CN1294517A - Immunoglobulin molecules having synthetic variable region and modified specificity - Google Patents

Immunoglobulin molecules having synthetic variable region and modified specificity Download PDF

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CN1294517A
CN1294517A CN98813117A CN98813117A CN1294517A CN 1294517 A CN1294517 A CN 1294517A CN 98813117 A CN98813117 A CN 98813117A CN 98813117 A CN98813117 A CN 98813117A CN 1294517 A CN1294517 A CN 1294517A
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modified immunoglobulin
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antigen
variable region
cdr
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R·M·布尔克
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Euro Celtique SA
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Abstract

The invention provides modified immunoglobulin molecules, particularly antibodies, that immunospecifically bind a member of a binding pair which immunoglobulins have a variable domain containing one or more complimentary determining regions that contain the amino acid sequence of a binding site for that member of the binding pair, which site is derived from the other member of the binding pair and is not naturally found in the complementary determining region. The invention further provides for therapeutic and diagnostic use of the modified immunoglobulin.

Description

Immunoglobulin molecules with synthetic variable region and modified specificity
The cross reference of related application
The application requires temporary patent application series number 60/065, November 4 1997 716 submission date and temporary patent application series number 60/081,403, the right in April 10 1998 submission date, described 2 patents are incorporated herein only for reference as a whole.
1. invention field
The immunoglobulin molecules that the present invention relates to modify, especially antibody, these molecules with combine a right member in conjunction with and have a complementary determining region (CDR) at least, this complementary determining region contains at the aminoacid sequence of this combination to member's binding site, and described binding site derives from conjunction with other right member.The present invention also relates to use modified antibodies of the present invention, treatment, diagnosis or screening are expressed relevant disease and imbalance with this combination to the member, especially the method for cancer or communicate illness.The present invention also relates to contain the Pharmaceutical composition and the diagnostic kit of modified antibodies of the present invention.
2. background of invention
2.1 antibody and immune system
Antibody is the albumen of contactin.Immunoglobulin superfamily comprises TXi Baoshouti, B-cell receptor, cell surface adhesion molecule, as the constant region of co-receptor CD4, CD8, CD19 and MHC molecule.With its soluble form, antibody is by being also referred to as the glycoprotein that plasmacytic mature B cell produces.In all animal and human's classes were replied exotic antigen, antibody was secreted in blood and other extracellular fluids, at the whole machine body internal recycle.
Antibody has two main functions.First function be identification or in conjunction with exotic antigen.Second function is to mobilize immune other factors to destroy exotic.Receptor on the immune effector cell surface is designed for the cell surface marker on identification antigen and other cells.This identifying transmission whether these signs belongs to self or nonself information, and is a kind of relating to regulate the key factor of immune system when replying there being antigen.
Be called as its antigenic determinant or epi-position with the antigen part of antibodies, some antigen can excite immunne response, and other antigens as autoinducer molecule by immune system recognition.Can excite the antigen of immunne response to be called immunogen, they are molecular weight 5000 daltonian macromole usually at least, as albumen, nucleic acid, saccharide and lipid.Be called haptenic less non-immunogen molecule, when with a larger vector molecule coupling, also can excite immunne response.
2.2 the structure of antibody
Substantially the complete unit of antibody is one four a chain y-type structure (Fig. 1).Early stage in the 1970's, Wu and Kabat have spliced the aminoacid sequence of a large amount of collected antibody and have illustrated the structure of antibody, in fact the member of all immunoglobulin superfamilies is made up of the rack area of the relative semi-rigid βZhe Die of guarding with 4 of 1 constant region, hypermutation sequence area alternate wherein (Wu and the Kabat in district (CDRs) determined in 3 short relatively complementations of knowing, 1970, The Journal of Experimental Medicine, 132 (2): 211-250; Wu and Kabat, 1971, institute of NAS newspaper 68 (7): 1501-1506).This prediction is confirmed (Poljak etc., 1973, institute of NAS newspaper 70 (12): 3305-3310 by the crystallization spectroscopy study of antibody structure; Disenhofer etc. 1976, Hopper Seylers Z Physiol Chem (West Germany) 357 (10): 435-445; Disenhofer etc. 1976, Hopper Seylers ZPhysiol Chem (West Germany) 357 (10): 1421-1434).
Fig. 1 represents the overall structure of antibody molecule.Antibody is linked together by disulfide bond and 2 long heavy chains by 2 short light chains and forms, and is linked together by disulfide bond between the heavy chain.As shown in Figure 2, the heavy chain of antibody protein and light chain comprise a plurality of districts, about 110 amino acid residues of the length in each district.Every the light chain and the heavy chain of antibody have a variable region (being called VL and VH) at its amino terminal; The specificity of conjugated antigen is given in the variable region of antibody just.A variable region of heavy chain and a variable region of light chain form single antigen binding site together, and therefore, basic immunoglobulin unit has 2 antigen binding sites.
Multiformity in the variable region of light chain and heavy chain is limited in 3 " hypermutation " district or CDRs.Be total up to 6 CDRs (Fig. 2) in each antibody molecule, each CDR contains about 5～10 aminoacid, when CDR with endogenous mechanism in conjunction with the time can reach about 20 aminoacid, this situation is common in some antibody types.3 CDRs of every light chain and variable region of heavy chain form ring bunch together, and are linked to the 4th of variable region and are called framework region (" FRS ") remainder, and framework region is conservative relatively in antibody molecule.In general, the multiformity of antibody changes generation by the sequence of CDRs.
The variable region is all different and constant region has higher conservative concerning every kind of antibody.Light chain only has a constant region domain, and CH includes a plurality of domains, called after CH1, CH2, CH3 ... CHx.Constant region is born various antibody mediated effect device functions in conjunction with the territory, for example the complement combination and with the combining of Fc receptor, the Fc receptor is expressed by lymphocyte, granulocyte, monokaryon pedigree cell, the killer cell that stimulates B cell proliferation, mastocyte and other immune effector cells.Other effector functions are that differentiation, the activation of complementation cell cracking system, opsonic action, macrophage are lured.Different isotype antibodies have different constant regions, and therefore have different effector functions.The darkest isotype of research is IgG and IgM.
All animal species are expressed several different antibody types.According to structural difference, number as immunoglobulin unit in single antibody molecule, the diversity of the structure of each unit disulfide bond and chain length and sequence, people's antibody can be divided into 5 types (IgG, IgA, IgM, IgD and IgB), in these types to be divided into various five classes.Up to now, IgG antibody is the most frequently used kind during diagnosis and treatment pharmacy are used, although from the use of the antibody of other type in some purposes as seen.
2.3 antibody engineering
The exploitation of the monoclonal antibody technique that is disclosed first by Kohler and Milstein (1975, natural 256:495-497) can allow to produce endless specificity accurately and the antibody that can repeat to produce.The program of Koh1er and Milstein comprises the splenocyte and not dead myeloma cell line fusion that obtains from the animal of immunity, and produces hybridoma.From these hybridomas, pick out and produce clone then with required specific antibody.The monoclonal antibody that hybridoma produces, their characteristic and specificity are homogeneous.
Up to now, be used to diagnose discriminating and generation with the suitable antibodies of therapeutic use to depend on stochastic process.The generation that produces the hybridoma of antibody comprises uses the antigen immune mice, and perhaps another kind of method is added to antigen in the external splenocyte prepared product.Therefore splenocyte colony and cell colony with concrete specific potential monoclonal antibody depend on animal to this antigenic immunoreation.
As alternative method, developed the additive method that is used for judgement and therapeutic use and produces antibody to the immunization method of above-described effort.A kind of method must be antibody cloning to phage virus, as at Clackson etc., and 1991, nature, 352:624; Marks etc., 1992, molecular biology magazine 222:581:Zebedee etc., 1992, institute of NAS reports 39:3175; Gram etc., 1992, the method that institute of NAS reports 89:3576 to describe is expressed the strand variable region at virus surface.Use the phage library technology, people can produce a large amount of libraries of expressing a large amount of intrinsic genetic diversities.Yet above-mentioned library is still limited by the antibody library in their sources.Still in other method, according to the description (1997, high-quality antibody library, the 8th antibody engineering international conference summary) of Pack, the gene of random mutagenesis and expression variable region also produces a large amount of libraries.Although two kinds of methods generally, are compared with traditional immunization method being successful aspect the very big multiformity of formation, they are more not successful aspect the useful antibody of discriminating, produce the CDR sequence because these methods depend on random fashion.In addition, the application of antibody in the human treatment that produces by immune mouse is restricted.Because concerning the people, mouse monoclonal antibody is an external source, thereby has immunogenicity, and they induce human anti-mouse antibody (HAMA) to reply (Shawler etc., 1985, Journal of Immunology, 135:1530; Chatenaucl etc., 1986, Journal of Immunology 137:830).
2.4. medicine according to the intermolecular interaction operation
Efficiency of drugs often derives from this medicine can enhancing, antagonism or simulate a kind of molecule and another kind of molecule, the bonded ability of part and its receptor, pathogen and cell receptor for example, thereby obtained some physiology and pharmacologically active is used for disease prevention or elimination.Up to date, medicine still is subject to synthetic or natural product serendipitous, and is the bonded micromolecule effector of part of the natural generation of simulation.Even when the structural information that obtains about part or their binding sites, present available method is not used to develop effective medicine easily.Such as according to the application of ligand receptor, and, do not prove reliable with the screening technique of peptide combinatorial library or natural extracts and receptors bind in conjunction with the molecular model method of right crystalline texture data design micromolecule analog.In addition, these synthesize or natural product does not always have difference bonded affinity of receptor subtype and specific ability, and this may cause owing to the deficiency to pharmacodynamics effect is controlled the adverse side effect that produces.
A kind of method of needs can more directly produce again or suppress natural interactional effect, and can design special-purpose pharmaceutical formulation, and these medicinal agents energy and concrete combination interact to the member and also simulate the behavior of the part of natural generation more accurately.
The list of references of quoting before this should not be interpreted as admitting that above-mentioned list of references is with respect to prior art of the present invention.
3. the present invention's general introduction
The present invention is according to the inventor's observed result.In conjunction with the binding site that comprises another member among the right member, this binding site can be transferred at least one CDR of immunoglobulin molecules, to give immunoglobulin molecules to the specificity of combination to second member.
The purpose of this invention is to provide a kind of method, have specific specific immunoglobulin, especially antibody with design, this method has overcome inscrutable immunity and the screening process that is used to separate specific antibody at present.Specifically, promptly made up and combined the right bonded synthesis modification antibody of member's immunologic opsonin, the variable region of this modified antibody has one or more CDRs like this, CDRs contains at this binding sequence in conjunction with this right member, and described binding sequence derives from conjunction with other right members.Therefore, this method is simplified the process of differentiating suitable antibodies widely and is effectively produced because immunologic tolerance or the unavailable many antigenic antibody of mysterious expression.
Therefore, the immunoglobulin molecules that the invention provides modification is antibody especially, these molecular immunes specifically with combine first right member's combination, this combination is to by first member and second member composition, described antibody comprises a variable region, wherein at least one CDR contains at the aminoacid sequence of combination to first member's binding site, and this binding site derives from conjunction with the second right member.Of the present invention preferred aspect, in natural CDR, the aminoacid sequence of binding site does not have discovery.
In conjunction with to may be any can specificity interactional 2 kinds of molecules.In specific embodiment, this is the antigen molecule of infectious disease cause of disease surface expression (promptly) of the cancer antigen molecule of cancerous cell surface expression (promptly), infectious disease cause of disease or at the cell receptor of infectious disease cause of disease in conjunction with first right member.Above-mentioned cancer antigen comprises the epi-position (PEM) or the human colon carcinoma associated protein antigen of table mucin antigen on human milk fat bead antigen (HMFG), the multiform.The antigen of above-mentioned infectious disease cause of disease comprises Brambell receptor (FCRB) and HSV-2, gonococcus, Tyreponema pallidum, chlamydia trachomatis or human papillomavirus's antigen.In other specific embodiments, this combination to be a kind of receptors ligand in conjunction with to the time, be bradykinin receptor wherein for example in conjunction with first right member.
The present invention further provides treatment or prevention method with modified immunoglobulin of the present invention.For example, following modified antibodies can be used for the treatment of or the antigen of prevention and particular cancer antigen or infectious disease cause of disease or at the relevant cancer or the infectious disease of expression of the cell receptor of infectious disease cause of disease; Described modified antibodies has one or more containing at the antigen of cancer antigen or infectious disease cause of disease or corresponding to the CDRs of the binding site of the cell receptor of infectious disease cause of disease.
The present invention further provides the method for utilizing modified immunoglobulin of the present invention to screen or detect or diagnose.For example, have one or more modified antibodies that contain at the CDRs of following antigenic binding site, can be used for the antigen of particular cancer antigen or infectious disease cause of disease or corresponding to the relevant cancer of the expression of the cell receptor of zymad or the (screening of infectious disease, detect and diagnosis), the binding site that contains on the CDRs is at the antigen of cancer antigen or infectious disease cause of disease
The present invention also provides treatment and diagnostic kit and the Pharmaceutical composition that contains modified immunoglobulin of the present invention.
The present invention further provides the method that produces synthetic modification immunoglobulin of the present invention.
Hereinafter the 6th joint is described the synthetic method of synthetic modification antibody, and in the method, one of CDRs contains from Kallidin I and comprises aminoacid sequence at the binding sequence of bradykinin receptor.This embodiment illustrate that this synthetic modification antibody mediated immunity combines with bradykinin receptor specifically and with the combining of Kallidin I competition and bradykinin receptor.The activity of synthetic modification antibody is by the active antagonist of Kallidin I institute antagonism.
4. accompanying drawing summary
Fig. 1. show the summary figure of light chain immunoglobulin or heavy chain structure, (form with being positioned at immunoglobulin carboxyl terminal district by constant region COOH) by the variable region that is positioned at immunoglobulin amino terminal district (H2N-) for every chain.
The summary figure of Fig. 2 .IgG is presented at 4 framework regions (FR1, FR2, FR3 and FR4) and 3 complementary determining regions (CDR1, CDR2 and CDR3) that (are labeled as VL and VH respectively) in light chain and the variable region of heavy chain.The constant region domain is denoted as CL corresponding to constant region of light chain, and is denoted as CH1, CH2 and CH3 corresponding to 3 domains of CH.The part of Fab displaying antibody fragment, this antibody fragment comprise variable region domain and the CL and the CH1 domain of light chain and heavy chain.Fc represents to contain the constant region fragment of CH2 and CH3 domain.
The structure of Fig. 3 A-C. (A) expression vector pMRRO10.1, this carrier contain a people K constant region of light chain sequence.(B) structure of expression vector pGammal, this carrier contain the sequence of coding human IgG1's constant region (CH1, CH2, CH3) heavy chain and hinge region sequence.(C) structure of expression vector pNEPuDGV, this carrier contain the constant region of encoded K constant region of light chain and heavy chain and the sequence of hinge region.All 3 kinds of carriers are seen Bebbington etc., 1991, and Enzymology method 2:136-145.
Fig. 4 A-H, the aminoacid and the nucleotide sequence of coding light chain and variable region of heavy chain domain, these domains have a CDR who contains the Kallidin I sequence and corresponding to the heavy chain and the variable region of light chain consensus sequence of synthetic antibody.All these sequences also contain targeting sequencing.(A) aminoacid sequence of the total variable region of light chain ConVL1 of coding and corresponding nucleotide sequence.(B) aminoacid of encoded light chain variable region BKCDR1 and corresponding nucleotide sequence, wherein CDR1 contains the Kallidin I sequence, (C) aminoacid of encoded light chain variable region BKCDR2 and corresponding nucleotide sequence, wherein CDR2 contains the Kallidin I sequence.(D) aminoacid of encoded light chain variable region BKCDR3 and corresponding nucleotide sequence, wherein CDR3 contains the Kallidin I sequence.(E) aminoacid of the total variable region of heavy chain ConVH1 of coding and corresponding nucleotide sequence.(F) aminoacid of encoding heavy chain variable region BKCDR4 and corresponding nucleotide sequence, wherein CDR4 contains the sequence of Kallidin I.(G) aminoacid of variable region of heavy chain BKCDR5 and corresponding nucleotide sequence, wherein CDR5 contains a Kallidin I sequence.(H) aminoacid of variable region of heavy chain BKCDR6 and corresponding nucleotide sequence, wherein CDR6 contains the Kallidin I sequence.
Fig. 5 coding contains the summary diagram of general step of cydorge gene assembling of the synthetic modification antibody of Kallidin I sequence.The oligonucleotide that is used to assemble this gene is expressed as " oligol "～" oligo10 ".
Fig. 6 A and B. (A) are used to the nucleotide sequence of the oligonucleotide of the Kallidin I assembling total light chain (ConVl1) and contain variable region of light chain by the scheme that shows among Fig. 5.(B) nucleotide sequence as shown in fig. 5, as to be used to assemble total variable region of heavy chain (ConVH1) and to contain the oligonucleotide of variable region of heavy chain Kallidin I.
Fig. 7 A-C. (A) stimulates the synthetic of PGE2 by Kallidin I in the SV-T2 cell, the each processing represented with PGE2 ng/ hole.Among the figure below, symbol "-" expression cell incubation when not having the factor, and "+" expression cell incubation when having the factor, promptly 1nM Kallidin I (row of going up) or, 1nM HOE 140, a kind of antagonist of Kallidin I (row down).(B) stimulate PGE2 synthetic by some synthetic modification antibody that has the CDRs that contains the Kallidin I sequence, results of stimulation represents with pg/ hole PGE2, synthetic antibody BKCDR3 (having real quadrate line), BKCDR4 (having real leg-of-mutton line), and the dilution factor of BKCDR5 (having real rhombohedral line), total variable region of heavy chain (line) and culture medium contrast (line) with open circles with filled circles as function.(C) existence or shortage Kallidin I are (in the figure bottom, be expressed as "+" or "-" respectively) under the situation, SV-T2 cell and have the antibody of BKCDR3, BKCDR4 or BKCDR5 variable region or have the PGE2 results of stimulation (amount of representing PGE2 with the Pg/ hole) of the antibody incubation of the total variable region (ConVH) of heavy chain, the result represents with bar diagram respectively.
5. Xiang Shu of the present invention
the present invention relates to the immunoglobulin (Ig) molecule of Xiu Shi, (for example where method Que determines immunologic opsonin to Yong mensuration well known in the art Zhen to the Ren of the antibody binding specificity of Qi antigen Yu being combined the antibody of first right member's combination on Qi Shi immunologic opsonin of You ground, for example, the method of Xia Mian 5.7 joint Miao Shu, and this Mian Yi Te Yi removes non-specific combination in conjunction with Pai, but optional to the cross reactivity that the antibody that Tian Ran occurs is often observed), and Zhe Xie antibody is Zhi Shao Yong You Yi complementary determining region (CDR) that contains from the amino acid sequence of second member in conjunction with right, the amino acid sequence Shi Yi Zhong Zhen of Zhe Xie molecule is to combination being listed as in conjunction with Xu first member. in conjunction with to can 2 molecules of Shi Ren Yi, comprise mutual between Zhi the interactional albumen of Neng, nucleic acid, Tang class or Zhi class, although You Xuan in conjunction with Pei to molecule (binding site derives from this molecule) Shi Yi Zhong albumen molecule. the Shi Shi scheme Zhong of Zai You Xuan, this antibody contains Yi Zhong Zhen to cancer antigen (being the molecule on cancer or Zhong oncocyte surface), infectious disease antigen (being the molecule on Zai infectious disease cause of disease surface), in conjunction with Xu, being listed as for cause of disease or Shou Ti or Pei Ti cell receptor (You Xuan Shou Ti-Pei Ti is in conjunction with right Shou Ti or Pei Ti, and this Pei Ti of Qi Zhong is combined Yu Shou Ti and this Yin Qi Sheng of You ought to answer).
The Zhi treatment method of also Ti Ying Yong of the present invention Xiu Shi of the present invention immunoglobulin (Ig), for example, Xian Shi mode processed not, the Xiu Shi antibody of Yi CDR of Yong You Zhi Shao can be used for Zhi and treats or Yu anti-cancer or infectious disease, the Te Zheng Shi of Zhe Xie disease exists the antigen Tong of Te Yi to cross this antigen the infectious disease cause of disease is combined Yu the Te isoreceptor, and the CDR of Xiu Shi antibody Yong You contains Yi Zhong Zhen in conjunction with Xu, being listed as the cell receptor of the antigen of Te Yi cancer antigen or infectious disease cause of disease or Xiang Ying Yu infectious disease cause of disease.
The method of also Ti of the present invention and Shai Xuan disconnected for Xiu Shi immunoglobulin (Ig) Zhen of the present invention, for example, Xian Shi mode processed not, the Xiu Shi antibody capable Yong Yu of Yi CDR of Yong You Zhi Shao detects take Te hapten or infectious disease cause of disease Yu the Te isoreceptor is cancer or the infectious disease of Te Zheng, and the CDR of this Xiu Shi antibody Yong You contains Yi Zhong Zhen in conjunction with Xu, being listed as the antigen of Te Yi cancer antigen or infectious disease cause of disease.
Wei Qing Chu ground Pi reveal Nei Rong of the present invention but not Shi Xian the present invention processed, with Xia row be divided into each Xiao joint Nei Rong Zhong and describe the present invention in detail.
5.1 the immunoglobulin (Ig) molecule of Xiu Shi
The invention provides Xiu Shi immunoglobulin (Ig) molecule, Qi Shi antibody of You, Zhe Xie molecule Yu be combined right first member Mian Yi Te Yi ground in conjunction with (being that Tong crosses mensuration Zhen well known in the art where method Que is fixed to the Ren of the antibody binding specificity of Qi antigen, for example, Ru Xia Mian 5.7 joint Zhong the method for Miao Shu), CDRs Zhong Zhi the Shao Yi of Qi Zhong antibody contains Zhen to the binding site in conjunction with to first member, and this binding site derives from conjunction with the amino acid sequence to Qi its member. Aspect the Yi of Zai You Xuan of the present invention, Zai Ran CDR Zhong of the amino acid sequence Zai Wei of binding site find.
Where method is differentiated but the amino acid sequence Tong of binding site crosses Ren well known in the art. For example, Zai Xie Qing Xing Zhong, Yi connect Yu are combined Qi right Ta member the pass that is combined with in conjunction with the Xu row Zhi to Mou Yi member through Que is fixed. Xia this Qing condition of Zai, the Xu row Neng Yong Yu of Shang Shu builds the CDR of synthetic antibody, and this synthetic antibody Te Yi ground Shi is not in conjunction with Qi right Ta member. Ru fruit Zai in conjunction with to Yi member to combination to Xia the amino acid sequence Wei Zhi Qing condition of Qi Ta member's binding site, it is fixed that but Tong crosses the method Que of what Shu Zhi of this area Ren, such as, but be not limited to molecule modeling method or experimental technique, Ru measure the part (as Tai class) of Zhen to this member of being attached to Qi its member, or this member Zhong Tu of Zai becomes and measures the method for the disconnected combination of Na Zhong Tu variable resistance.
In conjunction with to can Shi Ren what 2 Zhong molecule, comprise the synergistic protide of Neng, nucleic acid, Tang class or Zhi class, although You Xuan in conjunction with Pei to molecule (binding site derives from this) Shi Yi Zhong albumen molecule. The Shi Shi scheme Zhong of Zai You Xuan, Xiu Shi immunoglobulin (Ig) contain Zhen to the cell receptor of cancer antigen, infectious disease antigen, cause of disease or participate in Shou Ti-Pei Ti in conjunction with right Shou Ti or Pei Ti.
Zai specific embodiment Zhong, in conjunction with to Shi protein-protein interaction pair, Ta Shi Tong Xing Xiang mutual effect (that is, the Xiang mutual effect between Zhi 2 Zhong Xiang Tong albumen) or Yi Xing Xiang mutual effect (that is, the Xiang mutual effect between Zhi the different albumen of 2 Zhong).
In one specific embodiment, first member be ligand-receptor in conjunction with a right member, preferred part is attached to receptor and excites the receptor-ligand of the physiologic response of transmitting such as signal in the cell in conjunction with a right member then.Mode as an example rather than restriction the present invention, part or receptor can be hormone, autacoid somatomedin, cytokine or neurotransmitter or corresponding to the receptor of hormone, autacoid, somatomedin, cytokine or neurotransmitter or relate to any receptor or the part of signal transduction.(about the summary of signal transduction pathway, as see Campbell, 1977, department of pediatrics magazine 131:S42-S44; Hamilton, 1977, J.Leukoc.Biol.62:145-155; Soede-Bobok ﹠amp; Touw, 1977, molecular medicine magazine 75:470-477; Heldin, 1995, cell 80:213-223; Kishimoto etc., 1994, cell 76:253-262; Miyajima etc., 1992, academic year is commented 10:295-331 in immunity; With Cantley etc., 1991, cell 64:281-302).) in specific embodiment, in conjunction with a right member is part, such as, but be not limited to 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 corresponding to any receptor of these parts.In other embodiments, in conjunction with a right member is receptor, for example, but be not limited to opioid receptor, glucose transporter, glutamate receptor, the only plain receptor of Vulpes, erythropoietin receptor, Insulin receptor INSR, tyrosine kinase (TK) receptor, KIT stem cell factor receptor, trk C, IGF-1, granulocyte colony stimulating factor receptor, the growth hormone receptor, derive from the neurotrophic factor acceptor or the gp39 receptor of glial cell, G-protein receptor class or β-adrenergic receptor, or in conjunction with the part of any these receptors.In another embodiment, in conjunction be the ion channel of part gate to one of member, such as, but be not limited to calcium channel, sodium channel or potassium channel.In some embodiments, the invention provides immunity specifically with the modified immunoglobulin of receptors bind, and these albumen are the ligand antagonists in conjunction with described receptor, such as, but be not limited to the antagonist of endorphins, enkephalin or injury peptide.In other embodiments, the invention provides the immunity synthetic modification antibody of bind receptor specifically, these antibody and be the agonist of described receptor are such as, but be not limited to endorphins, enkephalin or injury peptide receptor.In an embodiment preferred, modified immunoglobulin is not joined on the fibronectin receptor.In another embodiment preferred, this binding sequence is not Arg-Gly-Asp, many bodies that neither binding sequence, and preferably be not many bodies of sequence A rg-G1y-Asp.
In other specific embodiment, modified immunoglobulin has a CDR, and this CDR contains the binding site corresponding to a transcription factor.Aspect preferred, modified immunoglobulin does not combine with specific DNA sequence, does not especially combine with transcription factor binding site point.
In preferred embodiments, modified immunoglobulin has a CDR at least, this CDR contains at the aminoacid sequence of the binding site of cancer antigen or tumor antigen (as: describing in detail in the following 5.3.1 joint), and preferred antigen is human colon carcinoma related antigen or epidermis mucin antigen.In other embodiments, modified immunoglobulin CDR contains aminoacid sequence at the binding site of the little ball receptor of human milk fat at least.In other embodiments, modified immunoglobulin has at least one CDR, described CDR contains the aminoacid sequence at the antigenic binding site of following tumor, and these tumors are the lymphocytic tumor of mammary gland, ovary, uterus, prostate, bladder, lung, skin, pancreas, colon, gastrointestinal tract, bone-marrow-derived lymphocyte or T.
In other preferred embodiments of the present invention, at least one CDR of modified antibodies at the aminoacid sequence of the binding site of the antigenic binding site of infectious disease cause of disease or infectious disease pathogenic cell receptor (for example contains, describe in detail in the following 5.3.2 joint), the preferred combination site is not plasmodial antigenic aminoacid sequence, or is not binding site Asn-Ala-Asn-Pro or Asn-Val-Asp-Pro.In other embodiment, modified antibodies has the CDR that contains directed toward bacteria or viral enzyme binding site.
Modified immunoglobulin molecule of the present invention can derive from the immunoglobulin molecules of any kind, such as, but be not limited to antibody, TXi Baoshouti, B-cell receptor, such as the immutable district of cell surface adhesion molecule and the MHC molecule of cofactor CD4, CD8, CD19.In embodiment preferred of the present invention, this modified immunoglobulin molecule is a kind of antibody, and it can be the antibody of any kind of, and as IgG, IgE, IgM, IgD or IgA, preferred antibody is IgG.In addition, antibody can be the subclass of any special type antibody.In another specific embodiment, the modified immunoglobulin molecule is a kind of TXi Baoshouti.
For producing the immunoglobulin of modified, adorned immunoglobulin can be any available immunoglobulin molecules, preferred monoclonal antibody or synthetic antibody.Adorned antibody can be the antibody of natural generation or preexist or synthetic according to the consensus sequence of knowing, for example at the consensus sequence of the light and variable region of heavy chain shown in Fig. 4 A and the B, or any other antibody consensus sequence or embryonal system (genome sequence that does not promptly have reorganization) sequence (for example, at Kabat etc., 1991, the sequence of immunology destination protein, the 5th edition, NIH publication number 91-3242, described those antibody consensus sequences of 2147-2172 page or leaf and embryonal system sequence).
Such as explained above, every kind of antibody molecule has 6 CDR sequences, there are on 3 and the heavy chain 3 on the light chain, and 5 of these CDRs be embryonal system CDRs (promptly, do not pass through any reorganization, be directed to the germline gene group sequence of this animal) and of these CDRs be non-embryonal system CDR (that is, produce with the different sequence of this animal germline gene group sequence) by the reorganization of embryonal system sequence.Whether a CDR is embryonal system or non-embryonal system sequence, can be by measuring the CDR sequence, then with as Kabat etc. (1991, the sequence of immune destination protein, the 5th edition, NIH publication number 91-3242, relatively the back is definite for 2147-2172) listed known embryonal system sequence.Show that with the significant change of known embryonal system sequence this CDR is a kind of non-embryonal system CDR.
Correspondingly, in other embodiments of the present invention, the CDR that contains the binding site aminoacid sequence is embryonal system CDR or is non-embryonal system CDR in addition.
Binding site can be inserted among any CDRs of described antibody, and binding site be inserted among the different CDRs of antibody that screening then produces can with combine the right bonded modified antibodies of special member, this technology those skilled in the art have in one's pocket, and saves the technology of being discussed as following 5.7.Thus, people can determine the most suitable binding site that contains of which kind of CDR.In specific embodiment, modify the CDR of heavy or variable region of light chain, make it contain the aminoacid sequence of binding site.In another specific embodiment, modified antibodies contains a variable region, and wherein, first, second of first, second of variable region of heavy chain or the 3rd CDR or variable region of light chain or the 3rd CDR contain the aminoacid sequence of binding site.In another embodiment of the invention, contain the aminoacid sequence of binding site more than 1 CDR, each contains perhaps a plurality of 1 CDR at the different binding sites of same molecular or contains different binding sites at different molecular.Especially in the embodiment, engineered 2,3,4,5 or 6 CDRs contain at the binding site of combination to first member it.In an embodiment preferred, one or more CDRs contain at involutory to first member binding site and one or more CDRs contain at such as but be not limited to the binding site of T cell, B cell, NK cell, K cell, til cell or neutrophil's immunocyte surface molecular.For example, have a binding site and the modified antibodies at the binding site of immunocyte surface molecular at cancer antigen or infectious disease cause of disease, can be used for that immunocyte is directed to load has the antigenic cancerous cell of cancer or is directed to the infectious disease cause of disease.
In specific embodiment of the present invention, the aminoacid sequence of binding site is not replace any aminoacid sequence of CDR itself and be inserted among the CDR, or the aminoacid sequence of binding site replaces the aminoacid sequence of all or part of CDR.In specific embodiment, the aminoacid sequence of binding site replaces 1,2,5,8,10,15 or 20 aminoacid of CDR sequence.
The aminoacid sequence of the binding site among the CDR is and combines right member in conjunction with essential minimum binding site (it can by any method measuring well known in the art); In addition, binding site can be than with to combine right member bigger in conjunction with essential minimum binding site.In specific embodiment, the aminoacid sequence of binding site be at least length be 4 aminoacid or at least length be 6,8,10,15 or 20 aminoacid.In other embodiments, the length amino acid sequence of binding site is no more than 10,15,20 or 25 aminoacid, or length is 5-10,5-15,5-20,10-15, a 10-20 or 10-25 aminoacid.
In addition, the total length of CDR (that is, the merging length of the remainder of binding site sequence and CDR sequence) should have suitable amino acid number, to allow this antibodies to antigen.Table 1 provides observed CDRs to contain total number of atnino acid purpose scope and observed CDRs magnitude range (abbreviation by table 2 explanation is indicated).
Table 1CDR residue number L1 10-17L2 7L3 7-11H1 5-7H2 9-12H3 2-25
(by Kabat and Wu, 1971, the data of the annual report 190:382-93 of NYAS page or leaf is write)
Though many CDR H3 heads of district are the 5-9 residue, it is longer than this to have observed some CDR H3 district.Especially, a lot of antiviral antibody has the long heavy chain CDR H3 district of 17-24 residue that is.
Correspondingly, in specific embodiment of the present invention, the CDR scope that contains binding site is among the magnitude range of the relevant special CDR that table 1 provided, that is, if it is first CDR of light chain, L1, this CDR are 10～17 amino acid residues; If it is second CDR of light chain, L2, this CDR are 7 amino acid residues; If it is the 3rd CDR of light chain, L3, this CDR are 7～11 amino acid residues; If it is first CDR of heavy chain, H1, this CDR are 5～7 amino acid residues; If it is second CDR of heavy chain, H2, this CDR are 9～12 amino acid residues; And if it is the 3rd CDR of heavy chain, H3, this CDR are 2～25 amino acid residues.In other specific embodiments, the CDR length that contains this binding site is 5-10,5-15,5-20,11-15,11-20, a 11-25 or 16-25 aminoacid.In other embodiments, the CDR 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.
In specific embodiment, modified immunoglobulin of the present invention contains a variable region part, that is, wherein light or heavy chain contains than framework region and 3 CDRs still less, such as, but be not limited to variable region wherein containing 1 or 2 CDRs, and preferably contain the intervention framework region.
In specific embodiment, this modified antibodies immunity combines (for example, but be not limited to the modified antibodies that following the 6th joint is described) specifically with bradykinin receptor.Especially, this embodiment provides a kind of modified antibodies, and wherein at least 1 CDR contains the aminoacid sequence of Arg-Pro-Pro-Gly-Phe-Gly-Phe-Ser-Pro-Phe-Arg.
In other specific embodiments, the modified antibodies immunity combines with human milk fat bead antigen specifically, and the CDRs of modified antibodies contains at least one and is selected from following aminoacid sequence: (i) Ala-Tyr-Trp-Ile-Glu (SEQID NO:4); (ii) Glu-Ile-Leu-Pro-Gly-Ser-Asn-Asn-Ser-Arg-Tyr-Asn-Glu-Lys-Phe-Lys-Gly (SEQ ID NO:5); (iii) Ser-Glu-Asp-Ser-Ala-Val-Tyr-Tyr-Cys-Ser-Arg-Ser-Tyr-Asp-Phe-Ala-Trp-Phe-Ala-Tyr (SEQ ID NO:6); (iv) Lys-Ser-Ser-Gln-Ser-Leu-Leu-Tyr-Ser-Ser-Asn-Gln-Lys-Ile-Tyr-Leu-Ala (SEQ ID NO:7); (v) Trp-Ala-Ser-Thr-Arg-Glu-Ser (SEQ ID NO:8); And (vi) Gln-Gln-Tyr-Tyr-Arg-Tyr-Pro-Arg-Thr (SEQ ID NO:9).
In a more particular embodiment, the CDRs of variable region of heavy chain contains the aminoacid sequence of above-mentioned (ⅰ)-(ⅲ), and the aminoacid sequence that the CDRs of variable region of light chain contains above-mentioned (ⅳ)-(ⅵ).
In specific embodiment, the invention provides a kind of and human colon carcinoma related antigen immunity specific bond and comprise the modified antibodies of a variable region, this variable region has a CDR:Thr-Ala-Lys-Ala-Ser-Gln-Ser-Val-Ser-Asn-Asp-Val-Ala (SEQ ID NO:10) who contains one of following amino acid sequences at least; Ile-Tyr-Tyr-Ala-Ser-Asn-Arg-Tyr-Thr (SEQ ID NO:11); Phe-Ala-Gln-Gln-Asp-Tyr-Ser-Ser-Pro-Leu-Thr (SEQ ID NO:12); Phe-Thr-Asn-Tyr-Gly-Met-Asn (SEQ ID NO:13); Ala-Gly-Trp-Ile-Asn-Thr-Tyr-Thr-Gly-Glu-Pro-Thr-Tyr-Ala-Asp-Asp-Phe-Lys-Gly (SEQ ID NO:14); Or Ala-Arg-Ala-Tyr-Tyr-Gly-Lys-Tyr-Phe-Asp-Tyr (SEQID NO:15).
After structure contains the antibody of modifying CDRs, can further change over the screening modified antibodies, have more high-affinity or specific antibody to select.Can produce and select by any method well known in the art target antigen is had more high-affinity or specific antibody.Such as, but be not limited to following method, the nucleic acid of coding synthetic modification antibody can be chemistry or direct mutagenesis randomly or make special mutation method at the nucleic acid ad-hoc location of coding modified antibodies and produce, and screens the antibody that the target antigen binding affinity is derived from the mutant nucleic acid molecule then and obtains.By the library of antibody molecule of detect expressing one by one or screening mutant nucleotide sequence, as by display technique of bacteriophage (as seeing U.S. Patent number 5,223,409; 5,403,484 and 5,571,698, all patents are finished by people such as Ladner; PCT publication WO 92/01047 patent or any other display technique of bacteriophage well known in the art that PCT is finished by people such as McCafferty), finish screening process.
Correspondingly, in one specific embodiment, described modified antibodies has higher specificity or affinity in conjunction with the antibody of same antigen to antigen specifically than the immunity of natural generation.In another embodiment, modified antibodies shows as at least 2 * 10 7The antigenic stable bond of M.
Modified antibodies of the present invention also can further be modified with any method of modified antibodies well known in the art, as long as further modification does not stop or suppress combining of modified antibodies and specific antigen.Especially, except aminoacid sequence insertion or replacement CDR sequence with binding sequence, modified antibodies of the present invention can have one or more aminoacid to replace, lack or insert.Above-mentioned aminoacid is replaced, lacked or insert can be any type of replacement, disappearance or insertion, but can not stop or suppress the immune specific bond of modified antibodies and target antigen.For example, above-mentioned aminoacid is replaced the replacement that comprises the amino acid residue that function furthers.For example, one or more amino acid residues can be by polar phase seemingly, and the other aminoacid that function is close is replaced, and the result produces reticent the change.Amino acid whose replacement can be under this aminoacid be selected other members of classification.For example, nonpolar (hydrophobic) aminoacid comprises alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.Polar neutral amino acid comprises glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.Positively charged (alkalescence) aminoacid comprises arginine, lysine and histidine.Electronegative (acidity) aminoacid comprises aspartic acid and glutamic acid.
In addition, one or more amino acid residues can be replaced by nonclassical amino acid in the sequence, or chemical amino acid analogue imports in the immunoglobulin sequences with replacement or addition manner.Nonclassical amino acid is including, but not limited to the D-heterogeneous body of common amino acid, α-An Jiyidingsuan, the 4-aminobutyric acid, Abu, the 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminocaprolc acid, Aib, the 2-aminoisobutyric acid, the 3-alanine, ornithine, nor-leucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteine, tert-butyl group glycine, tert-butyl group alanine, phenylglycine, Cyclohexylalanine, Beta-alanine, fluorine-based aminoacid, the aminoacid such as the Beta-methyl aminoacid of design, C α-methylamino acid, N α-methylamino acid and general amino acid analogue.In addition, aminoacid can be D (dextral) or L (left-handed).
In a specific embodiments of the present invention, this modified immunoglobulin is further modified, to strengthen the ability that its induced anti-idiotype is replied, for example by by the U.S. Patent Application Serial of Burch application _ _, exercise question " enhancing excites the modified antibodies of antiidiotype responsibility ", November 13 1998 submission date, (agent's file 6750-015) described method was carried out, and this patent is incorporated herein only for reference as a whole.Use above-mentioned method of modifying, for example remove or reduce in the chain or interchain disulfide bond, to reduce conformation restriction immune globulin variable region.Particularly, modified immunoglobulin is further modified, and the amino acid residue that one or more variable region cysteine residues that form disulfide bond are not so had sulfydryl replaces.
The cysteine residues of discriminating formation disulfide bond can be finished by any method well known in the art in the variable region of specific antibody.For example, but also unrestricted herein description, the cysteine residues that forms intrachain disulfide bond is conservative at various antibody and mutual species camber, and this fact is well-known in this area.Therefore, by with known which residue form disulfide bond other antibody molecules sequence relatively, the cysteine residues that the disulfide bond forms (consensus sequence that provides of Fig. 4 A and E for example can differentiate be provided, or in Kabat etc., 1991, the sequence of immune destination protein, the 5th edition, healthy and the human service of the U.S., Bei Suda, Maryland).
Table 1 provides the list of locations that forms the cysteine residues of disulfide bond at many antibody molecules.
Table 1
(source of data is in Kabat etc., 1991, the sequence of immune destination protein, the 5th edition, U.S. HHS, Bei Suda, Maryland)
Figure 9881311700361
Represent that by the positional number that () comprises this albumen is not sequence number corresponding to this position, but this residue sequence quantity is by coming out with known array comparison inference.
It should be noted that whole antibody molecules that his-and-hers watches 1 are listed, the cysteine residues that forms intrachain disulfide bond is variable region of light chain 23 and 88 s' residue and variable region of heavy chain 22 and 92 s' a residue.This positional number refer to consensus sequence in the corresponding residue of this residue, by Kabat (1991, the sequence of immunity destination protein, the 5th edition, healthy and the human Section of Radio of the U.S., Bei Suda, Maryland) determines or represent (weight that specific antibody sequence and consensus sequence or Fig. 4 A and E are described or the series arrangement of variable region of light chain are determined) respectively at Fig. 4 A with heavy chain and variable region of light chain sequence that E describes together.
Correspondingly, in an embodiment of the present invention, the modified immunoglobulin molecule is further modified, and light chain 23 and/or 88 s' residue is replaced with the amino acid residue that does not contain sulfydryl like this, and/or 22 and/or 92 residue is replaced with the amino acid residue that does not contain sulfydryl.
The amino acid residue of replacing the cysteine that forms disulfide bond is any amino acid residue that does not contain sulfydryl, as alanine, arginine, asparagine, aspartate (or aspartic acid), glutamine, glutamate, Glu (or glutamic acid), glycine, isoleucine, histidine, leucine, lysine, phenylpropyl alcohol ammonia salt, proline, serine, threonine, tryptophan, tyrosine or valine.In an embodiment preferred, cysteine residues is replaced by glycine, serine, threonine, tyrosine, asparagine or glutamine residue, is most preferably replaced by alanine residue.
In addition, form disulfide bond cysteine residues can by as top listed nonclassical amino acid that does not contain sulfydryl or chemical amino acid analogue.
In specific embodiment, replacing the residue that forms disulfide bond is at variable region of heavy chain or at variable region of light chain or heavy chain and variable region of light chain.In other specific embodiments, residues that are substituted (or disappearance) or two special disulfide bond of formation that form special disulfide bond residue are substituted (or disappearance).
In other specific embodiments, the invention provides the functional activity fragment of modified immunoglobulin.The functional activity fragment be meant described polypeptide can with target antigen immunity specific bond, this any known method (as by the described method of following 5.7 joints) of measuring immune specific bond in conjunction with available this area is determined.For example, above-mentioned fragment is including, but not limited to F (ab ') 2 fragments (containing heavy chain and variable region of light chain), constant region of light chain and heavy chain CH1 domain (this fragment produces by pepsin digested antibody) and Fab fragment (being produced by reduction F (ab ') 2 segmental disulfide bond) (Fig. 1; King etc., 1992, journal of biological chemistry, 281:317), and Fv fragment, the Fv fragment promptly contains fragment (Reichmann and Winter, 1988, the molecular biology magazine 203:825 of heavy chain and variable region of light chain domain; King etc., 1993, journal of biological chemistry 290:273).
The present invention also comprises single-chain antibody (SCA) (United States Patent (USP) 4,946,778; Bird, 1988, science 242:423-426; Huston etc., 1988, institute of NAS reports 85:5879-5883; With Warol etc., 1989, natural 334:544-546).The weight and the light chain segments that meet the Fv district through the aminoacid bridging form single-chain antibody, and the result produces single chain polypeptide.In addition, the present invention also provides heavy chain and light chain dimer and diabodies.
The present invention further provides modified antibodies, this antibody can be the modified antibodies of chimeric or humanized antibody.Chimeric antibody is a kind of antibody molecule, the different piece of this antibody molecule derives from the molecule of different animals species, as have one and derive from the variable region of Mus mAb and those molecules of a constant region that derives from human normal immunoglobulin's constant region, as humanized antibody.Developed the technology of producing chimeric antibody (Morrison etc., 1984, institute of NAS reports 81:6851-6855; Neuberger etc., 1984, natural 312:604-608; Takeda etc., 1985, natural 314:452-454; International patent application no PCT/GB85/00392 (Neuberger etc. and Celltech company limited)), this technology be from the gene of the specific mouse antibodies molecule of suitable antigen with from suitable bioactive human antibody molecules gene splicing together.In one specific embodiment, synthetic modification antibody is the chimeric antibody of a kind of non-human antibody of containing variable region and people's antibody constant region.
In a preferred embodiment, modified antibodies is a kind of humanized antibody, especially in this antibody, the CDRs of antibody (except containing one or more CDRs of binding sequence) derives from non-human animal's antibody, and framework region and constant region derive from people's antibody (by all U.S. Patent numbers 5 of Winter, 225,539).Anti-various antigenic above-mentioned transplanting CDR antibody have successfully been made up, for example the antibody of the anti-IL-2 receptor of (1989, institute of NAS report 86:10029) description such as Queen; The antibody of the anti-CAMPATH-cell surface receptor that Riechmann etc. (1988, natural 332:323) describe; The antibody of the anti-hepatitis B virus that Co etc. (1991, institute of NAS newspaper) describe; And the antigenic antibody of the anti-breathing cyton virus described of Tempest etc. (1991, biotechnology 9:267).
Make in all sorts of ways, as (Jones etc., 1986, natural 321:522; Riechmann etc., 1988, natural 332:323) side-directed mutagenesis of describing, and the assembled in vitro technology of complete transplanting CDR variable region (Queen etc., 1989, institute of NAS reports 86:10029); And use PCR synthetic graft CDR gene methods such as (Daugherty etc., 1991, nucleic acids research 19:2471), make up and transplant the CDR variable region gene.The framework region of the CDRs of mouse monoclonal antibody being transplanted to people's antibody produces the antibody of transplanting CDR.After the transplanting, most of antibody additional amino acid from framework region change to be benefited, and can keep affinity, might this be essential because of the framework region residue to keeping the CDR conformation, and to have illustrated some residue of framework region be the part of antigen binding site.Therefore, in specific embodiments of the present invention, described modified antibodies comprises a variable region, and wherein at least 1 framework region has the different amino acid residue of residue at this position in the framework region of one or more and natural appearance.
In a preferred embodiment of the present invention, this modified antibodies derives from human monoclonal antibodies.Might produce human monoclonal antibodies completely by the applying transgene mice.The transgenic mice that the personnel selection immunoglobulin loci replaces the mouse immunoglobulin genes seat provides affine sexual maturity mechanism in the body, to produce the human normal immunoglobulin.
In some embodiments, this modified immunoglobulin (or its fragment) through covalent bond (for example, but be not limited to peptide bond) terminal at N-end or C-be not that the aminoacid sequence of the albumen (or its part, preferably at least 10,20 or 50 amino acid moieties) of modified immunoglobulin merges with another.Preferably, this modified immunoglobulin is terminal covalently bound with other albumen at the N-of this constant region for immunoglobulin.In preferred embodiments, the invention provides described modified immunoglobulin matter and growth enhancer part or the covalently bound fusion rotein of immunostimulation factor part, the factor that is connected comprises interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-7, IL-10 INTERLEUKIN-10, il-1 2, interleukin-15, G-colony stimulating factor, tumor necrosis factor, perforin, gamma interferon and NK cellular antigens or MHC derived peptide.
For example, can further modify this modified immunoglobulin, as long as above-mentionedly covalently boundly do not stop or suppress this immunoglobulin to its target antigen immunity specific bond by the molecule of covalently bound any kind.For example; but not only to be limited to following method; described modified immunoglobulin is for example further modified through following method, and these methods comprise glucosidesization, acetylation, Pegylation, phosphorylation, amidatioon, by known protection/blocking group derivatization, Proteolytic enzyme cutting, be connected etc. with cell ligand or other albumen.Can carry out any of a large amount of chemical modification methods by known technology, these methods are synthetic etc. including, but not limited to the metabolism of special chemical cleavage, acetylation, formylated, tunicamycin.In addition, this modified antibodies can contain 1 or a plurality of as the above-mentioned listed nonclassical amino acid of this section.
In specific embodiment of the present invention, this modified immunoglobulin (or its fragment) covalently is connected with a treatment molecule, for example, treating molecular guide to special cell type or in organizing, for example cancer or tumor cell.The treatment molecule that described treatment molecule can be an any kind known in the art is such as, but be not limited to chemotherapeutic, toxin, antisense oligonucleotide, radionuclide, antibiotic, antiviral agents or antiparasitic etc. as ricin.
5.2 produce the method for described modified immunoglobulin
Any method, especially chemical synthesis or recombinant expressed method by synthetic immunoglobulin known in the art can produce modified immunoglobulin of the present invention, preferred recombination and expression techniques production.
Modified immunoglobulin of the present invention or its segmental recombinant expressed nucleic acid that needs to make up the coding modified immunoglobulin.Use any method known in the art, for example recombinant technique or chemical synthesis (as seeing Creighton, 1983, " protein: structure and molecular principle " W.H.Freeman ﹠amp; Co.N.Y.34-49 page or leaf and Sambrook etc., 1989, molecular cloning, laboratory manual, cold spring port publishing house, New York), or use known immunoglobulin gene PCR method is produced the nucleotide sequence that coding contains the CDR sequence of binding site with through engineering approaches, can produce the above-mentioned nucleotide sequence that contains the modified immunoglobulin of encoding.
Correspondingly, the invention provides the nucleic acid that contains coding modified immunoglobulin of the present invention or the segmental nucleotide sequence of its functional activity.
Preferably, the nucleic acid of coding modified immunoglobulin can chemosynthesis oligonucleotide assemble (as, according to Kutmeier etc., 1994, the method that biotechnology 17:242 describes), in brief, this method comprises that a synthetic cover contains this immunoglobulin sequences part of coding and the oligonucleotide of overlapping, annealing and be connected these oligonucleotide, then as the oligonucleotide that connected of following the 6th joint PCR method amplification of being demonstrated.
Correspondingly, the invention provides a kind of method that produces the nucleic acid of coding modified immunoglobulin, described method comprises: (a) synthetic cover oligonucleotide, this cover oligonucleotide comprises the nucleotide segment sequence that contains coding synthetic modification immunoglobulin and contains oligonucleotide with the complementary partial nucleotide sequence of nucleotide sequence of coding synthetic modification immunoglobulin, and except those contained the oligonucleotide of nucleotide sequence of the terminal and C-end portion of the N-of coding synthetic modification immunoglobulin, described every kind of oligonucleotide had and the eclipsed end sequence of another oligonucleotide of this cover; (b) make these oligonucleotide hybridization or annealing mutually; And (c) connect the oligonucleotide hybridized, produced the nucleic acid of the nucleotide sequence that contains coding synthetic modification immunoglobulin like this.
Perhaps, from containing the antibody molecule for example or at least the nucleic acid of the nucleotide sequence of the variable region of antibody molecule of encoding, make up the nucleic acid of the nucleotide sequence that contains the modified immunoglobulin of encoding.For example, with to the special probe hybridization method of special antibody molecule or with synthetic can with 3 ＇ of this sequence and the primer of the terminal hybridization of 5 ＇, then pcr amplification goes out the method for this sequence, can be from the clone of existing antibody molecule or variable region, or the nucleic acid acquisition that separates encoding antibody molecule or variable region from suitable source contains the nucleic acid of the nucleotide sequence of encoding antibody molecule, these preferred cDNA libraries of originating, for example from the antibody dna library of the cell or tissue of expressed antibody molecule storehouse or synthetic antibody libraries preparation or the cDNA library (as seeing, Clackson etc., 1991, natural 352:624; Hane etc., 1997, institute of NAS reports 94:4937).
Contain the nucleic acid of the nucleotide sequence of a variable region of encoding antibody molecule at least in case be cloned into, this binding site sequence just can be inserted in the nucleotide sequence of one or more CDRs of coding so.The engineering operation of above-mentioned special CDR coded sequence can be finished by conventional recombinant DNA technology known in the art.For example, use the method for PCR-based, external direct mutagenesis method etc., the nucleotide sequence that is contained the CDR of specific bond site sequence by coding can replace the nucleotide sequence of described coding CDR.If there is conventional restriction endonuclease sites in the nucleotide sequence of CDR, this sequence can digest by being limited property restriction endonuclease so, and the nucleic acid fragment that contains the nucleotide sequence of the binding site of encoding then can be connected on the restriction site.The nucleic acid fragment that contains binding site can obtain from coding contains the proteic all or part of nucleic acid of binding site, perhaps produces from the synthetic oligonucleotide of the sequence that contains coding binding site and its reverse complement.
The nucleic acid of coding modified antibodies optionally contains the nucleotide sequence of the leader peptide sequences of encoding, and this leader peptide sequences instructs the secretion of synthetic modification antibody molecule.
The nucleic acid of modified antibodies variable region at least in case obtained to encode, it just can import in the carrier of the nucleotide sequence that contains the encoding antibody constant region (as seeing PCT publication WO 86/05807; PCT publication WO 89/01036; With U.S. Patent number 5,122,464).Contain complete light chain or heavy chain carrier can with described nucleic acid coexpression, with the antibody molecule of The expressed, and these carriers are well-known in this area, as, pMRRO10.1 and pGammaI (also see Bebbington, 1991, Enzymology method 2:136-145).
By routine techniques surperficial carrier is transferred to host cell then, cultivate this transfectional cell with routine techniques then, to produce antibody of the present invention.Particularly, in case produce the nucleic acid of modified antibodies variable region, the method expression that this modified antibodies can for example be given an example through the 6th joint (also see Bebbington, 1991, Enzymology method 2:136-145).For example, described method comprises the expression vector transient transfection of coding modified immunoglobulin in the COS cell, cultivate the suitable time of this cell so that immunoglobulin expression is collected the cell culture supernatant from COS then, this supernatant contains the modified immunoglobulin of secreting, expressing.
The host cell that is used to express recombinant antibodies of the present invention can be such as colibacillary bacterial cell, and these cells are particularly suited for the expressing recombinant antibody fragment, perhaps eukaryotic cell preferably, and these cells are particularly suited for the expressing recombinant antibody molecule.Especially, is (the Foecking etc. of effective expression system that are used for immunoglobulin such as mammalian cells such as Chinese hamster ovary cell (CHO) or COS cells combining from the antibody expression vector under the main control of early gene promoter sub-element at once of human cytomegalic inclusion disease virus, 1986, gene 45:101; Cockett etc., 1990, biotechnology 8:662).
Multiple host expresses carrier system can be used for expressing the antibody of sequential coding of the present invention.Above-mentioned host expression system representative can produce the purpose coded sequence by carrier and follow the carrier of purification, and also available suitable nucleotide coding sequence transforms or transfection produces cell, and this cell in-situ is expressed antibody product of the present invention.These systems including, but not limited to such as the microorganism of antibacterial (as, escherichia coli, Bacillus subtillis), yeast (as saccharomyces cerevisiae belong to, pichia), insect cell system, plant cell system or mammal cell line system (for example, COS, CHO, BHK, 293 and the 3T3 cell), wherein transform microorganism with the reconstitution cell phage DNA that contains antibody coding sequence, plasmid DNA or cosmid DNA expression vector; With the reason group Yeast expression carrier transformed yeast that contains antibody coding sequence; With the recombinant virus expression vector that contains antibody coding sequence (as baculovirus) infected insect cell system; With the recombinant virus expression vector that contains antibody coding sequence (as, cauliflower mosaic virus, CaMV; Tobacco mosaic virus (TMV) TMV) infects or with recombinant plasmid expression vector (as Ti-plasmids) plant transformed cell system; Have contain derive from the genomic promoter of mammalian cell (as, metallothionein promoter) or from the promoter of mammalian virus (as, the mammal cell line system of recombinant expression construct body gland virus stage starting, pox bacterium virus 7.5k promoter).
In bacterial system, according to the application purpose for the treatment of expressing antibodies, can preferred various expression vectors.For example, when when producing the antibody Pharmaceutical composition and will produce a large amount of above-mentioned albumen, the carrier that instructs high level expression and be easy to the fusion protein product of purification meets the requirements.Above-mentioned carrier includes but not limited to, coli expression carrier pUR278 (Ruther etc., 1983, EMBOJ.2:1791) (in pUR278, antibody coding sequence can individually be connected in the carrier with lacZ coding region, so that fusion rotein produces); PIN carrier (Inouye ﹠amp; Inouye, 1985, nucleic acids research 13:3101-3109; Van Heeke ﹠amp; Schuster, 1989, journal of biological chemistry 264:5503-5509) etc.Also available pGEX carrier has the allogenic polypeptide of the fusion rotein of glutathione-S-transferase (GST) with expression.Generally speaking, above-mentioned fusion rotein is soluble, and utilizes its absorption and be attached on substrate glutathion-sepharose 4B, then with the elution process that contains free glutathione, and this albumen of purification easily from cell lysis.The pGEX carrier design has the thrombin of comprising or factor Xa protease cutting site, and such target gene product of cloning discharges from the GST component.
In the insecticide system, lucerne place Autographa spp nucleopolyhedrosis virus (AcNPV) is used as carrier with expression alien gene.This virus is grown in the fall army worm cell.Antibody coding sequence can singlely be cloned into the nonessential region (for example polyhedrosis gene) of virus and be placed under the control of AcNPV promoter (for example polyhedral body promoter).
In mammalian host cell, available many expression systems based on virus.As under the situation of expression vector, the purpose antibody coding sequence can be connected to adenovirus and transcribe/translate on the control complex, as late promoter and tripartite leader[at adenovirus.This mosaic gene can be inserted in the adenoviral gene group by recombinant technique in external or the body then.The insertion in (as E1 or E3 district) can be lived infecting prominent produce in main in virus genomic nonessential region, and recombinant virus that can expressing antibodies is (as seeing Logan ﹠amp; Shenk, 1984, institute of NAS newspaper).Effectively translate for the antibody coding sequence that makes insertion, also need special enabling signal.These signals comprise ATG start codon and contiguous sequence.And this start codon must have the reading frame of required coded sequence simultaneously, to protect the whole segmental translation of complete insertion.The translation control signal of these external sources and start codon can be natural and synthetic various origins.The efficient of expressing strengthens and can strengthen element, transcription terminator etc. and carry out (see Bittner etc., 1987, Enzymology method 153:516-544) by comprising suitable transcribing.
In addition, can select host cell strain, regulating insertion sequence, or modify and the processed gene product in the mode of specific needs.Method of modifying of above-mentioned protein product (as glucosidesization) and processing method (as cutting) may be important to proteic function.For the translation post-treatment and the modification of albumen and gene outcome, different host cells have its distinctive and special mechanism.Can select suitable cell line or host system, to guarantee the correct modification and the processing of expressed foreign protein.For reaching this purpose, available eukaryote host cell, these cells have the suitable processing to initial transcription, the cell mechanism of the glucosidesization of gene outcome and phosphorylation.Above-mentioned mammalian host cell is including, but not limited to CHO, VERO, BHK, Hela, COS, MDCK, 293,3T3, WI38.
For extended high rate amount ground produces recombiant protein, stably express is preferred.For example, but engineering is handled the cell line of stably express antibody.The expression vector that need not contain the virus replication original series, use by suitable expression control element (as, promoter, enhancer, sequence, transcription terminator, polyadenous glycosidation site etc.) and the DNA transformed host cell of selectable marking of control.After importing foreign DNA, engineering cell can be grown 1-2 days in nutritious culture medium, transferred to then and selected in the culture medium.But the selected marker in recombiant plasmid is given the selection resistance, and allows cell plasmid stably to be incorporated on their chromosome, and growth forms transforming focus then, and cell line is cloned and be expanded into to this transforming focus as a result.This method is of value to the engineering cell system of this antibody of construction expression.Above-mentioned through engineering approaches cell line is in screening and estimate in direct or indirect and the interactional chemical compound of antibody particularly useful.
Available many selective systems, these systems are including, but not limited to being respectively applied for the herpes simplex virus thymidine kinase (Wigler etc. of tk-, hgprt-or aprt-cell, 1977, cell 11:223), hypoxanthine-guanine phosphoribosyl transferase (Szybalska ﹠amp; Szybalski, 1962, institute of NAS reports 48:2026) and adenine phosphoribosyl transferase (Lowy etc., 1980, cell 22:817) gene.In addition, available antimetabolite resistance as the selection basis of following gene: dhfr give to the resistance of ammonia first psychopsid (Wigler etc., 1980, institute of NAS reports 77:3567; O ＇ Hare etc., 1981, institute of NAS reports 78:1527); Gpf gives (the Mulligan ﹠amp of the resistance to mycophenolic acid; Berg, 1981, institute of NAS reports 78:2072); Neo gives and gives the resistance to hygromycin (Santerre etc., 1984, gene 30:147) to the resistance of aminoglycoside G-418 (Colberre-Garapin etc., 1981, molecular biology magazine 150:1) and hygo.
(summary is seen Bebbington and Hentschel to increase the expression of synthetic modification antibody by the carrier TRAP, in dna clone based on the purposes of gene amplification method carrier of expression cloning gene in mammalian cell, the 3rd volume (academic press, New York, 1987)).When the labelling in the carrier system of expressing immunoglobulin can increase, the inhibitor level in the raising host cell culture will increase the copy number of marker gene.Because the gene that is increased is connected with immunoglobulin gene, the generation of immunoglobulin also will increase (Crouse etc., 1983, molecular cytobiology 3:257).
Described host cell can be with the cotransfection of 2 kinds of expression vectors of the present invention, and first vector encoded derives from the polypeptide of heavy chain and second vector encoded derives from the polypeptide of light chain.But these 2 kinds of carriers can contain consistent selected marker, this labelling is expressed heavy chain and light chain polypeptide equivalent, or, use single carrier, this vector encoded heavy chain and light chain polypeptide in these cases, light chain should place before the heavy chain, to avoid excessively (Proudfoot of deleterious free heavy chain, 1986, natural 322:562; Kohler, 1980, institute of NAS reports 77:2197).The coded sequence of heavy chain and light chain can comprise cDNA or genomic DNA.
The invention provides a kind of reconstitution cell that contains the carrier of encoding synthetic antibody, this antibody has a CDR district, and this district is contained from the aminoacid sequence of combination to a member's active binding site.
5.3. the therapeutic use of synthetic modification antibody
The present invention also provides by using medicine of the present invention (at this term for " medicine "), is used for the treatment of or prevention and special molecular are expressed the method for diseases associated and imbalance.Above-mentioned medicine comprises modified immunoglobulin of the present invention and its functional activity fragment (5.1 joints are described for example), and code book invention modified immunoglobulin and the segmental nucleic acid of its functional activity (5.2 joints are described for example).
In general, use and have identical On the Origin of Species with the experimenter or species reactive products method is preferred.Therefore, in preferred embodiments, Therapeutic Method of the present invention is with a kind of modified antibodies that derives from people's antibody; In other embodiments, method of the present invention is with a kind of modified antibodies that derives from chimeric or humanized antibody.
Particularly, contain the Pharmaceutical composition of immunologic opsonin ground, can be used for the treatment of or prevention and special molecular such as antigen presentation diseases associated or imbalance in conjunction with the modified antibodies of the present invention (or its functional activity fragment) of special molecular.Aspect concrete, in the embodiment that discusses in more detail in the following sub title, immunity is specifically in conjunction with the antigen of tumor or cancer antigen or infectious disease cause of disease or at the modified antibodies of the cell receptor of infectious disease cause of disease, can be used for the treatment of or tumor, cancer or infectious disease that prevention is relevant with the expression of specific antigen.The modified immunoglobulin of immune binding partner specifically or receptor can be used for the treatment of or prevent to increase or reduce the defective diseases associated of the quantity of sepcific ligands receptor.In some embodiments, described modified immunoglobulin is used for the treatment of or prevents self-disease, and these diseases are including, but not limited to rheumatic arthritis, lupus, ulcerative colitis or psoriasis.This modified immunoglobulin also can be used for the treatment of allergy.
Can be used for experimenter of the present invention can be any mammal or invertebrate species, and these species are including, but not limited to cattle, horse, sheep, pig, fowl (as chicken), goat, cat, Canis familiaris L., hamster, mice, rat, monkey, rabbit, chimpanzee and the mankind.In an embodiment preferred, the experimenter is human.
5.3.1 treatment for cancer and prevention
The invention provides treatment or prevention and it is characterized by existence in conjunction with the antigenic method for cancer of special cancer to the member.This method comprise the experimenter to above-mentioned treatment of needs or prevention use medicine of the present invention, for example immunity specifically with the bonded synthetic modification antibody of special cancer antigen (or its functional activity fragment), this antibody comprises a variable region with CDR, and described CDR contains the aminoacid sequence at the antigenic binding site of cancer.
By using synthetic modification antibody of the present invention, can treat or prophylaxis of cancer, these cancers including, but not limited to malignant tumor, tumor, metastatic tumo(u)r or any be the disease or the imbalance of feature with uncontrolled cell proliferation, the relevant antigen combination of cancerous cell of the cancer of to be treated with one or more the specifically or prevention of this modified antibodies immunity.Whether special medicine can be determined by any known method in this area to treating or preventing some cancer effective, such as, but be not limited to those methods described in following 5.6 joints.
For example, but not restriction mode of the present invention, contain and the CDR sequence can discern the antigenic synthetic antibody of the present invention of following cancer by using, energy treatment or prevention cancer and the tumor relevant: KS 1/4 full cancer antigen (Perez and Walker 1990 with described antigen, Journal of Immunology 142:3662-3667:Bumal, 1998, hybridoma Z (4): 407-415), ovarian cancer antigen (CA125) (Yu etc., 199l cancer research 51 (2): 468-475), prostanoic acid phosphoric acid (Tailor etc., 1990, nucleic acids research 18 (16): 4928), prostate specific antigen (Henttu and Vihko, 1989, biochemistry biophysical research communication 160 (2): 903-910; Israeli etc., 1993, cancer research 53:227-230), melanic related antigen p97 (Estin etc., National Cancer Institute's magazine 81 (6): 445-446), melanoma-associated antigen gp75 (Viiayasardahl etc., 1990, The Journal of Experimental Medicine 171 (4): 1375-1380), high molecular melanoma-associated antigen (HMW-MAA) (Natali etc., 1987, cancer 59:55-63; Mittelman etc., 1990, Journal of Clinical Investigation 86:2136-2144), prostatic specific membrane antigen, carcinoembryonic antigen (CEA) (Foon etc., 1994, the journal 13:294 of American Society of Clinical Oncology), multiform epidermis mucin antigen, the human milk fat human leucocyte antigen, the colorectal carcinoma related antigen is as CEA, TAG, 72 (Yokata etc., 1992, cancer research 52:3402-3408), GICA 19-9 (Herlyn etc., 1982, clinical immunology magazine 2:135), CTA-1 and LEA, Burkitt ＇ s lymphoma antigen 38.13.CD19 (Ghetie etc., 1994, blood 83:1329-1336), people B-lymphoma antigen-CD20 (Reff etc., 1994, blood 83:435-445), CD33 (Sgouros etc., 1993, nucleic acid medical journal 34:422-430), melanoma specific antigen such as ganglioside GD2 (Saleh etc., 1993, Journal of Immunology, 151,3390-3398), Ganglioside, GD3 (Shitara etc., 1993, cancer immunity and immunization therapy 36:373-380), Ganglioside GM2 (Livingston etc., 1994, Journal of Clinical Oncology 12:1036-1044), Ganglioside GM3 (Hoon etc., 1993, cancer research 53:5244-5250), such as the tumor antigen of the virus induction that comprises following antigen dna oncovirus and the tomour specific transfevent cell surface antigens (TSTA) such as envelope antigen of RNA oncovirus, the CEA of cancer tire antigen-α-fetus albumen such as colon, fertile antigen (the Hellstrom etc. of tumor of bladder cancer, 1985, cancer research 45:2210-2188), differentiation antigen such as human lung cancer antigen L6, L20, (Hellstrom etc., 1986, cancer research, 46:3917-3923), fibrosarcoma antigen, human leukemia T cellular antigens-Gp37 (Bhattacharya-Chatterjee etc., 1988, immunologic opsonin magazine 141:1398-1403), neoglycoprotein, sphingolipid, breast cancer antigen such as EGFR (EGF-R ELISA), HER2 antigen (p185HER2), many types of epidermis mucin (PEM) (Hilkens etc., 1992, biochemistry science trend 17:359), malignant tumor human lymphocyte antigen-APO-1 (Bernhard etc., 1989, science 245:301-304), differentiation antigen (Feizi, 1985, natural 314:53-57) as in FE, the I type antigen that finds in the elementary entoderm, the adult erythrocyte, the I type antigen that finds among the preceding transplantability embryo, the I that in adenocarcinoma of stomach, finds (Ma), the M18 that finds at the mammary gland epidermis, M39, the SSEA-1 that in the class myelocyte, finds, the VEP8 that in straight colon cancer, finds, VEP9, Myl, VIM-D5, D156-22, the TRA-1-85 that in adenocarcinoma of colon, finds (blood group H), C14, the F3 that in adenocarcinoma of lung, finds, the AH6 that in gastric cancer, finds, the Y hapten that in the embryonal carcinoma cell, finds, Ley, the TL5 that in the A431 cell, finds (blood group A), the EGF receptor, the E1 series (blood group B) that in cancer of pancreas, finds, the FC10.2 that in the embryonal carcinoma cell, finds, adenocarcinoma of stomach antigen, the CO-154 that in adenocarcinoma, finds (blood group Lea), the NS-10 that in adenocarcinoma, finds, the CO-43 (blood group Leb) that in AC31 cell EGF receptor, finds, G49, in colon cancer, find 19.9, the gastric cancer mucin, the T5A7 that in myeloid cell, finds, the R24 that in melanoma, finds, the GD.3 that in the embryonal carcinoma cell, finds, D1.1, DFA-1, G M2, OFA-2, G D2With M1:22:25:8 and the SSEA-3 and the SSEA-4 that in 4～8 cell stage embryos, find.In one embodiment, antigen is a kind of TXi Baoshouti derived peptide that derives from T-cell lymphoma,cutaneous (see Edelson, 1998, cancer magazine 4:62).
In other embodiments of the present invention, accept the experimenter of described modified antibodies treatment, optionally accept such as other treatments of cancer such as operation, radiotherapy and chemotherapies.Particularly, be used for the treatment of or the medicine of the present invention of prophylaxis of cancer, can use together with a kind of chemotherapeutics of or cooperative programs, these chemotherapeutics are including, but not limited to methotrexate, paclitaxel, purinethol, thioguanine, hydroxyurea, cytosine arabinoside, cyclophosphamide, ifosfamide, nitroso ureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, ghost ethylidene glycosides, camptothecine, bleomycin, amycin, darubicin, daunorubicin, D actinomycin D, the fold mycin, the day care anthraquinone, asparaginase, vincaleucoblastine, vincristine, Vinorelbine, paclitaxel, docetotaxel etc.In a preferred embodiment, synthetic modification antibody combines with the toxin of chemotherapeutic or other type, as Ricin or radionuclide or other any other medicaments that kill cancer or tumor cell or anticancer growth effectively.In another preferred embodiment, this modified immunoglobulin has one and contains at the CDR1 of the antigenic binding site of cancer and another and contain CDR at the binding site of immunocyte surface molecular, and described immunocyte is such as, but be not limited to T cell, B cell, NK cell, K cell, til cell or neutrophil(e) cell.
In some embodiments of the present invention, wherein the CDR of synthetic modification antibody comprise a kind of immunity specifically with the bonded aminoacid sequence of human colon carcinoma associated protein antigen, it is preferred that described antibody has following properties: (ⅰ) identification antigenic protein component epi-position but the antibody of nonrecognition antigenic carbohydrates component epi-position; (ⅱ) in health adult tissue, detect less than antigen; (ⅲ) detect in the human cancer cell except colon cancer cell less than antigen.In other embodiments, the CDR of synthetic modification antibody comprise immunity specifically with the bonded aminoacid sequence of a kind of antigen, detect in the human cancer cell of this antigen except breast cancer cell less than.Still in other embodiments, the CDR of synthetic modification antibody comprise immunity specifically with the bonded aminoacid sequence of a kind of antigen, detect in the human cancer cell of this antigen except ovarian cancer cell less than.
5.3.1.1 malignant tumor
(about the summary of these imbalances, see Fishman etc., 1985 by using malignant tumor and the related disorder that medicine of the present invention can treat or prevent including, but not limited to those listed in table 2 kinds, medical science, second edition, J.B.Lippincott Co., Philadelphia):
Table 3
Malignant tumor and related disorder
Leukemia
Acute leukemia
Acute lymphoblastic leukemia
Acute myelocytic leukemia
Myeloblast property
Early young grain myelocyte
The monokaryon myelocyte
Mononuclear cell
Erythroleukemia
Chronic leukemia
Chronic myelocytic (mononuclear cell) leukemia
Chronic lymphocytic leukemia
True sexual cell increase disease
Lymphoma
Hodgkin
The non-hodgkin's disease
Multiple myeloma
Waldenstrom ' s macroglobulinemia
Heavy chain disease
Solid tumor
Sarcoma and cancer
Fibrosarcoma
Myxosarcoma
Liposarcoma
Chondrosarcoma
Osteogenic sarcoma
Chordoma
Angiosarcoma
Endotheliosarcoma
Lymphangiosarcoma
Lymphangioendothelioma
Synovioma
Mesothelioma
Ewing's tumor
Leiomyoma
Become 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
Adenocarcinoma of nipple
Cystadenocarcinoma
The medullary substance cancer
Bronchogenic carcinoma
Renal cell carcinoma
Hepatocarcinoma
Cancer of biliary duct
Choriocarcinoma
Spermocytoma
Embryonal carcinoma
Wilms' tumor
Cervical cancer
Uterus carcinoma
Testicular tumor
Pulmonary carcinoma
Small cell lung cancer
Bladder cancer
The epithelium sarcoma
Glioma
Astrocytoma
Medulloblastoma
Craniopharyngioma
Ependymoma
Pinealoma
Hemangioblastoma
Neuroma
Oligodendroglioma
Meningioma
Melanoma
Neuroblastoma
Become retina tumor
In specific embodiment, treatment or the prevention pernicious or propagation imbalance in ovary, bladder, mammary gland, colon, lung, skin, pancreas, prostate, uterus, gastrointestinal tract, bone-marrow-derived lymphocyte or T lymphocyte changes (as metaplasia and abnormal development) or hyper-proliferative imbalance.In other specific embodiments, can treat or prevent sarcoma, melanoma or leukemia.
5.3.1.2. precancer the state of an illness
Also can use medicine of the present invention with treatment state of an illness precancer and neoplasia or the malignant state of prevention including, but not limited to those listed imbalances of table 3.Known or be suspected to have the state of an illness that develops to neoplasia or cancer direction and use above-mentioned prevention or treatment, especially the non-neoplastic cell proliferation of forming by hypertrophy, metaplasia, or particularly abnormal development (summary about the above-mentioned abnormality proliferation state of an illness is seen, Robbins and Angell 1976, basic pathology, the 2nd edition, W.B.Saunders Co., Philadelphia, the 68-79 page or leaf).Hypertrophy be a kind of not obvious change structure or function but relate to tissue or organ in the controlled cell proliferation form that increases of cell quantity.As only in one embodiment, endometrial hyperplasia is often prior to carcinoma of endometrium.Metaplasia is the form that a kind of controlled cell is grown, and in this form, 1 type adult or complete noble cells is replaced 1 type adult cell in addition.Metaplasia can occur in epidermis or connective tissue cell.Atypical metaplasia comprises a bit disorderly metaplasia epidermis.Abnormal development is the omen of cancer normally, mainly finds in epidermis; This is the most disorderly form of non-neoplasia sexual cell growth, comprises losing of cell individual homogeneity and losing of cell spaces structure direction.Dysplastic cell often has the unusual big dark nucleus of dyeing, and shows as pleomorphism.Dysplastic feature is to exist the position of chronic stimulation or inflammation to occur, and finds in the cervix uteri of being everlasting, respiratory tract, oral cavity and the gallbladder.
In addition or except existing with hypertrophy, metaplasia or abnormal development is the abnormal cell growth of feature, from patient's cell sample, in vivo or external demonstration when having a kind or multiple conversion phenotype or malignant phenotype's feature, just show needs preventative/the therapeutic administration medicine.As mentioned above such, the feature of above-mentioned conversion phenotype comprises metamorphosis, more loose substrate tack, forfeiture contact inhibition, forfeiture grappling dependency, discharges protease, increases the sugar transportation, reduces the serum demand, expresses fetal antigen, 250,000 dalton's cell surface protein disappearance or the like is (about the feature relevant with conversion or malignant phenotype, also see the same, the 84-90 page or leaf).
In one specific embodiment, the hypertrophy of leukoplakia, the optimum appearance of epidermis or abnormal development damage or Bao circle disease, original position cancer are preceding neoplasia damage, show the preventative interventional therapy of needs.
In another embodiment, Fibrocystic disease (cystic hyperplasia, breast development are unusual, especially adenopathy (optimum epidermal hyperplasia)) shows the preventative interventional therapy of needs.
In other embodiments, show as following a kind or more kinds of patient who tends to virulent factor by the medicine treatment of using effective dose of the present invention: with pernicious relevant chromosome translocation (as the Philadelphia chromosome of chronic lymphocytic leukemia, t of follicular lymphoma (14:18) or the like), familial polyposis or Gardner's syndrome (omen of possible colon cancer), optimum monoclonal gamma globulin disease (may be the omen of multiple myeloma), with suffer from cancer or preceding carcinous disease and show as the relevant lineal relative's relation of the personnel of Mendel's formula (heritability) hereditary form, these Mendelian inheritance types are (as the familial polyposis of colon, Gardner's syndrome, the heritability exostosis, polyendocrinoma, medullary substance thyroid carcinoma with amyloid and pheochromoblastoma, the Peutz-Jeghers syndrome, Von Recklinghausen neurofibromatosis, become retina tumor, neck moves the body of gland tumor, the dermal melanin cancer, the ophthalmic malignant melanoma, xeroderma pigmentosum, ataxia telangiectasia, cut enlightening Acker-Dong syndrome, albinism, Fan Keni aplastic anemia and Bloom ＇ s syndrome, see Robbins and Angell, 1976, basic pathology, the 2nd edition, W.B.Saunders Co., Philaolephca 112-113 page or leaf) etc.).
In another specific embodiment, use the development of medicine of the present invention with prevention ovary, mammary gland, colon, pancreas, bladder, skin, prostate, colon, gastrointestinal tract, bone-marrow-derived lymphocyte, T lymphocyte or uterus carcinoma or melanoma or sarcoma to patient.
5.3.2 the treatment of infectious disease
By using medicine of the present invention, especially use and the synthetic modification immunoglobulin (or its functional activity fragment) of the antigen of the cause of disease that produces communicable diseases or the enzyme immunity specific bond expressed at the cell receptor of infectious disease cause of disease or by the infectious disease cause of disease method that the present invention also provides treatment or keeps off infection.As going through below, the infectiousness cause of disease is including, but not limited to virus, antibacterial, fungus, protozoacide or parasite.
In specific embodiment, by modified antibodies (or its functional activity fragment) prevention or the treatment infectious disease of using immunoglobulin, one of antigen of following infectious disease cause of disease is discerned in described modified antibodies immunity specifically: influenza virus hemagglutinin (gene bank registration number JO 2132, Air, 1981, institute of NAS reports 78:7639-7643; Newton etc., 1983, virusology 128:495-501), people's respiratory syncytial virus G glycoprotein (gene bank registration number Z 33429; Gareia etc., 1994, Journal of Virology; Collins etc., 1984, institute of NAS reports 81:7683), core protein, stromatin or other albumen (the gene bank registration number M 19197 of dengue virus; Hahn etc., 1988, virusology 162:167-180), Measles virus hemagglutinin (gene bank registration number M 81899; Rota etc., 1992, virusology 188:135-142), II herpes simplex virus type glycoprotein gB (gene bank registration number M 14923; Bzik etc., 1986, virusology 155:322-333), gray nucleus viroid I VP1 (Emini etc., 1983, the nature 304:699), HIV I by membrane glycoprotein (Putney etc., 1986, science 234:1392-1395), hepatitis B virus surface antigen (Itoh etc., 1986, natural 308:19; Neurath etc., 1986, vaccine 4:34), diphtheria toxin, diphtherotoxin (Audibert etc., 1981, nature 289:543), streptococcus 24M epi-position (Beachey, 1985, experimental medicine is made progress 185:193 biology), gonococcus pilin (Rothbard and Schoolnik, 1985, experimental medicine is made progress 185:247 biology), pseudorabies poison g50 (gpD), pseudorabies poison II (gpB), pseudorabies poison III (gpC), pseudorabies poison glycoprotein h, pseudorabies poison glycoprotein E, transmissible gastroenteritis glycoprotein 195, the transmissible gastroenteritis stromatin, porcine rotavirus glycoprotein 28, many hollow capsids of pig breast albumen, the little Serpentis bacterium of swine dysentery protective antigen, bovine viral diarrhoea glycoprotein 55, the sick viral hemagglutinin neuraminidase of neofield, the swine flue hemagglutinin, the swine flue neuraminidase, foot and mouth disease virus, swine fever virus, swine influenza virus, the African swine fever poison, mycoplasma hyopneumoniae, infectious bovine rhinotracheitis virus (as, infectious bovine rhinotracheitis viral glycoprotein E or glycoprotein G), or infectious laryngotracheitis virus (as infectious laryngotracheitis virus glycoprotein G or glycoprotein I), glycoprotein (the Gonzales-Scarano etc. of LaGrosse virus, 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 129:2763), punta foro virus (Dalryaple etc., 1981, duplicating of negative strand viruses, Bishop and Compans (writing), Elsevier, New York, 167 pages), murine leukemia virus (Steeves etc., 1974, Journal of Virology 14:187), MMT virus (Massey and Schochetman, 1981, virusology 115:20), (as the UK Patent Application book of seeing that on June 4th, 1980 published, number is GB 203423A for hepatitis B virus core protein and/or hepatitis B virus surface antigen or fragment or derivatives thereof; Ganem and Varmus, 1987, bioid academic year is commented 56:651-693; Tiollais etc., 1985, nature 317:489-495), the antigen of equine influenza virus or equine herpes virus (as, equine influenza virus A type/Alaska 91 neuraminidases, equine influenza virus A type/Miami 63 neuraminidases, equine influenza virus A type/Kentucky 81 neuraminidases, equine herpes virus I type Glycoprotein B and equine herpes virus I type glycoprotein D), the antigen of cattle respiratory syncytial virus or bovine influenza virus (as, bovine respiratory syncytial virus attachment protein (BRSVG), cattle respiratory syncytial virus fusion rotein (BRSV F), cattle respiratory syncytial virus nucleocapsid protein (BRSV N), 3 type bovine influenza virus fusion rotein and bovine influenza virus 3 type hemagglutinin neuraminidases), bovine viral diarrhea virus glycoprotein 48 or glycoprotein 53).
Guiding is used for the treatment of the cell receptor of infectious disease to be listed at table 3, also lists and the bonded cause of disease of cell receptor simultaneously.
Cause of disease Cell receptor
B-parent lymphocyte papovavirus (LPV) LPV receptor on the B cell
Bordetella pertussis Adenyl cyclase
Borna disease virus (BDV) The BDV surface glycoprotein
Bovine coronavirus N-acetyl-9-O-n acetylneuraminic acid n receptor
Choriomeningitis virus CD4+
Dengue virus High sulphation type heparin sulfate p65
Escherichia coli Contain the Galal-4Gal isoreceptor
Ebola virus CD16b
Echovirus
1 Integrin VLA-2 receptor
Echovirus-11 (EV) The EV receptor
Endotoxin (LPS) CD14
Intestinal bacteria The carbohydrate conjugates receptor
Enteric orphan virus α/β TXi Baoshouti
Enterovirus The speed factor acceptor that declines
Feline leukaemia virus Cell envelope membrane glycoprotein (Env-SU) receptor
Foot and mouth disease virus Immunoglobulin Fc receptor poxvirus M-T7
Gibbon ape leukemia virus (GALV) 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 ammonia polyose of candy receptor fibroblast growth factor acceptor
Cause of disease Cell receptor
HIV-1 CC-chemokine receptor CCR 5 CD11a CD2 G-G-protein linked receptor CD4
Human cytomegalic inclusion disease virus Heparin sulfate Dan Baijutang annexin I CD13 (Aminopeptidase N)
The human corona virus People's Aminopeptidase N receptor
Influenza virus A, B and C The hemagglutinin receptor
Legionnella CR3 receptor protein kinase receptor galactose N-acetylgalactosamine (the quenchable agglutinin receptor chemokine receptors of Gal/Gal/NAC
Leishmania mexicana The annexin I
Monocytosis Li Site bacterium Act A albumen
Measles virus The CD46 receptor
Meningococcus The meningococcus virulence Opa receptor of being correlated with
Measles virus The CD46 receptor
Mouse hepatitis virus The carcino-embryonic antigen family receptors carcino-embryonic antigen Bgla of family receptor
Murine leukemia virus By membrane glycoprotein
γ-murine herpetovirus Interferon-gamma receptor
The Mus retrovirus Glycoprotein gp70 Rmc-1 receptor
Mus coronavirus Mouse hepatitis virus The carcino-embryonic antigen family receptors
Bird type Mycobacterium tuberculosis-M Human beta 2 integrin receptor α V β 3
Cause of disease Cell receptor
Diplococcus gonorrhoeae Heparin sulfate proteoglycans Shou Ti CD66 Shou Ti integrin receptor Mo common factor Shou Ti CD46 GM1 GM2 GM3 CD3 ceramide
Ewcastle disease virus Hemagglutinin neuraminidase fusion protein
Assays for parvovirus B 19 Erythrocyte P antigen receptor
Plasmodium falciparum CD36 receptor alpha-Glycophorins receptor
Poxvirus The gamma interferon receptor
Rhodopseudomonas Kdel receptor
Rotavirus Guiding mucosa α 4 β 7 receptors
Salmonella typhimurium EGF-R ELISA
Shigella dysenteriae Alpha 5 beta 1 integrin
Streptococcus Non-glucosides J774 receptor
1 type helper T cell Comprise following chemokine receptors: 6.CXCR1-4 7.CCR1-5 8.CXCR3 9.CCR5
1 type parent T lymphocyte virus The gp46 surface glycoprotein
Vaccinia virus TNFRp55 receptor TNFRp75 receptor soluble interleukin-6-1 beta receptor
The disease of virosis that can be treated by method of the present invention or prevent including, but not limited to causing by following virus, these viruses are hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, chickenpox virus, adenovirus, herpessimplexvirustype (HSV-1), herpes simplex virus type 2 (HSV-II), rinderpest virus, rhinovirus, echo virus, rotavirus, the respiratory syncytial virus, papovavirus, cytomegalovirus, bundle shape virus, arbovirus, Hantaan virus, Coxsackie virus, mumps virus, Measles virus, rubella virus, poliovirus, I type human immunodeficiency virus (HIV-I) and II type human immunodeficiency virus (HIV-II), any picorna virus, enterovirus, the embedding Calicivirus, any Norwalk papova, togavirus, as dengue virus, Alphavirus, yellow fever virus, coronavirus, rabies virus, Marburg virus, Ebola virus, parainfluenza virus, influenza virus, arenavirus, reovirus, wheel shape virus, socket of the eye virus, I type human T-cell leukemia virus, II type human T-cell leukemia virus, the troglodyte immunodeficiency virus, slow virus, polyoma virus, parvovirus, Epstein-Barr virus, herpes virus hominis-6, I type cercopithecid herpesvirus (B virus) and poxvirus.
Can be by method of the present invention treatment or the bacterial disease that prevents by including, but not limited to the caused disease of following antibacterial, these antibacterials are mycobacterium, rickettsia, mycoplasma, Neisser mushroom (as Neisseria meningitidis and Diplococcus gonorrhoeae), Legionnella, vibrio cholera, Streptococcus such as streptococcus pneumoniae, diphtheria corynebacterium, Clostridium tetani, bordetella pertussis, haemophilus class (as the influenza bacterium), chlamydia class, enterotoxication escherichia coli etc.
Can be by method of the present invention treatment or the protozoan disease that prevents including, but not limited to by Plasmodium, eimeria, leishmaniasis, the cocoa disease that causes of Eimeria (kokzidioa) and trypanosoma together.
In specific embodiment of the present invention, described medicine can combine with the suitable antibiotic that is used for the treatment of or keep off infection, antifungal, antiviral agents or any other medicine and use.In an embodiment preferred, synthetic modified antibodies is connected with the chemical compound of infection disease pathogen effectively, and this synthetic modification antibody for example is directed to antibiotic, antifungal or antiviral agent on the cause of disease.In another preferred embodiment, modified immunoglobulin has one and contains at the CDR of the antigenic binding site of infectious disease cause of disease and another and contain at the CDR at the binding site of following immunocyte surface molecular, and these immunocytes are such as, but be not limited to T cell, B cell, NK cell, K cell, til cell or neutrophil(e) cell.
5.3.3 gene therapy
In one specific embodiment, use the nucleic acid of the sequence that comprises code book invention synthetic modification antibody, bonded specifically developed by molecule diseases associated of treatment or prevention and synthetic modification antibody mediated immunity or imbalance.
In this embodiment of the present invention, the treatment usefulness a kind of sequence of nucleic acid coding, this sequence in cell, (do not have homing sequence) or iuntercellular (having homing sequence) but production modified immunoglobulin of the present invention.
Any this area efficient gene Therapeutic Method can both be used according to the present invention.Typical method is described below.
General summary about gene therapy method.See Goldspiel etc., 1993, clinical pharmacy 12:488-505; Wu and Wu, 1991, Biotherapeutics method 3:87-95; Tolstoshev, 1993, pharmacology's toxicity academic year is commented 32:573-596; Mulligan, 1993, science 260:926-932, and Morgan and Anderson, 1993, bioid academic year is commented 62:191-217; May, 1993, TIBTECH 11 (5): 155-215).This area as everyone knows can with the method for recombinant DNA technology aspect see following description, Ausubel etc. (writing), 1993, molecular biology modernism, John Wiley﹠amp; Sons, New York; Kricgler, 1990, gene transfer and expression, the chamber handbook is tested in the chamber, Stockton publishing house, New York; And the 12nd and 13 chapters, Dracocoli etc. (writing) 1994, human genetics modernism, John Wiley and Sons, New York.
An aspect, treatment nucleic acid comprises a kind of expression vector, this expression vector is expressed modified immunoglobulin (or its fragment) in suitable hosts.Particularly, above-mentioned nucleic acid has an operability to be connected to modify the promoter on the coded sequence of synthetic antibody, described promoter can be induced or composition and selectable tissue-specific.In another embodiment, nucleic acid molecule used therefor, wherein promote the zone of homologous recombination to be positioned at antibody coding sequence and any other required sequence flank by site required in genome, intrachromosomal expression (Koller and the Smithes of modified antibodies are provided thus, 1989, institute of NAS reports 86:8932-8935; Zijlstra etc., 1989, natural 342:435-438).
It can be direct or indirect method that this nucleic acid is carried to patient, and in direct situation, patient directly contacts nucleic acid or carries the carrier of nucleic acid or carry complex; In non-direct situation, at first use nucleic acid vitro conversion cell, be transplanted in the patient body then.These 2 kinds of methods are respectively in the body of knowing or stripped gene therapy.
In one embodiment, directly in the body administration of nucleic acid at the site of administration expression of nucleic acid to produce antibody.This scheme can be finished by any of big metering method well known in the art, for example makes up its part as suitable nucleic acid expression vector, uses the method that the result becomes cell inner expression then; For example (see U.S. Patent number 4 with the retrovirus of defective or attenuation or the method for other viral carriers infection; 980; 286); or the exposed DNA method of direct injection; or use the microparticle ballistic method (as particle gun; Biolistic; Dupont); or use lipid or cell surface receptor or transfection agents, with the biopolymer bag by method (as, poly--β-1-＞4-N-acetylglucosamine polysaccharide; see U.S. Patent number 5; 635,493) liposome tunicaization; microparticle or microencapsulation method, or use after nuclear peptide is connected with known can entering; or after being connected, uses nuclear part with known can entering; or (for example see Wu and Wu, 1987 with using after the part that carries out receptor-mediated cell endocytic is connected; journal of biological chemistry, method such as 262:4429-4432).In another embodiment, can form the nucleic acid ligands complex, part comprises a kind of fusion viral peptide to destroy endosome in this complex, makes nucleic acid avoid the lysosome degraded.And in another embodiment, by leading special receptor, this nucleic acid can lead in vivo with the absorption of carrying out cell-specific and expression (as seeing, PCT publication WO 92/06180,1992 on April 6, (Wu etc.) of date of publication; WO 92/22635 date of publication December 23 days (Wilson etc.) in 1992; WO 92/20316 date of publication November 26 (Findeis etc.) in 1992; WO 93/14188 date of publication July 22 (Young) in 1993).In addition, by the homologous recombination method with in the nucleic acid transfered cell and be attached among the prominent chief cell DNA of expression (Kollen and Smithies, 1989, institute of NAS reports 86:8932-8935; Zijlstra etc., 1989, natural 342:435-438).
In addition, can use single-chain antibody, for example this method is utilized as those technology of Marasco etc. (Marasco etc., 1993, institute of NAS report 90:7889-7893) description are listed in the targeted cell population nucleotides sequence of the single-chain antibody of encoding to express.Adenovirus is the another kind of viral vector that can be used for gene therapy.Particularly gene is transported to them and causes that in the respiratory system epidermis of gentle disease, adenovirus is attractive carrier.Other target spots of adenovirus induction system are liver, central nervous system, endotheliocyte and muscle.Adenovirus has the advantage that can infect Unseparated Cell.Kozarsky and Wilson (1993, the modern viewpoint 3:499-503 of hereditism and growth) have delivered the summary based on the adenoviral gene treatment.Bout etc. (1994, human gene therapy 5:3-10) have illustrated adenovirus vector and have transmitted the purposes of gene to Rhesus Macacus respiratory system epidermis.Other examples of using adenoviral can be at Rosenfeld etc., 1991, science 252:431-434 in gene therapy; Rosenfeld etc., 1992, cell 68:143-155 and Mastrangeli etc., 1993, find among the Journal of Clinical Investigation 91:225-234.Also the someone advises being used for gene therapy (Walsh etc., 1993, the journal 204:289-300 of the biomedical association of experiment) with adeno associated virus (AAV).
Envision the seriousness, patient condition of the form of used treatment nucleic acid and type that dosage depends on disease and required effect etc., and can determine by those skilled in the art.
5.3.4 vaccine virus immunization method
Modified antibodies of the present invention can be used as vaccine to be used in this experimenter to the experimenter, need be at the immunity of special molecular or antigen binding site.Vaccine of the present invention and method can be used for prevent disease or imbalance, perhaps treat disease or imbalance, and it is useful wherein replying in treatment and prevention to the antiidiotype of special synthetic antibody.
Method and composition of the present invention can be used for exciting the body fluid of the synthetic antibody of anti-vaccine in the experimenter and/or cell-mediated replying.In one specific embodiment, this method and composition excites the humoral response of the anti-synthetic antibody of being used among the experimenter.In another specific embodiment, this method and composition excites the cell-mediated response of the anti-synthetic antibody of being used in the experimenter.In an embodiment preferred, this method and composition excites body fluid and cell-mediated replying.
5.4 medicinal prepared product and application process
5.4.1 preparation and application process
Use the therapeutic combination contain modified immunoglobulin according to the present invention, available a kind or multiple physiology's acceptable carrier or excipient are made with the method for any routine.
Therefore, modified immunoglobulin (or its functional activity fragment or encoding antibody or segmental nucleic acid) and the acceptable salt of they physiologys and solvent can be made preparation, by inhalation or insufflation (by mouth or nose) or oral cavity, buccal, intestinal is outer or rectal administration.
About oral administration, medicine is prepared into by conventional method with pharmaceutically acceptable excipient and for example can makes tablet or capsular form, these excipient such as bonding agent (as corn starch, polyvinylpyrrolidone or the hydroxypropyl emthylcellulose of gelatinization in advance); Filler (as lactose, microcrystalline Cellulose or calcium hydrogen phosphate); Lubricant (as magnesium stearate, Pulvis Talci or Silicon stone), disintegrating agent (as potato starch or sodium starch glycolate); Or wetting agent (as sodium laurylsulfate).Tablet can add the bag quilt by method well-known in the art.The liquid prepared product that is used for oral administration can be made into the form as solution, syrup or suspending agent, perhaps they is made desciccate, water or the dissolving of other suitable carriers before using.The aforesaid liquid prepared product can be by pharmaceutically acceptable additive preparation of conventional method, these additives such as suspending agent (as the edible fat of sorbitol syrups, cellulose derivative or hydrogenation); Emulsifying agent is (as lecithin or arabic gum; Nonaqueous carrier (as almond oil, grease, ethyl hexanol or fractionated vegetable oil); And antiseptic (as methyl or propyl group P-hydroxybenzoic acid or sorbic acid).Prepared product also can contain suitable buffer salt, flavouring agent, toner and sweeting agent.
Can suitably produce and be used for medicinal preparation for oral administration, so that the controlled release of reactive compound.
Be used for oral administration, this medicine can adopt the form of tablet or lozenge, makes with conventional method.
Use as for inhalation, use suitable propellant according to the present invention, as dichlorodifluoromethane, Arcton 11, dichlorotetra-fluoroethane, carbon dioxide or other suitable gas, medicine of the present invention is carried from pressure vessel or aerosol apparatus easily with the form that aerosol spray presents.Under the aerocolloidal situation of pressure, dosage unit is by providing the valve of measuring quantity to determine.Be used for the mixture of powders of the chemical compound that inhalant or insufflation capsule and Catridgel such as gelatin make and such as the preparation of the suitable powder substrate of lactose or starch.
Through injection such as bulk injection or continuous infusion, medicine of the present invention can be made the preparation that intestinal is used (being intravenous or intramuscular) outward.The preparation of injection is with the form of unit dose, is placed in the container as ampoule or multiple dose with the antiseptic that adds.Said composition can adopt the form as suspending agent, solution or Emulsion in oil and water carrier, and can contain the prescription reagent such as suspending agent, stabilizing agent and/or dispersant.In addition, active component can form of powder, suitable carriers such as aseptic pyrogen-free water dissolution before using.
This medicine also can be made the rectal compositions such as suppository or delay enema, as contains conventional suppository base such as cupu oil or other glyceride.
Except previously described preparation, this medicine also can be made the storage prepared product.The preparation of above-mentioned long duration of action can be used by implantation (as subcutaneous or intramuscular) or intramuscular injection.Therefore, for example, this chemical compound is made preparation with suitable polymers or hydrophobic material (as the Emulsion in acceptable oil) or ion exchange resin or a spot of soluble derivative such as a small amount of soluble salt.
Modified immunoglobulin of the present invention can be used as independently compositions or single compositions, uses with the antibody more than a kind that is connected by conventional chemical method or molecular biology method.In addition, the diagnosis of antibody of the present invention and therapeutic value can be united with radionuclide or such as the toxin of ricin or as the chemotherapeutic of methotrexate and used and increase.
If necessary, said composition also can contain wetting agent or the emulsifying agent or the pH buffer agent of trace.Said composition can be aqueous solution, suspending agent, emulsifying agent, tablet, pill, capsule, slow releasing agent or powder.Oral formulations can comprise mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate of the carrier of standard such as pharmaceutical grade etc.
In general, with the form independence of unit dose or mix each component is provided, for example as lyophilization powder or anhydrous being concentrated in the sealed container, in the ampoule or folliculus (sachetle) as lined out activity agent quantity.If said composition is used by injection, the sterile diluent of an ampoule amount should be provided, each component can be mixed before using like this.
The present invention also provides a kind of and comprises 1 or the medicinal bag or the medicinal reagent box of multiple container, is equipped with 1 or the component of multiple bacterin preparation of the present invention in the container.Appended on above-mentioned these containers is points for attention, and its form is manufacturing, the use of the medicine worked out by government organs or biological product or sells rule that these points for attention show that the mechanism of the manufacturing, use or the sale that are applied to the people permits through approval.
If necessary, said composition can be placed in a medicated bag or the dispensation apparatus, can contain 1 or a plurality of unit dosage form of active component.This medicated bag for example can comprise metal or plastic foil, as the foaming bag.The description that this medicated bag or dispensation apparatus can attachedly be used.Comprise compound compositions of the present invention and also can make compositions, be placed in the suitable containers with compatible pharmaceutical carriers, and the indication condition of labeled for treatment.
Can import bacterin preparation of the present invention in many ways; These methods in oral, brain, intradermal, intramuscular, intraperitoneal, subcutaneous, intranasal passage and through the immunization route of scarification (as scratch the top layer of skin with fork needles) or any other standard.
The exact dose that is used to this modified immunoglobulin molecule in the preparation also will depend on the approach used and patient's characteristics, determine according to the clinical technology of standard and according to doctor's diagnosis and each patient's situation then.Effectively immunity amount refers to the amount of using this vaccine production thing in the host, is enough to produce the quantity that synthetic antibody mediated immunity is replied.Derive in the dose response curve that effective dose also can obtain from animal model mensuration system.
5.4.2 effective dose
Chemical compound of Miao Shuing and nucleotide sequence are used to patient in the present invention, and its treatment effective dose will be treated some disease or the imbalance such as cancer or infectious disease.The amount that a kind of therapeutic effective dose refers to chemical compound is enough to make among the experimenter who treated and obtains health advantages.
The toxicity of chemical compound and therapeutic efficacy can be determined in cell culture or laboratory animal by standard pharmaceutical procedures, for example measure LD50 (to the lethal dosage of 50% colony) and ED50 (the effective dosage of treatment in 50% colony).The ratio of toxicity and treatment effect is therapeutic index, and it can be expressed as the ratio of LD50/ED50.It is preferred showing as the exponential chemical compound of big treatment.Though can be with the chemical compound of toxic side effect, should careful design a kind of with the above-claimed cpd induction system of action site of affected tissue that leads, so that the potential damage of non-infected cells is reduced to minimum level, and therefore reduce side effect.
The data that obtain from cell culture mensuration and zooscopy can be used to be identified for human dosage range.Preferably, the dosage of above-claimed cpd is positioned at and comprises few or do not have among the circulation composition scope of toxic ED50.Change within this scope of dosage form that this dosage can adopt depending on and used route of administration.In method of the present invention, used any chemical compound, begin from cell culture is measured, to estimate the treatment effective dose.Systematically determine a kind of dosage to obtain the circulating plasma concentration range in animal model, this scope is included in the IC50 value of measuring in the cell culture (promptly obtaining maximum symptom one compound concentrations that half is measured that suppresses).Above-mentioned data is used for determining more accurately the mankind's doses available.For example can measure level in the blood plasma by the high performance liquid chroma-tography technology.
5.4.3 bacterin preparation and using
The present invention also provides the bacterin preparation that contains medicine of the present invention, and this bacterin preparation is applicable to be used to excite anti-some antigenic protective immunity (body fluid and/or cell-mediated) to reply, and for example is used for the treatment of and prevent disease.
The suitable prepared product of above-mentioned vaccine comprises the injectable agent as liquid solution or suspension; Also can be prepared into the solid form that is applicable to solution, suspension, liquid before the injection.Also emulsifying prepared product, or this polypeptide is with the lipid capsuleization.The active immne ultimate constituent often and pharmaceutically acceptable and with the mixed with excipients of active component compatibility.Suitable excipient is the isotonic aqueous buffer etc. and the conjugate of water, salt, butterfat salt, glucose, glycerol, ethanol, sterilization for example.In addition, if necessary, the vaccine production thing also can comprise the auxiliary substance of trace, as the adjuvant of wetting agent or emulsifying agent, pH buffer agent and/or enhancing vaccine effectiveness.
Effectively the example of adjuvant is including, but not limited to aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thrMDP), N-acetyl-just-muramyl-L-alanyl-D-isoglutamine, the different glutamy of N-acetyl muramyl-alanyl-D--L-alanine-2-(1 ＇-2 ＇-two palmityls-Sn-glyceryl-3-hydroxyl phosphorus acyloxy)-ethamine.
Can directly resist the effect of the injecting immune globulin made from special adjuvant adjuvant to determine the effectiveness of adjuvant by mensuration induced anti-idiotype antibody.
Said composition can be liquid solution, suspending agent, Emulsion, tablet, pill, capsule, slow releasing agent or powder.Oral formulations can comprise mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate of standard vector such as pharmaceutical grade etc.
In general, with the form independence of unit dose or mix each component is provided, for example as in lyophilization powder or the anhydrous container that is concentrated in sealing, in the ampoule or folliculus (sachette) that show activating agent quantity.If said composition is used by injection, the sterile diluent of an ampoule should be provided, can mix each component before using like this.
In specific embodiment, lyophilization modified immunoglobulin of the present invention is provided in first container, second container comprises diluent, is made up of 50% glycerol, 0.25% phenol and antibacterial (viride nitens as 0.005%).
The present invention also provides medicinal bag or the medicinal reagent box that comprises a kind or multiple container, and the composition of a kind or multiple bacterin preparation of the present invention is housed in these containers.With points for attention, its form is manufacturing, the use of the medicine worked out of government organs or biologic or sells rule that this rule represents that the mechanism that is applied to human manufacturing, use or sale obtains the permission approval on the said vesse.
If necessary, said composition can be placed in medicated bag or the dispensation apparatus, wherein contains one or more unit dosage forms of active component.Medicated bag can comprise for example metal or plastic foil, as the foaming bag.This medicated bag or dispensation apparatus can attachedly be used description.Also can prepare the compositions that comprises chemical compound of the present invention and make, be placed in the suitable containers with compatible pharmaceutical carrier, and the condition of accusing of of labeled for treatment.
The experimenter who uses this vaccine is mammal preferably, most preferably human, but also can be the animal of non-human, these animals be including, but not limited to cow, horse, sheep, pig, birds (as chicken), goat, cat, Canis familiaris L., hamster, mice and rat.
Can import bacterin preparation of the present invention in many ways; These methods in oral, brain, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal approach and through insufflation (as scratch the top layer of skin with fork needle) or any other standard immunoassay approach.In one specific embodiment, use scarification.
The exact dose that is used in this modified immunoglobulin molecule in the preparation also will depend on route of administration and patient's characteristics, and determine according to doctor's diagnosis and each patient's situation according to the clinical technology of standard.Effectively immunity amount is meant in the host who has used the vaccine production thing, is enough to produce the quantity of the modified immunoglobulin molecular immune being replied (being anti-idiotype reaction).Effective dose also can derive from the dose response curve that animal model mensuration system obtains.
5.5 diagnostic method
With combine member's special molecular bonded modified immunoglobulin, especially antibody (with its functional activity fragment), can be used as diagnostic agent and Prognosis agent according to the method described in the present invention.In various embodiments, the invention provides in conjunction with assay method and the purposes of said determination method in clinical use to the member.Modified immunoglobulin of the present invention for example can be used for the antigen in the detection of biological sample, therefore can to modified immunoglobulin with it bonded molecule abnormal level and/or exist the patient of the anomaly pattern of above-mentioned molecule to detect, " abnormal level " means with respect to increasing or reduce from the part of health or from level or representative standard level that the experimenter's who does not suffer from this imbalance similar sample exists at least.Modified antibodies of the present invention also can comprise and be used for diagnosing or the reagent of prognosis technology test kit.
In specific embodiments of the present invention, modified antibodies of the present invention with the antigen immune specific bond of the antigen of cancer or tumor or infectious disease cause of disease can be used for diagnosing, prognosis detection or screening and the antigen of cancer or tumor or relevant cancer or the tumor or the infectious disease of antigen presentation of infectious disease cause of disease.Aspect preferred, the invention provides the method for a kind of diagnosis or screening cancer or cancer development trend, it is characterized in that the cancer antigen that exists increases, this antigen be by described first member and second member composition in conjunction with the first right member, described method comprises measures modified antibodies and the immune specific bond level that derives from experimenter's sample among the experimenter, wherein said modified antibodies immunity combines with described cancer antigen specifically and this modified antibodies comprises a variable region, this variable region has 1 at least and contains described second member's partial C DR, and described part contains one at the antigenic binding site of this cancer and do not find under natural CDR environment.Wherein, with respect to from cancer stricken not or do not occur developing in experimenter's the similar sample of cancer trend should the immunity specific bond level, the level increase of described immune specific bond shows the trend that has cancer or the cancer of developing into is arranged.
Another preferred aspect, the invention provides the method that there are the infectious disease cause of disease in a kind of diagnosis or screening, it is characterized in that existing the antigen of described infectious disease cause of disease, this antigen be by first member and second member composition in conjunction with the first right member, described method comprises the level that is determined at modified antibodies among the experimenter and the sample immunity specific bond that derives from this experimenter, wherein said modified antibodies immunity comprises a variable region in conjunction with described antigen and this modified antibodies specifically, this variable region has 1 CDR that contains at least 4 amino acid moieties of described second member at least, described part contains at described antigenic binding site and does not find under natural CDR environment, wherein, with respect to from the immune specific bond level described in the experimenter's that the infectious disease cause of disease is not arranged the similar sample, the increase of this immunity specific bond shows and has described infectious disease cause of disease.
In another embodiment preferred, the invention provides the method for sepcific ligands in the sample that a kind of detection derives from the experimenter or receptor abnormality level, this method will be compared in conjunction with the immune specific bond that the immune specific bond of the modified antibodies of sepcific ligands or receptor and this modified antibodies temple have the sample of the part of normal level or receptor at sample.
Mensuration is by the method for the bonded molecule of modified antibodies, can be to detecting disease relevant among the experimenter and/or disease being carried out by stages with this molecule, the above-mentioned disease of screening in colony in the difference diagnosis and the influence of monitoring Drug therapy to the experimenter of experimenter's physiological condition, all has value.
Design following algoscopy with the molecule of detection with this modified antibodies immunity specific bond.
In specific embodiment, available these diagnostic methods detect the unusual of gene expression doses, or unusual in the Subcellular Localization of structure and/or temporary structure, tissue, cell or special molecular to be determined.
In general tissue to be analyzed or cell type comprise the kind of the expression special molecular that those are known or suspicious.In the method for this used protein isolate can be those methods of describing such as Harlow and Lane (Harlow, E and Lane, D.1988, " antibody: laboratory manual " publishing house of cold spring harbor laboratory, cold spring port, New York) for example.Institute's isolated cells can derive from cell culture or from patient.In addition, be used for modified antibodies of the present invention (or its functional activity fragment), available Histological method is used in immunofluorescence or the immunoelectron microscope, with this molecule of in situ detection.By from patient, pipetting histological specimens, as the specimens paraffin embedding slices of affected tissue, add the method for the modified antibodies of the present invention that labelling is crossed then, can finish in situ detection.The application process of preferred modified antibodies (or its functional activity fragment) is that the modified antibodies that labelling is crossed is covered on the biological sample.If the bonded with it molecule of antibody is arranged in Cytoplasm, for example, the permeable method of cell membrane meets the requirements by being imported to modified antibodies in the cell.By using said method, might not only determine to exist special molecule but also determine its distribution in the tissue of being checked.Utilize the present invention, general technical staff can easily see in order to obtain above-mentioned in situ detection result, can do modification to detect in the multiple Histological method any.(for example colouring method).
Immunoassay to special molecular typically comprises the steps, in the presence of modified antibodies that detectable labelling is crossed with as the cell of biological fluid, tissue extract, fresh separated or the sample incubations such as lysate of cultured cell, then with the bonded antibody of any detection of many technology well-known in the art.
Biological sample can contact and be fixed on the solid support or carrier as celluloid, or can fixed cell, on other solid supports of cell granulations or soluble protein.Wash this holder with a suitable buffer then, then the modified antibodies of crossing with detectable label is handled.Wash this solid support for the second time with this buffer then, to remove unconjugated antibody.Detect the quantity of the binding label on solid support then with conventional method.
At least " solid support or carrier " refers to the holder of any energy conjugated antigen or antibody.Well-known holder or carrier comprise cellulose, polyacrylamide, gabbro powder, the magnetite powder of glass, polystyrene, polypropylene, polyethylene, glucosan, nylon, amylase, natural and modified.The characteristics of carrier can be soluble or insoluble in a way with regard to purpose of the present invention.Support material in fact can have any possible node configuration, as long as molecular energy and the antigen or the antibodies of institute's coupling connection.Therefore, the configuration of holder can be spherical as pearl shape.Or cylindrical, as at the inner surface of test tube or the outer surface of post.In addition, this surface can be planar as thin slice, mensuration band etc.Preferred holder comprises polystyrene bead.Those skilled in the art can know many other suitable binding antibodies or antigenic carrier, maybe can use daily experimental technique and determine identical carrier.
Can determining according to well-known method of given modified antibodies in conjunction with activity.Those skilled in the art can measure definite can the operation and the suitableeest condition determination to each according to using the normal experiment method.
A kind of method in the method for the modified antibodies of detectable label is that same molecule is connected with enzyme, be used in (Voller in the enzyme immunoassay (EIA) (EIA) then, A " enzyme linked immunological absorption measurement method (ELISA) ", 1978, diagnostic level 2:1-7 microorganism association season publication, Walkersville, MD); Voller etc., 1978, clinical pathology magazine 31:507-520:Butler, 1981, Enzymology method 73:482-523; Maggio, E (writing), 1980, enzyme immunoassay, CRC publishing house, Boca Raton, FL; Ishikawa etc. (writing), 1981, enzyme immunoassay, Kgaku Shoin, (Tokyo).React with bonded enzyme of modified antibodies and suitable substrate, preferred chromogenic substrate can pass through as spectrophotography with the reaction that above-mentioned mode produces, and fluorophotometric method or visual method can detect chemical constituent.The enzyme that is used to detection property labelling modified antibodies is including, but not limited to malic dehydrogenase, staphylococcal nuclease, δ-5 steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphatase, dehydrogenase, triose-phosphate isomerase, horseradish peroxidase, alkali phosphatase, asparaginase, glucoseoxidase, beta galactosidase, ribonuclease, urine enzyme, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.After the chromogenic substrate reaction of use and this enzyme reaction, colorimetry is finished detection.Also can relatively finish detection to the degree naked eyes of the reference material of similar preparation by the degree of relatively substrates enzymes reaction.
Any that also can use various other immunoassays finished detection.For example, rely on the synthetic antibody or the fragment of radioactivity labelling, might detect by using radioimmunoassay (RIA) to detect antibody wants bonded albumen (for example to see, Weintraub, 1986, the radioimmunoassay principle, the 7th training class of radioligand determination techniques, endocrine association).Use γ-enumerator or liquid flashing counting device or autoradiography to detect this radiosiotope by said method.
Also might use this modified antibodies of fluorescent chemicals labelling.When the antibody of fluorescence labelling with the following time of light that is exposed to suitable wavelength, owing to exist fluorescence so just can detect its existence.In the most frequently used fluorescent labeling chemical compound Fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, different phycocyanin, phthalic aldehyde and fluorescamine.
Use the fluorescent emission metal as 152Eu or other group of the lanthanides series also can detect with this modified antibodies of labelling.These metals are with being connected on this antibody as diethylenetriamine benzene pentaacetic acid (DTPA) or ethylenediaminetetraacetic acid metal-chelating groups such as (EDTA).
Modified antibodies is coupled on the chemiluminescence compound, also can detects and use this modified antibodies.Then, by detecting the chemiluminescent existence that produces in the chemical reaction process, determine that the antibody of chemiluminescent labeling exists.Useful especially chemiluminescent labeling examples for compounds is luminol, different luminol, thermotaxis acridinium ester (theromatic acridinium ester), imidazoles, acridinium salt and oxalate.
Similarly, also available bioluminescent compound labelling synthetic modification antibody of the present invention.Bioluminescence is a kind of chemiluminescent type of finding in biosystem, but the efficient of wherein a kind of catalytic protein enhanced chemiluminescence reaction.Exist by the definite bioluminescent protein that detects the chemiluminescence existence.The important luminophor that is used for labelling is luciferin, luciferase and aequorin.
5.6 the proof of therapeutic use
Before being used for the mankind, preferable methods is in the external body then of medicine of the present invention, detects its required treatment or prophylactic activity.For example, being used to determine to use special medicine denys that effective external test method comprises cell in vitro cultivation assay method, in the method, otherwise contact with medicine or the administering therapeutic medicine from cell line or from the suitable cell of the patient's who suffers from special disease or imbalance cultured cell, observe the influence of medicine pair cell then.
If this medicine is a kind of modified immunoglobulin of discerning cancer or tumor antigen, by medicine is contacted with cultured cell (from patient or cultured cells system), measure surviving or growing state of cell with any method well known in the art then, just can measure the latent effectiveness of this modified immunoglobulin, for example, cell proliferation can by measure the mixing of 3H-thymus pyrimidine, directly cell counting, detect transcriptional activity such as knowns such as proto-oncogene (as fos, myc) or cell cycle labels and change and measure; Cell survival rate is estimated with trypan blue dyeing, according to the methods such as naked eyes evaluate differentiation of metamorphosis.
If this medicine is a kind of antigen of discerning the infectious disease cause of disease or at the modified antibodies of the cell receptor of infectious disease cause of disease, by this medicine is contacted with the cultured cell of crossing with the infectious disease pathogen infection (from patient or cultured cell system), measure the reduction that these cells reduce physiology's indication of infectious disease cause of disease or infectious disease pathogen infection then, can measure the latent effectiveness of this antibody.Perhaps, by medicine be subject to the infectious disease pathogen infection but cell (from cultivating behind the patient or cultured cell system) that not infected disease pathogen infects contacts, allow described cell contact with the infectious disease cause of disease then, whether the cell infection rate of determining contacted medicine then is than the infection rate with the contacted cell of medicine is not lower.The available any method known in the art of the infection of infectious disease cause of disease pair cell is measured.
If this medicine is a kind of to special receptor or the special modified immunoglobulin of part, by medicine is contacted with the cultured cell (from patient or cultured cells system) of expressing in conjunction with to the receptor member, determine then whether this medicine stops combining of part and receptor and/or receptor signal conducts, the perhaps method of this medicine costimulatory receptor signal conduction whether can be measured the latent effectiveness of this modified immunoglobulin.With known any method of this area mensuration ligand-receptor combination and receptor signal transmission (as in the 6th joint method for example), can measure these indications.
The usefulness of medicine also can be measured among the clinical trial and the mankind at suitable animal model.Any method in available this area is determined the usefulness of medicine.For example, after giving animal model or giving human experimenter's administering therapeutic medicine, estimate the disease of any this medicine plan treatment among animal or human's class experimenter or the indication of imbalance.For example, before treatment, during the treatment or treatment back reasonable time at interval, be determined at the direct acting molecular amounts level of this modified antibodies among animal model or the human experimenter, can estimate the usefulness of this medicine.Can identify any variation of this molecular amounts or lack to change, and determine the influence of treatment the experimenter.Whether the variation of described molecular amounts level to be, can determine by any method well known in the art, for example, in the above in 5.5 joints or any method of the method for immunity described of following 5.7 joints.
In others, by improvement or the recovery situation of monitoring experimenter, can measure the usefulness of this modified antibodies from the special disease or the state of an illness, the described disease or the state of an illness are relevant with the direct acting molecule of synthetic modification antibody.When this modified antibodies directly acted on tumor or cancer antigen, the diagnosis or the screening technique of available monitoring cancer that any is known of the special tumor or the improvement of cancer or tumor came trace detection.For example, but not method for limiting, by measuring in experimenter's serum or injection of labelled special at behind this antigenic antibody, the level of special cancer or tumor antigen (or another kind of and special cancer or relevant antigen of tumor) can be monitored the improvement of cancer or tumor.In addition, other image technology as computer tomography (CT) or sonogram or any other image method, can be used for monitoring the progress of cancer or tumor.Biopsy also can be used.Before above-mentioned test is carried out, can in the animal model of special cancer or tumor, carry out the potency test of modified immunoglobulin in the mankind.
If this medicine specific action is in the antigen of infectious disease cause of disease or the cell receptor of infectious disease cause of disease, by giving experimenter's (human experimenter or ill animal model) this modified antibodies, monitor the level of special infectious disease cause of disease or the symptom of special infectious disease then, can measure the usefulness of this modified antibodies.The level of infectious disease cause of disease can determine by any method well known in the art, as under the situation of virus, surveying virus titer, or bacteria levels (for example, with the sample of cultivating from patient), or the like.Also can measure the direct acting antigen levels of this modified immunoglobulin, determine the level of infectious disease cause of disease.The reduction of infectious disease cause of disease level or the improvement of infectious disease symptom show that this modified antibodies is effective.
Make vaccine if medicine is applied,, can determine to contain the immunity of the bacterin preparation of modified antibodies of the present invention by replying with the antiidiotype of monitoring test animal behind the vaccine immunity.The humoral response that occurs can be considered the symbol of total immunne response, other reply composition, especially cell-mediated immunity may be important to prevent disease.Experimental animal can comprise mice, rabbit, chimpanzee, is the human experimenter at last.The vaccine that the present invention the makes sexuality that can experimentize is dyed chimpanzee.Yet; because chimpanzee is protected species; the antibody response of vaccine of the present invention can be studied in the cheap animal earlier with many less, purpose be find 1 or 2 kind be used for the optimal candidate immunoglobulin molecules of chimpanzee effect research or the best combination of immunoglobulin molecules.
The immunne response of animal subject can be by the whole bag of tricks analysis.As the reactivity of immune serum that obtains and antibody, can pass through known technical measurement, as enzyme-linked immunosorbent assay (ELISA), immunoblotting, radioimmunoassay precipitation etc.; Perhaps prevention infection and/or alleviation are by immune host's disease symptoms.
Measuring vaccine combination of the present invention in rabbit induces the ability that the antiidiotype of this modified immunoglobulin molecule is replied to can be used as a suitable zooperal example.For example, the adult new zealand white rabbit of the male youth of available specific pathogen free (SPF) is tested.Each of test group rabbit is accepted the vaccine of effective dose.The matched group rabbit is accepted to use 1mM Tris-HCl, and pH9.0 contains the vaccine injection of natural generation antibody.Per 1 or 2 weeks were extracted the rabbit blood samples, with as in radioimmunoassay (Abbott joint laboratory) serum analysis to the anti-idiotype antibody of this modified immunoglobulin molecule with to the anti-anti-idiotype antibody of the direct acting antigen-specific of modified antibodies.Also available ELISA method is measured the existence of anti-idiotype antibody.Because the outbreeding of rabbit may produce the variation of replying, it is helpful to measure replying of vaccine in mice.
5.7. the algoscopy of modified immunoglobulin
Structure have 1 or a plurality of immunoglobulin that contains at the CDRs of the binding site of specific molecular after, any binding assay well known in the art all can be used for estimating resulting modified antibodies and special molecular in conjunction with situation.These algoscopys also can be used for selecting showing as has more high-affinity or specific antibody to specific antigen.
For example, but not the restriction assay method, this modified antibodies can be measured with various immunoassays well known in the art with the situation that combines of special molecular, these methods are measured system including, but not limited to using such as the competition of technology such as radioimmunoassay or with noncompetitive, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, the immune radiating measurement method, gel diffusion precipitation reaction, the immunodiffusion algoscopy, the original position immunoassay (for example, is used gold colloidal, enzyme or labelled with radioisotope), Western blot, precipitation, the agglutination algoscopy is (as gel agglutination algoscopy, hemagglutinin reaction assay method), the complement fixation algoscopy, immunofluorescence assay, protein A algoscopy and immunoelectrophoresis algoscopy etc.In one embodiment, the marker detection antibodies situation by detection of primary antibody.In another embodiment, secondary antibodies or the reagent that is incorporated on the primary antibody by detection comes detection of primary antibody.In further embodiment, the labelling secondary antibodies.Many methods are well known in the immunoassay of detection in conjunction with situation, and belong among the scope of the present invention.
A kind of useful external test system is described below, and this system is used to estimate the situation that combines of modified antibodies and target molecule.In brief, the mixed reaction solution of this modified antibodies and test sample is incubation under relevant condition, the effect certain hour is so that two kinds of component fully effects mutually, as mutually combine, form a species complex like this, it represents a kind of temporary complex, can be removed and/or be detected in reaction mixture.These mensuration can in all sorts of ways and carry out.For example, a kind of method of carrying out said determination comprises this modified antibodies or material to be checked is anchored on the solid formation that reaction finishes the back and detects the antibody/molecular complex that is anchored on the solid phase.In an embodiment of said method, direct or indirect labelling modified antibodies, and in solid phase surface grappling test sample.During practical operation, microtitration plate can be used as solid formation easily.The component of institute's grappling can be by non-covalent or covalently bound fixing.Solution with test sample is coated on solid phase surface drying then simply, can finish non-covalent connection.
Revocable component can join the bag that contains the grappling component and be measured by the surface of crossing.After reaction is finished, remove unreacted component (for example, ablution) under relevant condition, any so formed complex still keeps and is fixed on solid phase surface.The complex that is anchored on solid phase surface can detect in many ways.If revocable component is a labelling in advance, the detection that is fixed on the label on surface just shows and has formed complex.If revocable component is not a labelling in advance, indirect labels can be used for detecting the complex that is anchored on the surface so.
In addition, reaction can be carried out in liquid phase, and reaction product isolated from unreacted component detects complex then.
5.8 transgenic animal
The present invention also provides at the transgenic of modified immunoglobulin of the present invention (or its functional fragment) animal of (promptly containing code nucleic acid).The animal of any species including, but not limited to mice, rat, rabbit, Cavia porcellus, sheep, pig, miniature pig, goat with as non-human primates such as baboon, monkey and chimpanzees, can be used for producing transgenic animal of the present invention.
Therefore, in specific embodiment, the invention provides the reorganization non-human animal, this animal contains a kind of recombinant nucleic acid, this recombinant nucleic acid contains a kind of nucleotide sequence of code book invention modified immunoglobulin, especially the non-human transgenic animal of recombinating contains the nucleic acid of the modified antibodies of encoding, and this modified antibodies combines specifically with cancer or tumor antigen immunity; Or containing the reorganization non-human transgenic animal of the nucleic acid of the following modified antibodies of encoding, this modified antibodies combines specifically with the antigen of infectious disease cause of disease or the cell receptor immunity of infectious disease cause of disease.
Any technology known in the art can be used for the antibody transgenic is imported in the animal, to produce the original seed system of transgenic animal.Above-mentioned technology is including, but not limited to protokaryon microinjection (Hoppe and Wagner, 1989, U.S. Patent number 4,873,191); Embryonic lineage (Van der Putten etc., 1985, institute of NAS reports 82:6148-6152) is gone in the gene transfer of retrovirus-mediated method; The gene targeting of embryonic stem cell (Thonpson etc., 1989, cell 56:313-321); Embryo's electroporation (Lo, 1983, molecular cytobiology 3:1803-1814); Change (Lavitrano etc., 1989, cell 57:717-723) over to the gene of sperm mediation; Etc. technology.The summary of relevant above-mentioned technology is seen Gordon, 1989, and transgenic animal, international cytology summarizes 115:171-229, introduces only for reference as a whole at this.
The invention provides changes the transgenic animal that basic zooblast all carries the nucleotides sequence of coding modified antibodies, and all carries the transgenic transgenic animal in some cell rather than all cells, promptly inlays animal.Transgenic can be integrated into single transgenic or with the form of concatemer, as head to head arrange or head to the tail arranged in series.With the following method method introduced of Lasko etc. (Lasko etc., 1992, institute of NAS reports 89:6232-6236) for example, transgenic optionally imports in the specific cell type and in this cell and activates.The special activatory required adjusting sequence of above-mentioned cell type is relevant with specific target cell type, and these are apparent to one skilled in the art.When needing the genetically modified nucleotide of encoding synthetic antibody to be incorporated into the chromosomal foci of endogenous immunoglobulin, the preferred gene shooting method.In brief, when using above-mentioned technology, for realize integrating the carrier that must design contains with homologous some nucleotide sequence of endogenous immunoglobulin, through with the homologous recombination of chromosome sequence, with vector integration in the nucleotide sequence of endogenous immunoglobulin gene and destroy the function of this gene.Method according to introducing as (Gu etc., 1994, science 265:103-106) such as Gu can import to the transgenic selectivity in the specific cell type, and the endogenous immunoglobulin of result is inactivation in this cell type only.It is relevant with special target cell type that the needs of the special inactivation of above-mentioned cell type are regulated sequence, and apparent to one skilled in the art.
Generation has the method for transgenic animal that integrate to select the single copy in site, also well-known to one skilled in the art (as seeing, Bronson etc., 1996, institute of NAS reports 93:9067-9072).
In case produce transgenic animal, available standards technical measurement recombinant antibodies expression of gene.Whether take place to measure genetically modified integration by Southern engram analysis or round pcr analyze animal tissue, can finish and just cheat screening.Use following technology, also can judge the level of organizing the transfer gene mRNA expression of transgenic animal, these technology are including, but not limited to Northern engram analysis, in situ hybridization analysis and the RT-PCR of the tissue sample that obtains from animal.With the special antibody of antibody transgene product, also can estimate with immunocytochemistry the sample of expressing gene organization.
6. embodiment: the synthetic modification antibody that contains Kallidin I
Present embodiment describe immunity specifically with the structure of the bonded synthetic modification antibody of bradykinin receptor (BR).This bradykinin receptor combines with the native ligand that is called Kallidin I.The interaction of BR-Kallidin I is to can be used on the inventive method in conjunction with a right example, when the aminoacid that is called binding site in the Kallidin I contacts with bradykinin receptor, and the interaction of BR and Kallidin I.The synthetic modification antibody of present embodiment contains the aminoacid sequence from the Kallidin I binding site.Therefore, these synthetic modification antibody simulation Kallidin I parts, and can expect and be attached to (BR) on the bradykinin receptor.Made up 6 synthetic modification antibody that contain the Kallidin I sequence by following described method, and proof combines with BR.
The strategy that generation contains the synthetic modification antibody of Kallidin I binding sequence is summarized as follows:
1) uses oligonucleotide, handle variable region gene, make it contain a CDR with Kallidin I binding sequence by genetic engineering means;
2) will be inserted into by the variable region gene that genetic engineering means was handled in the mammalian expression vector that contains suitable constant region then;
3) usefulness contains a carrier transfection mammalian cell of light chain and heavy chain, and expresses this synthetic modification antibody; And
4) measure synthetic modification antibody to BR in conjunction with situation.
6.1 contain the structure of the variable region gene of Kallidin I binding site
In order to make up variable region gene that contains the CDR of Kallidin I binding site of coding, carry out following strategy.
At first, single stranded oligonucleotide annealing with produce the viscosity double chain DNA fragment (as Fig. 5, the diagram in the 1st step; Also see Kufemeier etc., 1994 biotechnology 17:242).Particularly, with the automatic technique of GenoSys Biotech Inc, composition length be 80 bases corresponding to the aim sequence oligonucleotide, have the overlay region of 20 bases.The distinguished sequence of these oligonucleotide is represented (making up light chain and variable region of heavy chain respectively) at Fig. 6 A and B.Fig. 6 A has listed and has been used for the oligonucleotide sequence that engineering makes up the chain variable region gene that contains one section Kallidin I binding sequence.Fig. 6 B has listed and has been used for the oligonucleotide sequence that engineering makes up the heavy chain variable region gene that contains one section Kallidin I binding sequence.The oligonucleotide combination and 2 consensuses (ConVL1 and ConVH1) that are used for engineering 6 Kallidin I CDRs of structure (BKCDR1, BKCDR2, BKCDR3, BKCDR4, BKCDR5, BKCDR6) are listed at table 5.
the oligonucleotides Ming that table 5 Yong Yu engineering builds the synthetic Xiu Shi antibody with bradykinin Xu row claims Oligo1, Oligo2, Oligo3, Oligo4, Oligo5, Oligo6, Oligo7, Oligo8, Oligo9, Oligo10, Oligo11, Oligo12ConV1, L1, BKLC1, BKLC2, BKLC3, BKLC4, BKLC5, BKLC6, BKLC7, BKLC8, BKLC9, BKLC10, L2BKCDR1, L1, BKLC1, BKCDR12, BKLC3, BKLC4, BKLC5, BKLC6, BKLC7, BKLC8, BKLCDR19, BKLC10, L2BKCDR2, L1, BKLC1, BKLC2, BKLCDR23, BKLC4, BKLC5, BKLC6, BKLC7, BKLCDR28, BKLC9, BKLC10, L2BKCDR3, L1, BKLC1, BKLC2, BKLC3, BKLC4, BKLCDR35, BKLCDR36, BKLC7, BKLC8, BKLC9, BKLC10, L2ConVH1, BKHC1, BKHC2, BKHC3, BKHC4, BKHC5, BKHC6, BKHC7, BKHC8, BKHC9, BKHC10BKCDR4, BKHC1, BKHDR42, BKHDR43, BKHC4, BKHC5, BKHC6, BKHC7, BKHC8, BKHDR49, BKHC10BKCDR5, BKHC1, BKHC2, BKHDR53, BKHC4, BKHC5, BKHC6, BKHC7, BKHDR58, BKHC9, BKHC10BKCDR6, BKHC1, BKHC2, BKHC3, BKHC4, BKHC5, BKHC6, BKHC7, BKHC8, BKHC9, BKHC10
For these oligonucleotide are combined into needed gene, with 10 or 12 kind of oligonucleotide group make up variable region gene by the method for following description.By following method 5 ＇ of every kind of oligonucleotide end phosphorylation: every kind of oligonucleotide of 25 μ l exists T4 polynucleotide kinase and 50mMATP, 37 ℃ of following incubations 1 hour.5 minutes cessation reactions of 70 ℃ of heating are used ethanol precipitation then.In case the phosphorylation of finishing, mix in the aseptic microcentrifugal tube by complementary oligonucleotide shown in Figure 5 (oligonucleotide 1+ oligonucleotide 10, oligonucleotide 2+ oligonucleotide 9, oligonucleotide 3+ oligonucleotide 8, oligonucleotide 4+ oligonucleotide 7, oligonucleotide 5+ oligonucleotide 6), 65 ℃ of water-baths are 5 minutes then, then cooling 30 minutes and annealing under the room temperature.Annealing obtains having the short double chain DNA fragment of sticky end.
Next step, the viscosity double chain DNA fragment connects into long two strands (Fig. 5, step 2-4), up to being assembled into the variable region gene that engineering makes up.Particularly, under the condition that has T4 dna ligase and 10mM ATP, the viscosity double chain DNA fragment connects 2 hours in the water-bath of 16 ℃ of constant temperature.Annealed oligonucleotide 1/10 and annealed oligonucleotide 2/9 mix, and annealed oligonucleotide 3/8 and 4/7 mixing of annealed oligonucleotide.The oligonucleotide that obtains is denoted as oligonucleotide 1/10/2/9 and oligonucleotide 3/8/4/7.Next step, oligonucleotide 3/8/4/7 is connected with oligonucleotide 5/6.The oligonucleotide 3/8/4/7/5/6 of Chan Shenging is connected on the oligonucleotide 1/10/2/9 then, obtains the variable region gene of a total length.
In addition, when being one group with 12 kinds of oligonucleotide, the order of interpolation is oligonucleoside acid number 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, the variable region gene of 1/12/2/11/3/10/4/9+5/8/6/7=total length.Made up 8 kinds of variable region genes with this method, wherein 4 kinds of genes are variable region of light chains and 4 kinds of genes are variable region of heavy chaines.The light chain gene that engineering makes up comprises ConVL1, a kind of total variable region of light chain that does not have the Kallidin I sequence, BKCDR1, a kind of variable region of light chain that contains the Kallidin I sequence in CDR1; BKCDR2, a kind of variable region of light chain that in CDR2, contains the Kallidin I sequence; And BKCDR3, a kind of variable region of light chain that in CDR3, contains the Kallidin I sequence.The heavy chain variable region gene that engineering makes up comprises ConVH1, a kind of variable region of heavy chain that does not have the Kallidin I sequence; BKCDR4, a kind of variable region of heavy chain that in CDR4, contains the Kallidin I sequence; BKCDR5, a kind of variable region of heavy chain that in CDR5, contains the Kallidin I sequence; And BKCDR6, a kind of variable region of heavy chain that in CDR6, contains the Kallidin I sequence.The sequence of the variable region gene of 8 kinds of engineering structures shows in Fig. 4 A to 4F.
Handle by following method by each cydorge gene that the combination oligonucleotide makes:
But obtain the engineering variable region gene with the gel electrophoresis purification.For removing excessive not connection oligonucleotide and other incomplete dna fragmentation, connect product on 1% low melting-point agarose gel, constant 110V electrophoresis 2 hours.Cutting-out contains the master tape of full length DNA product and is placed in the aseptic 1.5ml centrifuge tube.Digested 3 hours for 40 ℃ with the f3 beta-Agarase for making DNA discharge this gel slice from agarose.With 0.3M NaOAc and isopropyl alcohol-20 ℃ precipitation 1 hour, then 12,000rpm reclaimed this DNA in centrifugal 15 minutes.The DNA precipitation of purification is suspended in the 50ul TE buffer of pH8.0 again.Then through pcr amplification this project variable region gene.Particularly, the height fidelity thermally stable P fu archaeal dna polymerase of the engineering variable region gene of 100ng and 25mM dNTPs, 200ng primer and 5U mixes in buffer.Carry out 28 cyclic amplification DNA.The PCR product that obtains is analyzed on 1% agarose gel.
Then handle is inserted in the pUC19 bacteria carrier corresponding to the DNA of every kind of purification of engineering variable region gene, pUC19 has 2686 base pairs, the e. coli plasmid vector of high copy number, this carrier contain 54 the base pair polyclone connection site and the Amp selected marker that are arranged in the lacZ gene.In order to prepare the carrier that inserts the engineering variable region gene, 37 ℃ of linearization process 3 hours, the result obtained having the carrier of blunt end sequence 5 ＇ GTC to 10 μ gpUC19 with HincII (50U).For preventing reconnecting of carrier self, linear carrier DNA with the calf small intestinal alkaline phosphatase acid (CIP) of 25U 37 ℃ of dephosphorylations 1 hour.For the engineering variable region gene is inserted in the pUC19 carrier, about 0.5 μ g dephosphorylation wire carrier DNA mixes with the variable region gene of 3 μ g phosphorylations in the presence of T4 dna ligase (1000U), 16 ℃ of incubations 12 hours.
Then, with the bacteria carrier transform bacteria cell that contains the engineering variable region gene.Specifically, the pUC19 that contains the engineering variable region gene of the competence DH5 α cell 50 μ l of prepared fresh and 1 μ g mixes, and transfers to (0.2cm gap in the electroporation cuvette then; Bio-Rad).In electroporation apparatus (Bio-Rad gene pulse instrument), each cuvette applies pulse under 2.5kV/2000hm/25 μ F.In each cuvette, add 1ml SOC culture medium afterwards at once, and allow cell in centrifuge tube, recover 1 hour under 37 ℃.Pipette from each cell transformed equal portions, with dilution in 1: 100,100 μ l bed boards were to the LB flat board that contains ampicillin (Amp40 μ g/ml) then.This flat board is in the existence of 37 ℃ of following overnight incubation owing to the Amp labelling, and the transformant that only contains the pUC19 carrier is grown on the LB/Amp flat board.
The single transformant colony of picking constantly shakes 37 ℃ of following grow overnight in 3ml LB/Amp sterile glass tube.Plasmid DNA is separated with Easy Prep post (Pharmacia Biotech), is suspended in then in the 100 μ l TE buffer of pH7.5.For confirming in pUC19, to exist gene to insert, from 25 μ l plasmid DNA of each colony with nuclease in the Hinc II restriction endonuclease 37 ℃ of digestion 1 hour down, on 1% agarose gel, analyze then.By this method, contain the plasmid DNA of inserting gene, because forfeiture restriction site (5 ＇ ..GTCGAC..3 ＇), have resistance mobility and closed loop (CC) dna form mobility to further and those plasmids of not inserting can be cut to enzyme action, on gel with the simulation of linear (L) double chain DNA fragment mobility.
For correct gene sequence that confirms the engineering variable region gene and the probability of eliminating the non-required sudden change of generation in the structure operating process, automatically carrying out dna sequencing on the ABI 377 dna sequencing instrument, the primer (5 ＇ GAATTCATGGCTTGGGTGTG3 ＇) of order-checking at clone's M13/pUC reverse primer (5 ＇ AAC AGC TATGACCATG3 ＇) and terminal 20 bases of pcr gene product 5 ＇.All clones are proved and contain correct sequence.
With the method for present embodiment, make up 6 engineering variable region genes that contain the Kallidin I sequence.Table 6 show be the title of synthetic modification antibody and in variable region gene the location of corresponding Kallidin I binding sequence.For example, the synthetic antibody that is called hAb BKCDR1 contains Kallidin I binding sequence (BK) in the CDR1 of chain variable region gene (VL).This synthetic antibody has a consensus sequence (Con) in heavy chain variable region gene (VH).
Table 6. contains the synthetic modification antibody of Kallidin I
The title V of synthetic modification antibody LV HHAbBKCDR1 BKCDR1 ConVH1hAbBKCDR2 BKCDR2 ConVH1hAbBKCDR3 BKCDR3 ConVH1hAbBKCDR4 ConVL1 BKCDR4hAbBKCDR5 ConVL1 BKCDR5hAbBKCDR6 ConVL1 BKCDR6
The corresponding aminoacid sequence of each variable region of 6 synthetic modification antibody of present embodiment is listed at table 7.CDRs marks with black matrix.The aminoacid of Kallidin I binding site is: Arg ProPro Gly Phe Ser Pro Phe Arg, and mark in CDRs with underscore.Table 5 also illustrates human kappa light chain V LSubgroup I and people's heavy chain V HThe consensus sequence of subgroup I gene.Too short so that can not comprise under the situation of complete Kallidin I binding site sequence at total CDR, deletion is from the n terminal residue of Kallidin I binding site, because known carboxyl terminal residue more important in receptors bind (Stewart and Vavrek, the chemistry of peptide B2 brad ykinin antagonists, 5196 pages, Burch R.M. writes, brad ykinin antagonists, basis and clinical research, New York; MancalDekker, 1991, be incorporated herein only for reference).
The aminoacid sequence of table 7. engineering variable region gene
Ren Κ Qing chain VLSubgroups (Kabat et al, 1991) consensus sequence of the amino acid region BKCDR1 BKCDR2 BKCDR3 1 FR1 Asp Asp Asp Asp 2 Ile Ile Ile 3 Gln Gln Gln Gln 4 Met Met Met Met 5 Thr Thr Thr Thr 6 Gln Gln Gln Gln 7 Ser Ser Ser Ser 8 Pro Pro Pro Pro 9 Ser Ser Ser Ser 10 Ser Ser Ser Ser 11 Leu Leu Leu Leu 12 Ser Ser Ser Ser 13 Ala Ala Ala Ala 14 Ser Ser Ser Ser 15 Val Val Val Val 16 Gly Gly Gly Gly 17 Asp Asp Asp Asp 18 Arg Arg Arg Arg 19 Val Val Val Val 20 Thr Thr Thr Thr 21 Ile Ile Ile Ile 22 Thr Thr Thr Thr 23 Cys Cys Cys Cys 24 CDR1 Arg Arg Arg Arg 25 Ala Pro Ala Ala 26 Ser Pro Ser Ser 27 (AF) Gln Gln Gln Gln 28 Ser Phe Ser Ser 29 Ile Ser Ile Ile consensus sequence of the amino acid region BKCDR1 BKCDR2 BKCDR3 30 Ser Pro Ser Ser 31 Asn Phe Asn Asn 32 Tyr Arg Tyr Tyr 33 Leu Leu Leu Leu 34 Ala Ala Ala Ala 35 FR2 Trp Trp Trp Trp 36 Tyr Tyr Tyr Tyr 37 Gln Gln Gln Gln 38 Gln Gln Gln Gln 39 Lys Lys Lys Lys 40 Pro Pro Pro Pro 41 Gly Gly Gly Gly 42 Lys Lys Lys Lys 43 Ala Ala Ala Ala 44 Pro Pro Pro Pro 45 Lys Lys Lys Lys 46 Leu Leu Leu Leu 47 Leu Leu Leu Leu 48 Ile Ile Ile Ile 49 Tyr Tyr Tyr Tyr 50 CDR2 Ala Ala Pro Ala 51 Ala Ala Gly Ala 52 Ser Ser Phe Ser 53 Ser Ser Ser Ser 54 Leu Leu Pro Leu 55 Glu Glu Phe Glu 56 Ser Ser Arg Ser 57 FR3 Gly Gly Gly Gly 58 Val Val Val Val 59 Pro Pro Pro Pro 60 Ser Ser Ser Ser 61 Arg Arg Arg Arg 62 Phe Phe Phe Phe 63 Ser Ser Ser Ser 64 Gly Gly Gly Gly 65 Ser Ser Ser Ser 66 Gly Gly Gly Gly 67 Ser Ser Ser Ser 68 Gly Gly Gly Gly 69 Thr Thr Thr Thr 70 Arg Arg Arg Arg 71 Phe phe Phe Phe 72 Thr Thr Thr Thr 73 Leu Leu Leu Leu 74 Thr Thr Thr Thr amino acids regional consensus sequence BKCDR1 BKCDR2 BKCDR3 75 Ile Ile Ile Ile 76 Ser Ser Ser Ser 77 Ser Ser Ser Ser 78 Leu Leu Leu Leu 79 Gln Gln Gln Gln 80 Pro Pro Pro Pro 81 Glu Glu Glu Glu 82 Asp Asp Asp Asp 83 Phe Phe Phe Phe 84 Ala Ala Ala Ala 85 Thr Thr Thr Thr 86 Tyr Tyr Tyr Tyr 87 Tyr Tyr Tyr Tyr 88 Cys Cys Cys Cys 89 CDR3 Gln Gln Gln Arg 90 Gln Gln Gln Pro 91 Tyr Tyr Tyr Pro 92 Asn Asn Asn Gly 93 Ser Ser Ser Phe 94 Leu Leu Leu Ser 95 (AF) Pro Pro Pro Pro 96 Trp Trp Trp Phe 97 Thr Thr Thr Arg 98 FR4 Phe Phe Phe Phe 99 Gly Gly Gly Gly 100 Gin Gin Gin Gin 101 Gly Gly Gly Gly 102 Thr Thr Thr Thr 103 Lys Lys Lys Lys 104 Val Val Val Val 105 Glu Glu Glu Glu 106 Ile Ile Ile Ile 107 Lys Lys Lys Lys 108 Arg Arg Arg Arg 109 Thr Thr Thr Thr human heavy chain V...HSubgroups (Kabat et al, 1991) consensus sequence of the amino acid region BKCDR1 BKCDR2 BKCDR3 1 FR1 Asp Asp Asp Asp 2 Ile Ile Ile 3 Gln Gln Gln Gln 4 Met Met Met Met 5 Thr Thr Thr Thr 6 Gln Gln Gln Gln 7 Ser Ser Ser Ser 8 Pro Pro Pro Pro 9 Ser Ser Ser Ser 10 Ser Ser Ser Ser 11 Leu Leu Leu Leu 12 Ser Ser Ser Ser 13 Ala Ala Ala Ala 14 Ser Ser Ser Ser 15 Val Val Val Val 16 Gly Gly Gly Gly 17 Asp Asp Asp Asp 18 Arg Arg Arg Arg 19 Val Val Val Val 20 Thr Thr Thr Thr 21 Ile Ile Ile Ile 22 Thr Thr Thr Thr 23 Cys Cys Cys Cys 24 CDR1 Arg Arg Arg Arg 25 Ala Pro Ala Ala 26 Ser Pro Ser Ser 27 (AF) Gln Gln Gln Gln 28 Ser Phe Ser Ser 29 Ile Ser Ile Ile consensus sequence of the amino acid region BKCDR1 BKCDR2 BKCDR3 30 Ser Pro Ser Ser 31 Asn Phe Asn Asn 32 Tyr Arg Tyr Tyr 33 Leu Leu Leu Leu 34 Ala Ala Ala Ala 35 FR2 Trp Trp Trp Trp 36 Tyr Tyr Tyr Tyr 37 Gln Gln Gln Gln 38 Gln Gln Gln Gln 39 Lys Lys Lys Lys 40 Pro Pro Pro Pro 41 Gly Gly Gly Gly 42 Lys Lys Lys Lys 43 Ala Ala Ala Ala 44 Pro Pro Pro Pro 45 Lys Lys Lys Lys 46 Leu Leu Leu Leu 47 Leu Leu Leu Leu 48 Ile Ile Ile Ile 49 Tyr Tyr Tyr Tyr 50 CDR2 Ala Ala Pro Ala 51 Ala Ala Gly Ala 52 Ser Ser Phe Ser 53 Ser Ser Ser Ser 54 Leu Leu Pro Leu 55 Glu Glu Phe Glu 56 Ser Ser Arg Ser 57 FR3 Gly Gly Gly Gly 58 Val Val Val Val 59 Pro Pro Pro Pro 60 Ser Ser Ser Ser 61 Arg Arg Arg Arg 62 Phe Phe Phe Phe 63 Ser Ser Ser Ser 64 Gly Gly Gly Gly 65 Ser Ser Ser Ser 66 Gly Gly Gly Gly 67 Ser Ser Ser Ser 68 Gly Gly Gly Gly 69 Thr Thr Thr Thr 70 Arg Arg Arg Arg 71 Phe phe Phe Phe 72 Thr Thr Thr Thr 73 Leu Leu Leu Leu 74 Thr Thr Thr Thr amino acids regional consensus sequence BKCDR1 BKCDR2 BKCDR3 75 Ile Ile Ile Ile 76 Ser Ser Ser Ser 77 Ser Ser Ser Ser 78 Leu Leu Leu Leu 79 Gln Gln Gln Gln 80 Pro Pro Pro Pro 81 Glu Glu Glu Glu 82 Asp Asp Asp Asp 83 Phe Phe Phe Phe 84 Ala Ala Ala Ala 85 Thr Thr Thr Thr 86 Tyr Tyr Tyr Tyr 87 Tyr Tyr Tyr Tyr 88 Cys Cys Cys Cys 89 CDR3 Gln Gln Gln Arg 90 Gln Gln Gln Pro 91 Tyr Tyr Tyr Pro 92 Asn Asn Asn Gly 93 Ser Ser Ser Phe 94 Leu Leu Leu Ser 95 (AF) Pro Pro Pro Pro 96 Trp Trp Trp Phe 97 Thr Thr Thr Arg 98 FR4 Phe Phe Phe Phe 99 Gly Gly Gly Gly 100 Gin Gin Gin Gin 101 Gly Gly Gly Gly 102 Thr Thr Thr Thr 103 Lys Lys Lys Lys 104 Val Val Val Val 105 Glu Glu Glu Glu 106 Ile Ile Ile Ile 107 Lys Lys Lys Lys 108 Arg Arg Arg Arg 109 Thr Thr Thr Thr human heavy chain V...
35A????????Trp????????Phe???????Trp???????Trp
35B, Asn, Arg, Asn, Asn36, FR2, Trp, Trp, Trp, Trp37, Val, Val, Val, Val38, Arg, Arg, Arg, Arg39, Gln, Gln, Gln, Gln40, Ala, Ala, Ala, Ala41, pro, Pro, Pro, Pro42, Gly, Gly, Gly, Gly43, Gln, Gln, Gln, Gln44, Gly, Gly, Gly, Gly45, Leu, Leu, Leu, Leu46, Glu, Glu, Glu, Glu47, Trp, Trp, Trp, Trp48, Met, Met, Met, Met amino acid, Qu Yu, consensus sequence, BKCDR4, BKCDR5, BKCDR649, Gly, Gly, Gly, Gly50, CDR5, Trp, Trp, Trp, Trp51, Ile, Ile, Ile, Ile52, (A-C), Asn, Asn, Asn, Asn53, Gly, Gly, Gly, Gly54, Asn, Asn, Asn, Asn39, Lys, Lys, Lys, Lys40, Pro, Pro, Pro, Pro41, Gly, Gly, Gly, Gly42, Lys, Lys, Lys, Lys43, Ala, Ala, Ala, Ala44, Pro, Pro, Pro, Pro45, Lys, Lys, Lys, Lys46, Leu, Leu, Leu, Leu47, Leu, Leu, Leu, Leu48, Ile, Ile, Ile, Ile49, Tyr, Tyr, Tyr, Tyr50, CDR2, Ala, Ala, Pro, Ala51, Ala, Ala, Gly, Ala52, Ser, Ser, Phe, Ser53, Ser, Ser, Ser, Ser54, Leu, Leu, Pro, Leu55, Glu, Glu, Phe, Glu56, Ser, Ser, Arg, Ser57, FR3, Gly, Gly, Gly, Gly58, Val, Val, Val, Val59, Pro, Pro, Pro, Pro60, Ser, Ser, Ser, Ser61, Arg, Arg, Arg, Arg62, Phe, Phe, Phe, Phe63, Ser, Ser, Ser, Ser64, Gly, Gly, Gly, Gly65, Ser, Ser, Ser, Ser66, Gly, Gly, Gly, Gly67, Ser, Ser, Ser, Ser68, Gly, Gly, Gly, Gly69, Thr, Thr, Thr, Thr70, Arg, Arg, Arg, Arg71, Phe, Phe, Phe, Phe72, Thr, Thr, Thr, Thr73, Leu, Leu, Leu, Leu74, Thr, Thr, Thr, Thr75, Ile, Ile, Ile, Ile76, Ser, Ser, Ser, Ser77, Ser, Ser, Ser, Ser amino acid, Qu Yu, consensus sequence, BKCDR4, BKCDR5, BKCDR678, Leu, Leu, Leu, Leu79, Gln, Gln, Gln, Gln80, pro, Pro, Pro, Pro, 81, Glu, Glu, Glu, Glu82, Asp, Asp, Asp, Asp83, Phe, Phe, Phe, Phe84, Ala, Ala, Ala, Ala85, Thr, Thr, Thr, Thr86, Tyr, Tyr, Tyr, Tyr87, Tyr, Tyr, Tyr, Tyr88, Cys, Cys, Cys, Cys89, CDR3, Gln, Gln, Gln, Arg90, Gln, Gln, Gln, Pro55, Gly, Gly, Pro, Gly56, Asp, Asp, Pro, Asp57, Thr, Thr, Gly, Thr58, Asn, Asn, Phe, Asn59, Tyr, Tyr, Ser, Tyr60, Ala, Ala, Pro, Ala61, Gln, Gln, Phe, Gln62, Lys, Lys, Are, Lys63, Phe, Phe, Phe, Phe64, Gln, Gln, Gln, Gln65, Gly, Gly, Gly, Gly66, FR3, Arg, Arg, Arg, Arg67, Val, Val, Val, Val68, Thr, Thr, Thr, Thr, 69, Ile, Ile, Ile, Ile70, Thr, Thr, Thr, Thr71, Ala, Ala, Ala, Ala72, Asp, Asp, Asp, Asp73, Thr, Thr, Thr, Thr74, Ser, Ser, Ser, Ser75, Thr, Thr, Thr, Thr76, Ser, Ser, Ser, Ser77, Thr, Thr, Thr, Thr78, Ala, Ala, Ala, Ala79, Tyr, Tyr, Tyr, Tyr80, Met, Met, Met, Met81, Glu, Glu, Glu, Glu82, (A-C), Leu, Leu, Leu, Leu
82A???????Ser?????????Ser???????Ser???????Ser
82B???????Ser?????????Ser???????Ser???????Ser
82C, Leu, Leu, Leu, Leu amino acid, Qu Yu, consensus sequence, BKCDR4, BKCDR5, BKCDR683, Arg, Arg, Arg, Arg84, Ser, Ser, Ser, Ser85, Glu, Glu, Glu, Glu86, Asp, Asp, Asp, Asp87, Thr, Thr, Thr, Thr88, Ala, Ala, Ala, Ala89, Val, Val, Val, Val90, Tyr, Tyr, Tyr, Tyr91, Tyr, Tyr, Tyr, Tyr92, Cys, Cys, Cys, Cys93, Ala, Ala, Ala, Ala94, Arg, Arg, Arg, Arg95, CDR6, Ala, Ala, Ala, Ala96, Pro, Pro, Pro, Pro97, Gly, Gly, Gly, Gly98, Tyr, Tyr, Tyr, Phe99, Gly, Gly, Gly, Ser100, (A-K), Ser, Ser, Ser, Pro101, Asp, Asp, Asp, Phe102, Tyr, Tyr, Tyr, Arg103, FR4, Trp, Trp, Trp, Trp91, Tyr, Tyr, Pro, Pro92, Asn, Asn, Asn, Gly93, Ser, Ser, Ser, Phe94, Leu, Leu, Leu, Ser95, (A-F), Pro, Pro, Pro, Pro96, Trp, Trp, Trp, Phe97, Thr, Thr, Thr, Arg98, FR4, Phe, Phe, Phe, Phe99, Gly, Gly, Gly, Gly100, Gln, Gln, Gln, Gln101, Gly, Gly, Gly, Gly102, Thr, Thr, Thr, Thr, 103, Lys, Lys, Lys, Lys104, Val, Val, Val, Val105, Glu, Glu, Glu, Glu106, Ile, Ile, Ile, Ile107, Lys, Lys, Lys, Lys108, Arg, Arg, Arg, Arg109, Thr, Thr, Thr, Thr, 104, Gly, Gly, Gly, Gly105, Gln, Gln, Gln, Gln106, Gly, Gly, Gly, Gly107, Thr, Thr, Thr, Thr108, Leu, Leu, Leu, Leu chloro Suan, Qu Yu, consensus sequence, BKCDR4, BKCDR5, BKCDR6109, Val, Val, Val, Val110, Thr, Thr, Thr, Thr111, Val, Val, Val, Val112, Ser, Ser, Ser, Ser113, Ser, Ser, Ser, Ser
6.2. the engineering variable region gene is inserted in the mammalian expression vector
Article one, complete light chain of antibody has a variable region and a stable region.Article one, complete heavy chain of antibody contains a variable region, a constant region and a hinge region.In order to make up complete light chain and heavy chain, the modification variable region gene that above-mentioned engineering makes up can be inserted in the carrier that contains suitable constant region subsequently.The Kallidin I sequence is inserted the engineering variable region gene of light chain CDR, can be cloned in the pMRRO10.1 carrier (Fig. 3 A), and this carrier contains the people KConstant region of light chain.The engineering variable region of light chain is inserted into and obtains a complete sequence of light chain in this carrier.In addition, the Kallidin I sequence is inserted into the engineering variable region gene among the heavy chain CDR, is inserted in the pGAMMA1 carrier (Fig. 3 B), and this carrier contains human IgG1's constant region and hinge region sequence.The engineering heavy chain variable region gene is inserted into and obtains a complete sequence of heavy chain in this carrier.
In order to make up the mammal carrier of coding complete antibody, complete sequence of heavy chain and sequence of light chain be inserted in the single mammalian expression vector (Bebbington, C.R.1991 is in method one book: the matching method of Enzymology method, the 2nd volume, the 136-145 page or leaf).The light chain and the heavy chain of the vector encoded antibody that is produced, and be named as pNEPuDGV (Fig. 3 C).
6.3. the expression of synthetic modification antibody in mammalian cell
For checking the assembling production of antibodies, use the pNEPuDGV rotaring redyeing COS cell.COS cell (a kind of African green monkey kidney cell line, CV-1 obtain with the SV40 virus conversion that lacks initial point), the moment in short-term that is used to synthetic antibody expresses, because they can copy to very high copy number to the cyclic plasmid that contains the SV40 origin of replication.With calcium precipitation method (Sullian etc., FEBS Lett, 285:120-123,1991) the antibody expression vector transfection (is obtained this cell from American type culture collection) in the COS7 cell.Transformant was cultivated 72 hours in Dulbecco ＇ s improvement Eagle ＇ s culture medium, collected supernatant afterwards, the antibody of Kallidin I.With the IgG that assembles in the supernatant of ELISA method mensuration from rotaring redyeing COS cell.The ELISA method comprises on 96 orifice plates with anti-human IgG Fc bag quilt catches sample and reference material.With anti-people K chain that is connected to horseradish peroxidase (HRP) and substrate tetramethyl diphenylamines (TMB), detect bonded assembling IgG.The colour developing degree is directly proportional with the quantity of assembling antibody in sample.
6.4. contain the synthetic modification antibody simulation Kallidin I part of Kallidin I and be attached on the bradykinin receptor
Engineering makes up the synthetic modification antibody expection contain the Kallidin I binding sequence and can simulate the Kallidin I part and be attached on the bradykinin receptor (BR).In order to confirm these synthetic modification antibodies BR, measure synthetic antibody with the bradykinin receptor binding assay.Measure combining of synthetic antibody and BR with following method.The SV-T2 cell is a kind of fibroblast of conversion, about 3,000 bradykinin receptors of each cellular expression (BR).The receptor of stimulation on the SV-72 cell causes that PGE2 is synthetic to be increased rapidly, and increasing with the level that combines of Kallidin I of synthetic quantity is directly proportional.Therefore, the PGE2 that is discharged in the culture medium is the index of receptors bind.
Shown in Fig. 7 A, adding 1nM Kallidin I (part) stimulates 4 times of the synthetic approximately raisings of PGE2.The synthetic quantitative approach of PGE2 is that ELISA detects receptor antagonist HOE-140 also conduct in Fig. 7 A.Add HOE-140 and Kallidin I separately and do not cause that PGE2 is synthetic.
And shown in Fig. 7 B, its knot of the modified antibodies of expression is used to measure the ability of closing and stimulating bradykinin receptor.Use from stimulated the SV-T2 cell by the culture medium of the COS cell of antibody expression vector pNEPuDGV1 transfection, observe the expression of the bradykinin receptor on it, expression vector pNEPuDGV1 encode hABBKCDR3, hABBKCDR4, hABBKCDR5 or consensus sequence.Synthetic antibody with variable sequence BKCDR3 and BKCDR5 stimulates PGE2 synthetic in dosage dependence mode.BKCDR4, ConVH, single culture base, HOE-140 do not stimulate synthetic (Fig. 7 B) cell of PGE2 to contact with BKCDR4, and PGE2 is synthetic might be owing to the following fact, and CDR4 consensus sequence too weak point can not hold the whole Kallidin I binding sequence of adaptation.Table 6 has compared total cdr amino acid sequence and BKCDR sequence.The result shows that synthetic modification antibody BKCDR3 and BKCDR5 can combine bradykinin receptor with native ligand competitiveness.In conjunction with.Shown in Fig. 7 C, add Kallidin I and stimulate PGE2 synthetic 4 times (playing second frame 1 left).BKCDR3 or BKCDR5 join in advance can suppress the synthetic of PGE2 that Kallidin I stimulates in the cell that stimulated with natural Kallidin I.
The total CDR5:Trp Ile Asn Gly Asn Gly Asp Thr Asn Tyr Ala Gln Lys Phe Gln GlyBKCDR5:Trp Ile Asn Gly Arg Pro Pro Gly Phe Ser Pro Phe Arg Phe Gln Gly of the total CDR4:Ser Tyr Ala Ile Ser Trp AsnBKCDR4:Pro Gly Phe Ser Pro Phe Arg of the total CDR3:Gln Gln Tyr Asn Ser Leu Pro Trp ThrBKCDR3:Arg Pro Pro Gly Phe Ser Pro Phe Arg of table 8
In a word, these results show that the modified antibodies that contains the Kallidin I binding site can combine with bradykinin receptor.
Scope of the present invention is not limited to specified scheme as described herein.In fact, except those schemes described herein, to one skilled in the art, according to the description of front and appended legend, apparently various evolutionary approach can appear.Above-mentioned modification should be included within the scope of the appended claim of the present invention.
Quoted various lists of references at this, these disclosures are introduced only for reference as a whole.

Claims (122)

  1. One kind with combine the right bonded specifically modified immunoglobulin of first member's immunity, this combination is to by described one first member and one second member composition, described immunoglobulin comprises a variable region, this domain has a CDR who contains described second member's a part at least, described part contains first member's a binding site and in natural CDR discovery is not arranged, and described first member is a kind of cancer antigen.
  2. 2. the modified immunoglobulin of claim 1, it is a kind of antibody.
  3. 3. the modified immunoglobulin of claim 1, wherein said first member is a kind of tumor antigen.
  4. 4. the modified immunoglobulin of claim 3, wherein said tumor antigen is a multiform epidermis mucin antigen.
  5. 5. the modified immunoglobulin of claim 3, wherein said tumor antigen is a human colon carcinoma associated protein antigen.
  6. 6. the modified immunoglobulin of claim 5, wherein said part has a kind of aminoacid sequence that is selected from following each group: Thr-Ala-Lys-Ala-Ser-Gln-Ser-Val-Ser-Asn-Asp-Val-Ala, Ile-Tyr-Tyr-Ala-Ser-Asn-Arg-Tyr-Thr, Phe-Ala-Gln-Gln-Asp-Tyr-Ser-Ser-Pro-Leu-Thr, phe-Thr-Asn-Tyr-Gly-Met-Asn, Ala-Gly-Trp-Ile-Asn-Thr-Tyr-Thr-Gly-Glu-Pro-Thr-Tyr-Ala-Asp-Asp-Phe-Lys-Gly, and Ala-Arg-Ala-Tyr-Tyr-Gly-Lys-Tyr-Phe-Asp-Tyr.
  7. 7. the modified immunoglobulin of claim 3, wherein said tumor antigen are the relevant carbohydrate antigen of a kind of human colon carcinoma.
  8. 8. the modified immunoglobulin of claim 3, wherein said tumor antigen is a kind of people's butterfat bead antigen.
  9. 9. the modified immunoglobulin of claim 8, wherein said having part ownership is selected from the aminoacid sequence of following each group: Ala-Tyr-Trp-Ile-Glu, Glu-Ile-Leu-Pro-Gly-Ser-Asn-Asn-Ser-Arg-Tyr-Asn-Glu-Lys-Phe-Lys-Gly, Ser-Glu-Asp-Ser-Ala-Val-Tyr-Tyr-Cys-Ser-Arg-Ser-Tyr-Asp-Phe-Ala-Trp-Phe-Ala-Tyr, Lys-Ser-Ser-Gln-Ser-Leu-Leu-Tyr-Ser-Ser-Asn-Gln-Lys-Ile-Tyr-Leu-Ala, Trp-Ala-Ser-Thr-Arg-Glu-Ser, andGln-Gln-Tyr-Tyr-Arg-Tyr-Pro-Arg-Thr.
  10. 10. the modified immunoglobulin of claim 8, it further comprises a kind of second CDR that contains second member's part, second member has the aminoacid sequence that is selected from following each group: Ala-Tyr-Trp-Ile-Glu, Glu-Ile-Leu-Pro-Gly-Ser-Asn-Asn-Ser-Arg-Tyr-Asn-Glu-Lys-Ple-Lys-Gly, Ser-Glu-Asp-Ser-Ala-Val-Tyr-Tyr-Cys-Ser-Arg-Ser-Tyr-Asp-Phe-Ala-Trp-Phe-Ala-Tyr, Lys-Ser-Ser-Gln-Ser-Leu-Leu-Tyr-Ser-Ser-Asn-Gln-Lys-Ile-Tyr-Leu-Ala, Trp-Ala-Ser-Thr-Arg-Glu-Ser, and Gln-Gln-Tyr-Tyr-Arg-Tyr-Pro-
  11. 11. the modified immunoglobulin of claim 3, wherein said tumor antigen are the antigen of breast tumor, ovarian tumor, uterus tumor, tumor of prostate, tumor of bladder, lung tumor, cutaneous tumor, pancreas tumor, colon tumor, gastroenteric tumor, bone-marrow-derived lymphocyte tumor or T lymphocyte tumor.
  12. 12. the modified immunoglobulin of claim 1.Wherein said cancer antigen is selected from following each group, and they are KS1/4 vapour cancer antigen, ovarian cancer antigen, prostanoic acid phosphoric acid, prostate specific antigen, melanic related antigen p97, melanoma-associated antigen gp75, the high molecular melanoma-associated antigen, prostatic specific membrane antigen, carcinoembryonic antigen, multiform epidermis mucin antigen, human milk fat bead antigen, colorectal carcinoma related antigen TAG-72, CO17-1A, GICA19-9, CTA-1, LEA, Burkitt ＇ s lymphoma antigen 38.13, CD19, people B lymphoma antigens c D20, CD33, ganglioside GD2, Ganglioside, GD3, Ganglioside GM2, Ganglioside GM3, tomour specific transplantation type cell surface antigen, carcinoembryonic antigen-α-fetoprotein L6, human lung cancer antigen L20, human leukemia T cellular antigens-GP37, neoglycoprotein, sphingolipid, EGFR, HER2 antigen, multiform epidermis mucin, people's malignant lymphatic cellular antigens-Apo-1, I class antigen M18, M39, SSEA-1, VEP8, VEP9, Myl, VIM-D5, D156-22, TRA-1-85, C14, F3, AH6, the Y hapten, LeY, TL5, FC10.2, adenocarcinoma of stomach antigen, CP-514, NS-10, CO-43, MH2, in colon cancer, find 19.9, the 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 a kind of TXi Baoshouti derived peptide.
  13. 13. one kind with the modified immunoglobulin that combines right first member immunity specific bond, this combination is to by described first member and second member composition, described immunoglobulin comprises a variable region, this domain has a CDR who contains described second member's a part at least, described part contains at described first member's a binding site and in natural CDR discovery is not arranged, and described first member is a kind of antigen of infectious disease cause of disease.
  14. 14. the modified immunoglobulin of claim 13, it is a kind of antibody.
  15. 15. the modified immunoglobulin of claim 13, wherein said infectious disease cause of disease is a kind of antibacterial.
  16. 16. the modified immunoglobulin of claim 13, wherein said infectious disease cause of disease are a kind of virus.
  17. 17. the modified immunoglobulin of claim 13, wherein said infectious disease cause of disease are a kind of parasites.
  18. 18. the antigen of the modified immunoglobulin of claim 13, wherein said infectious disease cause of disease is selected from following each group, they are the antigen of the antigen of Brambell receptor, HSV-2, gonococcal antigen, treponema pallidum, the antigen of chlamydia trachomatis or human papillomavirus's antigen.
  19. 19. the modified immunoglobulin of claim 13; wherein the antigen of infectious disease cause of disease is selected from following each group, and they are influenza virus hemagglutinins; human respiratory syncytial precursor virus G glycoprotein; the core protein of dengue virus; the stromatin of dengue virus; the Measles virus hemagglutinin; II herpes simplex virus type glycoprotein gB; poliovirus I VP1; HIV1 by membrane glycoprotein; hepatitis B virus surface antigen; diphtheria toxin, diphtherotoxin; streptococcus 24M epi-position; the gonococcus pilin; pseudorabies poison g50; pseudorabies poison glycoprotein h; pseudorabies poison glycoprotein E; transmissible gastroenteritis glycoprotein 195; the transmissible gastroenteritis stromatin; porcine rotavirus glycoprotein 38; the pig parvoviral capsid protein; the little Serpentis bacterium of swine dysentery protective antigen; bovine viral diarrhoea glycoprotein 55; ewcastle disease virus hemagglutinin neuraminidase; the swine flue hemagglutinin; the swine flue neuraminidase; infectious bovine rhinotracheitis viral glycoprotein E; infectious laryngotracheitis virus glycoprotein G or glycoprotein I; the glycoprotein of La Crosse virus; the new calves diarrhea virus; hepatitis B virus core protein; hepatitis B virus surface antigen; equine influenza virus A type/Alaska 91 neuraminidases; equine influenza virus A type/Miami 63 neuraminidases; equine influenza virus A type/Kentucky 81 neuraminidases; I type equine herpes virus Glycoprotein B; I type equine herpes virus glycoprotein D; cattle respiratory syncytial virus attachment protein; cattle respiratory syncytial virus glycoprotein D; cattle respiratory syncytial virus nucleocapsid protein; bovine influenza virus 3 type fusion rotein; bovine influenza virus 3 type hemagglutinin neuraminidases; bovine viral diarrhea virus glycoprotein 48; bovine diarrhea virus glycoprotein 53.
  20. 20. the modified immunoglobulin of claim 15, wherein the infectious disease cause of disease is selected from following each group, and they are mycobacterium, rickettsia, mycoplasma, Neisser mushroom, shigella dysenteriae class, Legionnella, vibrio cholera, Streptococcus, diphtheria corynebacterium, clostridium tetani, bordetella pertussis, haemophilus class, chlamydia class and escherichia coli.
  21. 21. the modified immunoglobulin of claim 16, wherein the infectious disease cause of disease is selected from following each group, and they are hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, chickenpox virus, adenovirus, herpessimplexvirustype, herpes simplex virus type 2, rhinovirus, echovirus, rotavirus, the respiratory syncytial virus, papovavirus, parvovirus, cytomegalovirus, bundle shape virus, arbovirus, Hantaan virus, Coxsackie virus, mumps virus, Measles virus, rubella virus, poliovirus, I type human immunodeficiency virus, II type human immunodeficiency virus, picorna virus, enterovirus, the embedding Calicivirus, the Norwalk papova, togavirus, Alphavirus, yellow fever virus, coronavirus, rabies virus, Marburg virus, Ebola virus, parainfluenza virus, influenza virus, arenavirus, reovirus, wheel shape virus, socket of the eye virus, I type human T-cell leukemia virus, II type human T-cell leukemia virus, the troglodyte immunodeficiency virus, slow virus, polyoma virus, parvovirus, Epstein-Barr virus, herpes virus hominis-6, I type cercopithecid herpesvirus and poxvirus.
  22. 22. the modified immunoglobulin of claim 17, wherein the infectious disease cause of disease is selected from Plasmodium, eimeria, leishmaniasis, cocoa Eimeria (kokzidioa) and trypanosoma and fungus together.
  23. 23. one kind with the modified immunoglobulin that combines first member immunity specific bond, this combination is to by described first member and second member composition, described immunoglobulin comprises a variable region, this domain has a CDR who contains described second member's a part at least, described part contains a binding site at described first member, this binding site does not contain sequence A sn-Ala-Asn-Pro or Asn-Val-Asp-Pro and in natural CDR discovery is not arranged, and described first member is a kind of cell receptor at the infectious disease cause of disease.
  24. 24. the modified immunoglobulin of claim 23, it is a kind of antibody.
  25. 25. the modified immunoglobulin of claim 23, wherein, described infectious disease cause of disease is a kind of antibacterial.
  26. 26. the modified immunoglobulin of claim 23, wherein, described infectious disease cause of disease is a kind of virus.
  27. 27. the modified immunoglobulin of claim 23, wherein, described infectious disease cause of disease is a kind of parasite.
  28. 28. the modified immunoglobulin of claim 23, wherein cell receptor is selected from the albumen of following each group, and they are LPV receptor, adenosine cyclase, BDV surface glycoprotein, N-acetyl-9-O-n acetylneuraminic acid n receptor, CD4 -Height sulphation type heparin sulfate, p65, the isoreceptor that contains Gal-α l-4-Gal, CD16b, integrin VLA-2 receptor, the EV receptor, CD14, the carbohydrate conjugates receptor, α/β TXi Baoshouti, decay accelerated factor receptor, the cell envelope glycoprotein receptor, immunoglobulin FC receptor poxvirus M-T7, the GALV receptor, the CD14 receptor, lewis (b) blood group antigen receptor, TXi Baoshouti, heparin sulfate sugar is subjected to the polysaccharide receptor, fibroblast growth factor acceptor, CD11a, CD2, the G-protein coupling receptor, CD4, the heparin sulfate Dan Baijutang, the annexin II, CD13 (Aminopeptidase N), people's Aminopeptidase N receptor, the hemagglutinin receptor, the CR3 receptor, the protein kinase receptor, but galactose N-acetylgalactosamine inhibition agglutinin receptor, chemokine receptors, the annexin I, actA albumen, the CD46 receptor, the meningococcus virulence opa receptor of being correlated with, the CD46 receptor, the carcinoembryonic antigen family receptors, the Bg1a of carcinoembryonic antigen family receptor, interferon-gamma receptor, glycoprotein gp70, the rmc-1 receptor, human beta 2 integrin receptor α ν β 3, heparin sulfate Dan Baijutang receptor, the CD66 receptor, integrin receptor, membrane cofactor protein, 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, mucosa orientation alpha 4 β 7 receptors, EGF-R ELISA, alpha 5 beta 1 integrin, non-glucosides J774 receptor, the CXCR1-4 receptor, the CCR1-5 receptor, the CXCR3 receptor, the CCR5 receptor, the gp46 surface glycoprotein, the TNFRp55 receptor, the TNFp75 receptor, soluble interleukin-6-1 beta receptor.
  29. 29. modified immunoglobulin that combines right first member immunity specific bond with ligand-receptor, this combination is to being made up of described first member and second member, described immunoglobulin comprises a variable region, this domain have at least one contain the CDR of described second member's a part, described part contains a binding site at described first member, and in natural CDR discovery is not arranged.
  30. 30. the modified immunoglobulin of claim 29, it is a kind of antibody.
  31. 31. the modified immunoglobulin of claim 29, wherein said first member is a kind of receptor.
  32. 32. the modified immunoglobulin of claim 29, wherein said first member is a kind of part.
  33. 33. the modified immunoglobulin of claim 29, wherein said first member is a kind of receptor stimulating agent.
  34. 34. the modified immunoglobulin of claim 29, wherein said first member is a kind of receptor antagonist.
  35. 35. the modified immunoglobulin of claim 29, wherein said first member is a kind of bradykinin receptor.
  36. 36. the modified immunoglobulin of claim 35, wherein said part is made up of aminoacid sequence Arg-Pro-Pro-Gly-Phe-Gly-Phe-Ser-Pro-Phe-Arg.
  37. 37. the modified immunoglobulin of claim 31, wherein said receptor is selected from following one group, and they are opioid receptor, glucose transporter, glutamate receptor, the only plain receptor of Vulpes, erythropoietin receptor, Insulin receptor INSR, tyrosine kinase receptor, KIT stem cell factor receptor, trk C, IGF-1, granulocyte colony stimulating factor receptor, growth hormone receptor, the neurotrophic factor acceptor that derives from glial cell, gp39 receptor, G protein receptor class and beta 2-adrenergic receptor.
  38. 38. the modified immunoglobulin of claim 30, wherein said part is selected from following one group, and they are 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 and Endothelin.
  39. 39. claim 2,14,24 or 30 modified immunoglobulin, wherein said antibody is selected from IgG, IgE, IgM, IgD and IgA one type.
  40. 40. the fragment of a claim 2,14,24 or 30 modified immunoglobulin, wherein said fragment can combine specifically with described first member's immunity.
  41. 41. the fragment of claim 40, wherein said fragment are selected from Fab, (Fab ＇) 2, heavy chain homodimer, light chain dimer and Fv fragment.
  42. 42. claim 2,14,24 or 30 modified immunoglobulin, wherein said part is inserted within the described CDR.
  43. 43. claim 2,14,24 or 30 modified immunoglobulin, wherein said part replaces the aminoacid of one or more CDR.
  44. 44. claim 2,14,24 or 30 modified immunoglobulin, wherein containing described partial C DR is a kind of embryonal system CDR.
  45. 45. claim 2,14,24 or 30 modified immunoglobulin, wherein containing described partial C DR is a kind of non-embryonal system CDR.
  46. 46. claim 2,14,24 or 30 modified immunoglobulin, wherein said part is at least 4 aminoacid.
  47. 47. claim 2,14,24 or 30 modified immunoglobulin, wherein said part are 10-20 amino acid whose scope.
  48. 48. claim 2,14,24 or 30 modified immunoglobulin wherein contain described partial C DR and contain no more than 25 aminoacid.
  49. 49. claim 2,14,24 or 30 modified immunoglobulin, first CDR that wherein to contain described partial C DR be variable region of heavy chain.
  50. 50. claim 2,14,24 or 30 modified immunoglobulin, wherein containing described partial C DR is second CDR of variable region of heavy chain.
  51. 51. claim 2,14,24 or 30 modified immunoglobulin, wherein containing described partial C DR is the 3rd CDR of variable region of heavy chain.
  52. 52. claim 2,14,24 or 30 modified immunoglobulin, first CDR that wherein to contain described partial C DR be variable region of light chain.
  53. 53. claim 2,14,24 or 30 modified immunoglobulin, wherein containing described partial C DR is second CDR of variable region of light chain.
  54. 54. claim 2,14,24 or 30 modified immunoglobulin, wherein containing described partial C DR is the 3rd CDR of variable region of light chain.
  55. 55. claim 2,14,24 or 30 modified immunoglobulin, a wherein more than CDR contains the part of described binding site.
  56. 56. claim 2,14,24 or 30 modified immunoglobulin, wherein second CDR contains second binding site at other molecule outside described first member.
  57. 57. the modified immunoglobulin of claim 56, other molecule outside wherein said first member but a kind of molecule that is positioned at the immunocyte surface.
  58. 58. the modified immunoglobulin of claim 57, wherein said immunocyte are T cell, B cell, NK cell, K cell, til cell or neutrophil cell.
  59. 59. claim 2,14,24 or 30 modified immunoglobulin, this modified immunoglobulin is higher than the antibody with described first member immunity specific bond of natural generation to described first member's specificity.
  60. 60. claim 2,14,24 or 30 modified immunoglobulin, this immunoglobulin is higher than the antibody and the bonded specifically affinity of described first member's immunity of natural generation with described first member's affinity.
  61. 61. claim 2,14,24 or 30 modified immunoglobulin, the binding constant that it shows as described first member is at least 2 * 10 7M.
  62. 62. claim 2,14,24 or 30 modified immunoglobulin, the affinity costant that wherein said antibody has described first member is at least 2 * 10 7M.
  63. 63. claim 1,13,23 or 29 modified immunoglobulin, the one or more cysteine residues that form disulfide bond in the variable region of wherein said immunoglobulin are replaced by one or more amino acid residues that do not contain sulfydryl.
  64. 64. the modified immunoglobulin of claim 63, at least one is at 23 or 88 of variable region of light chain in wherein said one or more cysteine residues.
  65. 65. the modified immunoglobulin of claim 63, at least one is at 23 or 92 of variable region of heavy chain in wherein said one or more cysteine residues.
  66. 66. the modified immunoglobulin of claim 63, wherein said at least one not contain the mercaptoamino acid residue be alanine.
  67. 67. molecule, comprise and combine the right bonded specifically variable region of first member's immunity, this combination is to by described first member and second member composition, described variable region has at least one CDR that contains described second member's a part, described part contains a binding site at described first member, and in natural CDR discovery is not arranged, described first member is a kind of cancer antigen.
  68. 68. molecule, comprise and combine the right bonded specifically variable region of first member's immunity, this combination is to being made up of described first member and second member, described variable region has at least one CDR that contains described second member's a part, and in natural CDR, discovery is not arranged, described part contains a binding site at first member, and described first member is a kind of antigen of infectious disease cause of disease.
  69. 69. molecule, comprise and combine the right bonded specifically variable region of first member's immunity, this combination is to by described first member and second member composition, described variable region has at least one CDR that contains described second member's a part, described part contains a binding site at described first member, this binding site does not have sequence A sn-Ala-Asn-Pro or Asn-Val-Asp-Pro and in natural CDR discovery is not arranged, and described first member is a kind of cell receptor at the infectious disease cause of disease.
  70. 70. molecule, comprise and combine the right bonded specifically variable region of first member's immunity with ligand-receptor, this combination is to by described first member and second member composition, described variable region has at least one CDR that contains described second member's a part, and described part contains at described first member's a binding site and in natural CDR discovery do not arranged.
  71. 71. claim 67,68,69 or 70 molecule, wherein said molecule is a kind of single-chain antibody.
  72. 72. claim 67,68,69 or 70 molecule, it further comprises a constant region.
  73. 73. the molecule of claim 72, wherein except containing described partial C DR, this variable region is from mouse antibodies, and constant region is from people's antibody.
  74. 74. the molecule of claim 72, wherein except containing described partial C DR, the variable region has from the framework region of people's antibody with from the CDRs of mouse antibodies, and wherein constant region from people's antibody.
  75. 75. the molecule of claim 74, wherein with regard to the framework region of natural generation, described variable region has at least one framework region that has at least one aminoacid to change.
  76. 76. claim 67,68,69 or 70 molecule, it is fused on immunostimulation or growth enhancer or its functional fragment through covalent bond.
  77. 77. the molecule of claim 76, wherein the immunostimulation factor is selected from following each group, and they are interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-7, IL-10 INTERLEUKIN-10, il-1 2, interleukin-15, G-colony stimulating factor, tumor necrosis factor, perforin, gamma interferon and NK cellular antigens.
  78. 78. an isolating nucleic acid, it comprises the nucleotide sequence of coding claim 1,13,23 or 29 modified immunoglobulin.
  79. 79. an isolating nucleic acid, it comprises the nucleotide sequence of coding claim 67,68,69 or 70 molecule.
  80. 80. the isolating nucleic acid of claim 78, wherein said nucleic acid are a kind of carriers.
  81. 81. the isolating nucleic acid of claim 79, wherein said nucleic acid are a kind of carriers.
  82. 82. a cell that contains the nucleic acid of claim 78, this nucleic acid is recombinated.
  83. 83. a cell that contains the nucleic acid of claim 79, this nucleic acid is recombinated.
  84. 84. the non-human animal of a reorganization, this animal contains the nucleic acid of claim 78.
  85. 85. the non-human animal of a reorganization, this animal contains the nucleic acid of claim 79.
  86. 86. a pharmaceutical composition, it comprises the claim 1,13 of a kind of treatment or prevention effective dose, 23 or 29 modified immunoglobulin and a kind of pharmaceutically acceptable carrier.
  87. 87. a pharmaceutical composition, it comprises the claim 67,68 of a kind of treatment or prevention effective dose, 69 or 70 molecule and a kind of pharmaceutically acceptable carrier.
  88. 88. a Pharmaceutical composition, it comprises nucleic acid and a kind of pharmaceutically acceptable carrier of the claim 78 of a kind of treatment or prevention effective dose.
  89. 89. a Pharmaceutical composition, it comprises nucleic acid and a kind of pharmaceutically acceptable carrier of the claim 79 of a kind of treatment or prevention effective dose.
  90. 90. a Pharmaceutical composition, it comprises reconstitution cell and a kind of pharmaceutically acceptable carrier of the claim 82 of a kind of treatment or prevention effective dose.
  91. 91. a Pharmaceutical composition, it comprises reconstitution cell and a kind of pharmaceutically acceptable carrier of the claim 83 of a kind of treatment or prevention effective dose.
  92. 92. a vaccine combination, it comprises the claim 1,13 of q.s induce immune response, 23 or 29 modified immunoglobulin and a kind of pharmaceutically acceptable carrier.
  93. 93. a vaccine combination, it comprises the claim 67,68 of q.s induce immune response, 69 or 70 molecule and a kind of pharmaceutically acceptable carrier.
  94. 94. a vaccine combination, it comprises modified immunoglobulin and a kind of pharmaceutically acceptable carrier of the claim 63 that the q.s induced anti-idiotype replys.
  95. 95. differentiate in sample to be determined or measure or the antigenic method of detection cancer that this cancer antigen is that described method comprises following steps by first member in conjunction with centering of described first member and second member composition for one kind:
    (a) with immunity under relevant condition, contacting with sample to be determined with the bonded modified immunoglobulin of described cancer antigen specifically, described modified immunoglobulin comprises a variable region, this variable region has a CDR who contains described second member's a part at least, described part contains one at the antigenic binding site of cancer and in natural CDR discovery is not arranged, and special the combining of any described cancer antigen immune in described modified immunoglobulin and the sample can take place the result; Then
    (b) detect described modified immunoglobulin and antigenic any combination of described cancer;
    Wherein, detect described modified immunoglobulin and combine, be illustrated in and have described cancer antigen in the described sample with described cancer is antigenic.
  96. 96. the antigenic method in sample to be determined, differentiating or measure or detect the infectious disease cause of disease, this antigen be by described first member and second member composition in conjunction with the first right member, described method comprises following steps:
    (a) with immunity contacting with sample to be determined under relevant condition with the bonded modified immunoglobulin of described antigen specifically, described modified immunoglobulin comprises a variable region, this variable region has a CDR who contains described second member's a part at least, this part contains one at described antigenic binding site and in natural CDR discovery is not arranged, and any antigen immune specific bond in described modified immunoglobulin and the sample can take place the result; Then
    (b) detect described modified immunoglobulin and described antigenic any generation;
    Wherein, detecting described modified immunoglobulin and described antigenic associative list is shown in and has described antigen in the described sample.
  97. 97. the method for discriminating or mensuration or detector ligand in a sample to be determined, this part be by described first member and second member composition in conjunction with the first right member, described method comprises following steps:
    (a) with immunity under relevant condition, contacting with sample to be determined with the bonded modified immunoglobulin of described part specifically, described modified immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, this part contains one at the binding site of described part and in natural CDR discovery is not arranged, and the immune specific bond of any part in described modified immunoglobulin and the sample can take place the result; Then
    (b) any bonded generation of described modified immunoglobulin of detection and described part;
    Wherein, the associative list that detects described modified immunoglobulin and described part is shown in and has described part in the described sample.
  98. 98. the method in sample to be determined, differentiating or measure or detect receptor, this receptor be by described first member and second member composition in conjunction with the first right member, described method comprises following steps:
    (a) with can immunity under relevant condition, contacting with sample to be determined with the modified immunoglobulin of described receptors bind specifically, described modified immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, this part contains one at the binding site of described receptor and in natural CDR discovery is not arranged, and the immune specific bond of any receptor in described modified immunoglobulin and the sample can take place the result; Then
    (b) any bonded generation of described modified immunoglobulin of detection and described receptor;
    Wherein, the associative list that detects described modified immunoglobulin and described receptor is shown in and has described receptor in the sample.
  99. 99. one kind is detected the antigenic test kit of cancer, this cancer antigen be by described first member and second member composition in conjunction with the first right member, described test kit comprises in a container:
    (a) a kind of can immunity specifically with the modified immunoglobulin of described cancer antigen immune specific bond, described immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, and described part contains one at the antigenic binding site of described cancer and in natural CDR discovery is not arranged; And
    (b) described cancer antigen of a kind of detection and the bonded method of described immunoglobulin.
  100. 100. an antigenic test kit that detects the infectious disease cause of disease, this antigen be by described first member and second member composition in conjunction with the first right member, described test kit comprises in a container:
    (a) a kind of can immunity specifically with the bonded modified immunoglobulin of described antigen, described immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, and described part contains one at described antigenic binding site and in natural CDR discovery is not arranged; And
    (b) described antigen of a kind of detection and the bonded method of described immunoglobulin.
  101. 101. a detection is at the test kit of the cell receptor of infectious disease cause of disease, this cell receptor be by described first member and second member composition in conjunction with the first right member, described test kit comprises in a container:
    (a) a kind of can immunity specifically with the bonded modified immunoglobulin of described cell receptor, described immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, described part contains a binding site at described cell receptor, and this binding site does not have Asn-Lae-Asn-Pro or Asn-Val-Asp-Pro and in natural CDR discovery do not arranged; And
    (b) described cell receptor of a kind of detection and the bonded method of described immunoglobulin.
  102. 102. the test kit of a detector ligand, it be by described first member and second member composition in conjunction with the first right member, described test kit comprises in a container;
    (a) a kind of can immunity specifically with the bonded modified immunoglobulin of described part, described globulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, and described part contains at a binding site of part and in natural CDR discovery do not arranged; And
    (b) described part of a kind of detection and the bonded method of described immunoglobulin.
  103. 103. a test kit that detects receptor, it be by described first member and second member in conjunction with the first right member, described test kit comprises in a container:
    (a) a kind of can immunity specifically with the modified immunoglobulin of described receptors bind, described immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, and described part contains one at the binding site of described receptor and in natural CDR discovery is not arranged; And
    (b) described receptor of a kind of detection and the bonded method of described immunoglobulin.
  104. 104. a diagnosis or the method that the screening cancer exists or cancer development tends to, the feature of this cancer is that cancer antigen increases, it be by described first member and second member composition in conjunction with the first right member, described method comprises modified immunoglobulin among the experimenter and the level from experimenter's sample immunity specific bond of being determined at, wherein said modified immunoglobulin immunity combines with described cancer antigen specifically and described modified immunoglobulin comprises a variable region, this domain has at least one CDR that contains described second member's a part, described part contains one at the antigenic binding site of described cancer and in natural CDR discovery is not arranged, wherein with respect to from cancer stricken not or there is not the level of the immune specific bond described in experimenter's the similar sample of cancer development trend, the increase of this immunity specific bond level shows and has cancer or the cancer development trend is arranged.
  105. 105. diagnose or screen the method that has the infectious disease cause of disease for one kind, it is characterized in that existing the antigen of described infectious disease cause of disease, this antigen be by described first member and second member composition in conjunction with the first right member, described method comprises modified immunoglobulin among the experimenter and the level from experimenter's sample immunity specific bond of being determined at, wherein said modified immunoglobulin immunity combines with described antigen specifically and this modified immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, described part contains one at described antigenic binding site and in natural CDR discovery is not arranged, wherein with respect to the level from the immune specific bond in the experimenter's of not containing the infectious disease cause of disease the similar sample, the level increase of described immune specific bond shows and has the infectious disease cause of disease.
  106. 106. the treatment or the method for prophylaxis of cancer, this cancer is a feature there to be cancer antigen, this experimenter needs above-mentioned treatment or prevention, described cancer antigen be by described first member and second member composition in conjunction with the first right member, this cancer antigen is by modified immunoglobulin immunity combination specifically, described immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, described part contains one at the antigenic binding site of described cancer and in natural CDR discovery is not arranged, and said method comprises the described modified immunoglobulin of using a kind of treatment or prevention effective dose to the experimenter.
  107. 107. a treatment or the method that keeps off infection, this infectious disease is a feature there to be the infectious disease cause of disease, this experimenter needs above-mentioned treatment or prevention, described antigen be by described first member and second member composition in conjunction with the first right member, and this antigen is by modified immunoglobulin immunity combination specifically, described immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, described part contains one at described antigenic binding site and in natural CDR discovery is not arranged, and this method comprises the described modified immunoglobulin to experimenter's administering therapeutic or prevention effective dose.
  108. 108. a treatment or prophylactic method, this disease is caused with combining of cell receptor by the infectious disease cause of disease, this experimenter needs above-mentioned treatment or prevention, described cell receptor be by described first member and second member composition in conjunction with the first right member, and this cell receptor is by modified immunoglobulin immunity combination specifically, described immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, described part contains a binding site at described cell receptor, this binding site does not have sequence A sn-Ala-Asn-Pro or Asn-Val-Asp-Pro and in natural CDR discovery is not arranged, and described method comprises the described modified immunoglobulin to experimenter's administering therapeutic or prevention effective dose.
  109. 109. regulate in conjunction with to first member's active method for one kind, this combination is to by first and second member compositions, described method comprises uses claim 1,13,23 or 29 modified immunoglobulin.
  110. 110. method of producing modified immunoglobulin, this immunoglobulin immunity combines with cancer antigen specifically, this cancer antigen by described first member and second member composition in conjunction with the first right member, described modified immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, described part contains one at the antigenic binding site of described cancer and in natural CDR discovery is not arranged, described method comprises cultivates a kind of reconstitution cell that contains a kind of nucleic acid, this nucleic acid comprises a kind of nucleotide sequence of this modified immunoglobulin of encoding, the result is reclaimed then and is obtained expressed modified immunoglobulin by the modified immunoglobulin of this cellular expression coding.
  111. 111. method of producing modified immunoglobulin, this modified immunoglobulin immunity combines with the antigen of infectious disease cause of disease specifically, this antigen be by described first member and second member composition in conjunction with the first right member, described modified immunoglobulin comprises a variable region, this variable region has a CDR who contains described second member's a part, described part contains a kind of at described antigenic binding site and in natural CDR discovery is not arranged, this method comprises cultivates a kind of reconstitution cell that contains a kind of nucleic acid, this nucleic acid comprises a kind of nucleotide sequence of the modified immunoglobulin of encoding, the result is reclaimed then and is obtained expressed modified immunoglobulin by the modified immunoglobulin of this cellular expression coding.
  112. 112. method of producing modified immunoglobulin, this modified immunoglobulin combines specifically with cell receptor immunity at infectious disease, this cell receptor be by described first member and second member composition in conjunction with the first right member, described modified immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, described part contains a kind of binding site at described cell receptor, this binding site does not contain sequence A sn-Ala-Asn-Pro or Asn-Val-Asp-Pro and in natural CDR discovery is not arranged, this method comprises cultivates a kind of reconstitution cell that contains a kind of nucleic acid, this nucleic acid comprises a kind of nucleotide sequence of the modified immunoglobulin of encoding, the result is reclaimed then and is obtained expressed modified immunoglobulin by the modified immunoglobulin of this cellular expression coding.
  113. 113. method of producing modified immunoglobulin, this modified immunoglobulin combines specifically with the part immunity, this part be by described first member and second member composition in conjunction with the first right member, described modified immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, described part contains one at the binding site of described part and in natural CDR discovery is not arranged, described method comprises cultivates a kind of reconstitution cell that contains a kind of nucleic acid, this nucleic acid comprises a kind of nucleotide sequence of the modified immunoglobulin of encoding, the result is reclaimed then and is obtained expressed modified immunoglobulin by the modified immunoglobulin of this cellular expression coding.
  114. 114. method of producing modified immunoglobulin, this modified immunoglobulin combines specifically with the receptor immunity, this receptor be by a kind of by described first member and second member composition in conjunction with the first right member, described modified immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, this part contains one at the binding site of described receptor and in natural CDR discovery is not arranged, described method comprises cultivates a kind of reconstitution cell that contains a kind of nucleic acid, this nucleic acid comprises a kind of nucleotide sequence of the modified immunoglobulin of encoding, the result is reclaimed then and is obtained expressed modified immunoglobulin by the modified immunoglobulin of this cellular expression coding.
  115. 115. a method of producing nucleic acid, this nucleic acid coding claim 1,13,23 or 29 modified immunoglobulin, this method comprises:
    (a) a synthetic cover oligonucleotide, described a whole set of oligonucleotide comprises the oligonucleotide of the partial nucleotide sequence that contains this modified immunoglobulin of encoding, oligonucleotide with the complementary partial nucleotide sequence of nucleotide sequence that contains this modified immunoglobulin of encoding, and except those contained the oligonucleotide of nucleotide sequence of the terminal and C-end portion of the N-of the modified immunoglobulin of encoding, every kind of described oligonucleotide had and the eclipsed end sequence of the another kind of oligonucleotide of this cover;
    (b) allow the mutual cross of oligonucleotide phase; And
    (c) connect the oligonucleotide of hybridizing, the result has produced the nucleic acid of the nucleotide sequence that contains this modified immunoglobulin of encoding.
  116. 116. method of producing modified immunoglobulin, this modified immunoglobulin with combine first right member's immunity combination specifically, described immunoglobulin comprises a variable region, this variable region has at least one CDR that contains described second member's a part, described part contains one at described first member's binding site and in natural CDR discovery is not arranged, described first member is a kind of cancer antigen, and described method comprises:
    (a) cultivate the reconstitution cell of the nucleic acid that a kind of method that contains by claim 115 produces, the result is by the modified immunoglobulin of this cellular expression coding; And
    (b) reclaim the expressed modified immunoglobulin of acquisition.
  117. 117. method of producing modified immunoglobulin, this modified immunoglobulin with combine first right member's immunity specifically in conjunction with, this combination to by described first member and second member composition, described antibody comprises a variable region, this variable region has at least one CDR that contains described second member's a part, described part contains one at described first member's binding site and in natural CDR discovery is not arranged, described first member is a kind of antigen of infectious disease cause of disease, and described method comprises:
    (a) cultivate the reconstitution cell of the nucleic acid that a kind of method that contains by claim 115 produces, the result is by the modified immunoglobulin of this cellular expression coding; And
    (b) reclaim the expressed modified immunoglobulin of acquisition.
  118. 118. method of producing modified immunoglobulin, this modified immunoglobulin with combine first right member's immunity combination specifically, this combination is to by described first member and second member composition, described antibody comprises a variable region, this domain has described second member's of containing of at least one CDR a part, described part contains a binding site at first member, this binding site does not contain sequence A sn-Ala-Asn-Pro or Asn-Val-Asp-Pro and in natural CDR discovery is not arranged, described first member is a kind of cell receptor at the infectious disease cause of disease, and described method comprises:
    (a) cultivate a kind of reconstitution cell that contains the nucleic acid that is produced by claim 115, the result is by the modified immunoglobulin of this cellular expression coding.
    (b) reclaim the expressed modified immunoglobulin of acquisition.
  119. 119. method of producing modified immunoglobulin, this modified immunoglobulin with combine first right member's immunity combination specifically, this combination is to by described first member and second member composition, described antibody comprises a variable region, this variable region has at least one CDR that contains second member's a part, described part contains one at described first member's binding site and in natural CDR discovery is not arranged, described first member is a kind of part, and described method comprises:
    (a) cultivate the reconstitution cell of the nucleic acid that a kind of method that contains by claim 115 produces, the result is by the modified immunoglobulin of this cellular expression coding; And
    (b) reclaim the expressed modified immunoglobulin of acquisition.
  120. 120. method of producing modified immunoglobulin, this modified immunoglobulin with combine first right member's immunity combination specifically, this combination is to by described first member and second member composition, described antibody comprises a variable region, this variable region has the CDR variable region of at least one part that contains described second member, described part contains one at described first member's binding site and in natural CDR discovery is not arranged, described first member is a kind of receptor, and described method comprises:
    (a) cultivate the reconstitution cell of the nucleic acid that a kind of method that contains by claim 115 produces, the result is by the modified immunoglobulin of this cellular expression coding; And
    (b) reclaim the expressed modified immunoglobulin of acquisition.
  121. 121. an isolating nucleic acid, it is produced by the method for claim 115.
  122. 122. the isolating nucleic acid of claim 121, it is a kind of carrier.
CN98813117A 1997-11-14 1998-11-13 Immunoglobulin molecules having synthetic variable region and modified specificity Pending CN1294517A (en)

Applications Claiming Priority (4)

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US6571697P 1997-11-14 1997-11-14
US8140398P 1998-04-10 1998-04-10
US60/081,403 1998-04-10
US60/065,716 1998-04-10

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JP (2) JP2001526021A (en)
KR (2) KR20010015818A (en)
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BR (2) BR9815580A (en)
CA (2) CA2309990A1 (en)
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101970500B (en) * 2008-03-12 2013-08-14 伊姆克罗尼责任有限公司 Anti-tyrp1 antibodies
CN103275914A (en) * 2013-06-03 2013-09-04 中国人民解放军军事医学科学院微生物流行病研究所 Bacterial ghost presenting protective antigens and application thereof
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Families Citing this family (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MXPA01004778A (en) * 1998-11-13 2003-07-21 Euro Celtique Sa Contraceptive antibody vaccines.
DK1092779T3 (en) * 1999-10-11 2010-02-15 Pasteur Institut Lentivirus virus vectors for the preparation of immunotherapeutic preparations
AU2001261628A1 (en) * 2000-05-16 2001-11-26 Euroceltique S.A. Cd28 synthebody for the modulation of immune responses
ATE414720T1 (en) 2000-12-05 2008-12-15 Alexion Pharma Inc RATIONALLY DESIGNED ANTIBODIES
US7396917B2 (en) 2000-12-05 2008-07-08 Alexion Pharmaceuticals, Inc. Rationally designed antibodies
GB0103174D0 (en) * 2001-02-08 2001-03-28 Smithkline Beecham Plc Novel method of treatment
WO2002078613A2 (en) * 2001-04-02 2002-10-10 Purdue Pharma L.P. Immunoglobulin construct containing anti-mucin variable domain sequences for eliciting an anti-idiotype anti-tumor response
AU2002365926A1 (en) * 2001-10-25 2003-09-02 Euro-Celtique S.A. Compositions and methods directed to anthrax toxin
ATE382367T1 (en) * 2002-04-09 2008-01-15 Univ Missouri TREATMENT OF TYPE 1 DIABETES BEFORE AND AFTER EXPRESSION OF PREDIPOSITION MARKERS
EP2316922B1 (en) 2002-05-24 2013-05-22 Merck Sharp & Dohme Corp. Neutralizing human anti-IGFR antibody
AR040778A1 (en) * 2002-08-06 2005-04-20 Glaxo Group Ltd ALTERED ANTIBODIES OR FUNCTIONAL FRAGMENTS THAT BIND MAG (GLICOPROTEIN ASSOCIATED WITH HONEY).
CA2499300A1 (en) 2002-10-31 2004-05-21 Genentech, Inc. Methods and compositions for increasing antibody production
GB0306309D0 (en) 2003-03-19 2003-04-23 Glaxo Group Ltd Method of treatment
WO2005021595A1 (en) * 2003-08-28 2005-03-10 Euro-Celtique S.A. Methods of antibody engineering using antibody display rules
ATE514783T1 (en) 2003-11-12 2011-07-15 Schering Corp PLASMID SYSTEM FOR EXPRESSING MULTIPLE GENES
AR046639A1 (en) 2003-11-21 2005-12-14 Schering Corp ANTI-IGFR1 ANTIBODY THERAPEUTIC COMBINATIONS
JP4452839B2 (en) * 2004-03-09 2010-04-21 国立大学法人京都大学 Pharmaceutical composition containing a CXCR3 inhibitor
US7811562B2 (en) 2004-12-03 2010-10-12 Schering Corporation Biomarkers for pre-selection of patients for anti-IGF1R therapy
GB0706070D0 (en) 2007-03-28 2007-05-09 Scancell Ltd Nucleic acids
WO2010126972A1 (en) * 2009-04-29 2010-11-04 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Erg monoclonal antibodies
CN102816240B (en) * 2011-03-08 2014-02-12 中国人民解放军第三军医大学第二附属医院 Fusion protein and fusion protein expression vector thereof
US9611321B2 (en) 2011-05-27 2017-04-04 Fox Chase Cancer Center Rationally-designed anti-mullerian inhibiting substance type II receptor antibodies
CN108864291A (en) * 2011-11-23 2018-11-23 拜奥文斯瑞有限公司 Recombinant protein and its therapeutical uses
JP5437525B1 (en) 2012-12-28 2014-03-12 株式会社ナード研究所 Tyrosine derivative and method for producing tyrosine derivative
US20160206732A1 (en) * 2013-05-14 2016-07-21 Shanghai Hycharm Inc. Epitope vaccine for low immunogenic protein and preparing method and usage thereof
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
CN112646029B (en) * 2020-12-30 2022-07-29 深圳清华大学研究院 Antibody of mature brain-derived neurotrophic factor, application thereof and diagnostic kit
CN115856296B (en) * 2022-12-16 2023-07-25 华北理工大学 Monoclonal antibody for resisting shigella and application thereof in detection

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5688657A (en) * 1988-03-31 1997-11-18 International Bio-Immune Systems, Inc. Monoclonal antibodies against human colon carcinoma-associated antigens and uses therefor
ATE130765T1 (en) * 1989-02-24 1995-12-15 Univ California GENETICALLY MODIFIED IMMUNOGLOBULINS.
GB9019553D0 (en) * 1990-09-07 1990-10-24 Unilever Plc Specific binding agents
GB9109645D0 (en) * 1991-05-03 1991-06-26 Celltech Ltd Recombinant antibodies
GB9223084D0 (en) * 1992-11-04 1992-12-16 Imp Cancer Res Tech Compounds to target cells
AU6396494A (en) * 1992-11-16 1994-06-08 Cancer Research Fund Of Contra Costa Peptides and anti-sense peptides with broad neoplastic specificity
WO1994014848A1 (en) * 1992-12-24 1994-07-07 The Regents Of The University Of California Genetically engineered immunoglobulins
WO1994014847A1 (en) * 1992-12-24 1994-07-07 The Regents Of The University Of California Genetically engineered immunoglobulins
US5476784A (en) * 1993-04-06 1995-12-19 Rice; Peter A. Gonococcal anti-idiotypic antibodies and methods and compositions using them
US5653977A (en) * 1993-09-09 1997-08-05 Uab Research Foundation Anti-idiotypic antibody that mimics the GD2 antigen
US5624904A (en) * 1993-11-17 1997-04-29 Massachusetts Institute Of Technology Method for treating gram positive septicemia

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CN111978382A (en) * 2020-09-03 2020-11-24 吉林大学第一医院 Preparation method and application of recombinant protein of Sporothrix globosum Gp70

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