or receptor, 1997, J. department of pediatrics magazine .131:S42-S44; Hamilton, 1997, J.Leukoc.Biol.62:145-155; Soede-Bobok﹠amp; Touw, 1997, J.Mol.Med.75:470-477; Heldin, 1995, cell 80:213-223; Kishimoto et al., 1994, cell 76:253-262; Miyajima etal., 1992, Annu.Rev.Immunol.10:295-331; Cantley et al., 1991, cell 64:281-302), but be not limited to several aspects for example.In specific embodiments, be aglucon in conjunction with the first right component, as cholecystokinin, galanin, IL-1, IL-2, IL-4, IL-5, IL-6, IL-11, chemotactic factor, leptin, protease, neuropeptide tyrosine, neurokinine-1, neurokinin-2, neurokinin-3, bombesin, gastrin, corticotropin releasing hormone, Endothelin, melatonin, somatostatin, vasoactive intestinal peptide, epidermal growth factor, tumor necrosis factor, dopamine, Endothelin, or the receptor of these aglucons, but be not limited to given example.In other specific embodiments, in conjunction with the first right component is receptor, as Opioid Receptors, glucose transporter, glutamate receptor, lonely plain receptor, erythropoietin receptor, Insulin receptor INSR, tyrosine kinase receptor, KIT stem cell factor receptor, trk C, IGF-1, granulocyte clone stimulating factor receptor, growth hormone receptor, the neurotrophic factor acceptor or the gp39 receptor in glial cell source, G protein receptor class or beta 2-adrenergic receptor or in conjunction with the aglucon of these receptors, but be not limited to given example.In other specific embodiments, be the ion channel of ligand-gated in conjunction with the first right component, as calcium channel, sodium-ion channel, or potassium-channel, but be not limited to given example.In the embodiment of determining, the invention provides modified immunoglobulin, the antagonist of the aglucon of immunologic opsonin bind receptor and receptors bind, such as, endorphins, enkephalin or the plain antagonist of injury, but be not limited to given example.In other embodiments, the invention provides synthetic modification antibody, but this antibody immunologic opsonin bind receptor is the agonist of this receptor, as endorphins, enkephalin or the plain receptor of injury, but be not limited to given example.In the preferred embodiment, modified immunoglobulin is the binding fiber protein receptor not; In another preferred embodiment, binding sequence is not Arg-Gly-Asp, polymer that neither binding sequence, polymer that preferably neither sequence A rg-Gly-Asp.
In other specific embodiments, modified immunoglobulin contains a CDR district, and this district contains the transcription factor binding site point, and wherein preferred one side is that modified immunoglobulin is not in conjunction with special DNA sequence, especially not in conjunction with the binding site of transcription factor.
In preferred embodiments, have the aminoacid sequence (as: hereinafter describing in detail among the 5.2.1) that contains tumor antigen or cancer antigen binding site in the CDR district in the modified immunoglobulin at least, preferred antigen is human colon carcinoma related antigen or epidermis mucin antigen.In other embodiments, at least one CDR district contains the aminoacid sequence of human milk fat bead receptor binding site in the modified immunoglobulin.In other embodiments, modified immunoglobulin has at least a CDR district to contain mammary gland, ovary, uterus, prostate, bladder, lung, skin, colon, pancreas, gastrointestinal tract, the aminoacid sequence of B or T lymphocyte antigen binding site.
In other preferred embodiment, the aminoacid sequence that at least one CDR district of modified immunoglobulin contains infectious disease cause of disease binding site (sees 5.22 for details, hereinafter), or the aminoacid sequence of the cell receptor of infectious disease cause of disease, the preferred combination site is not the aminoacid sequence of plasmodium antigens, or is not binding site Asn-Ala-Asn-Pro or Asn-Val-Asp-Pro.In the other embodiments, modified antibodies has a CDR district, contains the binding site of antibacterial or viral enzyme.
Synthetic (being that the binding site sequence is inserted the CDR district) of antibody is based on the sequence of natural or existing antibody molecule, or the consensus sequence of synthetic known antibodies molecule, the consensus sequence of the weight chain variable region as shown in Fig. 7 A and B, or other antibody consensus sequence or plant system's (promptly not the genome sequence of reorganization) sequence (as described antibody consensus sequence such as Kabat and kind is sequence, 1991, the sequence of immunity destination protein, the 5th edition NIH publication 91-3242,2147-2172 page or leaf).
Each antibody molecule has 6 CDR sequences, and light chain has 3, and heavy chain has 3, wherein 5 is that kind is the CDR district, (promptly derive from the genome of animal, recombinate), one is that non-kind is CDR (being the CDR district of genome sequence with being different from kind promptly, is that the sequence reorganization produces by kind).Whether be kind to be CDR, can by to the order-checking of CDR district and with known kind be CDR region sequence comparison (sequence of immune destination protein, the 5th edition NIH publication 91-3242,2147-2172 page or leaf).With known kind is tangible different showing of CDR region sequence, and this CDR district is that non-kind is.The CDR district of CDR that correspondingly, to contain CDR district binding site or the antigen aminoacid sequence be kind of system or non-kind system.
Binding site or antigen sequence can be inserted into any CDR district of antibody molecule, and the technology of this area can be inserted into binding site in the different CDR district of antibody fully at present.Detect modified antibodies then in conjunction with the ability of specifying component in conjunction with centering, described in hereinafter 5.5, perhaps modified antibodies is induced the immunoreation to antigen site, described in hereinafter 5.5.So just can determine to comprise the suitableeest CDR district of binding site or antigen site.In specific embodiments, the CDR of heavy chain or light chain admits to contain binding site or antigen sequence after can being modified.In other specific embodiments, first, second of the variable region of heavy chain or light chain or the 3rd CDR district admit the sequence of binding site or antigen site.In another embodiment of the invention, the sequence of binding site or antigen site is contained in a more than CDR district, the different binding site sequences with a part are all contained in a perhaps more than CDR district, and the different binding site sequences of different molecular are contained in a perhaps more than CDR district.Particularly, 2,3,4,5 or 6 CDR districts comprise the binding site sequence in conjunction with centering first component after transforming.In preferred embodiments, one or more CDR district contains the binding site in conjunction with centering first component, and one or more CDR district contains immunocyte such as surface molecular binding sites such as T cell, B cell, NK cell, K cell til cell or neutrophilic granulocyte, but is not limited to cited several cells.For another example, modified antibodies contains tumor antigen binding site or infectious disease pathogen antigen binding site and immunocyte surface molecular binding site, and this antibody makes the immunocyte orientation in the tumor cell that contains tumor antigen or act on the infectious disease cause of disease.
In specific embodiments of the present invention, binding site or antigenic aminoacid sequence can be under the prerequisites of the aminoacid sequence that needn't replace any CDR district itself, be inserted into the CDR district, or replace the aminoacid in all or part of CDR district itself with binding site or antigenic aminoacid.In specific embodiments, binding site aminoacid replaces in the CDR district 1,2,5,8,10,15 or 20 aminoacid.
In the CDR district, the binding site of performance or antigen aminoacid sequence be in conjunction with to one of component in conjunction with essential minmal sequence, or induce at the essential minmal sequence of antigenic immunoreation (this can by any known experimental technique in this area through verification experimental verification); Alternative embodiment is, than in conjunction with in conjunction with to bigger binding site or the antigen aminoacid sequence of the essential minmal sequence of one of component, or than inducing at the big sequence of the essential minmal sequence of antigenic immunoreation.In specific embodiments, binding site or antigen amino acid length are 4 aminoacid at least, perhaps are 6,8,10,15 or 20 aminoacid at least.In other embodiments, the binding site amino acid length is not less than 10,15,20 or 25 aminoacid, or 5-10,5-15,5-20,10-15, a 10-20 or 15-20 aminoacid.
In addition, CDR district total length (being remaining sequence sum of binding site sequence and CDR district) is fit to antibody and antigenic bonded appropriate length.Table 2 has been enumerated the total number of atnino acid scope that has in the known CDR district and the size of CDR district (indicating among Fig. 2).
Table 2
CDR residue number
L1????10-17
L2????7
L3????7-11
H1????5-7
H2????9-12
H3????2-25
(data is from Kabat and Wu, and 1971, the annual report 190:382-9 of NYAS)
Most CDR H3 section length are 5-9 aminoacid, and the CDR H3 section length that has is long, and particularly antiviral antibody heavy chain CDRH3 section length majority is a 17-24 aminoacid.
Correspondingly, in the specific embodiments of the present invention, the CDR section length scope that contains binding site or antigen part within the CDR district institute how that table 2 is listed, i.e. a CDR district of light chain, L1 is between 10-17 aminoacid; The 2nd CDR district of light chain, L2 is between 7 aminoacid; The 3rd CDR district of light chain, L3 is between 7-11 aminoacid; The one CDR district of heavy chain, H1 is between 5-7 aminoacid; The 2nd CDR district of heavy chain, H2 is between 9-12 aminoacid; The 3rd CDR district of heavy chain, H3 is between 2-25 aminoacid.In other specific embodiments, the CDR section length that contains binding site is: 5-10,5-15,5-20,11-15,11-20, a 11-25 or 16-20 aminoacid.In other embodiments, the CDR section length that contains binding site is at least 5,10,15 or 20 aminoacid or no more than 10,15,20,25 or 30 aminoacid.
After building the antibody that contains modification CDR district, can further revise, screening is to detect the antibody of high-affinity or immunologic opsonin.High-affinity or immunologic opsonin can obtain and screen in conjunction with the antibody of target antigen by any known method in this area.Such as, the nucleotide sequence of first synthetic modification antibody, random mutation is chemistry or rite-directed mutagenesis then, perhaps concrete site mutation in the nucleotide sequence of coding modified antibodies screens high-affinity in conjunction with concrete antigenic antibody then from sudden change antibody.Detection method can be to measure one expressed antibody molecule, or screening sudden change library, shows the library as phage.(the U.S. Patent number 5,223,409 that provides referring to Ladner; With 5,403,484 and 5,571,698; The PCT publication WO92/01047 that McCafferty provides, or any known Display Technique in other this area).
In specific embodiment, the invention provides functional activity fragment, derivant or the analog of modified immunoglobulin molecule.That functional activity fragment, derivant or analog can be induced is anti--anti--idiotype antibody (promptly three grades antibody or Ab3), and this antibody recognition functional activity fragment, derivant or the analog antigen (method described in 5.5) that antibody is discerned of originating.Particularly, in preferred embodiments, the idiotype antigen of immunoglobulin molecules can hold the sequence of discerning antigenic special section to immunologic opsonin to strengthen by N in deletion framework region and the CDR district.Be to determine and the bonded CDR of antigen district, synthesize the antibody that contains the CDR region sequence earlier, utilize in this area any known detection and antigenic associativity method just can determine then.Correspondingly, in preferred embodiments, the amino acid residue that the cysteine that forms a disulfide bond among the present invention in the immunoglobulin molecules in the variable region is not contained sulfydryl substitutes, and deleted to the sequence of discerning between the concrete antigenic CDR from N-terminal in the CDR district.
In other embodiment of the present invention, comprise the fragment of modified antibodies of the present invention, as obtaining to comprise the variable region with the pepsin digested antibody molecule, the F in the CH1 district of the constant region of light chain and heavy chain (ab ') 2And reduction F (ab ') 2Disulfide bond further obtain the Fab fragment, but be not limited to cited example.The weight chain homodimer of modified antibodies of the present invention also is provided in the present invention, or such as the minimal segment of Fvs and single-chain antibody (SCAs) (as U.S.PatentNo.4,946,778; Bird, 1988, science 242:423-42; Huston et al., 1988, institute of NAS reports USA85:5879-5883; Wards et al., natural 334:544-54) or other have other molecule of the immunologic opsonin of modified antibodies.
By shearing the suitable antigenic gene of specific recognition in the mouse antibodies, then with human antibody molecules in suitable bioactive fragment gene splicing, develop into and produce chimeric antibody (Morrison et al., 1984 institutes of NAS newspaper, 81:851-855; Neuberger et al., natural 312:604-608; Takeda et al., natural 314:452-454) technology.Chimeric antibody is a hybrid molecule, and its different fragments derives from different animal kind systems, be derived from the Mus monoclonal antibody as the variable region, and constant region is derived from people's immunoglobulin, as humanized antibody.
In preferred embodiments, modified immunoglobulin of the present invention is a humanized antibody, preferred antibody is that middle frame district, variable region also is people's antibody, the CDR district is not people's antibody, mouse antibodies (see international patent application no PCT/GB8500392, Neubergeret al and Celltech Limited provide) preferably.
Transplant in the CDR district is the method for another kind of humanized antibody.Relate to the refigure of murine antibody, purpose is that antigenic specificity and the binding affinity that it is whole goes to people's antibody framework district (Winter et al., U.S. Patent number 5,225,539.).Successfully made up grafted antibody at the antigenic CDR of difference district, as Queen1989, the anti-IL-2 receptor that provides (institute of NAS newspaper, 86:10029), anti-cell surface receptor-CAMPATH (1988 that Riechmann provides, nature, 332:323), and Cole anti-hepatitis virus antigen-antibody and viral antigen (institute of NAS newspaper, 88:2869), the antibody of Tempest trachea syncytial virus (1991, biotechnology 9:267).The preparation method of CDR district grafted antibody is to transplant the CDR district the pure man antibody of mouse monoclonal antibody.Because the framework region residue is that to keep CDR district conformation necessary, and the antigen binding site of having evidence suggests some framework region subparticipation, thus after the transplanting, most antibody capables from framework region aminoacid change to affinity keep benefit.Yet, be unlikely to introduce the neoantigen site in order to ensure framework region, need by computer simulation, and relatively make up sequence and stable kind is a sequence.
In other embodiments, the invention provides modified immunoglobulin is fusion rotein (or functional activity fragment), such as passing through covalence key mode (as peptide bond), it is merged to another non-modified immunoglobulin (or part wherein, at least 10,20 or 25 amino acid whose protein fragments preferably) N-terminal or C-terminal.Preferably covalently is connected modified immunoglobulin or its fragment of other proteic constant region N-terminal.In preferred embodiments, fusion modified immunoglobulin provided by the invention is covalently bound to IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, gamma interferon, MHC derived peptide, G-CSF, TNF, punching is plain, the modified immunoglobulin on NK cellular antigens or the cell internalizing receptor.
Modified immunoglobulin of the present invention comprises the analog and the derivant of modification, and promptly covalently bound molecule to any kind, random length, these molecules do not hinder modified immunoglobulin induced anti-idiotype reaction (as: hereinafter method described in 5.5 is identified).Such as; the derivant of modified immunoglobulin; analog needs further to modify, and comprises such as glycosylation, acyl groupization; Pegylation; phosphorylation, the derivatization of amidatioon or known protection or blocking groups, Proteolytic enzyme cutting; be connected etc. with cell aglucon or other are proteic, but be not limited to these modifications.By known technical method, can realize most chemical modification, include but not limited to special chemical cleavage, acyl groupization methylates, and the metabolism of tunicamycin is synthetic etc.In addition, analog or derivant can comprise one or more atypia aminoacid, as: the aminoacid that this part is listed.
Modified immunoglobulin, fragment, the generation method of analog and derivant describe in detail in 5.4 hereinafter.
5.2 treatment is used
The invention provides, the method that produces anti-idiotype antibody, anti-anti-idiotype antibody is induced in application of treatment agent in a certain experimenter (called after therapeutic agent here).These therapeutic agents comprise modified immunoglobulin of the present invention, functional activity fragment, analog and thus obtained derivant (described in front 5.1), with code book invention modified antibodies, the functional activity fragment, the gene of analog and thus obtained derivant (as, front 5.1 is described).
Generally speaking, preferably use the provenance consistent or plant active product with the experimenter.Therefore, preferred embodiment, the modified antibodies that the inventive method is used derives from people's antibody.In other embodiments, the modified antibodies of the inventive method use derives from chimeric antibody or humanized antibody.
Particularly, the vaccine combination that comprises modified antibodies of the present invention, use induced anti-idiotype antibody (being anti-idiotype antibody or Ab2) to the experimenter, the idiotype of this anti-idiotype antibody identification modified antibodies, this antibody induces the anti-anti-idiotype antibody (Ab3) of specific recognition Ab2 idiotype again then, and Ab3 just has identification consistent or similar with modified antibodies and combination activity like this.
The invention provides to the experimenter and use modified antibodies, the method for induced anti-idiotype reaction promptly produces Ab2 and Ab3 antibody.After the present invention also provides and used modified antibodies to the experimenter, induce to produce and separate Ab2, use Ab2 to produce Ab3 antibody for then second experimenter.
Accordingly, the invention provides the method that in the experimenter, induces anti-idiotype reaction, method is to use first immunoglobulin (or the functional activity fragment of capacity, analog or derivant) induce, wherein said first immunoglobulin contains a variable region, and this immunoglobulin is in the variable region the one or more amino acid replacement, remaining is consistent with second immunoglobulin molecules, said second immunoglobulin molecules can immunologic opsonin conjugated antigen, said one or more amino acid replacements are with the one or more cysteine on the relevant position that forms disulfide bond in amino acid replacement second immunoglobulin molecules that does not contain sulfydryl.In another embodiment, further provide the method for the anti-idiotype antibody that separates this second immunoglobulin molecules idiotype of identification, used for then second experimenter.
In the particular embodiment of discussing in detail in the present invention's part subsequently, modified immunoglobulin can be used to induce infection disease pathogen or abnormal cell, such as antibacterial, parasite, virus, the anti-idiotype reaction of tumor and cancer, certainly, application facet is not limited to that these are several.Use modified immunoglobulin of the present invention to induce, and then can treat or prevent other disease or imbalance concrete antigenic anti-anti-idiotype reaction.
In other embodiments, modified antibodies can be used for treating autoimmune disease, such as rheumatoid arthritis, and lupus erythematosus, ulcerative colitis, or psoriasis, perhaps allergic treatment, these are several but application facet is not limited to.Method of the present invention and vaccine combination can be used for inducing body to produce the body fluid of modified immunoglobulin and/or cell-mediated reaction.In one embodiment, method of the present invention and vaccine combination are used to induce the humoral immunity of organism reaction.In one embodiment, method of the present invention and vaccine combination are used to induce body to produce cell-mediated immunoreation.In preferred embodiments, method of the present invention and vaccine combination are used to induce body to produce the body fluid of modified immunoglobulin and/or cell-mediated reaction.
Can use experimenter of the present invention, can be mammal or vertebrates, includes, but are not limited to following several animal, cattle, horse, sheep, pig, poultry (as chicken), goat, cat, Canis familiaris L., hamster, mice, rat, monkey, rabbit, chimpanzee and people.In the preferred embodiment, the experimenter is the people.Method and composition of the present invention can be used in prevent disease or imbalance, or treats concrete disease or imbalance, and wherein the anti-idiotype reaction at concrete immunoglobulin molecules is effectively reaction in treatment or prevent disease or the imbalance.
5.2.1 the prevention of cancer and treatment
Cancer, include, but are not limited to the biological tumor of chela, it is the disease or the imbalance of feature that metastatic tumor or other are grown out of control with cell, can use modified immunoglobulin of the present invention (functional activity fragment, derivant or analog) prevent or treat, or use coding modified immunoglobulin molecule (functional activity fragment, derivant or analog) nucleotide sequence, wherein modified immunoglobulin derives from the relevant one or more antigen molecules of cancer cell that immunologic opsonin identification will prevent or treat.Whether concrete therapeutic agent can effectively prevent or treat the cancer of certain particular type, and any known method is determined in available this area, such as, the method that describes in detail in 5.5, but be not limited to these methods.
Modifying the antigenic antibody of anticancer disease by method of the present invention, can be used for preventing or the cancer associated antigens for the treatment of cancer is exemplified below, but be not limited to given example: KS1/4 is general-cancer antigen (Perez﹠amp; Walker, 1990, Journal of Immunology .142:32-37; Burn, 1989, hybridoma 7 (4): 407-415), ovarian cancer antigen (CA125) (Yu et al., 1991, cancer research.51 (2): 48-475), prostanoic acid phosphoric acid (Tailor et al., 1990, nucleic acids research communication 18 (1): 4928), prostate specific antigen (Henttu and Vihko, 1989, biochemistry biophysical research communication 10 (2): 903-910; Israeli et al., 1993, cancer research 53:227-230), melanic related antigen p97 (Estin et al., 1989, National Cancer Institute's magazine 81 (6): 445-44), melanoma-associated antigen p75 (Vijayasardahl et al., 1990, The Journal of Experimental Medicine 171 (4): 1375-1380), high molecular melanoma-associated antigen (HMW-MAA) (Natali et al., 1987, cancer 59:55-3; Mittelman et al., 1990, Journal of Clinical Investigation 86:2136-2144), the membrane antigen of prostate-specific, carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc.Am.Soc.Clin.Oncol.13:294), polymorphic endotheliocyte mucin antigen, HMFG's proteantigen, colon cancer related antigen such as CEA, TAG-72 (Yokataet al., 1992, cancer research 52:3402-3408), CO17-1A (Ragnhammar et al., 1993, international journal of cancer 53:751-758), GICA19-9 (Herlyn et al., 1982, clinical immune magazine 2:135), CTA-1, LEA, Bert lymphomas antigen-38.13, CD19 (Ghetie et al., 1994, blood 83:1329-1 336), people B lymphoma antigen-CD20 (Reff et al., 1994, blood 83:435-445), CD33 (Sgouros etal., 1993, J.Nucl.Med.34:422-430), melanoma specific antigen such as ganglioside GD2 (Saleh et al J.Immunol., 151,3390-3398)., Ganglioside, GD3 (Shitara et al., Cancer Immunol.Immunother.36:373-380), Ganglioside GM2 (Livingston et al., 1994, Journal of Clinical Oncology 12:1036-1044)., Ganglioside GM3 (Hoon et al., 1993, cancer research 53:5244-5250), tumour immunity specificity transplantation type cell surface antigen (TSTA) comprises as the tumor antigen of virus induction: DNA oncovirus T antigen, the membrane antigens of RNA oncovirus, bladder cancer carcinoembryonic antigen (Hellstrom et a1., 1985 cancer research 45:2210-2188), differentiation antigen such as lung tumor antigen L6, L20, (Hellstrom et al., 1985 cancer research 46:3917-3923), fibroma antigen, human leukemia T cellular antigens-Gp37 (Bhattacharya-Chatterjec et al., 1988, immunity magazine 141:1398-1403), neoglycoprotein, sphingolipid, breast cancer antigen is as EGFR (EGF-R ELISA), HER2 antigen (p185 HER2), polymorphic epidermis mucin (PEM) (Hilkenset al., 1992, biochemistry trend Sci.17:359),, human malignant lesion lymphocyte antigen-APO-1 (Bernhard et al., 1989, science 245:301-304), the I antigen of differentiation antigen as in embryo's erythrocyte and elementary entoderm, finding, the I that in adenocarcinoma of stomach, finds (Ma), M18 that in the mammary gland epidermis, finds and M39, the SSEA-1 that in myeloid cell, finds, the D that in colorectal cancer, finds 156-22, VEP8, VEP9, Myl, VIM-D5 and, TRA-1-85 (blood group H), C14, the F3 that in adenocarcinoma of lung, finds, Y hapten, the AH6 that finds in the gastric cancer, the Le that finds in the embryo type cancerous cell y, TL5 (blood group A), the EGF receptor of in the A431 cell, finding, the E1 series of in cancer of pancreas, finding (blood group B), the FC10.2 that finds in embryo type cancerous cell and the adenocarcinoma of stomach, the CO-514 that finds in the adenocarcinoma (blood group LEa), NS-10, the C0-43 that in adenocarcinoma of colon, finds (blood group LEb), G49, EGF receptor, 19.9 of colon cancer discovery, the gastric cancer mucin, the T that in myeloid cell, finds 5A 7, the R that finds in the melanoma 24, 4.2, G D3, D1.1, OFA-1, G M2, OFA-2, G D2,, the M1:22:25:8 that in the embryo type cancerous cell, finds, the SSEA-3 that finds in the 4-8 embryonic cell phase, SSEA-4.Antigen in another program is the TXi Baoshouti that comes from the cutaneous T cell lymphoma peptide.(consult Edelson, 1998, cancer magazine 4:62)
In other embodiments of the present invention, use modified immunoglobulin treatment experimenter disease, optionally with other tumor therapeuticing method as operation, radiotherapy, chemotherapy combined use.Especially, therapeutic agent of the present invention can be united use with other one or more chemotherapy agents, and chemotherapy agents includes, but are not limited to methotrexate, taxol, purinethol, thio-purine, hydroxyl urine, cytosine arabinoside, cyclophosphamide, ifosfamide, nitroso-group urine, cisplatin, carboplatin, dacarbazine, procarbazine hydrochloride, Rhizoma Dysosmae Versipellis 2 V shapes, camptothecine, bleomycin, amycin, idarubicin, daunorubicin, D actinomycin D, plicamycin, mitoxantrone, asparaginase, vincaleucoblastine, vincristine, Vinorelbine, taxol, paclitaxel Docetaxel, mitomycin.
5.2.1.1 malignant tumor
Available therapeutic agent prevention of the present invention and treatment malignant tumor and relevant disease.Table 3 has been listed tumor and the relevant disease that can treat and prevent, but is not limited to these diseases.(, consulting) Fishman etc. for relevant disease, 1985, medical science, second edition J.B.Lippincotl Co., Philadelphia):
Table 3 tumor and relevant disease
Leukemia
Acute leukemia
Acute lymphoblastic leukemia
Acute myelocytic leukemia
Myeloblast
Promyelocyte
Myelomonocyte
Mononuclear cell
Erythroleukemia
Chronic leukemia
Chronic myelocytic (granule) leukemia
Chronic lymphocytic leukemia
Polycythemia vera
Lymphoma
Hokdkin disease
The Fei Hejiejinshi disease
Multiple myeloma
Macroglobulinemia Waldenstron
Heavy chain disease
Solid tumor
Sarcoma and cancer
Fibrosarcoma
Myxosarcoma
Liposarcoma
Chondrosarcoma
Osteogenic sarcoma
Chordoma
Angiosarcoma
Endotheliosarcoma
Lymphangiosarcoma
Lymphangioendothelial sarcoma
Synovioma
Mesothelioma
The You Wenshi tumor
Leiomyosarcoma
Rhabdomyosarcoma
Colon cancer
Cancer of pancreas
Breast carcinoma
Ovarian cancer
Carcinoma of prostate
Squamous cell carcinoma
Basal cell carcinoma
Adenocarcinoma
Syringocarcinoma
Sebaceous gland carcinoma
Papillary carcinoma
Papillary adenocarcinoma
Cystadenocarcinoma
Soft cancer
Bronchogenic carcinoma
Renal cell carcinoma
Hepatocarcinoma
Cancer of biliary duct
Choriocarcinoma
The spermatogonium cancer
Embryonal carcinoma
Wilms' tumor
Cervical cancer
Uterus carcinoma
Carcinoma of testis
Pulmonary carcinoma
Small cell lung cancer
Bladder cancer
Last Intradermal tumor
Glioma
Astrocytoma
Neural vasoblasts tumor
Craniopharyngioma
Ependymoma
Pinealoma
Hemangioblastoma
The auditory nerve tumor
Oligodendroglioma
Meningioma
Melanoma
Neuroblastoma
Retinoblastoma
In specific embodiments, ovary, bladder, mammary gland, colon, lung, skin, pancreas, the malignant tumor in the organ or tissue of uterus or breed not benign lesion (metaplasia and dysplasia), perhaps hypertrophy all can be treated or prevent.In other specific embodiments, sarcoma, melanoma, or leukemia can be prevented or treat.
5.2.1.2 precancer
Therapeutic agent of the present invention can be used for precancer or blocks its deterioration or enter tumor or the malignant tumor state, includes but not limited to the described disease of table 3.When known or suspection canceration, can carry out preventative or curative medication, especially nontumorous cell such as hypertrophy, (summarize about improper growth, when conversion or most dysplasia referring to Robbins and Angell, 197, the 2nd edition Ed. of basic pathology, W.B.SaundersCo., Philadelphia, propagation pp.8-79).Hypertrophy is a kind of control cell down that is in, organize and organ in quantitative increase, and do not have the change of tangible structure or function.The example of one of them exception is that endometrial hyperplasia often develops into adenomyoma.Conversion is to be in control cell proliferation down, wherein a kind of maturation or the cell replacement that breaks up fully another kind of sophisticated cell.Transform and often occur in endothelium or conjunctive tissue.The pathologic epithelium that atypical conversion relates in a way transforms.Dysplasia is the prelude of canceration normally, how to find in epithelial tissue; Most non-tumor cell pathological changes hypertrophy relates to the forfeiture of individual cells concordance and cellularity polarization.The common cell of hypogenetic cell increases, and nuclear staining is darker, the cellular morphology variation.The distinctive position that occurs in chronic Pruritus and inflammation of dysplasia is common in cervix uteri, trachea, oral cavity and gallbladder.
In addition, in vivo, outward, occur in the patient blood specimen with hypertrophy, conversion or dysplasia are the improper hyperplasia of feature, appearance is converted into the abnormal cell proliferation or the malignization phenotype of feature with cell table shape, shows that needs use vaccine combination to prevent or treat.Above described cell phenotype conversion characteristic, comprise morphological change, substrate is in conjunction with loose, the contact inhibition forfeiture, grappling dependency disappearance, protease discharges, and the sugar transportation increases, and the serum demand reduces, carcinoembryonic antigen is expressed, and 250,000 daltonian cell surface proteins are lost etc.(referring to the same feature relevant) with conversion or tumor phenotypes.
In specific embodiments, leukoplakia, the hypertrophy of optimum phenotype or epidermodysplasia, or Bowen disease, primary tumor all shows it is the tumor omen in early stage that need carry out preventive usage.
In other embodiments, fibrocyte disease (cystic hyperplasia, mammary gland dysplasia), especially, adenopathy (optimum epidermal hyperplasia) all needs to carry out preventive usage.
In other embodiments, one of the characterization factor of following indication malignancy of tumorization is arranged, can treat with the therapeutic agent of effective dose of the present invention.Comprise: the chromosome translocation relevant with malignancy of tumorization is (as the Philadelphia chromosome of chronic leukemia, lymph node lymphoma t (14:18)), familial polyposis tumor or Jia Dena syndrome (the possible omen of colon cancer), optimum monoclonal gamma Globulin disease (the possible omen of multiple myeloma), those can the heredity tumor of (meeting mendelian inheritance) or lineal relative's (as: familial polyp of colon tumor of the early stage disease patient of tumor, Gardner's syndrome, the heritability exostosis, many secreting glands adenhomatosis is followed the bone medullary carcinoma of starch products, pheochromocytoma, Pu Shi-Jie Ge syndrome, the neurofibromatosis of Feng's recklinghausen's disease, retinoblastoma, carotid body tumor, the dermal melanin cancer, the ophthalmic malignant melanoma, xeroderma pigmentosum, ataxia telangiectasia, cut enlightening Acker-Dong syndrome, albinism, renewable aplastic anemia of model Buddhist nun and Bo Lumu syndrome; Referring to Robbins and Angell, 197, basic pathology, the 2nd edition, W.B.Saundrers Co., Philadelphia, pp.112-113)
In other specific embodiment, therapeutic agent of the present invention uses to patient, can prevent ovary, mammary gland, colon, lung, pancreas, skin, prostate, gastrointestinal tract, bone-marrow-derived lymphocyte, T lymphocyte or uterus carcinoma melanoma or sarcoma.
5.2.2 infectious disease treatment
The present invention also provides and uses therapeutic agent prevention of the present invention or treatment infectious disease, come from immunologic opsonin in conjunction with the pathogen antigen that causes infectious disease or in conjunction with modified immunoglobulin (or the functional activity fragment of the cell receptor of these cause of diseases but especially use, derivant or analog, or coding immunoglobulin or functional activity fragment, the nucleic acid of derivant or analog).With the infectious disease cause of disease that goes through, comprise virus as below, antibacterial, fungus, plasmodium and parasite, but be not limited to that these are several.
In specific embodiments, use immunoglobulin of the present invention (or functional activity fragment, derivant or analog, or these proteic nucleic acid of encoding), but this immunoglobulin comes from the following infectious disease pathogen antigen of specific recognition, comprising: influenza virus hemagglutinin (GenBank registration number J02132; Air, 1981, institute of NAS reports 78:739-743; Newton etal., 1983, virusology 128:495-501), people's trachea syncytial virus G glycoprotein (GenBank accession number Z33429; Garcia et al., 1994, J Virol.; Collinset al., 1984, institute of NAS reports 81:783; ), dengue virus core protein, stromatin and other albumen (GenBank accession number M19197; Hahn et al., 1988 virusology 12:17-180), Measles virus hemagglutinin (GenBank accession number M81899; Rotaet al., 1992 virusology 188:135-142), II herpes simplex virus type glycoprotein GB (GenBank accession number M14923; Bzik et al., 198 virusology 155:322-333), I type poliovirus VP1 (Emini et al., 1983, nature 304:99), I type human immune deficiency type viral envelope glycoprotein is as GP120 (Putney et al., 198, science 234:1392-1395), hepatitis B surface antigen (Itoh et al., 198, natural 308:19; Neurath et al., 198, vaccine 4:34), diphtheria toxin, diphtherotoxin (Audibert etal., 1981, nature 289:543), chain diplococcus 24M epi-position (Beachey, 1985, experiments experiment Medical Biology progress Biol.185:147), gonococcus pilin (Rothbard andSchoolnik, 1985, the biological progress of experimental medicine Biol.185:247), pseudorabies virus g50 (gpD), pseudorabies virus glycoprotein I I (gpB), pseudorabies virus glycoprotein gi II (gpC), the pseudorabies virus glycoprotein h, the pseudorabies virus glycoprotein E, transmissible gastroenteritis glycoprotein 195, the transmissible gastroenteritis stromatin, porcine rotavirus glycoprotein 38, pig piconavirus capsid protein, the little Serpentis bacterium of pig dysentery malaria protective antigen, bovine viral diarrhoea glycoprotein 55, ewcastle disease virus hemagglutinin-neuraminidase, the swine flue hemagglutinin, the swine flue neuraminidase, foot and mouth disease virus, hog cholera virus, swine influenza virus, afirican swine fever virus, the hyopneumoniae chlamydia, infectious Nasus Bovis seu Bubali trachea virus (as infectious Nasus Bovis seu Bubali trachea virus glycoprotein E or G), infectious pharynx tracheitis virus (as infectivity pharynx tracheitis viral glycoprotein G or I), LA CROSSE viral glycoprotein (Gonzales-Scarano et al., 1982, virusology 120:42), new calves diarrhea virus (Matsuno and Inouye, 1983, infect and immune 39:155), peste loca virus (Mathews and Roehrig, 1982, Journal of Immunology 129:273), punta toro virus (Darlrymple et al., 1981, exempt from duplicating of chain patient, Bishop and Compans (eds.)), murine leukemia virus (Steeves, et al., 1974, Journal of Virology 14:187), mouse mammary tumour virus (Massey and Schochetman, 1981 virusology 115:20), hepatitis B virus core albumen and/or surface antigen (GB Patent Application No. No.GB 2034323A, be published in 1980,6,4; Ganem and Varmus, 1987, bioid academic year is commented .5:51-93; Tiollais et al., 1985, nature 317:489-495), the antigen of equine influenza virus or equine herpes virus (equine influenza virus A type/Alaska 91 neuraminidases, equine influenza virus A type/Miami 63 neuraminic acids, equine influenza virus A type/Kentucky 81 neuraminic acids, I type equine herpes virus Glycoprotein B, I type equine herpes virus glycoprotein D), self-conceit pipe syncytial virus or bovine parainfluenza virus antigen (self-conceit pipe syncytial virus conjugated protein (BRSV G), the self-conceit pipe syncytial virus III of self-conceit pipe syncytial virus nucleocapsid protein (BRSV N) type fusion rotein, bovine parainfluenza virus III type hemagglutinin-neuraminidase), bovine viral diarrhea virus glycoprotein 48 or 53.In other specific embodiments, use modified immunoglobulin of the present invention (or functional activity fragment, derivant or analog, or these proteic nucleic acid of encoding) can treat or the infection prevention disease, the cell receptor of this immunoglobulin identification infection disease pathogen, such as, but be not limited to cited cause of disease of table 4 and corresponding receptor.
Table 4
Cause of disease Cell receptor
Have a liking for the bone-marrow-derived lymphocyte papovavirus B cell LPV receptor
Bordetella pertussis Adenyl cyclase
Borna virus (BDV) The BDV surface glycoprotein
Bovine coronavirus N-acyl group-9-acyl group ceramide acid acceptor
Choriomeningitis virus CD4 +
Dengue virus Height sulphation type heparin sulfate
Escherichia coli Contain lactose-α-1-4 lactose isoreceptor
Ebola virus CD16b
Chinese mugwort can virus-1 Integrate plain VAL-2 receptor
Chinese mugwort can virus-11 (EV) The EV receptor
Endotoxin (LPS) CD14
Intestinal bacterium The glycocide receptor
Intestinal bacterium T cell α/beta receptor
The intestinal Orphan virus The decay accelerating factor receptor
Feline leukaemia virus Outer after birth glycoprotein (Env-SU) receptor of born of the same parents
Foot and mouth disease virus The immunoglobulin Fc receptor
Troglodyte leukemia () The GALV receptor
Gram negative bacteria The CD14 receptor
Helicobacter pylori Lewis (b) blood group antigen receptor
Hepatitis B virus (HBV) TXi Baoshouti
Herpes simplex virus Heparin sulfate glucosaminoglycan receptor bfgf receptor
HIV-1 CC-chemokine receptor CCR 5 CD11 CD2 G protein coupled receptor CD4
Giant cell virus Heparin sulfate Dan Baijutang calphobindin II CD13 (Aminopeptidase N)
The human corona virus Human Aminopeptidase N receptor
Influenza virus A, B, C The hemagglutinin receptor
Legionnella The agglutinin receptor chemokine receptors of CR3 receptor protein kinase receptor galactose N acyl group galactose ammonia inhibition
The Mexicana leishmania Calphobindin I
Monocytosis Li Site bacterium ActA albumen
Measles virus The CD46 receptor
Meningococcus The Opa receptor that meningococcus is relevant
Measles virus The CD46 receptor
Murine hepatitis virus The carcinoembryonic antigen family receptors carcinoembryonic antigen Bgla of family receptor
Murine leukemia virus The after birth glycoprotein
The Mus gamma herpes viruses Interferon-gamma receptor
The Mus retrovirus Glycoprotein gp70 Rmc-1 receptor
Mus coronavirus murine hepatitis virus The carcinoembryonic antigen family receptors
Bird type Mycobacterium tuberculosis M Human integrin receptor α v β 3
Gonorrhea diplococcus Heparin sulfate proteoglycans acceptor CD66 acceptor integrin receptor membrane cofactor protein CD46 GM1 GM2
GM3 CD3 ceramide
Ewcastle disease virus Hemagglutinin-neuraminic acid enzyme fusion proteins
Parvovirus Erythrocyte P antigen receptor
Plasmodium falciparum CD36 receptor alpha-Glycophorins receptor
Poxvirus Interferon-gamma receptor
Pseudomonas Kdel receptor
Rotavirus Mucosa α 4 β 7 receptors of going back to the nest
EGF-R ELISA
Shigella α 5 β 1 integrate plain
Streptococcus Non-glycosylated J774 receptor
1 type t helper cell Chemokine receptors comprises: 6.CXCR1-4 7.CCR1-5 8.CXCR3 9.CCR5
Have a liking for T cell virus 1 The GP46 surface glycoprotein
Vaccinia virus TNFRp55 receptor TNFRp75 receptor soluble interleukin-6-1 beta receptor
The viral disease of using method of the present invention to treat or to prevent comprises: hepatitis A virus (HAV), hepatitis B virus, hepatitis C virus, influenza virus, chickenpox virus, adenovirus, I herpes simplex virus type (HSV-I), II herpes simplex virus type (HSV-II), vaccinia virus, rhinovirus, people's intestinal cell pathological changes arc virus, rotavirus, the trachea syncytial virus, human papillomavirus, papovavirus, giant cell virus,, thorn-like virus, arbovirus, Chinese tower virus, Coxsackie virus, popular mumps virus, Measles virus, rubella virus, poliovirus, I type human immune deficiency type virus (HIV-I), II type human immune deficiency type virus (HIV-II), any Pironavirus, enterovirus, calicivirus, promise whirlpool virus, togavirus (as dengue virus), A Er falls ill malicious, yellow fever virus, coronavirus, rabies virus, Marburg virus, Ebola virus, parainfluenza virus, influenza virus, this refined virus, arenavirus, arc reovirus virus, rotavirus, Orbivirus, human I type T chronic myeloid leukemia virus, human II type T chronic myeloid leukemia virus, troglodyte immunodeficiency virus, slow virus, polyoma virus, parvovirus, Epstein-Barr virus, the nerpes vinrus hominis-, I type cercopithecid herpesvirus (B virus), poxvirus, encephalitis but be not limited to these diseases.
The bacterial disease that uses method of the present invention to treat or to prevent, comprise: Gram-negative and positive bacteria, the mycobacterium rickettsia, mycoplasma, naphthalene plucked instrument Salmonella (as naphthalene Se Shi gonococcus, naphthalene Se Shi diplococcus), Legionnella, vibrio cholera, Streptococcus such as streptococcus pneumoniae, diphtheria corynebacterium, Clostridium tetani, bordetella pertussis, haemophilus (as influenza), chlamydia, dust Xi Shi escherichia coli, shigella, or syphilis or Lyme disease pathogen, but be not limited to these diseases.
The parasitic disease that uses method of the present invention to treat or to prevent comprises: Plasmodium, and eimeria, Leishmania, the inferior Eimeria in cocoa ground (Kokzidioa), trypanosoma and fungus such as candidiasis, but be not limited to these diseases.
In specific embodiments of the present invention, the therapeutic agent of use can with suitable antibiotic, antifungal, antifungal or the treatment or the useful other medicines of infection prevention disease unite use.
5.3 gene therapy
The gene therapy of prevention or treatment disease is fall ill and expresses relevant antigenic experimenter and use coding to modify the nucleic acid of property immunoglobulin molecules by giving, and said modified immunoglobulin derives from discerns said antigenic antibody.Such as, disease or imbalance may be to express special tumor or the antigenic tumor of cancer, or express the infectious disease of special infectious disease pathogen antigen, or in conjunction with this antigenic cell surface receptor.In embodiments of the invention, (nothing is led peptide) or born of the same parents' outer (contain and lead peptide) modified immunoglobulin molecule in the nucleic acid coding born of the same parents of therapeutic agent.
About the comprehensive review of gene therapy method, referring to Goldspiel, 1993, clinical pharmacy 12:488-505; Wu Wu, 1991, Biotherapeutics 3:87-95; Tolstoshev, 1993, pharmacology's toxicity academic year is commented 32:573-596; Mulligan, 1993, science 260:926-932; Morgan Anderson, 1993, bioid academic year is commented 62:191-127).Used well-known recombinant DNA technology in this area, referring to Ausbel, (eds) 1993 molecular biology modern technologies, John Wiley﹠amp; Sons, NY; Kriegler, 1990, group transfer and expression, laboratory manual, Stockton Press, NY; Dracopoli etal., (eds), 1994, human genetics modern technologies John Wiley﹠amp; Sons, NY the 12nd, 13 chapters).
On the one hand, as the nucleic acid of therapeutic agent, comprise the expression vector of expressing the modified immunoglobulin molecule.
Nucleic acid is got involved in patient's method, can be that directly in this case, the patient directly is exposed to nucleic acid or carries in the carrier of nucleic acid; Or the transmission complex, or claim round-about way, in this case, at first to give the patient with this cell transplantation then at external use nucleic acid transformant.These two kinds of Therapeutic Method are called vivo gene treatment and body gene therapy.
In the specific embodiments, nucleic acid is directly to use and produce in vivo antibody in vivo.Method used in the art is varied, as make up suitable nucleic acid expression vector, then in the transfered cell, as the retrovirus by deficiency or attenuation or other virus (referring to U.S. Patent number 4,980,286), or the exposed DNA of direct injection, perhaps use microsome (as particle gun; Biolistic, Dupont) or use liposome or cell surface receptor or transfection reagent parcel, biopolymerization body capsule (poly-β-1-4-N-acyl group glycosaminoglycans; Referring to U.S. Patent number 5,635,493) lipid somatocyst, microsome, microcapsule perhaps is connected on the peptide section that can enter in the nuclear, perhaps be connected on the aglucon that can enter in the nuclear, the aglucon that perhaps is connected to receptor-mediated endocytosis first-class (as Wu andWu, 1987, J.Biol.Chem.262:4429-4432).In another embodiment, make up a nucleic acid-aglucon complex, wherein aglucon contains the viral peptide gene of one section fusion, this gene expression product cleavable endoplasmic reticulum and avoid lysosomal degraded.In other embodiments, nucleic acid can be directed to the absorption of immunologic opsonin cell by special receptor and express (PCT publication WO92/06180, submission date 16,1992, April 16 (wu et al.); WO92/22635, dated, 1992 submission dates December 23 days (Wilson, et al.); WO92/20316, November 26 1992 submission date (Findeis et al.); WO93/14188, June 22 1993 submission date (Young).In the alternative embodiment, nucleotide sequence can be introduced in the cell, be integrated in the host cell DNA by homologous recombination and express (Koller and Smithies, 1989, institute of NAS reports 86:8932-8935; Zijlstra et al., 1989, natural 342:435-438).
Perhaps, also can use single-chain antibody, as neutralizing antibody in conjunction with epi-position in the born of the same parents.Using method is, such as, utilize the method (Marasco et al., 1993, institute of NAS reports 90:7889-7893) of Marasco etc. to make up the nucleic acid of coding single-chain antibody molecule, in target cell, express then.Adenovirus vector is that Another application is in Vectors in Gene Therapy.Adenovirus is a kind of carrier that haves a great attraction, and virus can be imported tracheal epithelium, and causes slight disease there.Other target of adenovirus vector is a hepatocyte, central nervous system, endotheliocyte and muscle cell.The advantage of adenovirus vector is to infect non-splitted cell.Kozarsky and Wilson etc. have delivered the one piece of modern heredity of summary and growth comment 3:499-503 that carries out gene therapy about adenovirus vector.Bout etc. have proved with adenovirus vector can change gene over to Rhesus Macacus tracheal epithelial cell 1994, human gene therapy 5:3-10.Other example that uses adenovirus vector to carry out gene therapy has: Rosenfeld et al., 1991, science 252:431-434; Rosenfeld et al., 1992, cell 68:143-155; Mastrangeli et al., 1993, Journal of Clinical Investigation 91:225-234. mention the example that uses adenovirus vector to carry out gene therapy (Walsh et al., 1993, Proc.Soc.Exp.Biol.Med.204:289-300)
Use the expection mode and the dosage of therapeutic agent nucleic acid, mainly depend on the preciseness of disease type and effect thereof, patient's state etc. can be by determining with those skilled in the art.
5.3 bacterin preparation and using
The invention provides the vaccine formulation that comprises therapeutic agent of the present invention, wherein bacterin preparation is applicable to and can induces at concrete antigenic protective immunological reaction (body fluid and/or cell effect), as treatment of diseases and prevention.
Make the preparation that vaccine is suitable for, comprise injectable liquid reagent or suspension; Solubilized becomes the solid preparation of solution or suspension before using.Preparation can also be the polypeptide emulsifying form or that pack liposome methods into.The component of active immne originality can mix use with pharmaceutically acceptable and compatible with active constituent forming agent usually.The forming agent that is suitable for is such as water, normal saline, buffer, glucose, glycerol, ethanol, the liquid buffer of sterile isotonic or kind solution and combined soln.In addition, be ready that bacterin preparation also comprises a spot of additament, as wetting agent or emulsifying agent, pH buffer reagent and/or can strengthen the adjuvant of vaccine effect.
Effectively adjuvant comprises: aluminium hydroxide, N-acetyl-muramyl-L threonyl-D-isoglutamine (thr-MDP), N-acetyl-muramyl-L alanyl-D-isoglutamine, the different glutamy of N-acetyl muramyl-L-alanyl-D--L-alanyl-2 (1 '-2 ' two palmityls-sn-glyceryl-3-hydroxyl phosphorus acyloxy)-ethamine, but be not limited to following example.
The effect measuring method of adjuvant is the reaction of the inductive anti-idiotype antibody of modified immunoglobulin behind the concrete adjuvant of mensuration use.
Compositions can be liquid solution, suspension, emulsifying agent, tablet, pill, capsule, spacetabs type or powder.Oral formulations can comprise the mannitol such as the pharmacy level, lactose, magnesium stearate, sucrose, cellulose, carriers such as magnesium carbonate.
In a word, the component of unit dose can be independent, also can be blended, as is contained in the lyophilization powder in the sealing peace bottle, or anhydrous spissated powder, and the lined out activity component content.When using the compositions of injection type, provide the solution that is contained in the dilution peace bottle, with dissolving before standby.
In specific embodiment, adorn refrigerated modified immunoglobulin preparation in first container; Second container dress diluent, it consists of 50% glycerol, 0.25% phenol and antibacterial (as 0.005% viride nitens).
The present invention also provides medicine box or test kit, and this medicine box comprises one or more containers, and one or more bacterin preparation components of the present invention are housed.Relevant with medicine box is production, the use of government organs' regulation or the clause of selling medicine or biological product, and this clause has reacted Approved by the production of body and function medicine, use or marketing organization.
Be ready that compositions can be to pack separately or the distributor packing, and one or many using dosage active ingredient is housed.Packing may be the packing that comprises metal or plastic sheeting, such as blister pack.Each packing or distributor packing should have a operation instruction.Preparation of the present invention is mixed with mixture with the carrier of medically accepting, is placed in the suitable containers, and the service condition of labeled for treatment.
Use the experimenter of vaccine of the present invention, preferred mammal, people preferably, the also cattle except that the people, horse, sheep, pig, poultry (as chicken), goat, cat, Canis familiaris L., hamster, Mus and rat, but be not limited to these animals.
Bacterin preparation using method of the present invention has multiple, comprise in oral, the brain, skin corium, muscle, intraperitoneal and intravenous administration is subcutaneous, intranasal, cut (scratching skin surface as the syringe needle with bifurcated) or other standard immunoassay approach, but be not limited to these methods.In specific embodiments, make the cut inoculation.
Use in the preparation, the exact dose of modified immunoglobulin depends on the approach of inoculation and each patient's situation, should reach the clinical criteria technology according to the doctor's diagnosis the diagnosis of patient is come medication.Effectively immunizing dose is enough to induce the immunoreation (be anti-idiotype reaction) of host to the modified immunoglobulin bacterin preparation of use.Effectively immunizing dose also can be inferred from the dose dependent curve of animal pattern pilot system.
5.4 produce the method for modified immunoglobulin:
The production method of the modified immunoglobulin among the present invention comprises any known immunoglobulin synthetic method in this area, especially with chemical synthesis or expression of recombinant proteins method, and preferred expression of recombinant proteins method.
Recombinant expressed modified immunoglobulin of the present invention or fragment derivant or analog, needs make up the nucleic acid of coding modified immunoglobulin.If the nucleotide sequence of coding modified immunoglobulin is known, can be by synthetic this sequence of the method for chemosynthesis (as Kutmeier etal., 1994, biotechnology 17:242 is described), in brief, wherein relate to the synthetic of coding modified immunoglobulin oligonucleotide, and there is partial sequence overlapping between the oligonucleotide, therefore annealed, connection of these oligonucleotide and pcr amplification, the gene of the immunoglobulin that can obtain to encode, as: the example of the 6th part hereinafter.
The another kind of method that obtains the nucleotide sequence of coding immunoglobulin is the nucleotide sequence that obtains the immunoglobulin molecules in modified immunoglobulin source earlier.The nucleic acid gene sequence of immunoglobulin if do not encode, but gene order is known, 3 ' and 5 ' primer by synthetic gene is used PCR method so, and (as the cDNA library of the cell or tissue of antibody cDNA library or expression immunoglobulin) obtains this gene from suitable source.
If the immunoglobulin of specific recognition specific antigen can't obtain the cDNA library of this immunoglobulin (maybe can't obtain encode), so, at first obtain the immunoglobulin of this specific antigen by any means known in this area, animal such as immune rabbit one class obtains polyclonal antibody, even more preferably obtain monoclonal antibody such as Kohler and Milstein (1975, nature 256:495-497) or Kozbon et al (1983, today immunology 4:72) or Cole etal (1985 monoclonal antibodies and treatment of cancer, Alan R.Liss, Inc, pp77-96) described.Another kind method is screening Fab expression library (Huse et al., 1989, science 246:1275-1281) or antibody library (Clackson et al., 1991, natural 352:624; Hane et al., 1997 institutes of NAS report 94:4937), obtaining is the segmental portion gene of Fab of coding immunoglobulin at least.
After obtaining to be the gene of coding immune globulin variable region at least, be inserted into cloning vehicle and contain (as: PCT PublicationWO86/05807 in the carrier of coding constant region for immunoglobulin gene; PCT Publication WO89/01036; US PatentNo5,122,1464; Bebbington, 1991, Enzymology method 2:136-145; PCTPublication WO86/05807).Also can obtain to comprise the expression vector of complete light chain gene or heavy chain gene, form the complete antibody molecule behind the coexpression.Then, substitute or deleting technique is modified the nucleotide sequence of coding immunoglobulin with nucleic acid, essential substitute (or deletion) is the cysteine that forms with disulfide bond in the amino acid replacement participation variable region medium chain that does not contain sulfydryl, other aminoacid can also be arranged or substitute deletion or insertion.These modifications, can introduce sudden change or nucleotide sequence deletion by any known method in this area, such as chemomorphosis, external rite-directed mutagenesis (Hutchinson et al., 1978, J.Biol.Chem..153:6551), realization such as PCR method, but be not limited to these methods.
In addition, the splicing mouse antibodies the immunologic opsonin fragment with have suitable bioactive human immunoglobulin gene technology, also can be used for obtaining chimeric antibody (Morrison et al., 1984, institute of NAS reports 81:851-855; Neuberg er et al., 1984, natural 312:604-608; Takeda et al., 1985, natural 314:452-454).As the above, chimeric antibody is a kind of molecule that comprises the different fragments of different plant species, as comes from the variable region of Mus monoclonal antibody and come from human normal immunoglobulin's constant region, as: humanized antibody.
Another kind of technology is preparation method (United States Patent (USP) 4,694,778 of single-chain antibody; Bird, 1988, science 242:423-42; Huston et al., 1988, institute of Proc. NAS reports 85:5879-5883; Ward et al., 1989, natural 334:544-54).The preparation method of single-chain antibody is, connects light, the heavy chain of Fv section and the single chain polypeptide that constitutes through the aminoacid key.Also having used escherichia coli expression has the Fv fragment (Skerra et al., 1988, science 242:1038-1041) of function.
The antibody fragment of identification specific antigen epi-position can obtain by known technological means.As the F that obtains antibody molecule with pepsin digestion (ab ') 2Fragment, reduction F (ab) 2Segmental disulfide bond obtains the Fab fragment, certainly more than these methods.
After obtaining the nucleotide sequence of coding modified immunoglobulin molecule, use the carrier that this area recombinant DNA technology can obtain to express immunoglobulin.Recombinant expressed then with separate modification after immunoglobulin, method is any method as known in the art, such as method described in the 6th part (also can be referring to Bebbington, 1991, Enzymology method 2:136-145).In brief, be exactly to encode in the expression vector instantaneity or non-instantaneity rotaring redyeing COS cell or other suitable cultured cell of modified immunoglobulin, cultivate reasonable time then and express immunoglobulin, culture supernatant is collected in being expressed in the culture supernatant of protein excretion then.
Technology well-known to those having ordinary skill in the art all can be used for making up and comprises immunoglobulin molecules gene order and the suitable expression vector of transcribing and translate control signal.These methods comprise: external DNA recombinant technique, synthetic technology and vivo gene recombinant technique, referring to, the technology of standards such as Sambrook (1990, molecular cloning, laboratory manual, the 2nd edition cold spring harbor laboratory, New York) and Ausubel et al., eds., 1988, molecular biology modern technologies, John Wiley﹠amp; Sons, New York).
Producing immunoglobulin of the present invention expression vector transfection host cell technology of using and the cell technology of cultivating the transfection success is routine techniques.
Expressing the host cell of recombinant antibodies of the present invention, can be the recombination immunoglobulin molecule of colibacillary bacterial cell preferred eukaryotic cell, especially The expressed.Particularly, mammalian cell resemble CHO and the carrier logotype that contains the utmost point early promoter element of people's giant cell virus are to express the immunoglobulin efficient system.Foecking et al., 198, gene 45:101; Cockett et al., 1990, biotechnology 8:2
Host-the expression vector system of expressing modified immunoglobulin of the present invention has multiple.These host-expression vector systems are embodied in: carrier can be expressed destination protein, subsequent purificn, host cell transform or nucleotide sequence that transfection is suitable after, but expressed in situ modified immunoglobulin of the present invention.These host-expression vector systems comprise: can transform recombinant phage dna, and plasmid DNA, the escherichia coli of cosmid DNA (as dust Xi Shi escherichia coli, bacillus subtilis), the expression vector of used conversion contains immunoglobulin coding sequence; Can transform yeast (saccharomyces cerevisiae genus, the pichia) system of recombinant yeast expression vector, the expression vector of used conversion contains immunoglobulin coding sequence; Can transform the insect cell system of recombinant virus expression vector (bar virus), the expression vector of used conversion contains immunoglobulin coding sequence; Can transform recombinant virus expression vector (as cauliflower mosaic virus; Tobacco mosaic virus (TMV)) or the expression vector of the used conversion of plant cell system of recombinant plasmid expression vector (Ti-plasmids) contain immunoglobulin coding sequence; Mammalian cell expression system (COS, CHO, BHK, 293, the 3T3 cell), these systems contain the promoter (as gland virus stage starting, vaccinia virus 7.5K promoter) that comes from genomic promoter of mammalian cell (as metallothionein promoter) or mammalian virus.
In bacterial system,, preferentially select for use a series of carrier to come expressing immunoglobulin molecule according to the purpose of expressing.Such as producing a large amount of this albumen, be used to produce the pharmaceutical composition of immunoglobulin molecules, so preferentially select for use the expressed fusion protein level high and be easy to the expression vector of purification.These carriers comprise coli expression carrier pUR278 (Ruther etal., 1983, EMBO J2:1791), and the gene order of coding immunoglobulin can be cloned into the LacZ coding region and come expressed fusion protein; PIN carrier Inouye﹠amp; Inyuoye, 1985, nucleic acids research 13:3101-3109; Van Heeke﹠amp; Chuster, 1989, journal of biological chemistry 24:5503-5509) etc.The pGEX carrier also can be used for expressing foreign protein, and expression product is the fusion rotein that merges with glutathione s-transferase.Generally speaking, fusion rotein is a solubility, after the lysis, with glutathion-polydextran gel pearl absorption, combination, simply gets final product purification with no glutathion eluting then.Also designing in the pGEX carrier has the carrier that comprises thrombin or Xa factor digestion site, so just can obtain not contain the destination protein of GST.
In the insecticide expression vector, with autographa california nuclear polyhedrosis virus (AcNPV) expression alien gene.This virus is at the fall army worm Intracellular growth, and the gene order of coding immunoglobulin can be cloned into the nonessential region (as polyhedron gene) of virus and place under the control of AcNPV promoter (as the polyhedrin promoter).
In the mammalian expression systems, the expression system that is based on virus mostly of use.If use adenovirus vector to be expression vector, the aim sequence of the immunoglobulin of encoding so can be connected to transcribing/translating under the control element of adenovirus, under late promoter and tripartite leader[.By in the body or extracorporeal recombination, this fusion gene can be inserted into the adenoviral gene group.Insert virus genomic nonessential region (as E1 and E3 district), will obtain activated recombinant virions and can in the host who infects, express immunoglobulin (referring to Logan﹠amp; Henk, 1984, institute of NAS reports 81:355-359).For the immunoglobulin gene that makes insertion is effectively translated, need to insert the initial signal of determining, this initial signal comprises ATG start codon and near nucleotide sequence, and the start codon frame should be consistent with the aim sequence frame of expressing, to guarantee to insert the translation of fragment total length.These ectogenic translational control signals and start codon can be various sources, comprise natural and synthetic.The raising of Enzymology method expression efficiency can be by the suitable transcriptional enhancer of introducing, transcription terminator etc. (referring to Bittner et al., 1987, Methods in Enzymology 153:51-544).
In addition, the selection of host cell strain should be the expression that can regulate and control target gene fragment, or modifies the processed gene product according to concrete requirement.This type of protein modified (as glycosylation) is very important with processing (as cutting) for proteic function.Different host cells has the albumen of uniqueness of unique characteristics and the translation post-treatment and the modified mechanism of product.Select suitable cell line or host system, can guarantee that the foreign protein of expressing can be by correct modification and processed.After all, can correct processing transcribe initial product, product is carried out glycosylation and the phosphorylation eukaryotic host cell is only optionally.This class mammalian host cell comprises CHO, VERO, and BHK, HeLa, COS, MDCK, 293,3T3, W138, but not merely have these several.
For the recombiant protein that needs efficiently express for a long time, preferred stably express.Such as, the cell line of stably express immunoglobulin molecules can be genetically engineered cell line.Compare with using the expression vector that contains virus replication, host cell can transform with DNA, and used DNA has suitable expression regulation element (as promoter, enhancer, sequence and transcription terminator and PolyA tailing site) and selected marker.After introducing exogenous DNA, genetically engineered cell was cultivated 1-2 days on nutritious culture medium, went to selective medium then.Selected marker in the recombiant plasmid is given cell resistance, allows the plasmid stable integration to host cell chromosome, and growth forms colony, and cell line can be cloned and expand into to these colonies again.Preferential these methods of using are set up the engineering cell of expressing immunoglobulin.In screening with when assessing those chemical compounds that directly or indirectly act on immunoglobulin molecules, these cell lines are particularly useful.
Multiple screening system is available, comprises the thymidine kinase (Wiggler et al.1977, cell 11:223) of herpes simplex virus, hypoxanthine guanine phosphoribosyltransferase (Szybalska﹠amp; Szybalski, 192, institute of NAS reports 48:202) and the phosphoribosyltransferase transferring enzyme can be at corresponding tk -, hgprt -Or aprt -Use in the cell.The metabolite resistance is the basis of following gene Selection: DHFR, gives the anti-methotrexate resistance of cell (Lowy et al., 1980, cell: 22:817); GPT, give cell anti-mildew phenolic acid resistance (Wiggler et al., 1980,, 77:357; O ' Hare et al., 1981, institute of NAS reports 78:1527); Neo gives the anti-neomycin G-418 of cell resistance (Mulligan ﹠amp; Berg, 1981, institute of NAS reports 78:2072 clinical pharmacology 12:488-505; Wu and Wu, 1991, Biotherapeutics 3:87-95; Tolstoshev, 1993, pharmacology's toxicity academic year comments, 32:573-596; Mulligan, 1993, science 260:926-932; Morgan and Anderson, 1993, bioid academic year is commented 62:191-217; May, 1993, TIBTECH 11 (5): 155-215).Ausubel etc. write the recombinant DNA technology of common-sense in this area (1988, molecular biology modern technologies, John wiley﹠amp; Sons, NY; Kriegler, 1990, gene transfer and expression, laboratory manual Stocktonpress, NY; Dracopoli et al., (eds), 1994, human genetics modern technologies the 12nd and 13 chapters, John Wiley﹠amp; Sons, NY; Colberre-Garapin etal., 1981, journal of biological chemistry 150:1).
Utilize the fusion rotein antibody of immunologic opsonin recognition expression, simply purified fusion protein.Such as a system of descriptions such as Janknecht, can be easy to the non-degeneration fusion rotein (Janknecht etal.1991, institute of NAS reports 88:8972-897) that purification is expressed the human cell line.This system be with the genes of interest sub-clone to the recombinant vaccinia carrier, merged the label of six histidine like this at the aminoterminal of its open reading frame, this label is that the substrate of purified fusion protein is in conjunction with the territory.Collect the cell extract that the transfection recombinant vaccinia is expressed, cross nitrilo-nickel acetate-agarose gel post, with the buffer that contains imidazoles eluting optionally, obtain to contain histidine-tagged albumen then.
The raising of the expression of immunoglobulin molecules, can (see summary Bebbington and Hentschel by the carrier amplification method, in dna clone, purposes cloning based on carrier expression cloning gene in mammalian cell of gene amplification, Vol.3, (academic press, New York, 1987)).When in the host cell culture medium, having a certain amount of inhibitive factor, the amplification of a certain marker gene will make the copy number of marker gene increase in the carrier system of expressing immunoglobulin molecule, and the gene region of amplification links to each other with immunoglobulin gene, and final immunoglobulin output also just raises.(Crouse et al., 1983, molecular cytobiology .3:257)
Host cell can be with two expression vector cotransfections of the present invention, wherein first carrier is to express the derive carrier of polypeptide of heavy chain, second is the carrier of expressing the derived light chain polypeptide, and two carriers contain identical selected marker thing, expression weight chain polypeptide that can equivalent.Alternative is that identical carrier encoded light heavy chain polypeptide is before light chain gene is positioned at heavy chain gene in this case, to avoid excessive expression (Proudfoot, 1986, the natural 322:52 of free toxicity heavy chain; Kohler, 1980, institute of NAS reports 77:2197).The gene order of encoding heavy chain and light chain should comprise cDNA or genomic DNA.
In case it is recombinant expressed that modified immunoglobulin of the present invention obtains, just can carry out purification with the method for any known purification immunoglobulin molecules in this area, such as: column chromatography is (as ion exchange, affinity chromatograph, particularly use the protein A purification, sieve chromatography), centrifugal, the standard technique of dissolubility difference or other any purifying protein.
5.5 treatment applicating example:
With the efficient that modified antibodies of the present invention detects treatment and prevents special disease, can there be several different methods to detect or test.
The first, the immunological competence that comprises the vaccine of modified immunoglobulin can detect in the experimental animal of vaccine immunity anti-idiotype reaction and determine.The situation of the humoral immune reaction that produces can be represented total immunoreation situation, and other immune component, especially cellular immunization are very important aspect the prevention of disease.Experimental animal comprises mice, rabbit, chimpanzee and final people.The vaccine that the present invention produces can be used for the tentative chimpanzee that catches.But chimpanzee is the protectiveness animal, so at first test on some little and cheap animal bodies at the antibody response of this vaccine, seeks the efficient studies that optimal candidate immunoglobulin molecules or best of breed molecule are used for chimpanzee.
The immunoreation method of testing experiment animal has number of ways, as the reactive available known enzyme linked immunosorbent assay (ELISA) of immune serum antagonist, immunoblotting, radioimmunoassay precipitation etc.; Or pass through prevention infection and/or alleviate immune host's disease symptoms.
One of animal testing example is with vaccine combination immune rabbit of the present invention, to test the ability that it induces anti-modified immunoglobulin idiotype reaction.Such as the new zealand white rabbit of growing up with male, anosis (SPF) youth.Every rabbit of test group is accepted the vaccine of capacity, matched group injection natural antibody, and this antibody is dissolved in the Tris-HCl pH9.0 of 1mM.Each or two weeks take a blood sample, the antigenic specific recognition that serum analysis is discerned modified antibodies the anti-idiotype reaction and the anti-anti-idiotype antibody of modified immunoglobulin molecule.Such as using radioimmunoassay detection method (Abbott Laboratories).Anti-idiotype antibody also can detect with the ELISA method.Because the reaction that the outbreeding strain of rabbit is caused fluctuation is also used mice instead and is tested.
In addition, modification antibody test method of testing of the present invention can also be, to the test animal medication, no matter is animal or people at first, separates the anti-anti-idiotype antibody (being Ab3 antibody) as an anti-idiotype reaction part then.Test it then in conjunction with specifying antigenic ability (as tumor antigen, the infectious disease pathogen antigen), method can be any known immunologic detection method in this area, detect ELISA, the reaction of sandwiching immunity detection method gel diffusion as radioimmunoassay, immunodiffusion test, immunoblotting, the sedimentation method, coagulation detection method, the complement combined techniques, immunofluorescence detects, protein A detection method, immunoelectrophoresis detection method or the like.
If modified antibodies combines with tumor or cancer antigen, so isolating Ab3 treatment tumor, cancer or other tumor disease efficient detection method are, cultivation is from patient's tumor or cancerous cell, detect combining of Ab3 antibody and cell then, and relatively in conjunction with the propagation or the survival rate of binding antibody cell are not determined its efficient.If the propagation or the survival rate of binding antibody cell are lower, just show Ab3 (inducing) effectively to this patient's tumor treatment by using modified antibodies of the present invention.The multiple detection method of propagation or survival rate can be used for assessment survival and/or propagation in this area; Such as usefulness 3The H thymus pyrimidine method of mixing is measured the propagation of cell, with directly awarding cell counting, detect with the active variation of known certain gene transcription, as proto-oncogene (as fos, myc) or the cell cycle marker gene; Cell available tongue is anyway expected blue dyeing judgement, or changes to determine cell differentiation etc. by morphocytology.If modified antibodies identification infectious disease cause of disease is estimated isolating Ab3 activity at external use in conjunction with the antigenic activity of appointment so.
In addition, modified antibodies of the present invention is also anti-can directly measure in vivo.Be the curative effect of monitoring medicine of the present invention, can be before treatment, in the treatment or treatment back proper spacing detects the bonded antigen amount of modified antibodies.The variation of antigen amount or no change can detect, and the variation of antigen amount or no change are relevant with patient's curative effect.
Tumor therapeutic agent particularly, the order of severity of antigen amount and tumor in the serum, directly related as breast carcinoma and poor prognosis.In a word, the reduction of antigen amount is relevant with effectively treating.
When modified antibodies was identification infectious disease cause of disease, its efficient also can be by before treatment, and in the treatment or treatment back proper spacing detects infectious disease cause of disease amount, and it is effective that the cause of disease amount reduces the prompting modified antibodies.
On the method, determine antigen levels on the preferred different time point, then with background ratio.The index amount that exists in background is meant normal, the anosis individuality; And/or the level before the treatment, or the level when alleviating, or the level of stable phase.These indexs are relevant with disease course and therapeutic outcome.
Can determine the antigen amount with method as known in the art.As: determine the antigen amount by known amynologic diagnostic method such as immunoblotting, immunoprecipitation.
At the immunoreation intensity of modified immunoglobulin, can determine in the body, detect as skin delayed hypersensitivity and external T LCA, but be not limited to this several method by any method as known in the art.
The skin delayed hypersensitivity is being estimated the total immune potentiality of body and to significant aspect the antigenic cellular immunization.The technology of dermoreaction test requires to use pro-antigen should keep in Dark Place in 4 ℃, faces and uses preceding preparation.Use No. 25 or No. 27 syringe needles are guaranteed medication at skin corium rather than subcutaneous, after the medication 24-48 hour, with the maximum gauge and the persistent period of ruler measurement erythema.Reactive method of testing that goes down of specifying antigen or antigen group is to use high concentration antigen, or under ambiguous situation, uses median dose retest method.
The method of test T LCA is, separation is from immune experimenter's T lymphocyte, as with the isolating lymphocyte of conventional lymphocyte separation medium, directly cultivate in containing 3 milliliters of RPMI culture medium of 10% hyclone, stimulate again with the antigenic cell of expressing modified antibodies identification.Require to contain 33% lymphocyte culture supernatant in some test or in culture medium, add IL-2 as the T cell growth factor.When measuring the first set reaction of immunity back toxic T lymphocyte, isolating T cell should be cultivated with containing or do not contain antigenic cell.After 6 days, supernatant carries out 4 hours 51Cr discharges detection, to detect toxicity.When immunity is effective, spontaneous 51Cr discharges index and is not less than 20%.(Heike et al., immunotherapy magazine 15:15-174)
On the other hand, modified immunoglobulin efficient can be replied or improvement from morbid state by the immune experimenter of monitoring, and perhaps the antigen situation of modified antibodies identification is measured.When modified antibodies identification tumor or cancer antigen, the course of disease of tumor or cancer can be determined by known diagnosis or monitoring tumor technology.Determine with level (or another kind of and this tumor or the relevant antigen of cancer) or this antigenic traget antibody of injection specific recognition of concrete tumor or cancer associated antigens such as measuring in the tumor patient serum.In addition, other image technology such as computerized tomography CT, ultrasonic or other image technology also can be used for the monitoring of the tumor or the cancer course of disease, certainly, are not limited to these methods.The biopsy method can be used for monitoring equally.But before doing these tests on the person, must on specific tumors or animal model for cancer, test the efficient of modified immunoglobulin earlier.
If the treatment infectious disease, the efficiency monitoring method of modified antibodies is to experimenter's medication (animal model of people or disease), to detect the level of special pathogen in the body or the symptom of this infectious disease then earlier.Infectious disease cause of disease method of testing can be any known assay method in this area, as uses virus titer when detecting virus, amount of bacteria (as cultivating patient specimen) etc.Also can measure the antigenic amount of modified immunoglobulin identification or another antigen of this infectious disease.The decline of infectious disease cause of disease or the alleviation of its symptom show that all modified antibodies is effective.
6. embodiment: induce resistive connection intestinal cancer idiotypic vaccine
Embodiment has narrated the modified antibodies (hybridoma of secretion Mab31.1 monoclonal antibody derives from American type culture collection, numbering HB12314) that makes up with monoclonal antibody Mab31.1.The antigen that Mab31.1 identification human colon carcinoma is expressed.On the Mab31.1 basis,, after the cysteine in the weight chain variable region of this antibody substituted with alanine, be transformed into modified antibodies of the present invention by genetic engineering.Therefore, the modified antibodies weight chain variable region intrachain disulfide bond of acquisition disappearance.
6.1 the structure of modified antibodies
The strategy that makes up modified antibodies is that the gene of structure encoded light, variable region of heavy chain wherein becomes alanine to forming the crucial specific cysteine of intrachain disulfide bond.In the antibody that comes from Mab31.1 antibody, substitute cysteine with alanine residue, the position is 23 and 92 of variable region of heavy chain, 23 and 88 of variable region of light chain.For making up these cydorge genes, assemble one group of oligonucleotide (as discussed below), be inserted into then in the suitable carrier that constant region is provided.
Make up variable region gene tactful as follows of the CDRs of no intrachain disulfide bond.
At first, single stranded oligonucleotide annealing forms the double chain DNA fragment (shown in Figure 10 step 1) of sticky end.Particularly, be to use the automatic synthesizer of GenoSysBiotech Inc company synthetic with the corresponding oligonucleotide sequence of aim sequence, its length is 80 nucleotide, it is overlapped that 20 bases are wherein arranged.Every section oligonucleotide all is specific, shown in Fig. 9 A and 9B.Fig. 9 A has listed one group of ten oligonucleotide fragment, is used to make up the heavy chain variable region gene that is called 2CAVHCOL1.2CAVHCOL1 compares no cysteine with heavy chain variable region gene.Fig. 9 B has listed one group of 12 oligonucleotide fragment, is used to make up the chain variable region gene that is called 2CAVLCOLl.2CAVLCOL1 compares no cysteine with chain variable region gene.For oligonucleotide is connected into genes of interest, 10 or 12 oligonucleotide connect according to the methods below, and as shown in figure 10, wherein the 1-10 oligonucleotide among Figure 10 has detailed description in table 5.In conjunction with before, every oligonucleotide 5 ' hold phosphorylation as follows: under the catalysis of T4 polynucleotide polymerase, the oligonucleotide of 25 μ l and the ATP37 of 30mM ℃ reaction one hour; Hatch 5 minutes cessation reactions, use ethanol precipitation then for 70 ℃.After the phosphorylation, complementary oligonucleotide (oligonucleotide 1+10, oligonucleotide 2+9, oligonucleotide 3+8, oligonucleotide 4+7, oligonucleotide 5+6) as shown in figure 10, in aseptic microcentrifugal tube, mix 65 ℃ of water-baths 5 minutes, annealing at room temperature 30 minutes.The result forms short, band sticky end double-stranded DNA.
Next step, the viscosity double chain DNA fragment connects the chain (as Figure 10, second step is to the 4th step) of growing up, until being assembled into the variable region cydorge gene.It is pointed out that the method for attachment of viscosity double chain DNA fragment is: under the effect of T4 dna ligase, 16 ℃ of water-baths are 2 hours in the presence of the ATP of 10mM; Oligonucleotide 1/10 after the annealing mixes with oligonucleotide 2/9, and oligonucleotide 3/8 mixes with oligonucleotide 4/7, and reacting final product is labeled as oligonucleotide 1/10/2/9 and 3/8/4/7.Oligonucleotide 3/8/4/7 is connected with oligonucleotide 5/6 then, forms oligonucleotide 3/8/4/7/5/6; Oligonucleotide 1/10/2/9 and 3/8/4/7/5/6 is connected to form the variable region gene of total length
Equally, with the heavy chain variable region gene of the synthetic total length of 12 oligonucleotide, reaction sequence is as follows: 1+12=1/12,2+11=2/11,3+10=3/10,4+9=4/9,5+8=5/8,6+7=6/7,1/12+2/11=1/12/2/11,3/10+4/9=3/10/4/9,5/8+6/7=5/8/6/7,1/12/2/11+3/10/4/9=1/12/2/11/3/10/4/9,1/12/2/11/3/10/4/9+5/8/6/7=total length variable region gene.The oligonucleotide that is used for making up gene is all listed at table 5.Heavy chain after variable region gene is modified is labeled as 2CAYHCOLl, and the light chain after variable region gene is modified is labeled as 2CAVLCOLl.
The variable region gene purification process of modifying is a gel electrophoresis.For removing the unnecessary oligonucleotide that does not connect and other non-complete dna fragmentation, connect product in 1% low melting-point agarose gel, constant voltage 110V electrophoresis 2 hours.Cut out the master tape of full length DNA then, put into the 1.5ml microcentrifugal tube.With I40 ℃ of effect of f3-beta-Agarase 3 hours, so that DNA discharges from agarose.Add 0.3M NaOAc and isopropyl alcohol, placed 1 hour for-20 ℃, 12, centrifugal 15 minutes of 000rpm, the DNA of deposition and purification also is resuspended in 50 μ lTE buffer, pH8.0.At last, by PCR method amplification variable region gene.Get 100ng engineering variable region gene and 25mMdNTPs, 200ng primer and 5U high-fidelity thermophilic enzyme pfu archaeal dna polymerase and buffer mix.The PCR product is with 1% agarose gel electrophoresis analysis.
The variable region dna fragmentation of purification inserts the pUC19 bacteria carrier.The pUC19 carrier, total length 2686 a penicillins base pair is the e. coli plasmid vector of a high copy, comprising the multiple clone site and the ammonia benzyl resistant gene of 54 bases on the LACZ gene.Before inserting variable region gene, it is as follows to prepare linearizing carrier method: with the pUC19 of HinCII enzyme (50U) processing 10 μ g, 37 ℃ act on 3 hours.For preventing the carrier recirculation, with the little intestinal alkaline phosphatase (CIP) of 25U, 37 ℃ of dephosphorylations 1 hour.The dephosphorylation linear carrier of getting 0.5 μ g mixes with the variable region gene fragment of 3 μ g phosphorylations, adds T4 ligase (1000U), and 16 ℃ were reacted 12 hours.
With inserting the segmental recombinant plasmid transformed antibacterial of engineering variable region gene.Concrete grammar is: the pVC19 plasmid mixing that contains the engineering variable region gene of prepared fresh DH-5 α competence antibacterial 50 μ l and 1 μ g is transferred to (0.2cm gap in the groove of electric conversion instrument; Bio-Rad).In electric conversion instrument (Bio-Rad Gene Pulset), impulse action under the 2.5kV/200ohm/25 μ F condition.After the processing, add the 1mlSOC culture medium in each groove immediately, cultivate for 37 ℃ and made the cell answer in 1 hour.Take out cell transformed, be applied to (40 μ g/ml) on the LB flat board that contains ampicillin, 37 ℃ of overnight incubation by getting 100 μ l after the dilution in 1: 100.The transform bacteria that only contains the pUC19 plasmid could be grown on flat board.
Select single bacterium colony, be inoculated in the 3ml LB/Atp aseptic culture medium 37 ℃ of shaking table overnight incubation.With purification column (Parmacia Biotech) plasmid purification, and be dissolved in the 100 μ lTE buffer pH7.5.Identify that gene inserts among the pUC19, each clone gets 25 μ l, and 37 ℃ of restrictions produced the endonuclease enzyme action 1 hour, used 1% agarose gel electrophoresis analysis then.After using this method, restriction enzyme site in the recombinant plasmid dna (5 ' ..GTCGAC..3 ') forfeiture, to the enzyme resistance, the approaching plasmid of its mobility and cyclisation (CC) DNA in glue.And recombiant plasmid can be not digested, and its mobility is close with linear dsdna fragment (L).
In order to ensure the gene order of inserting is the engineering variable region gene, and reject the sudden change of not expecting in building process, may occur, the primer (5 ' GAATTCATGGCTTGGGTGTG3 ') that contains 20 bases with M13/pUC reverse primer (5 ' AACAGCTATGACCATG3 ') and 5 ' end checks order, and sequenator is the ABI377DNA sequenator.Prove that all clones contain correct sequence.
Table 5 coding source is from the structure of the modified antibodies that contains CDRs of Mab31.1 antibody
Oligo1?Oligo2?Oligo3?Oligo4?Oligo5?Olig6?Oligo7?Oligo8?Oligo9?Oligo102CAVHCOLl?VHC1???VHC2???VHC3???VHC4???VHC5???VHC???VHC7???VHC8???VHC9???VHC102CAVLCOL1?VLC1???VLC2???VLC3???VLC4???VLC5???VLC???VLC7???VLC8???VLC9???VLC10
6.3 the genetic engineering variable region gene inserts mammalian expression vector
Complete light chain of antibody has variable region and constant region.Complete heavy chain of antibody comprises a variable region, a constant region and a hinge region.The variable region gene 2CAVHCOL1 of modified or 2CAVLCOL1 insert and contain in the suitable constant region genophore.The chain variable region gene of no cysteine inserts the pMRR10.1 carrier, and as Fig. 6 A, this carrier contains the constant region of people κ type light chain.Variable region gene constitutes complete light chain gene after inserting.Equally, the heavy chain variable region gene of no cysteine inserts the pGammal carrier, as Fig. 6 B.This carrier contains human IgG1's constant region and hinge region gene, and heavy chain variable region gene inserts the back and constitutes complete heavy chain gene.
The mammalian expression vector method that structure comprises light chain and heavy chain gene is: complete weight chain gene is inserted among the mammalian expression vector pNEPuDGV, (Bebbington shown in Fig. 6 C, C.R., 1991, In METHODS:A Companion To Methods InEnzymology, vol.2, pp:136-145).The light chain and the heavy chain of recombiant plasmid coding modified antibodies.Method: the correlation method of Enzymology method.
6.4 the expression of synthetic modification antibody gene in mammalian cell
For the antibody gene that detects assembling is expressed, with mammal carrier rotaring redyeing COS cell.COS cell (a kind of African green monkey kidney cell line, CV-1, be transformed by the SV-40 replication-defective virus) because making, this cell contains cyclisation plasmid replication that SV-40 duplicates promoter to very high copy number, can be used for the transient expression of synthetic antibody short-term.Antibody expression vector rotaring redyeing COS 7 cell (coming from American type culture collection).After the transfection, cell culture is in improvement Eagle ' s culture medium, and transfection is with calcium coprecipitation method (Sullivan etal .FEBS Lett.285:120-123).Transfectional cell was cultivated 72 hours, collected supernatant.Detect the IgG of assembling with the ELISA method.The ELISA method comprises: anti-human IgG Fc antibody the catching sample and standard substance of bag quilt on the 96 hole elisa plates.With the anti-people κ type light chain antibody of horseradish peroxidase (HRP) labelling, detect the assembling IgG that catches, substrate is tetramethyl benzidine (TMB).The reaction color depth is proportional to the antibody amount that exists in the sample.
6.5 the antigen of modified antibodies immunity specific bond human colon cancer cell and this cellular expression
The expression and purification of modified antibodies with LS-174TWiDR cell (a kind of CCL188), detects the adhesion and the specificity of modified antibodies and this cell and antigen expressed as described in 6.4.
The adhesion and the specificity method that detect Mab31.1 antibody are immunoblotting (seeing Figure 11).From the membrane sample of LS-174T cell preparation, (Bio-Rad) is loaded on nitrocellulose filter with sample with the Bio-Blot instrument.The film of point sample seals non-specific bond site with skim milk.Use biotinylation Mab31.1 antibody incubation then.0.003nM-20nM unlabelled antibody act on then.Unconjugated antibody by eluting, adds the anti-human IgG effect of alkali phosphatase enzyme mark by washing then.At last, add the alkaline phosphatase substrate effect, show black purple precipitation as a result on the film, the antibodies that shows labelling is on film.Figure 11 is the result of immunoblotting, and the result shows that traget antibody is bonded to the LS-74T cell.Equally, unlabelled antibody can with the antibody competition of labelling, show that the modified antibodies that comes from Mab31.1 can combine with the tumor-cell antigen specificity.
6.6 anti-idiotype reaction
Modified antibodies be the CBA method in conjunction with effect detection method.The LS-174T cell is the antigenic human colon cancer cell of expressing the Mab31.1 antibody recognition.Prepared a CDRS polypeptide of sequence that comprises Mab31.1 antibody.With these polypeptide and modification property antibody and control antibodies immune mouse, obtain the antiserum of anti-Mab31.1 CDRs district and antibody, two weeks of immunity back and blood sampling are all around made CBA with these antiserums then, the results are shown in Figure 12A and 12B.
Antiserum and biotinylated antibody and LS-174T cell adhesion have been detected.Shown in Figure 12 A and 12B, the antiserum in anti-CDR3 and CDR4 district is significantly competed combining of control antibodies (from Mab31.1) and LS-174T cell.In addition, the antiserum in anti-CDR1 and CDR2 district also can significantly be competed combining of control antibodies and LS-174T cell.And from the mouse resisting anteserum of the antibody of immune 2CAVHCOL1 and 2CAVLCOL1 (being the modified antibodies that substitutes cysteine in the variable region with alanine), with antiserum, stronger with the competitiveness of biotinylated antibody from immune Mab31.1 antibody.(seeing Figure 12 B).This results suggest: the modified antibodies that uses alanine in the variable region to substitute cysteine can induce the anti-idiotype antibody of identification colon cancer antigen, and to contain the anti-idiotype antibody that the antibody induction of disulfide bond goes out stronger in the variable ratio district.
Table 6 comes from the biotinylation labelling of the CDR sequence of Mab31.1 monoclonal antibody
Peptide sequence: COL311 L1 biotin-N-Thr-Ala-Ser-Gln-Ser-Val-Ser-Asn-Asp-
Val-AlaCOL311 L2 biotin-N-Ile-Tyr-Tyr-Ala-Ser-Arg-Tyr-ThrCOL311 L3 biotin-N-Phe-Ala-Gln-Glngaspltyr-Ser-Ser-Pro-
Leu-ThrCOL311 H1 biotin-N-Phe-Thr-Asn-Tyr-Gly-Met-AshCOL311 H2 biotin-N-Ala-Gly-Trp-Ile-Asn-Thr-Tyr-Thr-Gly-
Glu-Pro-Thr-Tyr-Ala-Asp-Asp-Phe-Lys-GlyCOL311 H3 biotin-N-Ala-Arg-Ala-Tyr-Tyr-Gly-Lys-Tyr-Phe-
Asp-Tyr
Embodiment 7 contains the expression of the synthetic modification antibody of HMFG-1 sequence
Made up the anti-idiotype antibody of immunologic opsonin in conjunction with HMFG-1 antibody.HMFG-1 be a kind of known can be in conjunction with antibody (Stewart etal., 1990, the Journal of Clinical Oncology 8:1941-50 of polymorphic epidermis cell mucin (PEM); Kosmas et al., 1994, cancer 73:3000-3010).The antigenic determinant sequence ProAspThrArgPro of PEM is inserted into the variable region of antibody with method of the present invention.This weak point sequence is high immunogenicity zone in the polymorphic epidermis cell mucin of people (Gendl er et al., 198, journal of biological chemistry 163:12820-12823).The 27A-27E of HMFG-1 (SerLeuLeuTyrSer) residue (table 6) replaces with ProAspThrArgPro with the described oligonucleotide synthesis method of the 6th part, sees Figure 10.With the weight chain variable region known sequence of HMFG-1 antibody, the production immunologic opsonin is in conjunction with the anti-idiotype antibody of HMFG-1 antibody.The oligonucleotide addition sequence is as follows: 1+8=1/8,2+7=2/7,3+6=3/6,4+5=4/5,1/8+2/7=1/8/2/7,3/6+4/5=3/6/4/5,1/8/2/7+3/6/4/5=1/8/2/7/3/6/4/5.Table 7 has been listed the comparison of HMFG-1 and different CDR district consensus sequence.Referring to WO09/05142 (Imperial Cancer Research Technology Ltd.), the humanized antibody data is referring to WO92/04380 (Unilever) about HMFG-1 and relevant monoclonal antibody data.
Polymerase chain reaction (PCR) be used to increase assembled base because of, see Figure 13.The variable region gene of coding HMFG-1 antibody sequence makes up sees Figure 13.The variable region gene that makes up inserts suitable carriers, as the pNEPuDGV carrier, and expressing antibodies, method is described with the 6th part.Other construction method is suitable equally, comprises Jayaraman et al., and 1989, nucleic acids research .17:4403; Sandhu et al, 1992, Biotechniques 12:14; Barnett andErifie, 1990, H., nucleic acids research .18:3094; Ciccarelli et al., 1991, nucleic acids research .19:6007; Michaels et al., 1992, the described method of biotechnology 12:45 is drawn here for referring to document, but the reference method that is not limited to enumerate here.
The comparison of table 7:HMFG-1 antibody and different CDRS consensus sequence
VH1 CDR1 sequence residue 31 32 33 34 35 35A 35B people I Ser Tyr Ala Ile Ser people II Ser Tyr Ser/Tyr Trp Ser Trp Asn people III Ser Tyr Ala Met Ser mouse IA Ser Tyr Tyr Trp Asn Asn Ser mouse IB Ser Tyr Gly Val His Val Ser mouse IIA Asp/Ser Tyr Tyr Met Asn Asn mouse IIB Ser Tyr Trp Met His mouse IIC Asp/Ser Tyr Tyr Met His mouse IIIA Asp/Ser Phe/Tyr Tyr Met Glu mouse IIIB Arg Tyr Trp Met Ser mouse IIIC Arn Tyr Trp Met Asn mouse IIID Ser Tyr Ala Met Ser mouse VA Ser Tyr Gly Ile Asn mouse VB Ser Tyr Gly Leu TyrHMFG-1 Ala Tyr Trp Ile GluVH1 CDR2 sequences
Residue 50 51 52 52A 52B 52C 53 54 55 56 57 58 59 60 61 62 63 64 65 people I Trp Ile Asn Pro Tyr Gly Asn Gly Asp Thr Asn Tyr Ala Gln Lys Phe Gln Gly people II Arg Ile Tyr Tyr Arg Ala Tyr Ser Gly Ser Thr Asp/ Tyr Asn Pro Ser Leu Lys Ser
Asn III Val Ile Ser Gly Lys Thr Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys GlyIA Tyr Ile Ser + Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys SerIB Val Ile Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met SerIIA Asp Ile Asn Pro Gly Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys GlyIIB Arg Ile Asp Pro Asn Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys SerIIC Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lyr Phe Gln GlyIIIA Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser Ala Ser Val Lys GlyIIIB Glu Ile Asn Pro Lys Ala Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu Lys AspIIIC Glu Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Ala Glu Ser Val Lys GlyIIID Thr Ile Ser Ser Lys Ser Gly Gly Gly Tyr Thr Tyr Tyr Pro Asp Ser Val Lys GlyVA Tyr Ile Asn Pro Gly Asn Gly Tyr Thr Lys Tyr Asn Glu Lys Phe Lys GlyVB Tyr Ile Ser Ser Ser Ser Ala Tyr Pro Asn Tyr Ala Gln Lys Phe Gln GlyHMFG-l Glu Ile Leu Pro Gly Ser Asn Asn Ser Arg Tyr Asn Glu Lys Phe Lys GlyVH1 CDR3
Residue 95 96 97 98 99 100 100A 100B 100C 100D 100E 100F 100G 100H 100I 100J 100K 101 102 people I Ala Pro Gly Tyr Gly Ser Gly Gly Gly Cys Tyr Arg Gly Asp Tyr+Phe Asp Tyr people II Glu Leu Pro Gly Gly Tyr+Gly Asp Asp Tyr Tyr Tyr++ Gly Phe Asp Val people III+Arg+++Ser Leu Ser Gly+Tyr Tyr Tyr Tyr His Tyr Phe Asp Tyr mouse IA Gly Gly Tyr Gly Tyr Gly Tyr Tyr Tyr Tyr Asp+Tyr Tyr Tyr Tyr Phe Asp Tyr mouse IB Asp Arg Gly Arg Tyr Tyr Tyr+Ser Gly+++Tyr Tyr Ala Met Asp Tyr mouse IIA Gly+Tyr Tyr Ser Ser Ser Tyr Met+Ala++ Tyr Tyr Ala Phe Asp Tyr
95 96 97 98 99 100 100A 100B 100C 100D 100E 100F 100G 100H 100I 100J 100K 101 102IIB Tyr Tyr Tyr Gly Gly Ser Ser + + Val Tyr + Tyr Trp Tyr Phe Asp TyrIIC Gly Tyr Tyr Tyr Tyr Asp Ser + Val Gly Tyr Tyr Ala Met Asp TyrIIIA Asp Tyr Tyr Tyr Gly Ser Ser Tyr Tyr Glu Gly Pro Val Tyr Trp Tyr Phe Asp ValIIIB Leu Gly Gly Tyr Gly Tyr Phe Gly Ser Ser Tyr Tyr Ala Met Asp TyrIIIC Gly Gly Tyr Gly Gly + Arg Arg Ser + Trp Phe Ala TyrIIID Gly Gly Tyr Tyr Tyr Leu + Gly Ser Ala Pro Phe Asp Tyr Ala Met Asp TyrVA Ser Asn Tyr Tyr Gly Gly Ser Tyr Tyr Tyr + Phe Ala Tyr Tyr Tyr Phe Asp TyrVB Arg Val Ile Ser Arg Tyr Phe Asp GlyHMFG-1 Ser Tyr Asp Phe Ala Trp Phe Ala TyrVL CDR1
Residue 24 25 26 27 27A 27R 27C 27D 27E 27F 28 29 30 31 32 33 34
People κ I Arg Ala Ser Gln Ser Leu Val++ Ser Ile Ser Asn/Ser Tyr Leu Ala
People κ II Arg Ser Ser Gln Ser Leu Leu His Ser+Asp Gly Asn/Asp Asn/Thr Tyr Leu+
People κ III Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala
People κ IV Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala
Mice κ I Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu
Mice κ II Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu Gln
Mice κ III Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His
Mice κ IV Ser Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His
Mice κ V Arg Ala Ser Gln Asp Asp Ile Ser Asn Tyr Leu Asn
Mice κ VI Ser Ala Ser Ser Ser Val Ser Tyr Met His
Mice κ VIIHMFG-1 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Ile Tyr Leu AlaVLCDR2 sequence
Residue 50 51 52 53 54 55 56
People κ I Ala Ala Ser Ser Leu Glu Ser
People κ II Leu Val Ser Asn Arg Ala Ser
People κ III Gly Ala Ser Ser Arg Ala Thr
People κ IV Trp Ala Ser Thr Arg Glu Ser mouse κ I Trp Arp Ser Thr Arg Glu Ser mouse κ II Lys Val Ser Asn Arg Phe Ser mouse κ III Ala Ala Ser Asn Leu Glu Ser mouse κ IV Arg Thr Ser Asn Leu Ala Ser mouse κ V Tyr Ala Ser Arg Leu His Ser mouse κ VI Asp Thr Ser Lys Leu Ala Ser mouse κ VIIHMFG-1 Trp Ala Ser Thr Arg Glu SerVLCDR3 sequence
Residue 89 90 91 92 93 94 95 95A 95B 95C 95D 95E 95F 96 97
People κ I Gln Gln Tyr+Ser Leu Pro Glu Trp Thr
People κ II Met Gln Ala Leu Gln+Pro Arg+Thr
People κ III Gln Gln Tyr Gly Ser Ser Pro Pro Leu Thr
People κ lV Gln Gln Tyr Tyr Ser Thr Pro+Thr mouse κ I Gln Asn Asp Tyr Ser Tyr Pro Leu Thr mouse κ II Phe Gln Gly Thr His Val Pro Pro Tyr Thr mouse κ III Gln Gln Ser Asn Glu Asp Pro Pro Trp Thr mouse κ IV Gln Gln Trp Ser Ser Tyr Pro+Gly Leu Thr mouse κ V Gln Gln Gly Asn Thr Leu Pro Pro Arg Thr mouse κ VI Gln Gln Trp Ser Ser Aro Pro Pro Met Pro Leu Thr
Residue 89 90 91 92 93 94 95 95A 95B 95C 95D 95E 95F 96 97 mice κ VII Leu Gln Tyr Asp Glu Phe Ala Tyr ThrHMFG-1 Gln Gln Tyr Tyr Arg Tyr Pro Arg Thr
The concrete scheme that the present invention is not limited to here on scope to be lifted.In fact, except these methods of the present invention,, obviously can make various modification of the present invention to one skilled in the art according to the description of front and appended diagram.
The disclosed pertinent literature of literature content quotes in full as document here.

Claims (98)

1. vaccine combination, comprise a certain amount of first immunoglobulin molecules that is enough to induced anti-idiotype reaction, this first immunoglobulin molecules contains one section variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin molecules, the wherein said second immunoglobulin immunologic opsonin conjugated antigen, said one or more amino acid whose replacement refer to replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with the aminoacid that does not contain sulfydryl; With pharmaceutically acceptable carrier.
2. according to the vaccine combination of claim 1, wherein said antigen is tumor antigen.
3. according to the vaccine combination of claim 2, wherein said antigen is cancer antigen.
4. according to the vaccine combination of claim 3, wherein said antigen is polymorphic endotheliocyte mucin antigen.
5. according to the vaccine combination of claim 3, wherein said antigen is colon cancer associated protein antigen.
6. according to the vaccine combination of claim 3, wherein said antigen is the relevant carbohydrate antigen of colon cancer.
7. according to the vaccine combination of claim 1, wherein said variable region is a variable region of light chain, said 23 or 88 amino acids that do not contain the mercaptoamino acid residue corresponding to second immunoglobulin light chain variable region.
8. according to the vaccine combination of claim 1, wherein said variable region is a variable region of heavy chain, said 22 or 92 amino acids that do not contain the mercaptoamino acid residue corresponding to second immunoglobulin heavy chain variable region.
9. according to the vaccine combination of claim 1,7 or 8, the wherein said mercaptoamino acid residue that do not contain is an alanine.
10. according to the vaccine combination of claim 1, wherein said second immunoglobulin is Mab31.1, Mab33.28 or Mab HMFG-1, wherein said one or more amino acid replacement comprise 23 and/or 88 the aminoacid that substitutes chain variable region gene with alanine.
11. vaccine combination according to claim 1, wherein said second immunoglobulin is Mab31.1, Mab33.28 or Mab HMFG-1, wherein said one or more amino acid replacement comprise 22 and/or 92 the aminoacid that substitutes heavy chain variable region gene with alanine.
12. according to the vaccine combination of claim 3, wherein said antigen is human milk fat bead antigen.
13. according to the vaccine combination of claim 3, wherein said antigen is breast carcinoma, ovarian cancer, uterus carcinoma, carcinoma of prostate, bladder cancer, pulmonary carcinoma, skin carcinoma, colon cancer, cancer of pancreas, gastrointestinal cancer, B cell carcinoma or T cell carcinoma antigen.
14. according to the vaccine combination of claim 3, wherein said antigen is selected from: KS1/4 is general-cancer antigen, ovarian cancer antigen, prostanoic acid phosphoric acid, prostate specific antigen, melanic related antigen p97, melanoma-associated antigen p75, the high molecular melanoma-associated antigen, the membrane antigen of prostate-specific, carcinoembryonic antigen, polymorphic endotheliocyte mucin antigen, human milk fat microglobulin antigen, colon cancer related antigen TAG-72, C017-1A, GICA 19-9, CTA-1, LEA, burkitt's lymphoma antigen-38.13, CD19, people B lymphoma antigen-CD20, CD33, ganglioside GD2, Ganglioside, GD3, Ganglioside GM2, Ganglioside GM3, tumour-specific transplantation type cell surface antigen, carcinoembryonic antigen-α-embryo's albumen L6, human lung cancer antigen L20, human leukemia T cellular antigens-Gp37, neoglycoprotein, sphingolipid, EGFR, HER2 antigen, polymorphic epidermis mucin, human malignant lesion lymphocyte antigen-APO-1, I antigen M18, M39, SSEA-1, VEP8, VEP9, Myl, VIM-D5, D 156-22, TRA-1-85, C14, F3, AH6, Y hapten, Le y, TL5, FC10.2, adenocarcinoma of stomach antigen, CO-514, NS-10, CO-43, MH2, in the colon cancer 19.9, gastric cancer mucin, T 5A 7, R 24, 4.2, G D3, D1.1, OFA-1, G M2, OFA-2, G D2, M1:22:25:8, SSEA-3, SSEA-4 and the peptide that is derived from TXi Baoshouti.
15. according to the vaccine combination of claim 1, wherein said antigen is the infectious disease pathogen antigen.
16. vaccine combination according to claim 15; wherein said antigen is selected from: influenza virus hemagglutinin; people's trachea syncytial virus G glycoprotein; the dengue virus core protein; the dengue virus stromatin; the Measles virus hemagglutinin; II herpes simplex virus type glycoprotein gB; I type poliovirus VP1; I type human immune deficiency type viral envelope glycoprotein; hepatitis B surface antigen; diphtheria toxin, diphtherotoxin; chain diplococcus 24M epi-position; the gonococcus pilin; pseudorabies virus g50; the pseudorabies virus glycoprotein h; the pseudorabies virus glycoprotein E; transmissible gastroenteritis glycoprotein 195; the transmissible gastroenteritis stromatin; porcine rotavirus glycoprotein 38, pig piconavirus capsid protein, the little Serpentis bacterium of swine dysentery protective antigen; bovine viral diarrhoea glycoprotein 55; ewcastle disease virus hemagglutinin-neuraminidase, swine flue hemagglutinin, swine flue neuraminidase; infectious Nasus Bovis seu Bubali trachea virus glycoprotein E; infectious pharynx tracheitis viral glycoprotein G or I, La Crosse viral glycoprotein, new calves diarrhea virus; hepatitis B virus core albumen; hepatitis B virus surface antigen, equine influenza virus A type/Alaska 91 neuraminidases, A type equine influenza virus/Miami 63 neuraminidases; A type equine influenza virus/Kentucky 81 neuraminidases; I type equine herpes virus Glycoprotein B, I type equine herpes virus glycoprotein D, self-conceit pipe syncytial virus attachment proteins; self-conceit pipe syncytial virus fusion rotein; self-conceit pipe syncytial virus nucleocapsid protein, III type bovine parainfluenza virus fusion rotein, III type bovine parainfluenza virus hemagglutinin-neuraminic acid pheron; bovine viral diarrhea virus glycoprotein 48, bovine viral diarrhea virus glycoprotein 53.
17. according to the vaccine combination of claim 1, wherein said antigen is the cell receptor of infectious disease cause of disease.
18. according to the vaccine combination of claim 17, wherein said cell receptor is selected from: LPV receptor, adenyl cyclase, BDV surface glycoprotein, N-acetyl-9-0-n acetylneuraminic acid n receptor, CD4 +, height sulphation type heparin sulfate, P65, contain Gal-α 1-4-isoreceptor, CD16b integrates plain VLA-2 receptor, the EV receptor, CD14, carbohydrate conjugates receptor, TXi Baoshouti α/β, decay accelerating factor receptor, born of the same parents' peplos glycoprotein receptor, the immunoglobulin Fc receptor, poxvirus M-T7, GALV receptor, the CD14 receptor, Lewis (b) blood group antigen receptor, TXi Baoshouti, heparin sulfate glycosaminoglycans receptor, bfgf receptor, CD11a, CD2, g protein coupled receptor, CD4, the heparin sulfate Dan Baijutang, calphobindin II, CD13 (neural Aminopeptidase N), the neural Aminopeptidase N receptor of people, hemagglutinin receptor, CR3 receptor, the protein kinase receptor, galactose N-acetylgalactosamine-inhibition agglutinin receptor, chemokine receptors, calphobindin I, actA albumen, CD46 receptor, the opa receptor that meningococcus toxicity is relevant, the CD46 receptor, carcinoembryonic antigen family receptors, the β glA of carcinoembryonic antigen family receptor, interferon-gamma receptor, glycoprotein gp70, rmc-1 receptor, people's integrin receptor α v β 3, heparin sulfate Dan Baijutang receptor, the CD66 receptor, integrin receptor, film cofactor receptor, CD46, GM1, GM2, GM3, CD3, ceramide, hemagglutinin-neuraminic acid pheron, erythrocyte P antigen receptor, the CD36 receptor, the alpha-Glycophorins receptor, interferon gamma receptor, kdel receptor, mucin α 4/ β 7 receptors of going back to the nest, EGF-R ELISA, α 5/ β 1 integrates fibroin, non-glycosylated J774 receptor, the CXCR1-4 receptor, the CCR1-5 receptor, CXCR3 receptor, CCR5 receptor, the gp46 surface glycoprotein, the TNFRp55 receptor, TNFp75 receptor, soluble interleukin-6-1 receptor β.
19. according to the vaccine combination of claim 15 or 17, wherein said infectious disease cause of disease is an antibacterial.
20. according to the vaccine combination of claim 19, wherein said antibacterial is selected from: mycobacterium rickettsia, mycoplasma, naphthalene plucked instrument Salmonella class, Legionnella, Shigella, vibrio cholera, Streptococcus, diphtheria corynebacterium, Clostridium tetani, bordetella pertussis, haemophilus class, chlamydia class, bacillus coli, or syphilis or feel seven color arthritis substance.
21. according to the vaccine combination of claim 15 or 17, wherein said infectious disease cause of disease is a virus.
22. according to the vaccine combination of claim 21, wherein said virus is selected from: hepatitis A virus (HAV), hepatitis B virus, hepatitis C virus, influenza virus, chickenpox virus, adenovirus, I herpes simplex virus type, II herpes simplex virus type, rinderpest virus, rhinovirus, Chinese mugwort can be viral, rotavirus, respiratory syncytial virus, human papillomavirus, papovavirus, giant cell virus, thorn-like virus, arbovirus, Chinese tower virus, Coxsackie virus, popular mumps virus, Measles virus, rubella virus, poliovirus, I type human immune deficiency type virus, II type human immune deficiency type virus, Pironavirus, enterovirus, calicivirus, Norwalk virus (Norwalk) group, togavirus, A Er are fallen ill malicious, yellow fever virus, coronavirus, rabies virus, Marburg virus, Ebola virus, parainfluenza virus, influenza virus, this refined virus, arenavirus, arc reovirus virus, rotavirus, Orbivirus, I type human T-leukemia virus, II type human T-leukemia virus, the troglodyte immunodeficiency virus, slow virus, polyoma virus, parvovirus, Epstein-Barr virus, nerpes vinrus hominis-6, I type cercopithecid herpesvirus, poxvirus.
23. according to the vaccine combination of claim 15 or 17, wherein said infectious disease cause of disease is a parasite.
24. according to the vaccine combination of claim 23, wherein said parasite is selected from: Plasmodium, eimeria, Leishmania, cocoa ground inferior Eimeria, trypanosoma and fungus.
25. according to the vaccine combination of claim 1, the wherein said first immunoglobulin molecules type is selected from: IgG, IgE, IgM, IgD and IgA.
26. vaccine combination, comprise a certain amount of first immunoglobulin molecules that is enough to induced anti-idiotype reaction, this first immunoglobulin fragment contains one section variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin, the wherein said second immunoglobulin immunologic opsonin conjugated antigen, said one or more amino acid whose replacement refer to replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with the aminoacid that does not contain sulfydryl; With pharmaceutically acceptable carrier.
27. according to the vaccine combination of claim 26, wherein said fragment is the strand immunoglobulin.
28. according to the vaccine combination of claim 26, wherein said fragment is the Fab fragment, (Fab ') 2Fragment, heavy chain homodimer, light chain dimer or Fv fragment.
29. according to the vaccine combination of claim 26, wherein said fragment also comprises constant region.
30. according to the vaccine combination of claim 26, wherein the variable region derives from rat immune globulin, constant region then derives from the human normal immunoglobulin.
31. according to the vaccine combination of claim 1, wherein there are frame structure district that comes from people's antibody and the immunoglobulin complementary determining region (CDRs) that comes from mice in the variable region.
32. vaccine combination according to claim 1, the wherein said first immune globulin antibody molecule is by in the aminoacid sequence of covalently bound extremely one of following albumen: IL-2, IL-4, IL-5, IL-, IL-7, IL-10 or MHC derived peptide, G-CSF, TNF, punching is plain, NK cellular antigens or cell content action receptor.
33. vaccine combination according to claim 26, the fragment of wherein said first immunoglobulin molecules is covalently bound to the aminoacid sequence that is selected from one of following albumen: IL-2, IL-4, IL-5, IL-, IL-7, IL-10 or, ν interferon MHC derived peptide, G-CSF, TNF, punching is plain, NK cellular antigens or cell content action receptor.
34. in the experimenter, produce the method for anti-idiotype reaction, comprise to the experimenter and use a certain amount of first immunoglobulin molecules that is enough to induced anti-idiotype reaction, this first immunoglobulin contains one section variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin molecules, the wherein said second immunoglobulin immunologic opsonin conjugated antigen, said one or more amino acid whose replacement refer to replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with the aminoacid that does not contain sulfydryl.
35. the method according to claim 34 also further comprises: separation antibody from this experimenter, the antibody of said second immunoglobulin molecules of described antibody recognition uses to second experimenter this antibody then.
36. according to the method for claim 34, wherein said antigen is tumor antigen.
37. according to the method for claim 34, wherein said antigen is cancer antigen.
38. according to the method for claim 36, wherein said antigen is polymorphic endotheliocyte mucin antigen.
39. according to the method for claim 36, wherein said antigen is human colon carcinoma associated protein antigen.
40. according to the method for claim 36, wherein said antigen is colon cancer associated carbon hydrate antigen.
41. according to the method for claim 35, wherein said variable region is a variable region of light chain, said 23 or 88 amino acids that do not contain the mercaptoamino acid residue corresponding to second immunoglobulin light chain variable region.
42. according to the method for claim 35, wherein said variable region is a variable region of heavy chain, said 22 or 92 amino acids that do not contain mercaptoamino acid corresponding to second heavy chain immunoglobulin.
43. according to the method for claim 34, the wherein said mercaptoamino acid residue that do not contain is an alanine.
44. according to the method for claim 34, wherein said second immunoglobulin is Mab31.1, Mab33.28 or Mab HMFG-1, and he substitutes 23 and/or 88 the aminoacid that comprises with the alternative chain variable region gene of alanine wherein said aminoacid.
45. according to the vaccine combination of claim 1, wherein said second immunoglobulin is Mab31.1, Mab33.28 or Mab HMFG-1, and he substitutes 22 and/or 92 the aminoacid that comprises with the alternative heavy chain variable region gene of alanine wherein said aminoacid.
46. according to the method for claim 34, wherein said antigen is the human milk fat microglobulin.
47. according to the method for claim 34, wherein said antigen is mammary gland, ovary, uterus, prostate, bladder, lung, skin, colon, pancreas, gastrointestinal tract, B cell or T cell carcinoma antigen.
48. according to the vaccine combination of claim 3, wherein said antigen is selected from: KS1/4 is general-cancer antigen, ovarian cancer antigen, prostanoic acid phosphoric acid, prostate specific antigen, melanic related antigen p97, melanoma-associated antigen p75, the high molecular melanoma-associated antigen, the membrane antigen of prostate-specific, carcinoembryonic antigen, polymorphic endotheliocyte mucin antigen, human milk fat microglobulin antigen, colon cancer related antigen TAG-72, C017-1A, GICA 19-9, CTA-1, LEA, burkitt's lymphoma antigen-38.13, CD19, people B lymphoma antigen-CD20, CD33, ganglioside 6D2, Ganglioside, GD3, Ganglioside GM2, Ganglioside GM3, tumour-specific transplantation type cell surface antigen, carcinoembryonic antigen-α-embryo's albumen L6, human lung cancer antigen L20, human leukemia T cellular antigens-Gp37, neoglycoprotein, sphingolipid, EGFR, HER2 antigen, polymorphic epidermis mucin, human malignant lesion lymphocyte antigen-APO-1, I antigen M18, M39, SSEA-1, VEP8, VEP9, Myl, VIM-D5, D 156-22, TRA-1-85, C14, F3, AH6, Y hapten, Le y, TL5, FC10.2, adenocarcinoma of stomach antigen, C0-514, NS-10, C0-43, MH2, in the colon cancer 19.9, gastric cancer mucin, T 5A 7, R 24, 4.2, G D3, D1.1, OFA-1, G M2, OFA-2, G D2, M1:22:25:8, SSEA-3, SSEA-4 and the peptide that is derived from TXi Baoshouti.
49. according to the method for claim 34, wherein said antigen is the infectious disease pathogen antigen.
50. vaccine combination according to claim 15; wherein said antigen is selected from: influenza virus hemagglutinin; people's trachea syncytial virus G glycoprotein; the dengue virus core protein; the dengue virus stromatin; the Measles virus hemagglutinin; II herpes simplex virus type glycoprotein gB; I type poliovirus VP1; I type human immune deficiency type viral envelope glycoprotein; hepatitis B surface antigen; diphtheria toxin, diphtherotoxin; chain diplococcus 24M epi-position; the gonococcus pilin; pseudorabies virus g50; the pseudorabies virus glycoprotein h; the pseudorabies virus glycoprotein E; transmissible gastroenteritis glycoprotein 195; the transmissible gastroenteritis stromatin; porcine rotavirus glycoprotein 38, pig piconavirus capsid protein, the little Serpentis bacterium of swine dysentery protective antigen; bovine viral diarrhoea glycoprotein 55; ewcastle disease virus hemagglutinin-neuraminidase, swine flue hemagglutinin, swine flue neuraminidase; infectious Nasus Bovis seu Bubali trachea virus glycoprotein E; infectious pharynx tracheitis viral glycoprotein G or I, La Crosse viral glycoprotein, new calves diarrhea virus; hepatitis B virus core albumen; hepatitis B virus surface antigen, equine influenza virus A type/Alaska 91 neuraminidases, A type equine influenza virus/Miami 63 neuraminidases; A type equine influenza virus/Kentucky 81 neuraminidases; I type equine herpes virus Glycoprotein B, I type equine herpes virus glycoprotein D, self-conceit pipe syncytial virus attachment proteins; self-conceit pipe syncytial virus fusion rotein; self-conceit pipe syncytial virus nucleocapsid protein, III type bovine parainfluenza virus fusion rotein, III type bovine parainfluenza virus hemagglutinin-neuraminic acid pheron; bovine viral diarrhea virus glycoprotein 48, bovine viral diarrhea virus glycoprotein 53.
51. according to the method for claim 34, wherein said antigen is the cell receptor of infectious disease cause of disease.
52. according to the vaccine combination of claim 17, wherein said cell receptor is selected from: LPV receptor, adenyl cyclase, BDV surface glycoprotein, N-acetyl-9-0-n acetylneuraminic acid n receptor, CD4 +, height sulphation type heparin sulfate, P65, contain Gal-α 1-4-isoreceptor, CD16b integrates plain VLA-2 receptor, the EV receptor, CD14, carbohydrate conjugates receptor, TXi Baoshouti α/β, decay accelerating factor receptor, born of the same parents' peplos glycoprotein receptor, the immunoglobulin Fc receptor, poxvirus M-T7, GALV receptor, CD14 receptor, Lewis (b) blood group antigen receptor, TXi Baoshouti, heparin sulfate glycosaminoglycans receptor, bfgf receptor, CD11a, CD2, g protein coupled receptor, CD4, the heparin sulfate Dan Baijutang, calphobindin II, CD13 (neural Aminopeptidase N), the neural Aminopeptidase N receptor of people, the hemagglutinin receptor, the CR3 receptor, protein kinase receptor, galactose N-acetylgalactosamine-inhibition agglutinin receptor, chemokine receptors, calphobindin I, actA albumen, CD46 receptor, the opa receptor that meningococcus toxicity is relevant, the CD46 receptor, carcinoembryonic antigen family receptors, the BglA of carcinoembryonic antigen family receptor, interferon-gamma receptor, glycoprotein gp70, rmc-1 receptor, people's integrin receptor α vβ 3, heparin sulfate Dan Baijutang receptor, CD66 receptor, integrin receptor, film cofactor receptor, CD46, GM1, GM2, GM3, CD3, ceramide, hemagglutinin-neuraminic acid pheron, erythrocyte P antigen receptor, CD36 receptor, alpha-Glycophorins receptor, interferon gamma receptor, kdel receptor, mucin α 4/ β 7 receptors of going back to the nest, EGF-R ELISA, α 5/ β 1 integrates fibroin, non-glycosylated J774 receptor, the CXCR1-4 receptor, CCR1-5 receptor, CXCR3 receptor, the CCR5 receptor, gp46 surface glycoprotein, TNFRp55 receptor, the TNFp75 receptor, soluble interleukin-6-1 receptor β.
53. according to the method for claim 49 or 51, wherein said infectious disease cause of disease is an antibacterial.
54. according to the vaccine combination of claim 19, wherein said antibacterial is selected from: mycobacterium rickettsia, mycoplasma, naphthalene plucked instrument Salmonella class, Legionnella, Shigella, vibrio cholera, Streptococcus, diphtheria corynebacterium, Clostridium tetani, bordetella pertussis, haemophilus class, chlamydia class, bacillus coli, or syphilis or infection color arthritis substance.
55. according to the method for claim 49 or 51, wherein said infectious disease cause of disease is a virus.
56. according to the vaccine combination of claim 21, wherein said virus is selected from: hepatitis A virus (HAV), hepatitis B virus, hepatitis C virus, influenza virus, chickenpox virus, adenovirus, I herpes simplex virus type, II herpes simplex virus type, rinderpest virus, rhinovirus, Chinese mugwort can be viral, rotavirus, respiratory syncytial virus, human papillomavirus, papovavirus, giant cell virus, thorn-like virus, arbovirus, Chinese tower virus, Coxsackie virus, popular mumps virus, Measles virus, rubella virus, poliovirus, I type human immune deficiency type virus, II type human immune deficiency type virus, Pironavirus, enterovirus, calicivirus, Norwalk virus (Norwalk) group, togavirus, A Er are fallen ill malicious, yellow fever virus, coronavirus, rabies virus, Marburg virus, Ebola virus, parainfluenza virus, influenza virus, this refined virus, arenavirus, arc reovirus virus, rotavirus, Orbivirus, I type human T-leukemia virus, II type human T-leukemia virus, the troglodyte immunodeficiency virus, slow virus, polyoma virus, parvovirus, Epstein-Barr virus, nerpes vinrus hominis-6, I type cercopithecid herpesvirus, poxvirus.
57. according to the method for claim 49 or 51, wherein said infectious disease cause of disease is a parasite.
58. according to the method for claim 57, wherein said parasite is selected from: Plasmodium, eimeria, Leishmania, cocoa ground inferior Eimeria, trypanosoma and fungus.
59. according to the method for claim 34, the wherein said first immunoglobulin molecules type is selected from: IgG, IgE, IgM, IgD and IgA.
60. in a certain experimenter, produce anti-idiotype reaction method, comprise a certain amount of first immunoglobulin molecules that is enough to induced anti-idiotype reaction, this first immunoglobulin contains one section variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin molecules, the wherein said second immunoglobulin immunologic opsonin conjugated antigen, said one or more amino acid whose replacement refer to replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with the aminoacid that does not contain sulfydryl.
61. according to the method for claim 60, also further comprise separation antibody from this experimenter, said second immunoglobulin molecules of described antibody recognition, and use to second experimenter this antibody.
62. according to the method for claim 60, wherein said antigen is tumor antigen.
63. according to the method for claim 60, wherein said antigen is cancer antigen.
64. according to the method for claim 60, wherein said antigen is polymorphic endotheliocyte mucin antigen.
65. according to the method for claim 62, wherein said antigen is human colon carcinoma associated protein antigen.
66. according to the method for claim 62, wherein said antigen is human colon carcinoma associated carbon hydrate antigen.
67. according to the method for claim 60, wherein said variable region is a variable region of light chain, said 23 or 88 amino acids that do not contain the mercaptoamino acid residue corresponding to second immunoglobulin light chain variable region.
68. according to the method for claim 60, wherein said variable region is a variable region of heavy chain, said 22 or 92 amino acids that do not contain the mercaptoamino acid residue corresponding to second heavy chain immunoglobulin.
69. according to the method for claim 60,67 or 68, the wherein said mercaptoamino acid residue that do not contain is an alanine.
70. according to the method for claim 63, wherein said antigen is the human milk fat microglobulin.
71. according to the method for claim 63, wherein said antigen is breast carcinoma, ovarian cancer, uterus carcinoma, carcinoma of prostate, bladder cancer, pulmonary carcinoma, skin carcinoma, colon cancer, cancer of pancreas, gastrointestinal cancer, B cell carcinoma or T cell carcinoma antigen.
72. according to the method for claim 60, wherein said antigen is the infectious disease pathogen antigen.
73. vaccine combination according to claim 15; wherein said antigen is selected from: influenza virus hemagglutinin; people's trachea syncytial virus G glycoprotein; the dengue virus core protein; the dengue virus stromatin; the Measles virus hemagglutinin; II herpes simplex virus type glycoprotein gB; I type poliovirus VP1; I type human immune deficiency type viral envelope glycoprotein; hepatitis B surface antigen; diphtheria toxin, diphtherotoxin; chain diplococcus 24M epi-position; the gonococcus pilin; pseudorabies virus g50; the pseudorabies virus glycoprotein h; the pseudorabies virus glycoprotein E; transmissible gastroenteritis glycoprotein 195; the transmissible gastroenteritis stromatin; porcine rotavirus glycoprotein 38, pig piconavirus capsid protein, the little Serpentis bacterium of swine dysentery protective antigen; bovine viral diarrhoea glycoprotein 55; ewcastle disease virus hemagglutinin-neuraminidase, swine flue hemagglutinin, swine flue neuraminidase; infectious Nasus Bovis seu Bubali trachea virus glycoprotein E; infectious pharynx tracheitis viral glycoprotein G or I, La Crosse viral glycoprotein, new calves diarrhea virus; hepatitis B virus core albumen; hepatitis B virus surface antigen, equine influenza virus A type/Alaska 91 neuraminidases, A type equine influenza virus/Miami 63 neuraminidases; A type equine influenza virus/Kentucky 81 neuraminidases; I type equine herpes virus Glycoprotein B, I type equine herpes virus glycoprotein D, self-conceit pipe syncytial virus attachment proteins; self-conceit pipe syncytial virus fusion rotein; self-conceit pipe syncytial virus nucleocapsid protein, III type bovine parainfluenza virus fusion rotein, III type bovine parainfluenza virus hemagglutinin-neuraminic acid pheron; bovine viral diarrhea virus glycoprotein 48, bovine viral diarrhea virus glycoprotein 53.
74. according to the method for claim 60, wherein said antigen is the cell receptor of infectious disease cause of disease.
75. according to the vaccine combination of claim 17, wherein said cell receptor is selected from: LPV receptor, adenyl cyclase, BDV surface glycoprotein, N-acetyl-9-0-n acetylneuraminic acid n receptor, CD4 +, height sulphation type heparin sulfate, P65, contain Gal-α 1-4-isoreceptor, CD16b integrates plain VLA-2 receptor, the EV receptor, CD14, carbohydrate conjugates receptor, TXi Baoshouti α/β, decay accelerating factor receptor, born of the same parents' peplos glycoprotein receptor, the immunoglobulin Fc receptor, poxvirus M-T7, GALV receptor, CD14 receptor, Lewis (b) blood group antigen receptor, TXi Baoshouti, heparin sulfate glycosaminoglycans receptor, bfgf receptor, CD11a, CD2, g protein coupled receptor, CD4, the heparin sulfate Dan Baijutang, calphobindin II, CD13 (neural Aminopeptidase N), the neural Aminopeptidase N receptor of people, the hemagglutinin receptor, the CR3 receptor, protein kinase receptor, galactose N-acetylgalactosamine-inhibition agglutinin receptor, chemokine receptors, calphobindin I, actA albumen, CD46 receptor, the opa receptor that meningococcus toxicity is relevant, the CD46 receptor, carcinoembryonic antigen family receptors, the BglA of carcinoembryonic antigen family receptor, interferon-gamma receptor, glycoprotein gp70, rmc-1 receptor, people's integrin receptor α vβ 3, heparin sulfate Dan Baijutang receptor, CD66 receptor, integrin receptor, film cofactor receptor, CD46, GM1, GM2, GM3, CD3, ceramide, hemagglutinin-neuraminic acid pheron, erythrocyte P antigen receptor, CD36 receptor, alpha-Glycophorins receptor, interferon gamma receptor, kdel receptor, mucin α 4/ β 7 receptors of going back to the nest, EGF-R ELISA, α 5/ β 1 integrates fibroin, non-glycosylated J774 receptor, the CXCR1-4 receptor, CCR1-5 receptor, CXCR3 receptor, the CCR5 receptor, gp46 surface glycoprotein, TNFRp55 receptor, the TNFp75 receptor, soluble interleukin-6-1 receptor β.
76. according to the method for claim 72 or 74, wherein said infectious disease cause of disease is an antibacterial.
77. according to the vaccine combination of claim 19, wherein said antibacterial is selected from: mycobacterium rickettsia, mycoplasma, naphthalene plucked instrument Salmonella class, Legionnella, Shigella, vibrio cholera, Streptococcus, diphtheria corynebacterium, Clostridium tetani, bordetella pertussis, haemophilus class, chlamydia class, bacillus coli, or syphilis or infection color arthritis substance.
78. according to the method for claim 72 or 74, wherein said infectious disease cause of disease is a virus.
79. according to the vaccine combination of claim 21, wherein said virus is selected from: hepatitis A virus (HAV), hepatitis B virus, hepatitis C virus, influenza virus, chickenpox virus, adenovirus, I herpes simplex virus type, II herpes simplex virus type, rinderpest virus, rhinovirus, Chinese mugwort can be viral, rotavirus, respiratory syncytial virus, human papillomavirus, papovavirus, giant cell virus, thorn-like virus, arbovirus, Chinese tower virus, Coxsackie virus, popular mumps virus, Measles virus, rubella virus, poliovirus, I type human immune deficiency type virus, II type human immune deficiency type virus, Pironavirus, enterovirus, calicivirus, Norwalk virus (Norwalk) group, togavirus, A Er are fallen ill malicious, yellow fever virus, coronavirus, rabies virus, Marburg virus, Ebola virus, parainfluenza virus, influenza virus, this refined virus, arenavirus, arc reovirus virus, rotavirus, Orbivirus, I type human T-leukemia virus, II type human T-leukemia virus, the troglodyte immunodeficiency virus, slow virus, polyoma virus, parvovirus, Epstein-Barr virus, nerpes vinrus hominis-6, I type cercopithecid herpesvirus, poxvirus.
80. according to the method for claim 72 or 74, wherein said infectious disease cause of disease is a parasite.
81. 0 method according to Claim 8, wherein said parasite is selected from: Plasmodium, eimeria, Leishmania, cocoa ground inferior Eimeria, trypanosoma and fungus.
82. according to the method for claim 60, wherein said fragment is the strand immune globulin antibody.
83. 2 method according to Claim 8, wherein said fragment is the Fab fragment, (Fab ') 2Fragment or Fv fragment.
84. according to the method for claim 60, wherein said fragment comprises constant region.
85. 4 method according to Claim 8, wherein the variable region derives from rat immune globulin, and constant region then derives from the human normal immunoglobulin.
86. according to the method for claim 60, wherein there are frame structure district that comes from people's antibody and the immunoglobulin complementary determining region (CDRs) that derives from Mus in the variable region.
87. carry out among the experimenter of treatment of cancer or prevention at a certain needs, carry out the method for cancer prevention and treatment, comprise to this experimenter and use vaccine combination, comprise a certain amount of first immunoglobulin molecules that is enough to induced anti-idiotype reaction, this first immunoglobulin molecules contains one section variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin molecules, the wherein said second immunoglobulin molecules immunologic opsonin conjugated antigen, said one or more amino acid whose replacement refer to replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with the aminoacid that does not contain sulfydryl; With pharmaceutically acceptable carrier.
88. carry out among the experimenter of the prevention of infectious disease and treatment at needs, prevention and the method for the treatment of infectious disease, comprise to this experimenter and use vaccine combination, comprise a certain amount of first immunoglobulin molecules that is enough to induced anti-idiotype reaction, this first immunoglobulin molecules contains one section variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin molecules, wherein said second immunoglobulin immunologic opsonin identification infectious disease pathogen antigen, said one or more amino acid whose replacement refers to replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with the aminoacid that does not contain sulfydryl; With pharmaceutically acceptable carrier.
89. the method for carrying out prevention and treatment infectious disease among the experimenter of infectious disease treatment or prevention at needs, comprise to this experimenter and use vaccine combination, comprise a certain amount of first immunoglobulin molecules that is enough to induced anti-idiotype reaction, this first immunoglobulin contains one section variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin molecules, wherein said second immunoglobulin immunologic opsonin identification infectious disease pathogenic cell bind receptor, said one or more amino acid whose replacement refers to replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with the aminoacid that does not contain sulfydryl; With pharmaceutically acceptable carrier.
90. 8 or 89 method according to Claim 8, wherein said infectious disease cause of disease is selected from: syphilis, gonorrhea, AIDS, malaria, shigella infection, Salmonella infection, hepatitis A, hepatitis C infects color arthritis, encephalitis, herpes, Gram-positive and gram positive bacterial infection, Diplococcus pneumoniae infects.
91. preparation is enough to the method for induced anti-idiotype reaction first immunoglobulin molecules, this first immunoglobulin contains one section variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin molecules, the wherein said second immunoglobulin immunologic opsonin conjugated antigen, said one or more amino acid whose replacement refers to replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with the aminoacid that does not contain sulfydryl, and said method comprises step: thus the nucleotide that (a) replaces one or more cysteine residues of coding in the second immunoglobulin nucleic acid by the nucleotide that does not contain one or more residue of sulfydryl with coding makes up the nucleic acid of described first immunoglobulin molecules of encoding; (b) the introducing cell of the nucleotide sequence that step (a) is made up, and by said first immunoglobulin of this cellular expression; (c) reclaim first immunoglobulin of expressing.
92. according to the method for claim 91, wherein replacement nucleic acid adopts rite-directed mutagenesis.
93. preparation is enough to the method for induced anti-idiotype reaction first immunoglobulin molecules, this first immunoglobulin contains one section variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin molecules, the wherein said second immunoglobulin immunologic opsonin conjugated antigen, said one or more amino acid whose replacement refers to replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with the aminoacid that does not contain sulfydryl, comprises step; (a) the synthetic nucleic acid that contains the artificial gene of described first immunoglobulin molecules of encoding; (b) nucleotide sequence that step (a) is made up is introduced cell, and by said first immunoglobulin of this cellular expression; (c) reclaim first immunoglobulin molecules of expressing.
94. according to the said second immune globulin antibody molecule of the method for claim 91 or 93 is Mab31.1, or Mab33.28
95. according to the method for claim 91 or 93, the wherein said second immune globulin antibody molecule is HMFG-1.
96. vaccine combination, comprise a certain amount of first immunoglobulin molecules that is enough to cause anti-idiotype reaction, this first immunoglobulin contains one section variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin molecules, the wherein said second immunoglobulin immunologic opsonin conjugated antigen, during said one or more aminoacid is replaced at least some refer to not replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with not containing the aminoacid of dredging base, if wherein amino acid replacement is not above-mentioned alternate words, it is unsettled then substituting, and above-mentioned replacement is meant in one or more site of one or more cysteine residues of the formation disulfide bond of the described second immunoglobulin correspondence and replaces with the aminoacid that one or more does not contain sulfydryl; With pharmaceutically acceptable carrier.
97. in a certain experimenter, produce the method for anti-idiotype reaction, comprise to described experimenter and use first immunoglobulin molecules that is enough to the induced anti-idiotype reaction amount, this first immunoglobulin contains one section variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin molecules, the wherein said second immunoglobulin immunologic opsonin conjugated antigen, during said one or more aminoacid is replaced at least some refer to not replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with not containing the aminoacid of dredging base, if wherein amino acid replacement is not above-mentioned alternate words, it is unsettled then substituting, and above-mentioned replacement is meant in one or more site of one or more cysteine residues of the formation disulfide bond of the described second immunoglobulin correspondence and replaces with the aminoacid that one or more does not contain sulfydryl.
98. first immunoglobulin molecules, comprise and contain one section variable region, one or more aminoacid are substituted in the variable region, remaining aminoacid is consistent with second immunoglobulin molecules, the wherein said second immunoglobulin immunologic opsonin conjugated antigen, during said one or more aminoacid is replaced at least some refer to not replace cysteine in one or more site of the correspondence of one or more cysteine of the formation disulfide bond of said second immunoglobulin molecules with not containing the aminoacid of dredging base, if wherein amino acid replacement is not above-mentioned alternate words, it is unsettled then substituting, and above-mentioned replacement is meant in one or more site of one or more cysteine residues of the formation disulfide bond of the described second immunoglobulin correspondence and replaces with the aminoacid that one or more does not contain sulfydryl.
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* Cited by examiner, † Cited by third party
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CN105705522A (en) * 2013-05-14 2016-06-22 上海亨臻实业有限公司 Epitope vaccine for low immunogenic protein and preparing method and usage thereof
CN105705522B (en) * 2013-05-14 2020-09-15 上海亨臻实业有限公司 Epitope vaccine aiming at low-immunogenicity protein and preparation method and application thereof
CN105693859A (en) * 2016-03-22 2016-06-22 苏州莱泰生物科技有限公司 Anti-human-G2A monoclonal antibody and kit for detecting human macrophage G2A expression quantity
CN105693859B (en) * 2016-03-22 2019-06-21 苏州莱泰生物科技有限公司 Anti-human G2A monoclonal antibody and the kit for detecting human macrophage G2A expression quantity
CN115856296A (en) * 2022-12-16 2023-03-28 华北理工大学 Anti-shigella monoclonal antibody and application thereof in detection

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