CN101490085A - Single-chain multivalent binding proteins with effector function - Google Patents

Abstract

Multivalent binding peptides, including bi-specific binding peptides, having immunoglobulin effector function are provided, along with encoding nucleic acids, vectors and host cells as well as methods for making such peptides and methods for using such peptides to treat or prevent a variety of diseases, disorders or conditions, as well as to ameliorate at least one symptom associated with such a disease, disorder or condition.

Description

Single-chain multivalent binding proteins with effector function

Technical field

The present invention relates generally to the field that multivalence binding molecule and treatment thereof are used.

Sequence table is submitted with textual form and with the pdf document of the application requiring that meets electronic filing.Sequence table is created on June 12nd, 2007.Sequence table is incorporated herein by reference in full.

Background technology

In healthy Mammals, the immunity system health that watches for animals is avoided the infringement of foreign body and pathogenic agent.But in some cases, immunity system can be made mistakes, and causes traumatic damage and/or disease.For example, the B-cell can produce the identification oneself protein but not the antibody of foreign protein, and causing producing with the autoantibody is the autoimmune disorders of feature, such as lupus erythematosus disease, rheumatoid arthritis and similar disease thereof.In other cases, immunity system is resisted the typical favourable effect counterproductive of foreign matter, such as following organ transplantation.Confirmed the effect of immune system (and especially human immunity system), and made great efforts to control this system with avoid or improve because of immunity system in unusual environment (for example organ transplantation) normal functionating or immunity system in other external homes (for example autoimmune disorders progress) unusual functionating to harmful result that health was caused.In addition, made great efforts to utilize immunity system that multiple target specific diagnosis and methods of treatment are provided, this decides on the identification of antibodies specific ground and in conjunction with the ability of the specific antigenicity target of tool.

Watch for animals a kind of mode of health of immunity system is to produce specialized cell, is called bone-marrow-derived lymphocyte or B-cell.The B-cell produces the destructive antibody be bonded to foreign body or pathogenic agent and mediate foreign body or pathogenic agent in some cases.But in some cases, human immunity system (and specific, the bone-marrow-derived lymphocyte of human immunity system) can make mistakes and cause disease.Exist multiple cancer to relate to the propagation out of control of B-cell.The B-cell of the antibody of the part that also exists multiple autoimmune disorders to relate to be bonded to the animal health and non-binding foreign body and pathogenic agent produces.In addition, exist the pathology of multiple autoimmune disorders and inflammatory diseases to relate to the B-cell, for example, present or via other approach that relate to the B-cell to T-cell improper via the B-cell antigen.For example, the easy autoimmunity mouse of shortage B-cell is not developed autoimmunity ephrosis, vasculitis or autoantibody.(people such as Shlomchik, J Exp.Med.1994,180:1295-306).What is interesting is, the described B-of having cell but lack easy autoimmunity mouse that immunoglobulin (Ig) produces and when experimental technique is induced, do not developing autoimmune disorders (people such as Chan, J Exp.Med.1999,189:1639-48), this shows that the B-cell has indispensable effect in the development of autoimmune disorders.

The B-cell can be discerned by the molecule on its cell surface.CD20 is that first kind of human B-cell spectrum by monoclonal antibody identification is the specific surfaces molecule.It is that the aminoterminal of this phosphorprotein and carboxyl terminal all are positioned at cell without glycosylated hydrophobicity 35kDa B-cell transmembrane phosphorprotein.People such as Einfeld, EMBO J.1988,7:711-17.CD20 is by all normal mature B-cell expressings, but not expressed by precursor B-cell or plasmocyte.The native ligand of CD20 is not also identified, and the function of CD20 in the B-cytobiology is still not exclusively understood.

Another kind of B-cell spectrum is that the specific cell surface molecular is CD37.CD37 is through serious glycosylated 40-52kDa albumen, and it belongs to the tetratransmembrane albumen family of cell-surface antigens.Form two extracellular loop and make its aminoterminal and carboxyl terminal be exposed to tenuigenin across cytolemma for its four times.CD37 is highly expressed on the B-cell that produces normal antibody (sIg+), but on preceding B-cell or plasmocyte without expression.The expression of CD37 on tranquillization T cell and activating T cell, monocyte and granulocyte is lower, and detects less than the expression of CD37 on NK cell, thrombocyte or red blood corpuscle.Referring to people such as Belov, Cancer Res., 61 (11): 4483-4489 (2001); People such as Schwartz-Albiez, J.Immunol., 140 (3): 905-914 (1988); Reach people such as Link, J.Immunol., 137 (9): 3013-3018 (1988).Remove normal B-extracellular, (people such as Moore, 1987 are expressed and all be positive to nearly all malignant tumour of B-origin of cell (comprising CLL, NHL and hairy cell leukemia) for CD37; Merson and Brochier 1988; People such as Faure, 1990).CD37 participates in the adjusting of B-cell function, has the serum IgG 1 of low levels and finds that it weakens virus antigen and the antigenic humoral response of model because find the mouse that lacks CD37.It seems to serve as the collaborative stimulation molecule of non-classical type or via directly influencing antigen presentation with II class MHC molecule forming composite.Referring to people such as Knobeloch, Mol.Cell.Biol., 20 (15): 5363-5369 (2000).

Study and medicament research and development based on following notion: B-cell spectrum be specific cell surface molecular (such as CD37 and CD20) but targeting antibodies itself, described antibody can be bonded to have CD37 and CD20 in its surface, cause the B-cell of Cancerous disease and autoimmune disorders and mediates the destruction of described B-cell.Be provided at the what is called that is bonded to CD37 or CD20 " immunotherapy " antibody (or based on prepared antibody) for preparing among the non-human animal to exhaust the B-cell that causes Cancerous disease or autoimmune disorders to the patient.

Monoclonal antibody technique and genetic engineering method help researching and developing the immunoglobulin molecules that is used to diagnose and treat human diseases.The structural domain structure of immunoglobulin (Ig) is with the part that conforms to of engineering design, and antigen binding domains and the structural domain of giving effector function can exchange between immunoglobulins and subclass.Immunoglobulin structure and function are to summarize the Antibodies:A Laboratory Manual that compiles in people such as for example Harlow, the 14th chapter, and Cold Spring HarborLaborato ry is among the Cold Spring Harbor (1988).Extensive introduction and details about all aspects of recombinant antibodies technology are found in textbook " Recombinant Antibodies (recombinant antibodies) " (John Wiley ﹠amp; Sons, NY, 1999) in.The comprehensive compilation of antibody engineering Design Laboratory scheme is found in R.Kontermann and S.D ü bel (volume) in detail, " The AntibodyEngineering Lab Manual (antibody engineering Design Laboratory handbook) " (Springer Verlag, Heidelberg/New York, 2000) in.

Immunoglobulin molecules (being abbreviated as Ig) is a kind of polyprotein, and it is usually by two identical light chain polypeptides and two identical heavy chain polypeptide (H ₂L ₂) form, described polypeptide connects into the giant molecule mixture by interchain disulfide bond (that is, the covalent linkage between the thiohydroxy of adjacent cysteine residues).Form definition five class human immunoglobulins based on heavy chain, and called after IgG, IgM, IgA, IgE and IgD.IgG class and IgA antibody-like further are divided into subclass, that is are respectively IgG1, IgG2, IgG3 and IgG4, and IgA1 and IgA2.Intrachain disulfide bond connects the different zones in the same polypeptide chain, causes forming ring, and described ring constitutes immunoglobulin domains with adjacent amino acid.Each light chain and each heavy chain have single variable region in amino-terminal part office, and the single variable region of different antibodies shows sizable difference on amino acid is formed.Variable region of light chain V _LHave single antigen binding domains and with variable region of heavy chain V _H(also containing single antigen binding domains) is in conjunction with the antigen binding site Fv with the formation immunoglobulin (Ig).

Except that the variable region, the full length antibody chain has the constant region that contains one or more structural domains separately.Light chain has the constant region that contains the single structure territory.Therefore, light chain has a variable domains and a constant domain.Heavy chain has the constant region that contains several structural domains.Heavy chain in IgG, IgA and the IgD antibody has three and is appointed as C _H1, C _H2And C _H3Structural domain; Heavy chain in IgM and the IgE antibody has four structural domain: C _H1, C _H2, C _H3And C _H4Therefore, heavy chain has a variable domains and three or four constant domain.In all known species, it should be noted that the constant formation of described structural domain, the constant region that wherein contains one or more structural domain is located on or near the light chain of immunoglobulin molecules and the C-end of heavy chain, and variable domains is towards the terminal location of the N-of light chain and heavy chain.Immunoglobulin structure and function are to summarize the Antibodies:A Laboratory Manual (antibody of compiling in people such as for example Harlow: laboratory manual), the 14th chapter, Cold Spring Harbor Laboratory is among the Cold Spring Harbor (1988).

The heavy chain of immunoglobulin (Ig) also can be divided into three functional zone: the Fd district (comprises V _HAnd C _H1The fragment in (that is two N-end structure territories of heavy chain)), hinge area and Fc district (" FC " district).Fc contain in the district with cell on immunoglobulin receptor interact and with the initiator elements interactive domains of complement cascade.Therefore, it is generally acknowledged that Fc district or fragment be responsible for the effector function of immunoglobulin (Ig), such as ADCC (cytotoxicity of antibody dependent cellular mediation), CDC (CDC) and complement-fixation reaction, be bonded to the Fc acceptor, with respect to lacking F _CThe polypeptide in district and transformation period in the body of Yan Gengchang, albumin A in conjunction with and perhaps in addition placenta shift.People such as Capon, Nature, 337:525-531, (1989).In addition, the polypeptide that contains the Fc district is allowed the dimerization/multimerization of polypeptide.Described term also can be used for the similar district of other immunoglobulin (Ig)s.

Although everyone immunoglobulin like protein homotype all contains the common identifiable structures, each homotype shows the effector function of different types.IgG (as non exhaustive property example) neutralizes a toxin and virus, conditioning, conjugated complement (CDC) and participation ADCC.On the contrary, among the IgM and blood-borne pathogens and participate in opsonization.IgA is secreted when combining with its secretory piece and is provided main defence at infected by microbes via mucous membrane; It also neutralizes a toxin and supports opsonization.IgE mediates Inflammatory response, relates generally in establishing the raising of other required cells of complete reaction.Known IgD is used to provide immunoloregulation function, the activation of control B cell.The described difference that can be present between the human homotype that is characterized as of homotype effector function provides non-comprehensive explanation.

The hinge area that is present in IgG, IgA, IgD and the IgE antibody-like serves as flexible spacer, makes the Fab part can move freely in the space.In immunoglobulins and subclass, the hinge arrangement territory is compared structurally different with constant region, and sequence and length all have nothing in common with each other.For example, the length of hinge area and flexibility have nothing in common with each other in the IgG subclass.The hinge area of IgG1 comprises amino acid 216-231, and because it can free deflection, thus the Fab fragment can around its symmetry axis rotation and can be between with two heavy chains first key of disulfide linkage be to move in the spheroid at center.IgG2 has the hinge shorter than IgG1, and the hinge of IgG2 has 12 amino-acid residues and four disulfide linkage.The hinge area of IgG2 lacks glycine residue, and is shorter relatively, and contains the rigidity polyproline duplex by disulfide linkage is firm between extra heavy chain.The flexibility of described characteristic limitations IgG2 molecule.The difference of IgG3 and other subclasses is the prolongation hinge area (four double-lengths of about IgG1 hinge) of its uniqueness, and this prolongation hinge area contains 62 amino acid (comprising 21 proline(Pro) and 11 halfcystines), forms inflexibility polyproline duplex.In IgG3, the Fab fragment is far away relatively apart from the Fc fragment, makes this molecule have bigger flexibility.Elongated hinges among the IgG3 also causes other subclasses of its molecular weight ratio higher.The hinge area of IgG4 is shorter and its flexible occuping in the middle of IgG1 flexibility and the IgG2 flexibility than the hinge area of IgG1.According to reports, the flexibility of hinge area is successively decreased according to following order: IgG3〉IgG1〉IgG4〉IgG2.These four kinds of IgG subclasses also differ from one another with regard to its effector function.This difference is relevant with textural difference (difference that comprises interaction, Fab fragment and constant Fc fragment aspect between the variable region).

According to crystallography research, immunoglobulin hinge region can further be further divided into three districts on function: upper hinge district, core area reach hinge area down.People such as Shin, 1992 ImmunologicalReviews 130:87.The upper hinge district comprises C _H1The amino acid of carboxyl terminal first residue that restriction is moved to the hinge (being generally first cysteine residues that between two heavy chains, forms interchain disulfide bond).The length in upper hinge district is flexible relevant with the section of antibody.The core hinge area contains disulfide linkage in the heavy chain, and hinge area connects C down _H2The N-terminal in territory and comprise C _H2In residue.Ditto.The core hinge area of IgG 1 contains sequence C ys-Pro-Pro-Cys, and therefore this sequence gives flexibility in that the fashionable formation cyclic octapeptide of dimerization causes this cyclic octapeptide can serve as pivot by forming disulfide linkage.Hinge area also can contain one or more glycosylation sites, and it comprises a plurality of sites that are used to connect carbohydrate with different types of structure.For example, IgA1 contains five glycosylation sites at 17 amino acid sections of hinge area, thereby makes the hinge area polypeptide have tolerance to ereptase, is considered to the advantageous feature of immunoglobulin,exocrine.

The structure of immunoglobulin hinge region polypeptide sequence and flexible topographical variations of being allowed also can influence the effector function of the Fc part of antibody.The three classes general effector function relevant with the Fc district comprises the activation of (1) classical complement cascade; (2) interact with the effector cell; And the compartmentation of (3) immunoglobulin (Ig).Different IgG subclasses differ from one another with regard to the relative effectivenes of the step of its conjugated complement or complement activation cascade and amplification complement cascade.Referring to for example Kirschfink, 2001Immunol.Rev.180:177; People such as Chakraborti, 2000 Cell Signal 12:607; People such as Kohl, 1999 Mol.Immunol.36:893; People such as Marsh, 1999 Curr.Opin.Nephrol.Hypertens.8:557; People such as Speth, 1999 Wien Klin.Wochenschr.111:378.

Be present in camellid (camel, dromedary camel and yamma; People such as Hamers-Casterman, 1993 Nature 363:446; People such as Nguyen, 1998 J.Mol.Biol 275:413), nurse shark (people such as Roux, 1998 Proc.Nat.Acad.Sci.USA 95:11804) and ratfish (people such as Nguyen, 2002 Immunogenetics 54 (1): the H that has conventional antibody in some homotype of the immunoglobulin (Ig) 39-47) ₂L ₂The exception of structure.Obviously, described antibody only uses variable region of heavy chain can form antigen binding domain, that is described functional antibodies is the homodimer of heavy chain (being called " heavy chain antibody " or " HCAb ") only.Though antibody technique has advantage in medical diagnosis on disease and treatment, research and development whole antibody technology still exist in as diagnosis and/or treatment reagent some unfavorable aspect.Whole antibody is the large protein structure, for example contains the isostructural special-shaped tetramer structure of IgG of two light chains and two heavy chains.Described macromole is subjected to sterically hindered in some applications.For example, in the treatment of noumenal tumour, whole antibody is not easy to infiltrate inside tumor.In addition, large-sized relatively whole antibody can provide the vivo medicine-feeding that excites to guarantee described molecule not bring out immune response.In addition, the generation of active antibody molecule is usually directed to cultivate the recombined eukaryotic cell that suitable translation post-treatment can be provided nascent antibody molecule, and described cell is difficult to cultivate and be difficult to use the commerce that active antibody is provided to use the mode of output to bring out.

Recently, the less immunoglobulin molecules of construction to overcome and the relevant problem of panimmunity sphaeroprotein method.The variable antibody fragment of strand (scFv) comprise the heavy chain of antibody variable domains that is connected with the light chain of antibody variable domains via small peptide (people such as Huston, Proc.Natl.Acad.Sci.USA, 1988,85:5879-83).Because the size of scFv molecule is little, so it is compared demonstration with the panimmunity sphaeroprotein and more effectively infiltrates tissue.Antitumor scFv compare with corresponding chimeric antibody show the infiltration of tumour faster and the more uniform distribution in tumor mass (people such as Yokota, Cancer Res.1992,52:3402-08).

Bring advantage though the scFv molecule is a serum treatment, still there are some shortcomings in this methods of treatment.ScFv can remove from the recycle system fast, thereby can reduce Normocellular toxic action, but this subliminal dose of removing obstruction fast is delivered to target tissue.Owing to express and separate the scFv difficulty and output is caused disadvantageous effect, therefore making the scFv that capacity is used to give the patient has become challenge.During expressing, scFv molecule deficient in stability and often because match the variable region of differing molecular assembles.In addition, in mammalian expression system, the yielding poorly of scFv molecule, this restriction is effectively made treatment with the potential of scFv molecule (people such as Davis, J Biol.Chem.1990,265:10410-18; People such as Traunecker, EMBO J 1991,10:3655-59).Probed into the strategy of improved production, comprise in the variable region, add glycosylation site (Jost, No. the 5th, 888,773, C.R. United States Patent (USP), people such as Jost, J.Biol.Chem.1994,69:26267-73).

Another shortcoming of using the scFv treatment is for lacking effector function.Do not have cytolysis function (such as the cytotoxicity (ADCC) and the CDC (CDC) of antibody dependent cellular mediation), invalid for the treatment disease with the constant region bonded scFv of immunoglobulin (Ig).Though before 12 years, began to research and develop the scFv technology, still do not had the scFv product at present and get permission to be used for the treatment of.

Perhaps, proposed to utilize specific antigens scFv and another molecule such as toxin to be merged so that toxin is delivered to target tissue in conjunction with active and undersized scFv.People such as Chaudary, Nature 1989,339:394; People such as Batra, Mol.Cell.Biol.1991,11:2200.Therefore, can be with the combining or merge of toxin and scFv as the alternative strategy that effective antigen-specific molecule is provided, but be subjected to the restriction of excessive and/or non-special toxicity (due to the toxin moiety in the described preparation) with described combination or mosaic administration.Toxic action can comprise rational rising of the excusing from death of liver enzyme and vascular leakage syndromes, and other undesirable actions.In addition, immunotoxin itself has high immunogenicity after giving the host, and limits the likely effectiveness of repetitive therapy treatment individuality at host's antibody that immunotoxin produced.

Non-surgery cancer therapy (such as external irradiation and chemotherapy) since described treatment to cancer cells show specificity lack, because of the toxic action effect to healthy tissues and cell limited.For overcoming this limitation, researched and developed the targeted therapy method with strengthen treatment to needs its cell and the specificity of tissue.This targeted approach is used for the example of purposes in the body for giving the antibodies body, and wherein antibody is designed to can discern the mark relevant with the cell or tissue that needs treatment specifically and this antibody is combined with therapeutical agent (such as toxin (under the situation of cancer therapy)).Antibody is capable of circulation to susceptibility and bad health compartment, such as marrow as the general medicament.In acute radiation injury, the destruction of lymph and hematopoiesis compartment is development septicemia and dead subsequently principal element.In addition, antibody is spherical large protein, and its tissue to the needs treatment can show bad infiltration.

Human patients and the non-human individuality of suffering from lysis in multiple latter stage need organ transplantation usually.Yet organ transplantation must be tackled receptor's improper immune response and suppress the receptor by the cytotoxic agent with the lymph part that influences hemopoietic system and other parts the cellular immunity of external organ is reacted the immunological rejection of avoiding implanting organ.The transplant acceptability is subjected to the restriction of receptor to the tolerance of described cytotoxicity chemical (wherein multiple and anticancer (anti proliferative) medicament is similar).Equally, when using cytotoxicity biocide (especially antiviral) maybe when using cytotoxic drug treatment autoimmune disorders, for example in treatment systemic lupus erythematosus disease, seriously be restricted to therapeutical agent to the marrow of health and the toxic action of hematopoietic cell.

Targeted therapies (such as the targeting antibodies combined therapy) use the site that need to be positioned effect by way of the therapeutical agent that is designed to make as far as possible maximum, and whether described therapy successfully embodies by the high relatively signal of therapeutical agent and the ratio of background values.The example of targeting antibodies comprises the diagnostic reagent combination or the therapeutical agent combination of antibody or antibody fragment, cell specific polypeptide or tissue specificity peptide and hormone and other receptors bind molecules.For example, used antibody to detect and treat multiple pathologic state or focus at the different determiners of being correlated with pathologic cell and normal cell and being correlated with pathogenic microbes.In described method, targeting antibodies directly is coupled to suitable detection agent or the therapeutical agent described in following document, people such as Hansen for example, United States Patent (USP) the 3rd, 927, No. 193 and Goldenberg, United States Patent (USP) the 4th, 331, No. 647, the 4th, 348, No. 376, the 4th, 361, No. 544, the 4th, 468, No. 457, the 4th, 444, No. 744, the 4th, 460, No. 459, the 4th, 460, No. 561, the 4th, 624, No. 846 and the 4th, 818, No. 709.

Directly a problem that is met with in the targeted approach (that is wherein diagnostic reagent or therapeutical agent (" promoting agent ") directly are coupled to the method for targeting moiety) is, in fact the relative smaller portions of conjugate are bonded to the target site, and most of conjugate keeps the function of the circulation and the target conjugate that detracts by every means.For guaranteeing the maximum location of promoting agent, give excessive target conjugate usually, thereby guarantee that some conjugate keeps the background content of unbound state and promotion promoting agent.Diagnostic conjugate (for example the radio-immuno-image conjugate of its target of debond or nuclear magnetic resonance conjugate) can keep circulation, thereby increases the resolution of background and reduction diagnostic techniques.Under the situation of therapeutic conjugate (toxin as promoting agent (for example radio isotope, medicine or toxic chemical) that circulating target part (such as antibody) was connected when this therapeutic conjugate had with length), the circulation conjugate can produce the unacceptable toxicity of host, poisons or systemic side effects such as marrow.

United States Patent (USP) the 4th, 782, No. 840 a kind of methods that reduce the effect of high radiation background amount at intra-operative of announcement.This method comprises using organizes the specific antibody of tool that the patient is injected to superfluous natural disposition, wherein through transformation period that the antibody of radio isotope (such as iodine-125) mark has suitable length.After radiolabeled antibody, will perform the operation postpones at least 7 to 10 days, preferred 14 to 21 days in injection, so that any unconjugated radiolabelled antibody can be removed until low background amount.

United States Patent (USP) the 4th, 932 is disclosed in the method that operation is used to reduce or revise non-specific radiation background in carrying out between detection period for No. 412.Described method comprises to accepting patient through radiolabeled primary antibody and gives contrast medium, subtracts the shadow agent or in conjunction with the secondary antibody of primary antibody.

Except that producing above-mentioned antibody, immunity system comprises multiple cell type with Johnson ﹠ Johnson's thing effect.During hematopoiesis, the differentiation of stem cells that comes from marrow becomes immune mature cell (" B " cell) or be divided into the sophisticated cell precursors in thymus gland of moving out (" T " cell) in marrow.

The B cell is formed most important for immunoreactive body fluid.The B cell is suitably presented activation and is become the antibody-secreting plasmocyte through antigenic; Antigen presentation also causes the clonal expansion of activating B cell.The B cell mainly is responsible for immunoreactive body fluid and is formed.Plasmocyte shows about 10 in its surface usually ⁵Individual antibody molecule (IgD and IgM).

The T lymphocyte can be divided into two classes.Cytotoxic T cell, Tc lymphocyte or CTL (CD8+T cell) kill the cell that carries the external surface antigen relevant with I class MHC and can kill the have cytozoon cell of (bacterium or virus), as long as institute's cells infected shows microbial antigen in its surface.Tc cell kill tumor cell, and explanation is to implanting the rejection of cell.Antigen on the Tc cell recognition target cell-I class MHC mixture is in contact with it, and the particle inclusion directly is released in the target cytolemma, thus dissolved cell.

The second class T cell is helper cell or Th lymphocyte (CD4+T cell), and it is created in the lymphokine that the B cell maturation is conduct " assisting " factor in the antibody-secreting plasmocyte.The Th cell also produces the active lymphokine of lymphocytic differentiation of some stimulatory effect T and scavenger cell.Antigen and (passing through IL-1) relevant with II class MHC on the Th1 cell recognition scavenger cell are activated to produce lymphokine, comprise the IFN-γ of activated macrophage and NK cell.The various aspects of described cell-mediated immune response (comprising the delayed-type hypersensitivity reaction).Th2 cell recognition antigen presenting cell or APC (for example transport property scavenger cell and dendritic cell) go up the antigen relevant with II class MHC and follow and produce be situated between white plain and stimulation specific b-cell and T-cell proliferation and active other materials.

Except that serving as the APC that causes T cell interaction, growth and propagation, scavenger cell also is involved in the expression of cell-mediated immunity, because its IFN-γ activation by being produced in the cell mediated immune response.Activated macrophage has the soluble substance that enhanced phagocytic cell potential and release cause inflammation and destroys various bacteria and other cells.Natural killer cell is the solubilized cell that has neoantigen (no matter whether it is the MHC type), and even solubilized some do not have the cytotoxic cell of the proteic cell of MHC.Natural killer T cell or NK cell define according to the ability that it kills the cell (for example tumour cell) that shows exotic antigen, no matter whether be the MHC type and no matter whether in advance sensitization (exposure) in antigen.The NK cell can be activated by IL-2 and IFN-γ, and with the mode dissolved cell identical with cytotoxic T lymphocyte.Some NK cell has the acceptor (for example CD16 or FC γ RIII) of the Fc structural domain of IgG antibody and therefore can be bonded to the Fc part of the IgG on the target cell surface and discharge the molten cell component that kills the target cell via the cytotoxicity of antibody dependent cellular mediation.

Another group cell is granulocyte or polymorphonuclear leukocyte (PMN).Neutrophil (PMN one type) killing bacteria invasion and attack person and engulf residue.Eosinophil is the another kind of type of PMN and contains the toxic particle of showed cell when discharging for another kind of cell (such as external cell).Basophilic leukocyte (the third type of PMN) is the important medium grain of the rational reaction of Johnson ﹠ Johnson (for example inflammation), and it brings into play its effect by discharging various biological active compound (such as organizing ammonia, thrombotonin, prostaglandin(PG) and leukotriene).The common ground of described all cells type is usually by killing and randomly remove the ability that harmful composition (such as external cell) is brought into play physiological action in organism.

Although multiple mammalian cell (comprising immune cell) can directly be brought into play physiological action (for example, being the cell killing effect of representative with Tc, NK, some PMN, scavenger cell and similar cell thereof), other cells are facilitated physiological action indirectly.For example, antigen is initially presented to immune naive T cell and need be ordered the MHC of cell-cells contacting to present.In addition, need usually between activating T cell and the antigen-specific b cells contact to obtain specific immunogenic response.In the immune response the third form of common cell-cells contacting be activating B cell with the folliculus dendritic cell between contact.Described cell-cells contacting requires to make separately the biologically active agent target to specify target to become complicated.

CDC (CDC) is considered to remove the important mechanisms such as the particular target cell of tumour cell.CDC activates a series of incidents of forming to each other by a large amount of enzymes with cascade system.Complement has vital role in removing antigen, this is realized by its four kinds of major functions: (1) local vessel expansion; (2) attract immunocyte, especially scavenger cell (chemotaxis); (3) the external organism of mark is used for phagolysis (opsonization); And (4) destroy the invasion and attack organism by membrane attack complex (MAC attack).Main molecules is a C3 albumen.It is cracked into two segmental enzymes for the component by classical pathway or alternative route.Classical pathway brings out by antibody (especially IgG and IgM), and alternative route stimulates by bacterial product (as lipopolysaccharides (LPS)) non-specificly.In brief, C3 cracked product comprises small peptide C 3a, and it has chemotaxis and cause local vessel expansion by impelling the C5a fragment to discharge in C5 for engulfing the type immunocyte.Another part C3b of C3 coat the lip-deep antigen of external organism and to organism performance opsonization with its destruction.C3b also with other components reaction of complement system to form the MAC that forms by C5b, C6, C7, C8 and C9.

In the human treatment, exist and the relevant problem of use antibody, this is to any antigenic reaction because of immunity system, even the simplest person, also be " polyclone " reaction, that is system be manufactured on its land with and the effect district in all have wide range of structures antibody.

Used two kinds of methods to attempt to reduce the problem of immunogenic antibodies.First method is the preparation chimeric antibody, wherein makes the antigen-binding portion thereof (variable region) of mouse monoclonal antibody and effect part (constant region) fusion of human antibodies.In the second approach, complementary determining region (CDR) is transplanted or the technology of " humanization " changes antibody by being called.This method through further improvement to comprise the variation that is called following each person: " reconstruct " (people such as Verhoeyen, 1988 Science239:1534-1536; People such as Riechmann, 1988 Nature 332:323-337; People such as Tempest, Bio/Technol 1991 9:266-271), " super chimeric " (people such as Queen, 1989 Proc NatlAcad Sci USA 86:10029-10033; People such as Co, 1991 Proc Natl Acad Sci USA88:2869-2873; People such as Co, 1992 J Immunol 148:1149-1154), and " facial ornament " (people such as Mark, in: Metcalf BW, Dalton BJ compiles Cellular adhesion:moleculardefinition to therapeutic potential.New York:Plenum Press, among the 1994:291-312).

From 1986, rise when promptly announcing after the monoclonal antibody 11 years, have every year on average less than a kind of therapeutic antibodies and introduce market.Between decade, there are five kinds of mouse monoclonal antibodies to introduce in the human medicine, comprise " Orthoclone OKT 3 (muromonab-CD3) " (OrthoClone of the acute rejection that is used for the organ transplantation body from 1986 to nineteen ninety-five

); " Edrecolomab (edrecolomab) " that is used for colorectal carcinoma

" Odulimomab (odulimomab) " that is used for the transplant rejection

And be used for " ibritumomab tiuxetan (ibritumomab) " of non-Hodgkin lymphomas (non-Hodgkin ' s lymphoma)

Yiuxetan).In addition, commercially available mono-clonal Fab " ReoPro (abciximab) "

Be used to prevent coronary artery to block again.Three kinds of chimeric mAbs have also been released: " Rituximab (rituximab) " that is used for the treatment of B cell lymphoma " basiliximab (basiliximab) " that is used for the transplant rejection

And be used for the treatment of " the sharp former times monoclonal antibody (infliximab) of English " of rheumatoid arthritis and Crohn disease (Crohn ' s disease)

In addition, " ReoPro "

(the 47.6kD Fab fragment of the mosaic type mankind-mouse monoclonal antibody) is commercially available to be used to prevent experience the patient's of percutaneous coronary interventional procedure heart ischemia complication as the adjuvant of percutaneous coronary interventional procedure.At last, seven kinds of " humanization " monoclonal antibodies have been released." Dary pearl monoclonal antibody (Daclizumab) "

It is the acute rejection that is used to prevent to transplant kidney; " palivizumab (palivizumab) "

Be to be used for RSV; " Herceptin (trastuzumab) "

In conjunction with HER-2 (a kind of somatomedin that is present on the breast cancer cell); " lucky trastuzumab (gemtuzumab) "

Be to be used for acute myelogenous leukemia (AML); And " alemtuzumab (alemtuzumab) "

Be to be used for lymphocytic leukemia; " adalimumab (adalimumab) " (

(D2E7)) be to be used for the treatment of rheumatoid arthritis; And " Ao Mazuo monoclonal antibody (omalizumab) " Be to be used for the treatment of persistence asthma.

Therefore, multiple antibody technique receives publicity in the work of research and development and more effective therapeutical agent of sale and negative catalyst.Regrettably, still have problems and destroy the prospect of described various therapies.For example, most of cancer patients's recurrence in about 6 to 12 months usually through rituximab treatment, and be reported in the Rituximab infusion and the reaction of mortality infusion take place in 24 hours.Also report, the acute renal failure that needs to dialyse under the situation that has the mortality result has the seriously mucocutaneous reaction of (fatal sometimes) in rituximab treatment.In addition, intravenous injection needs the Rituximab of high dosage, and this is because this molecule big (about 150kDa) and diffuse into the Lymphoid tissue that has the massive tumor cell and be restricted.

The Herceptin administration can cause taking place ventricle kakergasia, congestive heart failure and serious anaphylaxis (comprising severe allergy), infusion reaction and lung's incident.Dary pearl monoclonal antibody immunosuppressive therapy makes the risk that Lymphoid tissue hyperproliferative disorder and opportunistic infection take place increase.Be reported in and taken place among the patient who accepts lucky trastuzumab because of the death due to the liver failure, because of the death due to the serious hepatotoxicity and because of the death due to the venous occlusion disease (VOD).

Also report is accepted the intravital hepatotoxicity of patient of alemtuzumab.Serious (being fatefulue under some rare cases) pancytopenia/hypoplastic bone marrow, autoimmunity idiopathic thrombocytopenia and autoimmune hemolytic anemia have taken place in patient that accept the alemtuzumab therapy.Alemtuzumab also can cause serious infusion reaction and opportunistic infection.According to reports, in the patient of adalimumab treatment, the deterioration of clinical symptom and/or the radiograph sign of demyelination appear in severe infections and septicemia (comprising mortality severe infections and septicemia), and it is higher than the desired sickness rate of general groups to suffer from lymphadenomatous sickness rate through the patient of adalimumab treatment in clinical trial.The Ao Mazuo monoclonal antibody can bring out malignant tumour and severe allergy according to reports.

Cancer comprises the disease of broad range, involves the individuality of worldwide interior about 1/4th.Malignant cell fast and the propagation of not regulated be the multiple cancer types sign of (comprising malignant hematologic disease).Although the patient who suffers from malignant hematologic disease is the (people such as Multani that is benefited of the progress because of cancer therapy in 20 years in the past, 1998 J.Clin.Oncology 16:3691-3710), and remission is multiplied, but the disease of Most patients still can recur and death.The obstacle of curing for cytotoxic drug comprises for example high toxicity of tumour cell tolerance and chemotherapy, and described obstacle hinders the best administration to many patients.

According to reports, the patient who uses mosaic type CD20 mab treatment to suffer from low potential malignancy or folliculus B cell lymphoma partial reaction or the total overall reaction that can bring out the patient.People such as McLaughlin, 1996 Blood 88:90a (supplementary issue 1, summary); People such as Maloney, 1997 Blood 90:2188-95.Yet as mentioned above, tumor recurrence generally took place in six months to 1 year.Need further improvement serotherapy can effectively treat high-grade malignant lymphoma and other B cell diseases to bring out the more lasting reaction of for example low potential malignancy B cell lymphoma and to make.

Another kind method makes radio isotope target B cell lymphoma for using to the specific monoclonal antibody of CD20 tool.Be increased although treat validity according to reports, the toxicity relevant with the transformation period in the long body of radioactive antibody also strengthens, and needs the patient experience stem cell to save often.People such as Press, 1993 N.Eng.J.Med.329:1219-1224; People such as Kaminski, 1993 N.Eng.J.Med.329:459-65.Before connecting radio isotope, also with proteolytic enzyme with the monoclonal antibody cracking of CD20 to produce F (ab ') ₂Or Fab fragment.According to reports, this can improve the radio isotope combination and infiltrate in the tumour and can shorten the transformation period in the body, thereby reduces the toxicity to healthy tissues.Yet described molecule lacks effector function, comprises complement-fixation reaction and/or ADCC.

Autoimmune disorders comprises autoimmunity thyroid disease (comprising lattice RD (Graves ' disease) and struma lymphomatosa (Hashimoto ' s thyroiditis)).Only, there are 20,000,000 people to suffer from the autoimmune thyropathy of certain form approximately in the U.S..The autoimmune thyropathy is because of produce to stimulate Tiroidina to cause hyperthyroidism (lattice RD) or destroy due to the autoantibody that Tiroidina causes hypothyroidism (struma lymphomatosa).To thyroid stimulation be because of autoantibody in conjunction with and activation thyrotropic hormone (TSH) acceptor due to.To thyroid destruction is because of due to the reaction of autoantibody and other thyroid antigens.The current therapy of lattice RD comprises operation, radioiodine or antithyroid drug therapy.Radioiodine is extensive use of, and this is because antithyroid drug has remarkable side effect and palindromia rate height.Operation is specialized in when suffering from big patient strumous or needing extremely fast the normalizing thyroid function used.Still do not exist target to be responsible for stimulating the therapy of generation of the autoantibody of tsh receptor.The current therapy of struma lymphomatosa is levothyroxine sodium (levothyroxine sodium), and is contemplated to lifelong therapy owing to the possibility of remission is low.The inhibition therapy has proved the thyrocele that can reduce struma lymphomatosa, but the still unknown therapy that reduces the autoantibody generation with the target disease mechanisms.

Rheumatoid arthritis (RA) is for being the chronic disease of feature with the joint inflammation, and it causes swelling, pain and afunction.Estimate at 2,500,000 people in the U.S. and suffer from RA.RA is because of due to the composition of matter that comprises reaction of primary infection or damage, abnormal immune and inherited genetic factors.Although have autoreaction T cell and B cell among the RA, can utilize the high-content antibody (be called the rheumatoid factor) of detection of aggregation in the joint to diagnose RA.The current therapy of RA comprises the multiple medicine that is used for pain management and slows down progression of disease.Do not find to cure the therapy of this disease as yet.Medicine comprises non-steroidal anti-inflammatory drug (NSAIDS) and changes state of an illness resisting rheumatoid disease medicine (DMARDS).NSAIDS is applicable to benign disease, but can't stop the destruction of joint that develops into serious RA and unable.All with significantly side effect is relevant with DMARDS for NSAIDS.Only a kind of new DMARD leflunomide (Leflunomide) is got permission over 10 years.Leflunomide stops the generation autoantibody, reduces inflammation and slows down the RA progress.Yet this medicine also causes serious side effects, comprise nauseating, suffer from diarrhoea, shave one's head, fash and liver injury.

Systemic lupus erythematosus disease (SLE) is an autoimmune disorders, and it is because of due to the blood vessel generation repeatability in the multiple organ that comprises kidney, skin and the joint damage.Estimate that in the U.S. exceeding 500,000 people suffers from SLE.In suffering from the patient of SLE, the improper interaction between T cell and the B cell causes producing the autoantibody of attack cells nuclear.Described antibody comprises anti-double-chain DNA and anti--Sm antibody.Also have autoantibody approximately in half SLE patient's body, and described autoantibody causes vascular lesion and low blood counting in conjunction with phosphatide.Immunocomplex accumulates in SLE patient's kidney, blood vessel and the joint, causes inflammation and tissue injury.Do not find to cure the SLE therapy of this disease as yet.Severity on disease is decided, and can use NSAIDS and DMARDS treatment.The plasma clearance art of utilizing plasma exchange to remove autoantibody can make SLE patient obtain temporary improvement.Usually consistently think that autoantibody causes SLE, the novel therapy that therefore exhausts B cell spectrum and be (when being produced new B cell by precursor immunity system is recovered) is that SLE patient is benefited lastingly hope is provided.

Repairing Gram syndrome (Sjogren ' s syndrome) is the autoimmune disorders of feature for the destruction that produces body of gland with body fluids.Repair Gram syndrome and be one of the most general autoimmune disorder, estimate that in the U.S. nearly 4 million peoples suffer from this disease.Repair among the Gram syndrome patient that half also suffers from connective tissue disease approximately, such as RA, and second half is suffered from primary and repaiies Gram syndrome but do not suffer from other concurrency autoimmune disorderss.The autoantibody that comprises anti-cell antinuclear antibodies, the rheumatoid factor, anti-fodrin and anti-muscarinic receptor is present in usually to be suffered from the syndromic patient's body of the Gram of repairing.Routine treatment comprises reflunomide, and other more effective therapies are also useful.

Immunologic thrombocytopenic purpura (ITP) is to be bonded to thrombocyte and to cause due to it destroys because of autoantibody.Some situation of ITP is because of drug-induced, and other situations are and infection, conceived or relevant such as the autoimmune disease of SLE.Half can classify as the special property sent out origin approximately in all situations.The treatment of ITP is that the severity according to symptom decides.Although inhibitive ability of immunity medicine (comprising that reflunomide or intravenous infusion immunoglobulin (Ig) are to exhaust the T cell) in most of the cases is provided, need not treatment in some cases.The another kind that often causes platelet counts to increase is treated to removing spleen, and this organ destroys the thrombocyte that antibody coats.For the patient of serious situation, use more effective inhibitive ability of immunity medicine, comprise ciclosporin (cyclosporine), endoxan (cyclophosphamide) or azathioprine (azathioprine).By making patient's blood plasma remove second line treatment that autoantibody can be used as the patient who suffers from serious disease by the albumin A tubing string.Need other more effective therapies.

Multiple sclerosis (MS) also is an autoimmune disorders.It is characterized by the inflammation of central nervous system and the destruction of myelin, make that the neurocyte fiber in brain, spinal cord and the health is isolated.Although cause of disease the unknown of MS believes that extensively autoimmunity T cell mainly facilitates factor for this disease incidence mechanism.Yet, have high-load antibody in MS patient's the celiolymph, and the B cell response that some people's prophesy causes antibody to produce has vital role for this disease of mediation.MS patient is not studied B cell depleting therapy as yet, and still do not have MS and cure.Current therapy is a reflunomide, and it can reduce the time length and the severity of attack, but does not influence MS process in time.Novel biotechnology Interferon, rabbit (IFN) therapy of MS is granted recently, but still needs other more effective therapies.

Myasthenia gravis (MG) is for being the chronic autoimmunity neuromuscular deficiency disorder of feature with any muscle group weakness.About 40,000 people suffer from MG in the U.S..MG is bonded to due to the acetylcholine receptor of myoneural junction place through expressing because of autoantibody.Autoantibody reduces or blockage of acetylcholine receptor, stop signal by neurotransmission to muscle.Still the cure method of unknown MG.Common treatment comprises with reflunomide, ciclosporin, endoxan or azathioprine carries out immunosuppression.Usually utilize operation to remove thymus gland and weaken autoimmune reaction.The plasma clearance art that is used for reducing the autoantibody content of blood can be effective to MG, but is transience because of autoantibody continues to produce.The plasma clearance art was generally used before operation for serious muscle valetudinarian, invalid.Novel effectively therapy also is useful.

About five million peoples suffer from psoriasis, and it is characterized by the autoimmune inflammation of skin.In 30% patient, psoriasis also relevant (arthritic psoriasis) with sacroiliitis.Used multiple therapy, comprised steroid, UV-light retinoids, vitamin D-derivatives, ciclosporin and Rheumatrex (methotrexate), but obviously psoriasis also will be benefited from novelty and effective therapy.Scleroderma is the chronic autoimmune disorders of reticular tissue, also is called Sjogren's syndrome.The excess that scleroderma is characterized as collagen protein produces, and causes the skin thickening, and suffers from scleroderma about 300,000 people of the U.S., and scleroderma also will be benefited from novelty and effective therapy.

Apparent by above argumentation, need through improved compositions and method with treatment, improve or prevent multiple disease, illness and state, comprise cancer and autoimmune disorders.

General introduction

The present invention satisfies in the above-mentioned needs in this area at least one by following each person is provided: the albumen that contains at least two specificity binding domainss, wherein these two structural domains are to connect by the constant subprovince that is derived from antibody molecule, and the C-end of this antibody molecule is connected to the connexon that is called scorpion shape connexon herein; And nucleic acids encoding said proteins; And the preparation of described albumen and nucleic acid, diagnostic and therapeutic use.This constant subprovince comprises and is derived from immunoglobulin (Ig) C _H2The structural domain of structural domain and preferred source are from immunoglobulin (Ig) C _H3The structural domain of structural domain is not derived from or corresponding to immunoglobulin (Ig) C but do not contain _H1The structural domain of structural domain or district.Thought before that the constant region that will be derived from antibody placed active site of protein can disturb antibody function, such as effector function, by that analogy, the constant region with antibody places the C-terminal of antibody chain also can disturb antibody function usually.In addition, be to be different from the formation that naturally occurring immunoglobulin (Ig) constitutes in the terminal scorpion shape connexon (it can be immunoglobulin (Ig) hinge sample peptide) of arranging of the C-of constant subprovince.Yet, place polypeptide chain or protein chain inside to make the albumen of demonstration effect function and multivalence (monospecific or polyspecific) binding ability not be subjected to sterically hindered obstruction relatively constant subprovince (wherein scorpion shape connexon connects the C-end of constant region) according to the present invention.Apparent as those skilled in the art institute after considering this disclosure, described albumen is designed to module and can followingly be fabricated: by selecting any structure territory as binding domains 1 or binding domains 2 (or as any other binding domains that is present in the specific protein of the present invention) in multiple binding domains; By selecting to have the constant subprovince of effector function; And by selecting the scorpion shape connexon (for example II type C-agglutinin receptor stem district peptide) of hinge sample or non-hinge sample, wherein albumen shows the general formation of the constant subprovince of N-binding domains 1--scorpion shape connexon-binding domains 2-C.Those skilled in the art will further understand the albumen with this structure and will there be multiple application in this proteic nucleic acid of encoding, comprise medical science and veterinary applications.

An aspect of of the present present invention is about having the multivalence single strand binding protein of effector function or scorpion shape connexon (described term is used interchangeably), this conjugated protein first binding domains that is derived from immunoglobulin (Ig) (for example antibody) or immunoglobulin-like molecule that comprises; The constant subprovince of effector function is provided, and this constant subprovince is positioned at the C-end of first binding domains; Be positioned at the scorpion shape connexon of the C-end of constant subprovince; And being derived from second binding domains of immunoglobulin (Ig) (such as antibody) or immunoglobulin-like molecule, this second binding domains is positioned at the C-end of constant subprovince; Thereby constant subprovince is positioned between first binding domains and second binding domains.The polyspecific of single strand binding protein (for example dual specific) is that they can be in conjunction with two or more different targets, or it can have monospecific, has two binding sites at same target.In addition, these proteic all structural domains all are present in the strand, but this albumen can form homopolymer, for example form homopolymer by forming interchain disulfide bond.In certain embodiments, first binding domains and second binding domains are the variable regions that is derived from the light immunoglobulin chain and the heavy immunoglobulin chain of identical or different immunoglobulin (Ig) (for example antibody).Immunoglobulin (Ig) can derive from any vertebrates, such as Mammals, comprises the mankind, and can be mosaic type, humanization fragment, varient or the derivative of naturally occurring immunoglobulin (Ig).

The present invention contain wherein first and second binding domains be derived from identical or different immunoglobulin (Ig) (for example antibody) and wherein first and second binding domains discern the albumen of identical or different molecular target (for example cell surface marker, such as embrane-associated protein).In addition, first and second binding domains can be discerned identical or different epi-position.First and second molecular target can be relevant with first and second target cell, virus, carrier and/or object.In the preferred embodiment aspect this according to the present invention, each is derived from human immunoglobulin naturally first binding domains, second binding domains and constant subprovince, such as IgG antibody.In other embodiments, multivalent binding proteins with effector function has first binding domains that can discern at least a acellular molecular target (for example with the incoherent albumen of cell, such as deposition albumen or soluble proteins) and in second binding domains at least one.For example, acellular molecular target comprise with cell from incoherent albumen, institute's administered compound for example is such as albumen; And through secretion, cracking, be present in the exosome or discharge or isolating albumen from cell.

The target molecules of being discerned by first and second binding domains can be present on identical or different prokaryotic cell prokaryocyte, eukaryotic cell, virus (comprising bacillary phage), organic or inorganic target molecules carrier and the foreign body or be relevant with described object.In addition, this target molecules can be positioned at (for example two different eukaryotic cells, prokaryotic cell prokaryocyte, virus or carriers) on cell, virus, carrier or the object that type is identical, entity is different, or this target molecules can be positioned at (for example eukaryotic cell and virus) on dissimilar cells, virus, carrier or the object.The target cell is for those cells relevant with the target molecules of being discerned by binding domains and comprise endogenous cell or autogenous cell and foreign cell or external cell (infective micro-organisms cell, transplanting mammalian cell (comprising the blood transfusion cell)).The present invention includes the target that is used to be present in first and/or second binding domains on the target cell surface relevant with Mammals (such as the mankind's) disease, illness or state.Exemplary target cell comprises cancer cells, cell and the infectious cell (for example infectious bacteria) relevant with autoimmune disorders or illness.The cell of also containing infectious organism (such as the Mammals parasite) is as the target cell.In certain embodiments, albumen of the present invention is that the multivalence (for example polyspecific) with effector function is conjugated protein, and wherein at least one identification in first binding domains and second binding domains is selected from the target of the group of being made up of following each person: tumour antigen, the B-cell target, TNF receptor superfamily member, the Hedgehog family member, receptor tyrosine kinase, the proteoglycan associated molecule, the TGF-beta superfamily member, the Wnt associated molecule, receptors ligand, the T-cell target, the dendritic cell target, the NK cell target, monocyte/macrophage target and vasculogenesis target.

In above-mentioned proteic some embodiment, tumour antigen is to be selected from the group of being made up of following each person: squamous cell carcinoma antigen 1 (SCCA-1), (albumen T4-A), squamous cell carcinoma antigen 2 (SCCA-2), ovarian cancer antigen CA125 (1A1-3B) (KIAA0049), MUC-1 (tumour be correlated with Saliva Orthana), (cancer be correlated with Saliva Orthana), (polymorphism epithelium Saliva Orthana), (PEM), (PEMT), (epithelium Saliva Orthana), (tumour be correlated with EMA), (EMA), (H23AG), (the reactive uromucoid of peanut), (PUM), (breast cancer related antigen DF3), CTCL tumour antigen se1-1, CTCL tumour antigen se14-3, CTCL tumour antigen se20-4, CTCL tumour antigen se20-9, CTCL tumour antigen se33-1, CTCL tumour antigen se37-2, CTCL tumour antigen se57-1, CTCL tumour antigen se89-1, the prostate specific membrane antigen, 5T4 cancer embryo trophoderm glycoprotein, the Orf73 Kaposi sarcoma simplexvirus (Orf73 Kaposi ' ssarcoma-associated herpesvirus) of being correlated with, MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 antigen (MAGE-XP antigen) (DAM10), MAGE-B2 antigen (DAM6), MAGE-2 antigen, MAGE-4a antigen, MAGE-4b antigen, colon cancer antigen NY-CO-45, LuCA NY-LU-12 varient A, cancer relevant surfaces antigen, gland cancer antigen A RT1, the relevant brain of secondary tumour-Testiculo-cancer antigen (neural cancer antigen MA2; Secondary tumour neurone antigen), neural tumor veutro antigen 2 (NOVA2), hepatocellular carcinoma antigen gene 520, tumor associated antigen CO-029, tumor associated antigen MAGE-X2, synovia sarcoma, X breakpoint 2, squamous cell carcinoma antigen, the colon cancer antigen 1 of serology definition, the breast cancer antigen NY-BR-15 of serology definition, breast cancer antigen NY-BR-16, the Chromogranin A of serology definition by the T cell recognition; Parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA 72-4, CA 195 and L6.

The embodiment of aforesaid method comprises the B cell target that is selected from the group of being made up of following each person: CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138 and CDw150.

In other embodiments of aforesaid method, TNF receptor superfamily member is selected from the group of being made up of following each person: 4-1BB/TNFRSF9, NGFR/TNFRSF16, BAFFR/TNFRSF13C, protect ossein/TNFRSF11B, BCMA/TNFRSF17, OX40/TNFRSF4, CD27/TNFRSF7, RANK/TNFRSF11A, CD30/TNFRSF8, RELT/TNFRSF19L, CD40/TNFRSF5, TACI/TNFRSF13B, DcR3/TNFRSF6B, TNF RI/TNFRSF1A, DcTRAILR1/TNFRSF23, TNF RII/TNFRSF1B, DcTRAIL R2/TNFRSF22, TRAILR1/TNFRSF10A, DR3/TNFRSF25, TRAILR2/TNFRSF10B, DR6/TNFRSF21, TRAILR3/TNFRSF10C, EDAR, TRAILR4/TNFRSF10D, Fas/TNFRSF6, TROY/TNFRSF19, GITR/TNFRSF18, TWEAK R/TNFRSF12, HVEM/TNFRSF14, XEDAR, lymphotoxin-beta R/TNFRSF3,4-1BB part/TNFSF9, lymphotoxin, APRIL/TNFSF13, lymphotoxin-beta/TNFSF3, BAFF/TNFSF13C, OX40 part/TNFSF4, CD27 part/TNFSF7, TL1A/TNFSF15, CD30 part/TNFSF8, TNF-α/TNFSF1A, CD40 part/TNFSF5, TNF-β/TNFSF1B, EDA-A2, TRAIL/TNFSF10, Fas part/TNFSF6, TRANCE/TNFSF11, GITR part/TNFSF18, TWEAK/TNFSF12 and LIGHT/TNFSF14.

Aforesaid method also comprises that wherein the Hedgehog family member is the embodiment that is selected from the group of being made up of Patched and Smoothened.In other embodiments, the proteoglycan associated molecule is to be selected from the group of being made up of proteoglycan and conditioning agent thereof.

Other embodiments of this method are to be the method that is selected from the group of being made up of following each person: Axl about receptor tyrosine kinase wherein, FGF R4, C1q R1/CD93, FGF R5, DDR1, Flt-3, DDR2, HGF R, Dtk, IGF-I R, EGF R, IGF-II R, Eph, INSRR, EphA1, Regular Insulin R/CD220, EphA2, M-CSF R, EphA3, Mer, EphA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R α, EphA7, PDGFR β, EphA8, Ret, EphB1, ROR1, EphB2, ROR2, EphB3, SCF R/c-kit, EphB4, Tie-1, EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF R1, VEGF R1/Flt-1, FGF R2, VEGF R2/Flk-1, FGF R3 and VEGF R3/Flt-4.

In other embodiments of this method, transforming growth factor (TGF)-beta superfamily member is to be selected from the group of being made up of following each person: activin RIA/ALK-2, GFR α-1, activin RIB/ALK-4, GFR α-2, activin RIIA, GFR α-3, activin RIIB, GFR α-4, ALK-1, MIS RII, ALK-7, Ret, BMPR-IA/ALK-3, TGF-β RI/ALK-5, BMPR-IB/ALK-6, TGF-β RII, BMPR-II, TGF-β RIIb, endothelial factor/CD105 and TGF-β RIII.

Other embodiments of this method comprise the Wnt associated molecule that is selected from the group of being made up of following each person: Frizzled-1, Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1, Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6, sFRP-4, Frizzled-7, MFRP, LRP5, LRP6, Wnt-1, Wnt-8a, Wnt-3a, Wnt-10b, Wnt-4, Wnt-11, Wnt-5a, Wnt-9a and Wnt-7a.

In other embodiments of this method, this receptor part is to be selected from the group of being made up of following each person: 4-1BB part/TNFSF9, lymphotoxin, APRIL/TNFSF13, lymphotoxin-beta/TNFSF3, BAFF/TNFSF13C, OX40 part/TNFSF4, CD27 part/TNFSF7, TL1A/TNFSF15, CD30 part/TNFSF8, TNF-α/TNFSF1A, CD40 part/TNFSF5, TNF-β/TNFSF1B, EDA-A2, TRAIL/TNFSF10, Fas part/TNFSF6, TRANCE/TNFSF11, GITR part/TNFSF18, TWEAK/TNFSF12, LIGHT/TNFSF14, amphiregulin (Amphiregulin), NRG1 isotype GGF2, β cell growth factor (Betacellulin), NRG1 isotype SMDF, EGF, NRG1-α/HRG1-α, Epigen, NRG1-β 1/HRG1-β 1, epiregulin (Epiregulin), TGF-α, HB-EGF, TMEFF1/ soil is regulin (Tomoregulin)-1 not, neuregulin-3 (Neuregulin-3), TMEFF2, IGF-I, IGF-II, Regular Insulin, activin A, activin B, activin AB, activin C, BMP-2, BMP-7, BMP-3, BMP-8, BMP-3b/GDF-10, BMP-9, BMP-4, BMP-15, BMP-5, Dpp albumen (Decapentaplegic), BMP-6, GDF-1, GDF-8, GDF-3, GDF-9, GDF-5, GDF-11, GDF-6, GDF-15, GDF-7, Aunar bright (Artemin), the special unit of knob (Neurturin), GDNF, Pu Saifen (Persephin), TGF-β, TGF-β 2, TGF-β 1, TGF-β 3, LAP (TGF-β 1), TGF-β 5, potentiality TGF-β 1, potentiality TGF-β bp1, TGF-β 1.2, Lefty, Nodal, MIS/AMH, acid FGF, FGF-12, basic FGF, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, neural pilin-1 (Neuropilin-1), P1GF, neural pilin-2, P1GF-2, PDGF, PDGF-A, VEGF, PDGF-B, VEGF-B, PDGF-C, VEGF-C, PDGF-D, VEGF-D and PDGF-AB.

In other embodiments, the T-cell target is to be selected from the group of being made up of following each person: 2B4/SLAMF4, IL-2R α, 4-1BB/TNFRSF9, IL-2R β, ALCAM, B7-1/CD80, IL-4R, B7-H3, BLAME/SLAMF8, BTLA, IL-6R, CCR3, IL-7R α, CCR4, CXCR1/IL-8RA, CCR5, CCR6, IL-10R α, CCR7, IL-10R β, CCR8, IL-12R β 1, CCR9, IL-12R β 2, CD2, IL-13R α 1, IL-13, CD3, CD4, ILT2/CD85j, ILT3/CD85k, ILT4/CD85d, ILT5/CD85a, integrin alpha 4/CD49d, CD5, integrin alpha E/CD103, CD6, integrin alpha M/CD11b, CD8, integrin alpha X/CD11c, integrate plain β 2/CD18, KIR/CD158, CD27/TNFRSF7, KIR2DL1, CD28, KIR2DL3, CD30/TNFRSF8, KIR2DL4/CD158d, CD31/PECAM-1, KIR2DS4, CD40 part/TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CD83, leukotriene B4 R1, CD84/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, common γ chain/IL-2R γ, osteopontin (Osteopontin), CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1, L-selects plain (L-selects plain), CXCR3, SIRP β 1, CXCR4, SLAM, CXCR6, TCCR/WSX-1, DNAM-1, thymopoietin (Thymopoietin), EMMPRIN/CD147, TIM-1, EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas part/TNFSF6, TIM-4, Fc γ RIII/CD16, TIM-6, GITR/TNFRSF18, TNF RI/TNFRSF1A, particle cytolysin (Granulysin), TNF RII/TNFRSF1B, HVEM/TNFRSF14, TRAILR1/TNFRSF10A, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAIL R3/TNFRSF10C, IFN-γ R1, TRAILR4/TNFRSF10D, IFN-γ R2, TSLP, IL-1RI and TSLP R.

In other embodiments, the NK cell receptor is to be selected from the group of being made up of following each person: 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1, CD94, LMIR1/CD300A, CD69, LMIR2/CD300c, CRACC/SLAMF7, LMIR3/CD300LF, DNAM-1, LMIR5/CD300LB, Fc ε RII, LMIR6/CD300LE, Fc γ RI/CD64, MICA, Fc γ RIIB/CD32b, MICB, Fc γ RIIC/CD32c, MULT-1, Fc γ RIIA/CD32a, handle albumen-2/CD112, Fc γ RIII/CD16, NKG2A, FcRH1/IRTA5, NKG2C, FcRH2/IRTA4, NKG2D, FcRH4/IRTA1, NKp30, FcRH5/IRTA2, NKp44, class Fc acceptor 3/CD16-2, NKp46/NCR1, NKp80/KLRF1, NTB-A/SLAMF6, Rae-1, Rae-1 α, Rae-1 β, Rae-1 δ, H60, Rae-1 ε, ILT2/CD85j, Rae-1 γ, ILT3/CD85k, TREM-1, ILT4/CD85d, TREM-2, ILT5/CD85a, TREM-3, KIR/CD158, TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4/CD158d and ULBP-3.

In other embodiments, the monocyte/macrophage target is to be selected from the group of being made up of following each person: B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, the common beta chain, integrin alpha 4/CD49d, BLAME/SLAMF8, integrin alpha X/CD11c, CCL6/C10, integrate plain β 2/CD18, CD155/PVR, integrate plain β 3/CD61, CD31/PECAM-1, latex element (Latexin), CD36/SR-B3, leukotriene B4 R1, CD40/TNFRSF5, LIMPII/SR-B2, CD43, LMIR1/CD300A, CD45, LMIR2/CD300c, CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97, LMIR6/CD300LE, CD163, LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN/CD147, MGL2, endothelial factor/CD105, bone plain (Osteoactivin)/GPNMB alive, Fc γ RI/CD64, osteopontin, Fc γ RIIB/CD32b, PD-L2, Fc γ RIIC/CD32c, Siglec-3/CD33, Fc γ RIIA/CD32a, SIGNR1/CD209, Fc γ RIII/CD16, SLAM, GM-CSF R α, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-γ R1, TLR4, IFN-γ R2, TREM-1, IL-1RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k, TREML1/TLT-1,2B4/SLAMF4, IL-10R α, ALCAM, IL-10R β, aminopeptidase N/ANPEP, ILT2/CD85j, the common beta chain, ILT3/CD85k, C1q R1/CD93, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, integrin alpha 4/CD49d, CCR5, integrin alpha M/CD11b, CCR8, integrin alpha X/CD11c, CD155/PVR, integrate plain β 2/CD18, CD14, integrate plain β 3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4 R1, CD68, LIMPII/SR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97, LMIR2/CD300c, CD163, LMIR3/CD300LF, thromboplastin/tissue factor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR, endothelial factor/CD105, NCAM-L1, Fc γ RI/CD64, PSGL-1, Fc γ RIII/CD16, RP105, G-CSF R, L-selects plain, GM-CSF R α, Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-1, IL-6R, TREM-2, CXCR1/IL-8RA, TREM-3 and TREML1/TLT-1.

In other embodiments of this method, the dendritic cell target is to be selected from the group of being made up of following each person: CD36/SR-B3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-AI/MSR, CD5L, SREC-I, CL-P1/COLEC12, SREC-II, LIMPII/SR-B2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9,4-1BB part/TNFSF9, IL-12/IL-23p40,4-amino-1, the 8-naphthalimide, ILT2/CD85j, CCL21/6Ckine, ILT3/CD85k, 8-oxygen base-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5, integrin alpha 4/CD49d, Aag, integrate plain β 2/CD18, AMICA, langhans' cells differential protein (Langerin), B7-2/CD86, leukotriene B4 R1, B7-H3, LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, C1qR1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB, CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD84/SLAMF5, MMR, CD97, NCAM-L1, CD2F-10/SLAMF9, bone element/GPNMB alive, Chem23, PD-L2, CLEC-1, RP105, CLEC-2, Siglec-2/CD22, CRACC/SLAMF7, Siglec-3/CD33, DC-SIGN, Siglec-5, DC-SIGNR/CD299, Siglec-6, DCAR, Siglec-7, DCIR/CLEC4A, Siglec-9, DEC-205, Siglec-10, lectin-1/CLEC7A, Siglec-F, lectin-2/CLEC6A, SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, Fc γ RI/CD64, TLR3, Fc γ RIIB/CD32b, TREM-1, Fc γ RIIC/CD32c, TREM-2, Fc γ RIIA/CD32a, TREM-3, Fc γ RIII/CD16, TREML1/TLT-1, ICAM-2/CD102 and capsaicine R1 (Vanilloid R1).

In other embodiments of this method, the vasculogenesis target is to be selected from the group of being made up of following each person: angiogenin (Angiopoietin)-1, class angiogenin 2, angiopoietin-2, class angiogenin 3, angiogenin-3, class angiogenin 7/CDT6, angiogenin-4, Tie-1, class angiogenin 1, Tie-2, angiogenine (Angiogenin), iNOS, thromboplastin/tissue factor, nNOS, CTGF/CCN2, NOV/CCN3, DANCE, OSM, EDG-1, Plfr, EG-VEGF/PK1, proliferating agent (Proliferin), endostatin (Endostatin), ROBO4, erythropoietin (Erythropoietin), thrombostondin-1, kassinin kinin statin (Kininostatin), thrombostondin-2, MFG-E8, thrombostondin-4, nitrogen protoxide (Nitric Oxide), VG5Q, eNOS, EphA1, EphA5, EphA2, EphA6, EphA3, EphA7, EphA4, EphA8, EphB1, EphB4, EphB2, EphB6, EphB3, Ephrin-A1, Ephrin-A4, Ephrin-A2, Ephrin-A5, Ephrin-A3, Ephrin-B1, Ephrin-B3, Ephrin-B2, acid FGF, FGF-12, basic FGF, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, FGF R1, FGF R4, FGF R2, FGF R5, FGF R3, neural pilin-1, neural pilin-2, arm plate albumen 3A, arm plate albumen 6B, arm plate albumen 3C, arm plate albumen 6C, arm plate albumen 3E, arm plate albumen 6D, arm plate albumen 6A, arm plate albumen 7A, MMP, MMP-11, MMP-1, MMP-12, MMP-2, MMP-13, MMP-3, MMP-14, MMP-7, MMP-15, MMP-8, MMP-16/MT3-MMP, MMP-9, MMP-24/MT5-MMP, MMP-10, MMP-25/MT6-MMP, TIMP-1, TIMP-3, TIMP-2, TIMP-4, ACE, IL-13R α 1, IL-13, C1q R1/CD93, integrin alpha 4/CD49d, the VE-cadherins, integrate plain β 2/CD18, CD31/PECAM-1, KLF4, CD36/SR-B3, LYVE-1, CD151, MCAM, CL-P1/COLEC12, Fibronectin-2/CD112, thromboplastin/tissue factor, E-selects plain, D6, palatelet-selectin, DC-SIGNR/CD299, SLAM, EMMPRIN/CD147, Tie-2, endothelial factor/CD105, TNF RI/TNFRSF1A, EPCR, TNF RII/TNFRSF1B, erythropoietin R, TRAIL R1/TNFRSF10A, ESAM, TRAIL R2/TNFRSF10B, FABP5, VCAM-1, ICAM-1/CD54, VEGF R2/Flk-1, ICAM-2/CD102, VEGF R3/Flt-4, IL-1RI and VG5Q.

Other embodiments of this method provide multivalent binding proteins, and wherein at least one in binding domains 1 and the binding domains 2 is specifically in conjunction with being selected from the target of the group of being made up of following each person: prostate specific membrane antigen (folic acid lytic enzyme 1), EGF-R ELISA (EGFR), be used for the acceptor (RAGE of advanced glycosylation end product; Also be called advanced glycosylation end product acceptor or AGER), IL-17A, IL-17F, P19 (IL23A and IL12B), Dickkopf-1 (Dkk1), NOTCH1, NG2 (chondroitin sulfate proteoglycan 4 or CSPG4), IgE (IgHE or IgH2), IL-22R (IL22RA1), IL-21, kind of starch β oligomer (Ab oligomer), kind of starch β forerunner albumen (APP), NOGO acceptor (RTN4R), LDH receptor related protein 5 (LRP5), IL-4, muscle mass (GDF8), very late antigen 4, α 4, β 1 integrates plain (VLA4 or ITGA4), be present in the α 4 on the white corpuscle, β 7 integrates plain, and IGF-1R.For example, the VLA4 target can be discerned for the multivalent binding proteins that is derived from natalizumab (Natalizumab) binding domains (Antegren) by in wherein binding domains 1 and the binding domains 2 at least one.

In certain embodiments, cancer cells is transition hematopoietic cell or carcinous hematopoietic cell.In some described embodiment, at least one identification in first binding domains and second binding domains is selected from the target of the group of being made up of following each person: B-cell target, monocyte/macrophage target, dendritic cell target, NK-cell target and T-cell target (its separately as defined herein).In addition, at least one marrow discerned target in first binding domains and second binding domains includes but not limited to: CD5, CD10, CD11b, CD11c, CD13, CD14, CD15, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD29, CD30, CD31, CD33, CD34, CD35, CD38, CD43, CD45, CD64, CD66, CD68, CD70, CD80, CD86, CD87, CD88, CD89, CD98, CD100, CD103, CD111, CD112, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CDw123, CDw131, CD141, CD162, CD163, CD177, CD312, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5, B-B2, B-B8 and B-cell antigen receptor.

Other embodiments of the present invention are about multivalent binding proteins as described herein, and it comprises the sequence that is selected from the group of being made up of following each person: SEQ ID NO:2,4,6,103,105,107,109,332,333,334 and 345.Other embodiments are about comprising the multivalent binding proteins of the sequence that is selected from the group of being made up of following each person: SEQ ID NO:355,356,357,358,359,360,361,362,363,364 and 365.

In other embodiments, have that the multivalence of effector function and polyspecific are conjugated protein to have right first binding domains and second binding domains of target that identification is selected from the group of being made up of EPHB4-KDR and TIE-TEK.In described embodiment, this albumen has first binding domains of identification EPHB4 and second binding domains of identification KDR, or second binding domains of first binding domains of identification KDR and identification EPHB4.Similarly, this albumen can have first binding domains of identification TIE and second binding domains of identification TEK, or second binding domains of first binding domains of identification TEK and identification TIE.

In related fields, the invention provides multivalent binding proteins, wherein constant subprovince recognition effect cell F with effector function _CAcceptor (F for example _Cγ RI, F _Cγ RII, F _Cγ RIII, F _Cα R and F _Cε RI).In specific embodiments, constant subprovince identification is selected from effector cell's surface protein of the group of being made up of following each person: CD2, CD3, CD16, CD28, CD32, CD40, CD56, CD64, CD89, F _{C ε}RI, KIR, thrombostondin R, NKG2D, 2B4/NAIL and 41BB.Constant subprovince can comprise the C that is derived from identical or different immunoglobulin (Ig), antibody morphism or allelic variation body _H2Structural domain and C _H3Structural domain.In certain embodiments, C _H3Structural domain is the truncation type structural domain and comprises the C-end sequence that is selected from the group of being made up of following each person: SEQ ID NO:366,367,368,369,370 and 371.When connexon is when being derived from the hinge sample peptide of immunoglobulin (Ig), C _H2Structural domain and scorpion shape connexon preferably are derived from identical category or are derived from the immunoglobulin (Ig) of identical subclass.

Following some albumen is also contained in the present invention, described albumen further comprises and has the scorpion shape connexon that is connected at least about 5 amino acid, with constant subprovince and is connected with second binding domains, thereby scorpion shape connexon is positioned between the constant subprovince and second binding domains.Usually, scorpion shape connexon peptide length is between 5 to 45 amino acid.Scorpion shape connexon comprises the hinge sample peptide that is derived from immunoglobulin hinge region (such as IgG1, IgG2, IgG3, IgG4, IgA and IgE hinge area).Preferably, hinge sample scorpion shape connexon will keep at least one can form interchain disulfide bond under physiological condition halfcystine.The scorpion shape connexon that is derived from IgG1 can have 1 halfcystine or 2 halfcystines, and preferably will keep the halfcystine corresponding to the terminal hinge cysteine of N-of IgG1.In some solid yardage case, the prolongation for homoimmune sphaeroprotein hinge area of scorpion shape connexon, and in exemplary, comprise the sequence that is selected from by SEQID NO:351,352,353 and 354 groups of forming.Also contain non-hinge sample peptide as scorpion shape connexon, its restricted condition provides enough spaces and flexible to obtain forming the single chain protein of two binding domainss for described peptide, and this single chain protein is to locate towards each albumen end (N and C) with respect to the constant subprovince structural domain of more close positioned centrally.Exemplary non-hinge sample scorpion shape connexon comprises the peptide in the stem district (such as the stem district of CD69, CD72, CD94, NKG2A and NKG2D) that is derived from II type C-lectin.In certain embodiments, scorpion shape connexon comprises the sequence that is selected from by SEQ ID NO:373,374,375,376 and 377 groups of forming.

Described albumen also can comprise and has at least about 5 amino acid, the connexon that is connected and is connected with first binding domains with constant subprovince, thereby this connexon is positioned between the constant subprovince and first binding domains.In certain embodiments, connexon is one of to be present in constant subprovince and two binding domainss between the person, and this connexon can have identical or different sequence and can have identical or different length.

Proteic constant subprovince of the present invention provides at least a effector function.Contain as known in the art and the relevant any effector function of immunoglobulin (Ig) (for example antibody), such as the effector function that is selected from the group of forming by following each person: the transformation period that prolongs relatively in the cytotoxicity (ADCC) of antibody dependent cellular mediation, CDC (CDC), the body (with respect to the same molecular that lacks constant subprovince), FcR associativity, albumin A associativity and similar effector function thereof.In certain embodiments, the prolong half-life of albumen of the present invention in the mankind is at least 28 hours.Certainly, desire to give in this non-human individuality, will show the transformation period that prolongs relatively to the albumen of non-human individuality, then not necessarily so in the mankind.

Generally speaking, albumen of the present invention (comprising polypeptide and peptide) shows less than 10 in first binding domains and second binding domains at least one ^-9M or at least 10 ^-6The binding affinity of M.

Another aspect of the present invention is about comprising the composition pharmaceutically of albumen and pharmaceutically acceptable adjuvant, carrier or vehicle as described herein.Any adjuvant as known in the art, carrier or vehicle all are applicable in the composition pharmaceutically of the present invention.

Another aspect of the present invention provides a kind of preparation aforesaid proteic method, this method comprises: the nucleic acid of proteins encoded is introduced host cell and this host cell is preferably hatched under the condition of expressing protein being suitable for the amount of 1 mg/litre at least, thereby express this albumen.In certain embodiments, this method further comprises this albumen of following separation: with itself and its behind cell inner expression at least a albumen sepn of institute's bonded.Be suitable for express nucleic acid to prepare the host cell that proteic host cell of the present invention includes, but is not limited to be selected from the group of being made up of following each person: the VERO cell, HeLa cell (HeLa cell), Chinese hamster ovary celI, the COS cell, the W138 cell, bhk cell, the HepG2 cell, the 3T3 cell, the RIN cell, mdck cell, the A549 cell, the PC12 cell, the K562 cell, the HEK293 cell, the N cell, noctuid (Spodoptera frugiperda) cell is coveted on the meadow, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) cell, Pichia pastoris (Pichia pastoris) cell, any person among the multiple fungal cell and any person in the various bacteria cell (include, but is not limited to bacillus coli (Escherichiaco li), Bacillus subtilus (Bacillus subtilis), salmonella typhimurium (Salmonella typhimurium) and streptomycete (Streptomycete)).

The present invention also provides a kind of method for preparing aforesaid this proteic nucleic acid of coding, this method comprises: make coding source covalently bound with 5 ' end of the polynucleotide of the constant subprovince of coding from 3 ' end of the polynucleotide of first binding domains of immune globulin variable region, make the 5 ' end of polynucleotide of coding scorpion shape connexon covalently bound with 3 ' end of the polynucleotide of the constant subprovince of coding, and make coding source covalently bound with 3 ' end of the polynucleotide of coding scorpion shape connexon, thereby form the nucleic acid that coding has the multivalent binding proteins of effector function from 5 ' end of the polynucleotide of second binding domains of immune globulin variable region.Described coding region can be used as the part of single-stranded structure of the present invention separately and is separated by the coding region of connexon or hinge sample peptide.In certain embodiments, the polynucleotide of the single stranded form of this method preparation coding first binding domains, this first binding domains comprises the sequence that is selected from the group of being made up of following each person: SEQ ID NO:2 (anti-CD 20 variable region, V _L-V _HOrientation), SEQ ID NO:4 (anti--the CD28 variable region, V _L-V _HBe orientated) and SEQ ID NO:6 (anti--the CD28 variable region, V _H-V _LOrientation), however require the special-shaped polyprotein (comprising natural antibody) must be by assembling through encoded polypeptides respectively.The exemplary polynucleotide sequence of first binding domains of encoding is the polynucleotide that comprises SEQ ID NO:1, any person of 3 or 5.

This aspect of the present invention also provides the method for preparing coding nucleic acid, described coding nucleic acid further comprises the connexon polynucleotide between the polynucleotide of the polynucleotide that inserts in coding first binding domains and the constant subprovince of encoding, and this connexon polynucleotide encode has at least 5 amino acid whose peptide connexons.In addition, described method prepares the nucleic acid of the connexon polynucleotide between the polynucleotide that further comprises the polynucleotide that inserts in the constant subprovince of coding and second binding domains of encoding, and this connexon polynucleotide encode has at least 5 amino acid whose peptide connexons.Preferably, coded peptide connexon has the amino acid between 5 and 45.

Be present between BD1 and the EFD or be present in EFD and the consistence that is connected the subarea between the BD2 and be derived from other sequences so that homology-the Ig superfamily member is discerned.Be derived from the novel connexon of existing existing sequence in homology-Ig superfamily member in research and development, be used to make cell surface receptor through the substrate of protease cracking because described sequence is generally, therefore preferably avoid occurring being similar to the tract of those tracts between C-spline structure territory end and membrane spaning domain end with the formation soluble form.Between the different members of-Ig superfamily and subfamily, carry out sequence alignment, can compare between the molecule and to connect a plurality of V-spline structure territories or to connect similarity aspect the connexon sequence in V spline structure territory and C spline structure territory.Analyze as can be known thus, the conservative naturally occurring sequence pattern of type can occur; Described sequence should have stronger proteolytic enzyme tolerance as the connexon between the minor structure territory of multivalence fusion rotein the time, can facilitate suitably folding between the Ig ring district, and not have immunogenicity because of it betides in the extracellular domain of endogenous cell surface molecular.

Described nucleic acid itself constitutes another aspect of the present invention.Contain any proteic nucleic acid of coding the present invention as herein described.Therefore, nucleic acid of the present invention comprises the coding region of coding region, constant subprovince sequence and second binding domains of first binding domains according to 5 ' to 3 ' order.The nucleic acid of also containing the proteins encoded varient, wherein these two binding domainss and constant subprovince sequence have at least 80% and preferred at least 85%, 90%, 95% or 99% consistence altogether with the composite sequence of known immunoglobulin variable domain sequence and known constant subprovince sequence aspect aminoacid sequence.Perhaps, protein variants of the present invention is by encoding with the nucleic acid of the nucleic acid hybridization of coding unmanifest body protein of the present invention under 65-68 ℃, the stringent hybridization condition of 0.015M sodium-chlor, 0.0015M Trisodium Citrate or under the stringent hybridization condition of 42 ℃, 0.015M sodium-chlor, 0.0015M Trisodium Citrate and 50% methane amide.Varient nucleic acid of the present invention shows the hybridization ability under the condition that has above just defined, or shows 90%, 95%, 99% or 99.9% sequence identity with the nucleic acid of coding unmanifest body protein of the present invention.

In related fields, the invention provides the carrier that comprises aforesaid nucleic acid and comprise carrier as described herein or the host cell of nucleic acid.Can use any carrier as known in the art (for example plasmid, phagemid (phagemid), phagemid (phasmid), clay, virus, artificial chromosome, shuttle vectors and analogue thereof), and which kind of carrier those skilled in the art will understand and be particularly suitable for specifying purpose.For example, in preparation proteic method of the present invention, the expression vector that selection can be operated in selected host cell.Equally, contain any host cell that can carry out genetic transformation through nucleic acid of the present invention or carrier.Preferred host cell is the higher eucaryote host cell, but also contains lower eukaryotes (for example yeast) and prokaryotic organism (bacterium) host cell.

Another aspect of the present invention is the method for inducing the target cell injury about a kind of, and this method comprises makes the target cell contact with the albumen as described herein of treatment significant quantity.In certain embodiments, by having this organism that needs to come contact target cell in the body in albumen or coding nucleic acid.Containing multivalence single strand binding protein wherein in this aspect of the present invention can be to the method for the damage of target cell induction addition amount, the amount of damage that this amount is served as reasons and estimated because of the damage total amount due to the separation antibody that comprises one or another person in the described binding domains.Also contain multivalence single strand binding protein wherein can to the collaborative amount of target cell induction (with by comprising first binding domains but do not comprise the first antibody of second binding domains and comprise second binding domains but the second antibody institute inductive damage total amount that do not comprise first binding domains is compared) the method for damage.In certain embodiments, the multivalence single strand binding protein has polyspecific and comprises specifically identification that to be selected from the right binding domains of the antigen of the group of being made up of following each person right: CD19/CD20, CD20/CD21, CD20/CD22, CD20/CD40, CD20/CD79a, CD20/CD79b, CD20/CD81, CD21/CD79b, CD37/CD79b, CD79b/CD81, CD19/CL II (that is II class MHC), CD20/CL II, CD30/CL II, CD37/CL II, CD72/CL II and CD79b/CL II.

This aspect of the present invention also comprises that polyspecific multivalence single strand binding protein wherein can be to the method for the damage of target cell induction amount of suppression (with comprising first binding domains but not comprising the first antibody of second binding domains and comprise second binding domains but the second antibody institute inductive damage total amount that do not comprise first binding domains is compared).Exemplary comprises that wherein polyspecific multivalence single strand binding protein comprises the right right method of binding domains of antigen that identification specifically is selected from the group of being made up of following each person: CD20/CL II, CD21/CD79b, CD22/CD79b, CD40/CD79b, CD70/CD79b, CD72/CD79b, CD79a/CD79b, CD79b/CD80, CD79b/CD86, CD21/CL II, CD22/CL II, CD23/CL II, CD40/CL II, CD70/CL II, CD80/CL II, CD86/CL II, CD19/CD22, CD20/CD22, CD21/CD22, CD22/CD23, CD22/CD30, CD22/CD37, CD22/CD40, CD22/CD70, CD22/CD72, CD22/79a, CD22/79b, CD22/CD80, CD22/CD86 and CD22/CL II.

In related fields, the invention provides the method for a kind of treatment cell proliferation illness (for example cancer), this method comprises the albumen (as described herein) of treatment significant quantity or the organism that coding nucleic acid has these needs.Those skilled in the art's (comprising medical professional and veterinary science professional) know the organic discriminating for the needs treatment.The subject illness of The book of Changes that the present invention is contained comprises the illness that is selected from the group of being made up of cancer, autoimmune disorder, the infection of Lloyd's (Rous) sarcoma virus and inflammation.In certain embodiments, this albumen be by expression in vivo as described herein the nucleic acid of proteins encoded come administration.The present invention also comprises that the approach by being selected from the group of being made up of following each person gives albumen: intravenous injection, intra-arterial injection, intramuscular injection, subcutaneous injection, peritoneal injection and direct tissue injection.

Another aspect of the present invention is a method of improving the symptom relevant with the cell proliferation illness about a kind of, and this method comprises the organism that the albumen as described herein of treatment significant quantity is had these needs.Those skilled in the art also know for showing those illnesss easily stand the symptom improved or the discriminating of disease or state.In certain embodiments, this symptom is to be selected from the group of being made up of pain, heating, swelling and ankylosis.

Another aspect of the present invention is the method about a kind of treatment infection relevant with infectious agent, this method comprise will the treatment significant quantity albumen of the present invention the patient of these needs is arranged, wherein this albumen comprises specifically the binding domains in conjunction with the target molecules of infectious agent.According to this aspect of the present invention, the subject infectious agent of The book of Changes comprises prokaryotic cell prokaryocyte and eukaryotic cell, virus (comprising phage), exotic and infectious organism, such as parasite (for example Mammals parasite).

Related fields of the present invention are methods of improving the infection symptoms relevant with infectious agent about a kind of, this method comprises the patient who the albumen of the present invention of significant quantity is had these needs, and wherein this albumen comprises specifically the binding domains in conjunction with the target molecules of infectious agent.Haveing the knack of medical science and veterinary science operator can utilize normal experiment to judge proteic significant quantity based on individual example.

Another related fields of the present invention are a kind of reduction because of the method for the infection risk due to the infectious agent, this method comprise will the prevention significant quantity the patient of albumen of the present invention with risk of suffering from this infection, wherein this albumen comprises specifically the binding domains in conjunction with the target molecules of infectious agent.Haveing the knack of correlation technique person can utilize normal experiment to judge proteic prevention significant quantity based on individual example.

Another aspect of the present invention is about above-mentioned multivalence single strand binding protein, and wherein at least one in first binding domains and second binding domains is specifically in conjunction with the antigen that is selected from the group of being made up of following each person: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and the main II of histocompatibility complex class peptide.

In certain embodiments, in first binding domains and second binding domains one is specifically in conjunction with CD20, and in some described embodiment, another binding domains is specifically in conjunction with the antigen that is selected from the group of being made up of following each person: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and the main II of histocompatibility complex class peptide.For example, in one embodiment, first binding domains can be specifically in conjunction with CD20, and second binding domains can be specifically in conjunction with for example CD19.In another embodiment, first binding domains is in conjunction with CD19, and second binding domains is in conjunction with CD20.Also contain wherein two binding domainss all in conjunction with the embodiment of CD20.

In some other embodiment aspect this according to the present invention, in first binding domains and second binding domains one is specifically in conjunction with CD79b, and in some described embodiment, another binding domains is specifically in conjunction with the antigen that is selected from the group of being made up of following each person: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and the main II of histocompatibility complex class peptide.Exemplary comprises different polyspecific multivalence single strand binding proteins, wherein first binding domains: second binding domains is specifically in conjunction with CD79b:CD19 or CD19:CD79b.Also comprise multivalent binding proteins with first and second binding domains that can discern CD79b.

In some other embodiments, in first binding domains and second binding domains one is specifically in conjunction with the main II of histocompatibility complex class peptide, and in some described embodiment, another binding domains is specifically in conjunction with the antigen that is selected from the group of being made up of following each person: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and the main II of histocompatibility complex class peptide.For example, in one embodiment, the main II of the histocompatibility complex class peptide of first binding domains combination specifically, and second binding domains can be specifically in conjunction with for example CD19.In another embodiment, first binding domains is in conjunction with CD19, and second binding domains is in conjunction with the main II of histocompatibility complex class peptide.Also contain wherein two binding domainss all in conjunction with the embodiment of the main II of histocompatibility complex class peptide.

In other embodiments aspect this according to the present invention, in first binding domains and second binding domains one is specifically in conjunction with CD22, and in some described embodiment, another binding domains is specifically in conjunction with the antigen that is selected from the group of being made up of following each person: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and the main II of histocompatibility complex class peptide.Exemplary embodiment comprises different polyspecific multivalence single strand binding proteins, wherein first binding domains: second binding domains is specifically in conjunction with CD22:CD19 or CD19:CD22.Also comprise multivalent binding proteins with first and second binding domains that can discern CD22.

Related fields of the present invention are about above-mentioned multivalence single strand binding protein, and wherein this albumen has synergistic effect (with respect to the summation of the effect of each binding domains) to the target cell behavior.In certain embodiments, this albumen comprises specifically identification to be selected from the right binding domains of the antigen of the group of being made up of following each person right: CD20-CD19, CD20-CD21, CD20-CD22, CD20-CD40, CD20-CD79a, CD20-CD79b and CD20-CD81.

The present invention further comprises aforesaid multivalence single strand binding protein, and wherein this albumen has additive effect (with respect to the summation of the effect of each binding domains) to the target cell behavior.Comprise according to the embodiment of this aspect of the present invention and to comprise the right right polyspecific albumen of binding domains of antigen that identification specifically is selected from the group of being made up of following each person: CD20-CD23, CD20-CD30, CD20-CD37, CD20-CD70, CD20-CD80, CD20-CD86, CD79b-CD37, CD79b-CD81, the mainly II of histocompatibility complex class peptide-CD30 and the main II of histocompatibility complex class peptide-CD72.

Another related fields of the present invention are aforesaid multivalence single strand binding protein, and wherein this albumen has retarding effect (with respect to the summation of the effect of each binding domains) to the target cell behavior.In certain embodiments, this albumen has polyspecific and comprises the right binding domains of antigen that identification specifically is selected from the group of being made up of following each person: the CD20-II of main histocompatibility complex class peptide, CD79b-CD19, CD79b-CD20, CD79b-CD21, CD79b-CD22, CD79b-CD23, CD79b-CD30, CD79b-CD40, CD79b-CD70, CD79b-CD72, CD79b-CD79a, CD79b-CD80, CD79b-CD86, the CD79b-II of main histocompatibility complex class peptide, the main II of histocompatibility complex class peptide-CD19, the main II of histocompatibility complex class peptide-CD20, the main II of histocompatibility complex class peptide-CD21, the main II of histocompatibility complex class peptide-CD22, the main II of histocompatibility complex class peptide-CD23, the main II of histocompatibility complex class peptide-CD37, the main II of histocompatibility complex class peptide-CD40, the main II of histocompatibility complex class peptide-CD70, the main II of histocompatibility complex class peptide-CD79a, the main II of histocompatibility complex class peptide-CD79b, the main II of histocompatibility complex class peptide-CD80, the main II of histocompatibility complex class peptide-CD81, the main II of histocompatibility complex class peptide-CD86, CD22-CD19, CD22-CD40, CD22-CD79b, CD22-CD86 and the CD22-II of main histocompatibility complex class peptide.

Another aspect of the invention is at least one the method in the binding domains of the above-mentioned multivalence binding molecule of a kind of discriminating (such as the polyspecific binding molecule), this method comprises: (a) make anti-homotype antibody and discern the first antigenic antibody specifically and reach and discern the second antigenic antibody specifically and contact; (b) at least one the target that comprises in the described antigen is contacted with the composition of step (a); And (c) activity of measurement target drone, wherein this activity is at least one of binding domains that is used for differentiating the multivalence binding molecule.In certain embodiments, this target is a diseased cells, such as the B-cell of cancer cells (for example carcinous B-cell) or generation autoantibody.

In above-mentioned each method of the present invention, expect that this method can further comprise multiple multivalence single strand binding protein.In certain embodiments, the binding domains of the binding domains of the first multivalence single strand binding protein and the second multivalence single strand binding protein can be to target cell induction synergistic effect, additive effect or retarding effect, such as the damage to the collaborative amount of target cell induction, addition amount or amount of suppression.The synergistic effect of multiple multivalence single strand binding protein, additive effect or retarding effect are by the effect of described multiple protein is compared and judged with one antibody and the combined effect that comprises the antibody of another binding domains in comprising binding domains.

Related fields of the present invention are the compositions that comprise multiple aforesaid multivalence single strand binding protein about a kind of.In certain embodiments, said composition comprises multiple multivalence single strand binding protein, wherein the binding domains of the binding domains of first kind of multivalence single strand binding protein and second kind of multivalence single strand binding protein can be to target cell induction synergistic effect, additive effect or retarding effect, such as the damage to the collaborative amount of target cell induction, addition amount or amount of suppression.

The present invention further is the composition pharmaceutically that comprises above-mentioned composition and pharmaceutically acceptable carrier, thinner or vehicle about a kind of.In addition, the present invention includes a kind of test kit, this test kit comprises composition as described herein and one group about giving said composition the target cell is produced the specification sheets of effect (such as damage target cell).

At last, the present invention also comprises a kind of test kit, and this test kit comprises albumen as described herein and one group about giving this albumen with treatment cell proliferation illness or improve the specification sheets of the symptom of cell proliferation illness.

By more clearly understanding other features of the present invention and advantage with reference to following detailed description (comprising embodiment).

The accompanying drawing summary

Fig. 1 shows the synoptic diagram of the multivalence single chain molecule that the present invention is contemplated.Indivedual minor structures territory of expressing fusion protein box is by the independent shapes on the figure/square indication.BD1 is meant binding domains 1, and connexon 1 is meant any potential connexon or the hinge sample peptide between BD1 and " effector function structural domain " (with EFD indication).This minor structure territory is generally the engineered forms of the Fc structural domain of IgG 1, but can comprise other minor structure territories with one or more effector function as defined herein.Connexon 2 is meant and (if exist, then) is present in the C-terminal of EFD and the connexon sequence between the binding domains 2 (BD2).

Fig. 2 shows non-reduced proteic western blotting expressed in the COS cell.Make protein excretion in substratum, and after 48-72 hour by centrifugal from cellular segregation medium supernatant through transient transfection.The thick supernatant liquor of 30 microlitres (30 μ l) is written in each gel pore.Swimming lane sign: 1-molecular weight marker, numeral indication kilodalton (kilodalton); 2-2H7-IgG-STD1-2E12LH; 3-2H7-IgG-STD1-2E12HL, 4-2H7-IgG-STD2-2E12LH; 5-2H7-IgG-STD2-2E12 HL; 6-2E12LH SMIP; 7-2E12HL SMIP; 8-2H7SMIP." 2H7 " is meant the strand construct, wherein the CD20 specificity binding domains (2H7) of BD1 coding VLVH orientation; " 2E12 " is meant the specific binding domains of CD28 tool;-IgG-be meant have coding wherein all C all sport the strand construct of hinge of the sequence (sss) of S, and the CH2 structural domain of IgG1 and CH3 structural domain contain the sudden change (P238S and P331S) of getting rid of ADCC effector function and CDC effector function; " STD1 " is meant BD2 adjacent or the 2E12 (V that is inserted in the VL-VH orientation _L-V _H) the 20 amino acid connexons (in Fig. 7, being designated " STD1=20aa ") of adjacent." STD1-HL " is meant the following construct that is orientated for VH-VL with described similar but BD2V district just now: 2H7-sssIgG (P238/331S)-20 amino acid connexon-2E12 (V _H-V _L)." STD2-LH " is meant 2H7-sssIgG (P238/331S)-38 amino acid connexon-2E12 (V _L-V _H); " STD2-LH " is meant 2H7-sssIgG (P238/331SS)-38 amino acid connexon-2E12 (V _H-V _L); " SMIP " is meant little module immune drug; And " H " typically refers to V _H, and " L " typically refers to V _LExcept as otherwise noted, otherwise all albumen orientation is the N-end to the terminal orientation of C-.

Fig. 3 shows two width of cloth histograms, and its explanation is by the binding characteristic of the expressed 2H7-sssIgG of COS cell (P238S/P331S)-STD1-2e12 LH derivative and 2H7-sssIgG (P238S/P331S)-STD1-2e12HL derivative.Described experiment is with thick culture supernatants but not purified albumen carries out.To hatch with the cell (WIL-2S) of expressing CD20 or the cell (CD28CHO) of expressing CD28 without the serial dilution of the culture supernatants of 16 times of dilutions.With comparing of supernatant liquor in conjunction with active check sample (such as TRU-015 or 2e12VLVH or 2e12VHVL SMIP) with the associativity of testing relevant single specificity SMIP.The combination of each sample be to use Fluorescein Isothiocyanate (FITC) and the anti-IgG of goat combination, detect with the extent of dilution of 1:100.

Fig. 4 is the frequency distribution plan of the binding pattern that shows the proteic albumin A purifying type tested among Fig. 3 and WIL2-S cell." TRU015 " is to the specific SMIP of CD20 tool.Also to two kinds of conjugated protein analyses of polyspecific with effector function: " 2H7-2E12LH " has V _L-V _HThe orientation to the specific binding domains 2 of CD28 tool; " 2H7-2E12HL " has V _H-V _LThe orientation to the specific binding domains 2 of CD28 tool.Test the associativity under each comfortable 5 μ g/mL of described albumen, and with the anti-IgG of FITC goat, with 1:100 detection associativity.About the more complete description of institute's test molecule, referring to the explanation of above Fig. 2.

Fig. 5 shows two amplitude-frequency degree distribution plans, and its explanation has the conjugated protein associativity with the Chinese hamster ovary celI of expressing CD28 of the polyspecific through the albumin A purifying of effector function." 2H7-2E12LH " has V _L-V _HThe orientation to the specific binding domains 2 of CD28 tool; " 2H7-2E12HL " has V _H-V _LThe orientation to the specific binding domains 2 of CD28 tool.Test the associativity under each comfortable 5 μ g/mL of described albumen, and with the anti-IgG of FITC goat, with 1:100 detection associativity.About the more complete description of institute's test molecule, referring to the explanation of Fig. 2.

Fig. 6 A) explicit identification connects the form of the connexon of constant subprovince and binding domains 2.Connexon is to identify with title, sequence, recognition sequence number, sequence length and with sequence that binding domains 2 merges.B) form of the multiple construct of explicit identification, described construct identify the element in the illustration molecule of the present invention.Outside name identification multivalence binding molecule, also disclose element in those molecules with regard to binding domains 1 (BD1), constant subprovince (hinge and effector domain or EFD), connexon (about other information of connexon, referring to Fig. 6 A) and binding domains 2 (BD2).The sequence of multiple exemplary multivalent binding proteins is provided, and number identifies with recognition sequence in the drawings.Other multivalent binding proteins have element or the element order through changing, the change that can be come forecasting sequence by the sequence that is disclosed.

Fig. 7 shows the built-up type histogram, and it illustrates that purified albumen is bonded to WIL-2S cell of expressing CD20 and the Chinese hamster ovary celI of expressing CD28 with single in conjunction with concentration." H1-H6 " is meant to have H1-H6 connexon and V _H-V _LThe 2H7-sss-hIgG-Hx-2e12 molecule in the 2e12V district of orientation." L1-L6 " is meant to have L1-L6 connexon and V _L-V _HThe 2H7-sss-hIgG-Lx-2e12 molecule in the 2e12V district of orientation.All molecules are all tested under the concentration of 0.72 μ g/mL, and use FITC with the combination of the anti-IgG of goat, with 1:100 detection associativity.Then to the average candle power intensity mapping of each sample, it is two kinds of target cell types being tested paired histograms with respect to each multivalence construct (L1-L6 or H1-H6) of being tested.

Fig. 8 shows the photo of Ku Masi (Coomassie) dyeing irreducibility and reductibility SDS-PAGE gel.Described gel shows that varient connexon sequence/length on the 2H7-sss-hIgG-Hx-2e12HL albumen is to the influence of the amount that is revealed in two kinds of major protein bands on the gel.

Fig. 9 shows to use with the 2H7 specificity to have reactive (a) CD28mIgG or (b) [2H7-sss-hIgG-H6-2e12] fusion rotein that Fab surveyed and the western blotting of relevant single specificity SMIP.The result shows that the existence of H6 connexon causes producing the multivalence construct of cracking form, and it lacks the CD28 binding specificity.

Figure 10 shows the binding curve of the different connexon varients of [TRU015-sss-IgG-Hx-2e12HL] H1-H6 connexon form.First component shows the binding curve that is bonded to the WIL-2S cell of expressing CD20.Second component shows the multi-form binding curve that is bonded to the CD28CHO cell.The following generation of described binding curve: serial dilution detects associativity through the fusion rotein of albumin A purifying and the combination of use FITC and the anti-IgG of goat with 1:100.

Figure 11 shows the form of the SEC separating resulting of summarizing the 2H7-sss-IgG-2e12HL polyspecific fusion rotein with varient connexon H1-H7.Each enumerates the different connexon varients of [2H7-sss-IgG-Hx-2e12-HL] fusion rotein in the table.Residence time at the peak of paying close attention to (POI) and the fusion rotein that exists with POI percentage and also list in the table with the proteic percentage that other forms exist.Also enumerate whether cracking of molecule, the cracking degree is indicated in qualitative mode, wherein (is), is, is or is not four kinds of possible selections.

Figure 12 shows two binding curve figure of [2H7-sss-hIgG-Hx-2e12] polyspecific fusion rotein with varient connexon H3, H6 and H7 connexon and the cell of expressing CD20 or CD28.To hatch with the WIL-2S cell or the CD28 Chinese hamster ovary celI of expressing CD20 from the serial dilution of 10 μ g/ml to 0.005 μ g/ml through the fusion rotein of albumin A purifying.Use the combination of FITC and the anti-IgG of goat to detect associativity with 1:100.Panel A shows and the combining of WIL-2S cell, and the combining of component B demonstration and CD28 Chinese hamster ovary celI.

Figure 13 shows by the substituting result in conjunction with mensuration that molecule produced who is used for Figure 12.In the case, at first make fusion rotein be bonded to the WIL-2S cell of expressing CD20, and then detect associativity with 1:100 with CD28mIgG (5 μ g/ml) and the anti-mouse reagent of FITC.Described result shows be bonded to CD20 and CD28 simultaneously in a part.

Figure 14 shows the result who uses another kind of polyspecific fusion constructs varient to be obtained.In the case, can change the specificity of BD2, so that use the V district of G28-1 antibody to form CD37 specificity binding domains.Two shown width of cloth figure illustrate CD20 and/or CD37 antibody blocking [2H7-sss-IgG-Hx-G28-1] polyspecific fusion rotein and the Ramos or the bjab cell bonded relative capacity of expressing CD20 and CD37 target.Before the polyspecific fusion rotein is hatched, with each cell type with CD20 specific antibody (25 μ g/ml) or CD37 specific antibody (10 μ g/ml) or two kinds of reagent (described reagent is the human reagent of mouse anti) preincubate.Then detect the associativity of polyspecific fusion rotein with 1:100 with the anti-IgG reagent of FITC goat (preadsorption in mouse to get rid of cross reactivity).

Figure 15 shows the result who measures with BJAB target cell, PBMC effector cell and the ADCC that carried out as test agent with the CD20-hIgG-CD37 specific fusion protein.The complete description of this program is referring to suitable embodiment.This figure is a fusion rotein concentration with respect to the graphic representation of the % specificity kill ratio of under various dosage single specificity SMIP reagent and [2H7-sss-hIgG-STD1-G28-1] LH and HL varient being tested.For the dose-response effect of person one of in described single specificity or the polyspecific strand fusion rotein, to each DS mapping.

Figure 16 shows the form enumerate the co-culture experiments result, wherein at TRU 015, G28-1SMIP, two kinds of molecules cultivation PBMC together or in the presence of [2H7-sss-IgG-H7-G28-1HL] varient.Use the fusion rotein of 20 μ g/ml, and hatched 24 hours or 72 hours.Then sample is used in conjunction with the CD3 antibody of FITC and in conjunction with CD19 or the dyeing of CD40 specific antibody of PE, then stood flow cytometry.Then the percentage of cell in each lock is listed in the table.

Figure 17 is presented at [2H7-sss-hIgG-H7-G28-1HL] molecule or the single CD20 of contrast and/or CD37 specificity SMIP and hatches after 24 hours two width of cloth histograms to the influence of B cell line cell apoptosis alone or in combination.The percentage of annexin V (annexin V)-propidium iodide positive cell is mapped as the function of the test agent type that is used for hatching altogether experiment.Panel A shows the result who uses the Ramos cell to be obtained, and component B shows the result who uses the Daudi cell to be obtained.SMIP at each single CD20 or CD37 shows with prescribed concentration; In addition, if use two kinds of combination of agents, then show the relative quantity of all ingredients with bracket.Test with the concentration of 5 μ g/ml, 10 μ g/ml and 20 μ g/ml for polyspecific CD20-CD37 fusion rotein.

Figure 18 shows [2H7-hIgG-G19-4] molecular variant and itself and CD3 express cell (Jurkats) or CD20 express cell (WIL-2S) bonded two width of cloth figure.Described molecule comprises [2H7-sss-hIgG-STD1-G19-4HL], LH and [2H7-csc-hIgG-STD1-G19-4HL].Fusion rotein through the albumin A purifying passes through 20 μ g/ml titration to 0.05 μ g/ml, and uses the anti-IgG of FITC goat to detect associativity with 1:100.MFI (average candle power intensity) is mapped as the function of protein concentration.

Figure 19 shows the result that ADCC that the NK cell depleting type PBMC with whole human PBMC of [2H7-hIgG-STD1-G19-4HL] molecular variant, BJAB target cell and action effect cell with SSS hinge or CSC hinge or action effect cell is carried out measures.Kill ratio is scored as the function of polyspecific fusion rotein concentration.To compare with using G19-4, TRU015 or this two kinds of being seen kill ratios of combination of agents with the kill ratio that described molecule is observed., wherein % specificity kill ratio is mapped as the function of protein concentration to each DS mapping with respect to different test agent.

Figure 20 is presented at anti-CD 20 antibodies and exists or do not exist down (if add, then exist with 2 μ g/ml) each member in the substrate combination of B-cell antibody (2 μ g/ml) hatch overnight after, through the percentage of the Ramos B-cell of annexin V (Ann) and/or propidium iodide (PI) positive staining.Goat-anti-mouse secondary antibody always exists with the concentration ratio with respect to other antibody (independent matrix antibody, or matrix antibody and anti-CD 20 antibodies) twice.The matrix antibody (2 μ g/ml) and goat anti-mouse antibody (4 μ g/ml) represented on vertical striped post-X-axis.The matrix antibody of representing on horizontal stripe post-X-axis (2 μ g/ml), anti-CD 20 antibodies (2 μ g/ml) and goat anti-mouse antibody (4 μ g/ml)." the 2nd step " condition served as control and comprise goat anti-mouse antibody (vertical striped post) or the 8 μ g/ml (horizontal stripe post) that add 4 μ g/ml, and do not add matrix antibody or anti-CD 20 antibodies." CL II " among the figure (II class MHC) is meant with HLA DR, DQ and DP (that is, with II class MHC antigen) to have the monoclonal antibody of cross reactivity.

Figure 21 is presented at, and anti--CD79b antibody exists or does not exist down (if adds, then exist with 0.5 or 1.0 μ g/ml) each member in the substrate combination of B-cell antibody (2 μ g/ml) hatch overnight after, through the percentage of the Ramos B-cell of annexin V (Ann) and/or propidium iodide (PI) positive staining." CL II " reaches the explanation of the discriminating of " the 2nd step " sample referring to Figure 20.Vertical striped post-matrix antibody (2 μ g/ml) and goat anti-mouse antibody (4 μ g/ml); Horizontal stripe post-matrix antibody (2 μ g/ml), anti--CD79b antibody (1.0 μ g/ml) and goat anti-mouse antibody (6 μ g/ml); Strokes and dots post-matrix antibody (2 μ g/ml), anti--CD79b antibody (0.5 μ g/ml) and goat anti-mouse antibody (5 μ g/ml).

Figure 22 is presented at, and anti--CL II antibody exists or does not exist down (if adds, then exist with 0.25 or 0.5 μ g/ml) each member in the substrate combination of B-cell antibody (2 μ g/ml) hatch overnight after, through the percentage of the Ramos B-cell of annexin V (Ann) and/or propidium iodide (PI) positive staining." CL II " reaches the explanation of the discriminating of " the 2nd step " sample referring to Figure 20.Vertical striped post-matrix antibody (2 μ g/ml) and goat anti-mouse antibody (4 μ g/ml); Horizontal stripe post-matrix antibody (2 μ g/ml), anti--CL II antibody (0.5 μ g/ml) and goat anti-mouse antibody (5 μ g/ml); Strokes and dots post-matrix antibody (2 μ g/ml), anti--CL II antibody (0.25 μ g/ml) and goat anti-mouse antibody (4.5 μ g/ml).

Figure 23 is presented at, and anti--CD22 antibody exists or does not exist down (if adds, then exist with 2 μ g/ml) each member in the substrate combination of B-cell antibody (2 μ g/ml) hatch overnight after, through the percentage of the DHL-4B-cell of annexin V (Ann) and/or propidium iodide (PI) positive staining." CL II " reaches the explanation of the discriminating of " the 2nd step " sample referring to Figure 20.Solid post-matrix antibody (2 μ g/ml) and goat anti-mouse antibody (4 μ g/ml); Slanted bar line post-matrix antibody (2 μ g/ml), anti--CD22 antibody (2 μ g/ml) and goat anti-mouse antibody (8 μ g/ml).

The free CD20 SMIP (solid) of the caption that Figure 24 provides or monospecific CD20 * CD20 scorpion shape molecule (hollow) are to the direct growth restraining effect of lymphoma cell line Su-DHL6 (trilateral) and DoHH2 (square).

Pictorialization free anti--CD37 SMIP (solid) or the monospecific of Figure 25 be anti--and CD37 scorpion shape molecule (hollow) is to the direct growth restraining effect of lymphoma cell line Su-DHL-6 (trilateral) and DoHH2 (square).

The combination of pictorialization two kinds of different monospecific SMIP (solid) that Figure 26 provides or dual specific CD20-CD37 scorpion shape molecule (hollow) are to the direct growth restraining effect of lymphoma cell line Su-DHL-6 (trilateral) and DoHH2 (square).

The free CD20 SMIP of chart announcement of Figure 27 and the combination (solid) of CD37 SMIP or dual specific CD20xCD37 scorpion shape molecule (hollow) are to the direct growth restraining effect of lymphoma cell line Su-DHL-6 (trilateral) and WSU-NHL (square).

The histogram that Figure 28 provides shows the cell cycle effect of scorpion shape molecule.The sample of DoHH2 lymphoma cell is divided into: unprocessed, handle or handle through monospecific CD37 * CD37 scorpion shape molecule through SMIP 016.Blank post: Asia-G of cell cycle ₁Phase; Black post: G ₀/ G ₁Phase; Shade post: S phase; And striped post: G2/M phase.

The data drawing list proof that Figure 29 provides is handled lymphoma cell with scorpion shape molecule makes the signal transmissibility compare enhancing (as measured by calcium ionic current) with free SMIP.

The caption scorpion shape molecule dependent cell cytotoxicity that Figure 30 provides.

The numerator mediated CDC of Figure 31 data presented caption scorpion shape.

Figure 32 combines with the low affinity isotype (B) of Fc γ RIII (CD16) and the comparative ELISA of high-affinity isotype (A) with data presentation SMIP and the scorpion shape molecule that diagrammatic form provides.

The chart that Figure 33 provides proves in the presence of the target cell, SMIP and scorpion shape molecule and the low affinity allelotype (A) of Fc γ RIII (CD16) and combining of high-affinity allelotype (B).

The histogram of Figure 34 is presented in two experiments (flask 1 and flask 2), under six kinds of different culture condition, and the expression degree of CD20 * CD20 scorpion shape molecule.Filled black post: flask 1; Striped post: flask 2.

The histogram that Figure 35 provides shows the output of CD20 * CD37 scorpion shape molecule.

Figure 36 provides the SDS-PAGE gel image (under reduction and non-reduced condition) of SMIP and scorpion shape molecule.

The pictorialization scorpion shape molecule that Figure 37 provides keeps being bonded to the ability of target cell.Fill square: CD20 SMIP; Fill trilateral: CD37 SMIP; Fill circle: humanization CD20 (2Lm20-4) SMIP; Blank rhombus: CD37 * CD37 monospecific scorpion shape molecule; Square hollow: CD20 * CD37 dual specific scorpion shape molecule; And hollow triangle: humanization CD20 (2Lm20-4) * humanization CD20 (2Lm20-4) scorpion shape molecule.

The result of the pictorialization competitive binding assay that Figure 38 is contained proves that the N-end participates in the target cell with the terminal scorpion shape of C-molecule binding domains and combines.

Figure 39 has the dissociation rate lower than SMIP with the data presentation scorpion shape molecule that diagrammatic form provides.

The chart proof scorpion shape molecule that Figure 40 shows in vivo, in serum, have stability, be characterized as that scorpion shape molecule is renewable, circulating half-life is lasting.

Figure 41 provides the dose-response chart of CD20 x CD37 dual specific scorpion shape molecule, effect in the body of its explanation scorpion shape molecule administration.

Figure 42 shows combining of monospecific CD20xCD20 scorpion shape molecule (S0129) and sugared varient and target B-cell.

What the caption CD20xCD20 scorpion shape molecule (parental antibody and sugared varient) that Figure 43 provides was induced ADCC mediation kills BJAB B-cell.

The gel image that Figure 44 shows discloses because of changing the influence to scorpion shape stability of molecule that scorpion shape connexon (comprise the sequence that changes this connexon and prolong connexon by the H7 sequence is added into connexon) is caused, and this is indicated by "+" in the H7 line of gel below.

Figure 45 shows combining of CD20xCD20 scorpion shape molecule (S0129) and its scorpion shape connexon varient and WIL2S cell.

Figure 46 shows CD20xCD20 scorpion shape molecule and the CD20SMIP direct cell killing effect to multiple B-cell.

Figure 47 discloses the direct cell killing effect of monospecific CD20xCD20 scorpion shape molecule to other B-clones.

Figure 48 show two kinds of monospecific scorpion shape molecules (that is CD20 * CD20 and CD37 * CD37) and in the dual specific CD20xCD37 scorpion shape molecule the direct cell of each kill ability, the latter shows multi-form kill ratio curve.

Figure 49 describes the Su-DHL-6B-cell to each reaction in CD20xCD20 (S0129), CD37xCD37 and the CD20xCD37 scorpion shape molecule with diagrammatic form.

Figure 50 show dual specific CD19xCD37 scorpion shape molecule and

Directly kill the ability of Su-DHL-6B-cell.

Histogram that Figure 51 provides show multiple in conjunction with CD20 scorpion shape molecule and SMIP and

Directly kill DHL-4B-cell (as shown in FIG.).Blue post: viable cell; Each is to the maroon post on right side: annexin +/PI+.

The chart that Figure 52 provides describe various in conjunction with CD20 scorpion shape molecule and SMIP and

Direct cell killing effect (as shown in FIG.).

Figure 53 provide by various in conjunction with CD20 scorpion shape molecule and SMIP (as shown in FIG.) and

The active chart of the inductive ADCC of institute.

Figure 54 provide by various in conjunction with CD20 scorpion shape molecule and SMIP (as shown in FIG.) and

The active chart of the inductive CDC of institute.

The histogram that Figure 55 provides shows C1q and the CD20 associativity scorpion shape molecule bonded degree that is bonded to Ramos B-cell.

The scatter diagram of the facs analysis that Figure 56 provides shows, CD20 associativity scorpion shape molecule (2Lm20-4x2Lm20-4 and 011x2Lm20-4) and

Cause mitochondrial membrane potential with respect to contrast decline (last figure); Percentile histogram (breaking property MMP: the black post) be shown in figure below with cell of breaking property mitochondrial membrane potential.

The histogram that Figure 57 provides shows that CD20 associativity scorpion shape molecule (2Lm20-4x2Lm20-4 and 011x2Lm20-4), Rituximab (Rituximab), CD95 and contrast are to caspase 3 (caspase3) activatory relative deficiency.

Figure 58 provides with the combination of Western blotting to carrying out available from poly-(ADP-ribose) polysaccharase of B-cell and caspase 3,7 and 9 analyzing for four times, shows that any described albumen is bonded to cell degraded in a small amount because of CD20 associativity scorpion shape molecule.

Figure 59 is the gel electrophoresis figure of B-cell chromosome DNA, and it shows because of CD20 associativity scorpion shape molecule and is bonded to degree of crushing due to the cell.

Figure 60 is the gel electrophoresis figure with the immunoprecipitate that each obtained in anti-phosphorylated tyrosine antibody and the anti--SYK antibody.Immunoprecipitate is the molten born of the same parents' thing that contacts with CD20 associativity scorpion shape molecule available from the B-cell, as shown in FIG..

Figure 61 provides the combinatorial index graphic representation of the combination treatment of each in CD20 associativity scorpion shape molecule and hydroxyl daunoblastin (doxorubicin), vincristine(VCR) (vincristine) and the rapamycin (rapamycin).

Detailed Description Of The Invention

The invention provides the composition of relatively little peptide, described relatively little peptide has at least two lands or binding structural domain, described land or binding structural domain can provide one or more binding specificity, be derived from immunoglobulin (Ig) (such as antibody) variable binding structural domain, settle endways with respect to the effector domain that comprises at least part of constant region for immunoglobulin (that is source of constant subprovince as defined herein); And related nucleic acid, carrier and host cell in the described peptide of restructuring preparation; And in multiple diagnostic and therapeutic is used, use the method for described peptide combinations, described application comprises treatment illness and at least a symptom of improving this illness. Described peptide combinations is advantageously arranged the second binding structural domain C-end to effector domain, this arranges provides in this peptide at least two binding structural domains without the combination under the sterically hindered or weak steric hindrance unexpectedly, and simultaneously maintenance is placed in the effector function of the effector domain at center.

The first binding structural domain of multivalence peptide of the present invention and the second binding structural domain can be identical (that is, have uniformity or substantially conforming amino acid sequence and have monospecific) or different (and having polyspecific). Although the first binding structural domain and the second binding structural domain are different aspect primary structure, it can be identified and in conjunction with the identical epi-position of target molecules and therefore will have monospecific. Yet in multiple situation, described binding structural domain will have different structure and in connection with to different binding sites, produce multivalence polyspecific albumen. These different binding sites can be present on the single target molecules or on the different target molecules. Identify in the situation of different target molecules at two binding molecules, this target molecules can be present in (for example isocellular surface) on (for example) same structure or in the same structure, or this target molecules can be present on absolute construction or the position or is present in absolute construction or the position. For example, polyspecific of the present invention can have the binding structural domain that is bonded to specifically the lip-deep target molecules of different cell types in conjunction with albumen. Perhaps, binding structural domain can be bonded to the target on the cell surface specifically, and another binding structural domain can be bonded to specifically finds the target irrelevant with cell, such as structural (matrix) albumen in extracellular or free (for example solubility or matrix) albumen.

The first binding structural domain and the second binding structural domain are one or more districts of the protein structure (such as antibody molecule) that is derived from identical or different immunoglobulin (Ig). The first binding structural domain and/or the second binding structural domain can show the sequence consistent with the sequence in immunoglobulin (Ig) zone, or the modified forms that can be this sequence is to provide (for example) binding characteristic or stability through changing through changing. Describedly modify known in this area and comprise and directly cause the amino acid sequence of characteristic changing (changing such as associativity) to change, for example cause the secondary structural change of peptide or more the amino acid sequence that changes of higher structure change. Also contain the modified amino acid sequence that obtains by incorporating alpha-non-natural amino acid (such as non-natural conventional amino acid, unconventional amino acid and imino acid) into. In certain embodiments, change sequence and cause translating rear processing change, for example cause glycosylation pattern to change.

Contain any person for the multiple binding structural domain that is derived from immunoglobulin (Ig) or immunoglobulin-like polypeptide (for example acceptor) of scorpion shape molecule. The binding structural domain that is derived from antibody comprises V_LDomain and V_HThe CDR district of domain (referring to the context that for example uses the binding structural domain that is derived from humanized antibody). Comprise the complete V that is derived from antibody_LAnd V_HThe binding structural domain of domain can arbitrary orientation be organized. Scorpion shape molecule of the present invention can have described any binding structural domain herein. For the scorpion shape molecule of the binding structural domain with at least one identification B-cell, exemplary scorpion shape molecule has the binding structural domain that at least one is derived from following each person: CD3, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138 or CDw150. In certain embodiments, scorpion shape molecule is the multivalent binding proteins that comprises at least one binding structural domain, and this binding structural domain has the sequence that is selected from the group that is comprised of following each person: SEQ ID NO:2,4,6,103,105,107 and 109. In certain embodiments, scorpion shape molecule comprises the binding structural domain that contains the sequence that is selected from the group that is comprised of any following each person: SEQ ID NO:332-345. In certain embodiments, scorpion shape molecule comprises to contain and is derived from immunoglobulin (Ig) V_LAnd V_HThe binding structural domain of the sequence of domain, wherein this sequence is to be selected from the group of appointing whichever to form by among the SEQ ID NO:355-365. The scorpion shape molecule that comprises binding structural domain is further contained in the present invention, and this binding structural domain has can be by appointing sequence that whichever infers, have V among the SEQ ID NO:355-365_LWith V_HInverted orientation.

The district more than one that is derived from immunoglobulin (Ig) for any one or both in the binding structural domain wherein (Ig V for example_LDistrict and Ig V_HThe district) embodiment, a plurality of districts can connect by the connexon peptide. In addition, can use connexon that the first binding structural domain is connected with constant subprovince. Being connected of constant subprovince and the second binding structural domain (that is binding structural domain 2 towards the C-of scorpion shape molecule is terminal settle) can reach by scorpion shape connexon. Described scorpion shape connexon preferably has about 2 to 45 amino acid or 2 to 38 amino acid or 5 to 45 amino acid. For example, H1 connexon length is that 2 amino acid and STD2 connexon length are 38 amino acid. Except general consideration length, the scorpion shape connection subarea that is applicable in the scorpion shape molecule of the present invention comprises the antibody hinge region that is selected from the group that is comprised of IgG, IgA, IgD and IgE hinge and variant thereof. For example, scorpion shape connexon can be the antibody hinge region that is selected from the group that is comprised of IgG 1, IgG 2, IgG 3 and IgG 4 and variant thereof. In certain embodiments, scorpion shape connection subarea has the single cysteine residues that is used to form interchain disulfide bond. In other embodiments, scorpion shape connexon has two cysteine residues that are used to form interchain disulfide bond. in certain embodiments, it is to be derived from immunoglobulin hinge region or C-agglutinin stem district and to comprise the sequence that is selected from the group that is comprised of following each person that scorpion shape connects the subarea: SEQ ID NO:111,113,115,117,119,121,123,125,127,129,131,133,135,149,151,153,155,157,159,161,163,165,167,169,231,233,235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,287,289,297,305,307,309,310,311,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,346,351,352,353,354,373,374,375,376 and 377. More generally, when the sequence that is derived from hinge area is provided, contain any amino acid sequence of identifying in the sequence table as the scorpion shape connexon in the scorpion shape molecule of the present invention. In addition, the scorpion shape connexon that is derived from the Ig hinge is hinge sample peptide domain, and this peptide domain has at least one free cysteine that can participate in interchain disulfide bond. Preferably, the scorpion shape connexon that is derived from Ig hinge peptide remains with cysteine, and described cysteine is corresponding to the hinge cysteine towards the terminal arrangement of the N-of this hinge. Preferably, the scorpion shape connexon that is derived from the IgG1 hinge has one or have two corresponding to the cysteine of hinge cysteine. In addition, scorpion shape connexon is the stem district of II type C-agglutinin molecule. In certain embodiments, scorpion shape molecule comprises the scorpion shape connexon with the sequence that is selected from the group that is comprised of SEQ ID NO:373-377.

The constant subprovince that is placed in the center is the constant region that is derived from immunoglobulin (Ig). Constant subprovince normally is derived from the C of the immunoglobulin (Ig) in the abstract of invention_HThe C in district_H2Part is although it also can be derived from C_H2-C _H3Part. Constant subprovince randomly can be derived from the hinge-C of immunoglobulin (Ig)_H2Or hinge-C_H2-C _H3Part will place corresponding to the peptide of Ig hinge area the N-of constant subprovince terminal and be placed between constant subprovince and the binding structural domain 1. In addition, the part of constant subprovince can be derived from the C of different immunoglobulin (Ig)s_HThe district. In addition, can be with the peptide brachymemma corresponding to Ig CH3, the remaining C-terminal amino acid sequence that is selected from the group that is formed by SEQ ID NOS:366-371. Yet preferably, the scorpion hinge is to be derived from the embodiment of hinge sample peptide of immunoglobulin (Ig) hinge therein, and scorpion shape connexon and constant subprovince are the immunoglobulin (Ig)s that is derived from same type. Constant subprovince provides at least a and C immunoglobulin (Ig)_HThe activity that the district is relevant, such as the cytotoxicity (ADCC) of antibody dependent cellular mediation, CDC (CDC), albumin A associativity, with at least a F_CThe stability (with respect to for the albumen of the present invention not having constant subprovince) that the associativity of acceptor, recyclability detect, reach perhaps placenta metastatic, just as is known to the person skilled in the art, wherein the subculture of molecule of the present invention shifts as favourable. As above-mentioned binding structural domain, constant subprovince is to be derived from least a immunoglobulin molecules and to show consistent or consistent amino acid sequence substantially with one or more zone of at least a immunoglobulin (Ig). In certain embodiments, via at least a immunoglobulin (Ig) should or described sequence (by replacing one or more conventional or unconventional non-natural (for example synthetic) amino acid or imino acid) modified in constant subprovince, thereby produce and to make secondary structure or more higher structure changes, characteristic is also with the primary structure of its change, maybe can cause translating rear processing and change, such as glycosylation.

If described binding structural domain and constant subprovince and one or more immunoglobulin polypeptides show consistent or consistent amino acid sequence substantially, then can produce the molecule of modified (with respect to as the immunoglobulin (Ig) of modifying the basis) to the posttranslational modification of molecule of the present invention. For example, can utilize technology as known in the art, so that the polypeptide glycosylation pattern is modified host cell with respect to the mode that this polypeptide in not modified (for example CHO) host cell changes, CHO cell for example.

In possessing described molecule and body in its situation of method of restructuring preparation, developed the approach of novel targeting diagnosis and treatment, for example to allow that immune effector cell (for example cytotoxic T lymphocyte, natural killer cell and fellow thereof) target raises cell, tissue, medicament and the exotic that destroys or cut off to wish, such as cancer cell and infectious agent. Except therapeutic cells is positioned the therapentic part, described peptide is applicable to the locating therapy compound, such as through radiolabeled albumen. In addition, described peptide also is applicable to remove harmful composition, for example by making harmful composition (such as toxin) and cell (for example macrophage) combination that can destroy or get rid of this toxin. Molecule of the present invention is applicable to regulate the activity of binding partner molecule (such as cell surface receptor). This is shown among Figure 17, and wherein molecule of the present invention obviously strengthens the Apoptosis signal conduction via CD20 and/or CD37. The result of this signal conduction is the target cell death. Wherein getting rid of the restriction cell mass is that useful disease and state comprises infectivity and management of parasitic diseases, inflammatory and autoimmune state, malignant tumour and similar disease thereof. The Enhancement Method that it will be understood by a person skilled in the art that the conduction of Apoptosis signal is unrestricted. Mitosis signal conduction and the signal conduction that causes limiting differentiation, activation or the deactivation of cell mass can be induced via suitable selection binding partner molecule by molecule of the present invention. By considering that the following express definitions of used term herein will be conducive to the further understanding to disclosure of the present invention.

" single strand binding protein " is that covalently bound amino acid whose single this chain can be bonded to one or more binding partner specifically in abutting connection with arranging, and described partner shares the determinant that is enough to the binding site of single strand binding protein detectability ground combination. Exemplary binding partner comprises albumen, carbohydrate, lipid and little molecule.

For ease of explanation, it is just to describe with the difference of albumen of the present invention and/or polypeptide and/or peptide that " derivative " of albumen of the present invention, polypeptide and peptide reaches " variant ", and this means described derivative and variant (it is protein/polypeptide/peptide of the present invention) is different from of the present invention not deriving or unmanifest albumen, polypeptide or peptide with ad hoc fashion. It will be understood by a person skilled in the art that described derivative and variant are originally as albumen of the present invention, polypeptide and peptide.

" antibody " is endowed the most extensive definition consistent with its implication in the art, and comprise can be in conjunction with albumen, polypeptide and the peptide of at least one binding partner (such as albumen or non-albumen antigen). " antibody " comprises the member of the immunoglobulin superfamily of the member, strand of immunoglobulin superfamily of albumen of any species or the albumen that multichain forms as used herein, and variant, analog, derivative and the fragment of described molecule. Specific, " antibody " comprises any form of antibody as known in the art, includes, but is not limited to monoclonal and polyclonal antibody, mosaic type antibody, CDR grafted antibody, humanized antibody, single chain variable fragment, bispecific antibody, bifunctional antibody, antibody fusion and similar antibody thereof.

" binding structural domain " is regional in conjunction with one or more specific binding partner's peptide specifically, such as the polypeptide fragment that is derived from immunoglobulin (Ig) (for example antibody). If there are a plurality of binding partners, then this partner share be enough to detectability ground in conjunction with this binding structural domain in conjunction with determinant. This binding structural domain is preferably in abutting connection with amino acid sequence.

" epi-position " in this article given general sense has single antigen site for the material (for example albumen) in interact with the antibody specificity (for example by combination), that is antigenic determinant. Other terms of implication in immunoglobulin (Ig) (for example antibody) technology, have been obtained to determine, such as " variable region of light chain ", " variable region of heavy chain ", " constant region of light chain ", " CH ", " antibody hinge region ", " complementary determining region ", " framework region ", " antibody morphism ", " FC district ", " single chain variable fragment " or " scFv ", " bifunctional antibody ", " chimera ", " CDR grafted antibody ", " humanized antibody ", " shaping antibody ", " antibody fusion " and similar terms thereof, has separately definite implication as known in the art, unless specify in addition herein.

Unless clearly definition herein, otherwise the term that those skilled in the art's reference antibody technology is understood has the implication that obtains in this area. The example of described term is " V_L" reach " V_H", refer to be derived from respectively the variable land of light chain of antibody and heavy chain; And C_LAnd C_H, refer to " constant region for immunoglobulin ", that is be derived from respectively the constant region of light chain of antibody or heavy chain, should be appreciated that wherein latter's constant region can be further divided into C_H1、C _H2、C _H3And C_H4The constant region domain, this antibody morphism of originating on this district (IgA, IgD, IgE, IgG, IgM) is decided. CDR means " complementary determining region ". " hinge area " is to be derived from the C that inserts in the antibody strand_H1District and C_H2Between the district and connect the amino acid sequence in these two districts, be known in the art " hinge area " and provide flexible with " hinge " form to whole antibody.

" constant subprovince " is the term that defines herein, and it refers to corresponding to or be derived from peptide, polypeptide or the protein sequence of one or more constant region domain of antibody. Therefore, constant subprovince can comprise following any domain or all domains: C_H1Domain, hinge area, C_H2Domain, C_H3Domain (IgA, IgD, IgG, IgE and IgM) and C_H4Domain (IgE, IgM). Therefore, as defined herein constant subprovince can refer to corresponding to the complete constant region of antibody or the peptide zone of its part. Usually, the constant subprovince of polypeptide of the present invention or code nucleic acid have hinge, C_H2Domain and C_H3Domain.

The function relevant with antibody constant region or the function that provided by antibody constant region are provided " effector function ". The exemplary effect function comprises cytotoxicity (ADCC), complement activation and CDC (CDC), the F of antibody dependent cellular mediation_CIncrease receptor binding and plasma half-life and the placenta transfer. The effector function of the present composition has detectability; The activity specific of present composition activity specific preferred and wild type antibody with regard to this effector function with this function is roughly the same, that is for wild type antibody, any effector function is not preferably lost in the constant subprovince of multivalence binding molecule.

" connexon " is peptide or polynucleotide, its connection or connect other peptides or polynucleotide. Usually, the peptide connexon is for having about 2 to 50 amino acid whose oligopeptides, wherein typical polynucleotide connexon encode this peptide connexon and therefore length be about 6 to 150 nucleotides. Connexon makes the first binding structural domain be connected with constant subprovince domain. Exemplary peptide connexon is (Gly₄Ser) ₃ Scorpion shape connexon is to be connected with the second binding structural domain for the C-end with constant subprovince. Scorpion shape connexon can be derived from immunoglobulin hinge region or be derived from the stem district of II type C-agglutinin, such as more detailed description hereinafter.

" target " is endowed more than one implications, and environment for use defines the clear and definite implication in the various situations. With regard to its narrower sense, " target " is binding site, that is the binding structural domain of the binding partner of peptide combinations of the present invention. With regard to broad sense more, " target " or " molecular target " refers to present the complete binding partner (for example albumen) of binding site. Specificity target (such as " CD20 ", " CD37 " and similar target thereof) is endowed in the art acquired general sense of this term separately. " target cell " is any prokaryotic or the eukaryotic (no matter healthy or whether ill) relevant with target molecules of the present invention. Certainly, also find and the equal incoherent target molecules (that is acellular target) of any cell, or form relevant target molecules with other, such as viral (comprising bacteriophage), organic or inorganic target molecules carrier and exotic.

The example of the material relevant with target molecules comprises from body sexual cell (for example cancer cell or other diseased cells), infectious agent (for example infectious cell and infectious virus) and analog thereof. Target molecules can be combined with enucleate cell, cell membrane, liposome, cavernous body, gel, capsule, lozenge and analog thereof, no matter which kind of intended purpose (for example is used for drug therapy as the result of well-meaning or unconscious measure, or further be the biological threat of terrorism), it all can be used for sending, conveying or positioning target molecule. " acellular ", " virus-free ", " carrier-free ", " without object " and fellow thereof refer to the target molecules of not being combined with particular composition or material.

" binding affinity " refers to the intensity of the non-covalent combination of peptide combinations of the present invention and its binding partner. Binding affinity preferably refers to for the quantitative measurement of combination to attraction between the member.

" adjuvant " is for strengthening or promoting and its material in conjunction with the functional effect of the compound of (such as to comprise the composition forms combination pharmaceutically of activating agent and adjuvant). " excipient " is for being used as the inert substance of diluent in the composition preparation pharmaceutically. " carrier " is generally inert substance, and it is be used to the medium that provides to send composition pharmaceutically.

" host cell " refers to wherein exist any prokaryotic or the eukaryotic of polynucleotide of the present invention, albumen or peptide.

Nucleic acid or polynucleotide " introducing " host cell meaned by any mode as known in the art make nucleic acid or polynucleotide enter this cell, send transduction, the trajectory projectile that described mode includes, but is not limited to the precipitation of external salt mediation of nucleic acid/polynucleotide that naked nucleic acid/polynucleotide or carrier carry and other reformulationses, virus-mediated infection and randomly has or do not have " an assisting " molecule, coupling and similar fashion thereof.

" hatching " host cell means this cell is maintained under the environmental condition of be suitable for earmarking (such as gene expression) as known in the art. Described condition (comprising that temperature, ionic strength, oxygen tension, gas concentration lwevel, nutrients form and conditions of similarity) is in well known in the art.

" separation " compound (such as albumen of the present invention or peptide) mean with this compound from from being present in such as at least a different compound separation the host cell of expressing the separated compound of wish of its natural combination, the host cell of consumption culture medium from grow in this culture medium that for example will contain this compound separates.

" have this need organism " easily stands to treat or the risk of any disease, illness or the state of easy-to-use composition for improved of the present invention or suffer from any organism of any this disease, illness or state for having to suffer from, described disease, illness or state include, but is not limited to any person of any person of various cancer forms, multiple autoimmune disease, because of the radiation poisoning due to radioactive marker protein, peptide and the similar compound, the property taken in or inner property toxin and the similar type thereof of producing, this will become apparent after looking back whole disclosure. There is this organism that needs to be preferably human patients.

As known in the art, " improvement " of disease symptoms order of severity detectability ground of meaning this symptom of disease reduces. Exemplary symptom comprises pain, heating, swelling and anchylosis.

Unless offer some clarification in the context, otherwise term " albumen ", " peptide " reach " polypeptide " and be used interchangeably in this article, it refers to amino acid whose at least one adjacent chain separately. Similarly, unless offer some clarification on specific and not interchangeable implication in the context, term " polynucleotide ", " nucleic acid " reach " nucleic acid molecules " and are used interchangeably.

" pharmaceutically acceptable salt " refers to the salt of the compounds of this invention, and it is derived from the combination (acid-addition salts) of described compound and organic acid or inorganic acid or the combination (base addition salts) of described compound and organic base or inorganic base.

Hereinafter utilize term as hereinbefore defined to provide general explanation to various aspects of the present invention. After the generality explanation, describe work embodiment and think that operability of the present invention disclosed herein and applicability provide additional proof.

Proteins and peptides

In certain embodiments of the invention, any person in the described multivalent binding proteins (comprising the integrated structure domain-immunoglobulin fusion proteins) with effector function herein is provided, and the multivalent binding proteins or the peptide that wherein have effector function comprise two or more binding structural domain peptide sequences. In the binding structural domain peptide sequence each all can in conjunction with or specifically in conjunction with one or more targets, such as one or more antigens, wherein one or more targets or antigen can be identical or can be not identical. The binding structural domain peptide sequence can be derived from the antigen variable region, or it can be derived from the immunoglobulin-like molecule, the acceptor that for example folds in the mode of imitating immunoglobulin molecules. The antibody that binding structural domain is originated can be any other form of polyclonal antibody (comprising the monospecific polyclonal antibody), monoclonal antibody (mAb), recombinant antibodies, chimeric antibody, humanized antibody (such as the CDR-grafted antibody), human antibodies, single-chain antibody, catalytic antibody and antibody as known in the art, with and fragment, variant or derivative. In certain embodiments, each in the binding structural domain of albumen of the present invention all is the complete variable regions that are derived from immunoglobulin (Ig). In preferred embodiments, binding structural domain is based on human Ig variable region separately. In other embodiments, albumen is the fragment that is derived from the Ig variable region. In described embodiment, preferably, each binding structural domain peptide sequence is corresponding to each sequence in the complementary determining region of assigned I g variable region. The present invention is also contained corresponding to the binding structural domain than whole CDR CDR still less of assigned I g variable region, and its restrictive condition is that described binding structural domain keeps specifically the ability in conjunction with at least a target.

Multivalent binding proteins with effector function also has the constant subprovince sequence between two binding structural domains that are derived from constant region for immunoglobulin (preferred antibody CH), covalency and place the multivalent binding proteins with effector function.

Multivalent binding proteins with effector function also has the terminal scorpion shape connexon that is connected of the terminal N-with binding structural domain 2 of the C-that makes constant subprovince. Scorpion shape connexon is not for helical peptides and can be derived from antibody hinge region, be derived from the zone that connects immune globulin binding structural domain or the stem district that is derived from II type C-agglutinin. Scorpion shape connexon can be derived from the wild type hinge area of immunoglobulin (Ig), such as IgG1, IgG2, IgG3, IgG4, IgA, IgD or IgE hinge area. In other embodiments, the invention provides the multivalent binding proteins with the hinge through changing. Be suitable for being contained in the kind through changing hinge area in the multivalent binding proteins and be the hinge classification that the quantity that is involved in the cysteine residues (those Cys residues especially as known in the art) that the interchain disulfide bond in the immunoglobulin (Ig) homologue molecule with wild type hinge forms changes. Therefore, albumen can have the IgG1 hinge, wherein lacks three one that can participate in the Cys residue that interchain disulfide bond forms. Be to indicate through changing the cysteine substructure of hinge, show the Cys subsequence from N-end to C-end. Utilize this recognition system, the multivalent binding proteins that has through changing the IgG hinge comprises the hinge arrangement that is characterized by cxc, xxc, ccx, xxc, xcx, cxx and xxx. The Cys residue can lack, or can be through 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, and conservative replaces or non-conservation replaces thereby form. In certain embodiments, cysteine is replaced through serine. For the albumen with the scorpion shape connexon that comprises the IgG1 hinge, decreased number corresponding to the cysteine of hinge cysteine is 1 or 2, preferably, corresponding to terminal arrangement of N-of close this hinge of the one in those cysteines of hinge cysteine.

For the albumen with the scorpion shape connexon that comprises the IgG2 hinge, can there be 0,1,2,3 or 4 Cys residue. For comprising the scorpion shape connexon through changing the IgG2 hinge that contains 1,2 or 3 Cys residue, the institute of containing the Cys residue might subset. Therefore, for the described connexon with a Cys, multivalent binding proteins can have following Cys motif in hinge area: cxxx, xcxx, xxcx or xxxc. For the scorpion shape connexon that comprises the IgG2 hinge variant with 2 or 3 Cys residues, contain through the Cys residue of reservation and might making up of the Cys residue that is substituted (or disappearance). For having the multivalent binding proteins that comprises through changing IgG3 or the scorpion shape connexon through changing the IgG4 hinge area, contain Cys residue in the hinge area by 1 integral number that is reduced to less than the Cys residue, no matter this minimizing is to replace via disappearance or via conservative amino acid or non-conservation amino acid (for example serine). Contain equally the multivalent binding proteins with the scorpion shape connexon that comprises wild type IgA, IgD or IgE hinge, also contain the correspondence of the Cys residue (number of its number sum of existing Cys residue in 0 extends to less than corresponding wild type hinge) with decreased number through changing hinge area. In having some embodiment of IgG1 hinge, keep the terminal Cys residue of first or N-of this hinge. For having wild type hinge area or the albumen through changing hinge area, expect that described multivalent binding proteins is for can for example forming the single chain molecule of homopolymer (such as dimer) by disulfide bond. In addition, as disclosed herein, the hinge area end with the albumen that changes hinge can be through revising, for example terminal at the N-of designation area or domain (such as the hinge arrangement territory), C-is terminal or two terminal deletions or replace one or more amino acid residue.

In another exemplary embodiment, constant subprovince is to be derived from the constant region that comprises natural IgD hinge area or through engineering approaches IgD hinge area. The human IgD hinge of wild type have one can with natural IgD structure in light chain form the cysteine of disulfide bond. In certain embodiments, this IgD hinge cysteine through sudden change (for example disappearance) with produce as the bonding pad between the binding structural domain of for example bispecific molecule through changing hinge. Not producing other amino acid variations or the disappearance of bad hinge rigidity in the IgD hinge or revising is to belong to category of the present invention. Natural or the through engineering approaches IgD hinge area of other species also belongs to category of the present invention, the natural or through engineering approaches IgD hinge and also belong to category of the present invention from (other non-IgD) hinge area (such as yamma IgG2 hinge) of other mankind or non-human antibody's homotype from non-human species's humanization.

The present invention further comprises the constant subprovince that is connected with the scorpion shape connexon that can be derived from corresponding to the hinge of aforesaid hinges known district (such as IgG1 hinge or IgD hinge). (with respect to wild type) modified or modified hinge area can be contained in this constant subprovince, and wherein at least one cysteine residues of known participation interchain disulfide bond connection is replaced into another kind of amino acid with conservative replacement (for example Ser replaces Cys) or non-conservation replacement mode. This constant subprovince does not comprise corresponding to immunoglobulin (Ig) C_H1Peptide district or the peptide domain of domain.

The alternative hinge and the connexon sequence that can be used as the bonding pad are the parts that is derived from the cell surface receptor that connects immunoglobulin (Ig) V-spline structure territory or immunoglobulin (Ig) C-spline structure territory. Also contain district's (wherein cell surface receptor contains a plurality of tandem Ig V-spline structures territory) between the Ig V-spline structure territory and the district's (wherein cell surface receptor contains a plurality of tandem Ig C-samples district) between the Ig C-spline structure territory as the bonding pad. Hinge and connexon sequence usually length are 5 to 60 amino acid, and not only are mainly the tool flexibility, and more multiple stiffness feature also can be provided. In addition, connexon provides the sterically hindered minimized space of being convenient between the binding structural domain usually. Preferably, the structure of described hinge and connexon peptide is mainly the α spiral, and the β laminated structure is minimum. Preferred sequence has stability and the cutting of tolerance proteolysis in blood plasma and serum. Preferred sequence can contain naturally occurring motif or the motif through adding, such as giving disulfide bond to stablize the CPPC motif of dimer formation. Preferred sequence can contain one or more glycosylation site. The example of preferred hinge and connexon sequence includes, but is not limited to distinguish between the Ig V-sample district of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD150, CD166 and CD244 and the domain between the Ig C-sample district.

Constant subprovince can be derived from the camellid constant region, such as yamma or camel IgG2 or IgG3.

Specific, contain to have and be derived from any Ig classification or be derived from any IgG subclass, such as the C of IgG1 (for example IgG 1)_H2-C _H3The constant subprovince in district. In preferred embodiments, constant subprovince all is derived from identical Ig classification with the scorpion shape connexon that is derived from the immunoglobulin (Ig) hinge. In other preferred embodiments, constant subprovince all is derived from identical Ig subclass with the scorpion shape connexon that is derived from the immunoglobulin (Ig) hinge. Constant subprovince also can be the CH3 domain that is derived from any Ig classification or subclass (such as IgG1, for example IgG 1), and its restrictive condition is that it is relevant with at least a immunoglobulin (Ig) effector function.

Constant subprovince does not correspond to complete constant region for immunoglobulin (that is the C of IgG class_H1-hinge-C_H2-C _H3). Constant subprovince can be corresponding to the complete constant region for immunoglobulin of other classifications, also contain have sudden change rear or disappearance rear the IgA constant domain (such as IgA1 hinge, IgA2 hinge, IgA C_H2And IgA C_H3Domain) as constant subprovince. In addition, any light chain constant domain all can be used as constant subprovince, for example C_KOr any C_L Constant subprovince also can include or hingless JH or JK. Constant subprovince also can be corresponding to engineered antibody, wherein such as in this area understanding, for example a kind of transplant of encircling makes up to produce one for natural F by the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of selecting with the IgG framework_CAcceptor (CD16, CD32, CD64, F beyond the R_Cε R1) binding site. An exemplary constant subprovince of this type is a modified IgG C with CD89 binding site_H2-C _H3The district.

This aspect of the present invention provides a kind of multivalent binding proteins or peptide with effector function, comprise following, basically form or formed by following by following: (a) binding structural domain peptide sequence that is derived from immunoglobulin (Ig) that is placed in N-end, merge or be connected in the constant subprovince peptide sequence that (b) is derived from constant region for immunoglobulin, it preferably includes a hinge area sequence, wherein this hinge area polypeptide can be as described herein, and can comprise following, basically form or formed by following by following: alternative hinge area peptide sequence for example, this constant subprovince peptide sequence merge again or are connected in the second natural or through engineering approaches binding structural domain peptide sequence that is derived from immunoglobulin (Ig) that (c) is placed in the C-end.

This constant subprovince peptide sequence that is derived from constant region for immunoglobulin that is placed in the center can have at least a immunocompetence that is selected from by the following group that forms: the cytotoxicity of antibody dependent cellular mediation, CDC, complement fixation, and F_CReceptors bind, and described integrated structure domain polypeptide separately can in conjunction with or specifically in conjunction with target, such as antigen, wherein target can be identical or different, and can effectively be present in the identical physiological environment (for example isocellular surface) or varying environment (different cell surfaces for example, cell surface and acellular position are such as in solution) in.

This aspect of the present invention also comprises that the multivalence albumen with effector function with as disclosed herein particular sequence has at least 80% and variant albumen or the polypeptide of preferred 85%, 90%, 95% or 99% conforming demonstration effect function.

Polynucleotide

The present invention also provide coding albumen of the present invention or peptide polynucleotide (through separating or purified polynucleotide or pure polynucleotide), comprise the carrier (comprising cloning vector and expression vector) of described polynucleotide, and through polynucleotide of the present invention or carrier transforms or the cell (for example host cell) of transfection. Aspect coding albumen of the present invention or polypeptide, polynucleotide encode the first binding structural domain, the second binding structural domain and F_CDomain, described domain all is derived from immunoglobulin (Ig), and preferred source is from human immunoglobulin. Each binding structural domain can contain corresponding to total length variable region sequences (heavy chain and/or light chain) or corresponding to the sequence of its partial sequence, and its restrictive condition is that respectively this binding structural domain keeps the ability of specific binding. F_CDomain can have corresponding to the total length IgF_CDomain sequence or corresponding to the sequence of its partial sequence, its restrictive condition is F_CDomain shows at least a effector function as defined herein. In addition, each in the described binding structural domain all can via normal length at least 8 amino acid and preferably at least 13 amino acid whose connexon peptides be connected to F_CDomain. Preferred connexon sequence is based on Gly₄The sequence of Ser motif is such as (Gly₄Ser) ₃。

The present invention also comprises the variant of the multivalent binding proteins with effector function. Variant polynucleotide and one in the polynucleotide with defined nucleotide sequence as described herein have at least 90% and preferred 95%, 99% or 99.9% uniformity, or its can with those polynucleotides with defined nucleotide sequence in one hybridize at 65-68 ℃, 0.015M sodium chloride, 0.0015M natrium citricum or under the stringent hybridization condition of 42 ℃, 0.015M sodium chloride, 0.0015M natrium citricum and 50% formamide. The variant polynucleotide body keeps the ability that coding has the multivalent binding proteins of effector function.

Term " strictly " is used for referring to the usually strict condition that is interpreted as of this area. The concentration of hybridization stringency Main Basis temperature, ionic strength and denaturant (such as formamide) is judged. The example that is used for the stringent condition of hybridization and washing be under 65-68 ℃, 0.015M sodium chloride, 0.0015M natrium citricum, or under 42 ℃, 0.015M sodium chloride, 0.0015M natrium citricum and 50% formamide. Referring to people such as Sambrook, Molecular Cloning:A Laboratory Manual, the 2nd edition, Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y.1989).

Also can use stricter condition (such as higher temperature, more LIS, higher formamide or other denaturants); Yet hybridization speed will be affected. Relate to therein in the situation of hybridization of deoxidation oligonucleotides, other exemplary stringent hybridization conditions are included under 37 ℃ (for for the 14 base oligonucleotides), 48 ℃ (for for the 17 base oligonucleotides), 55 ℃ (for for the 20 base oligonucleotides) and 60 ℃ (for 23 base oligonucleotides) and wash in 6 times of SSC, 0.05% sodium pyrophosphate.

In related fields of the present invention, provide a kind of and (for example prepare polypeptide of the present invention or albumen or other constructs, comprise multivalent binding proteins or peptide with effector function) method, the method includes the steps of: (a) will be as described herein or the host cell that provides cultivate allowing under the condition of expressing this construct; And (b) expression product (multivalent binding proteins or the peptide that for example have effector function) is separated with host cell or host cell culture.

Construct

The present invention also is about the carrier that comprises separately polynucleotide of the present invention or nucleic acid and by the prepared construct of known carrier, particularly about being applicable to the recombinant expression construct body of gene therapy, comprise any person in the various known constructs, comprise and send construct, it comprises that coding as the multivalence with effector function (for example polyspecific comprises bispecific) that provides are in conjunction with any nucleic acid of albumen and polypeptide herein; Be about in heredity through the host cell of carrier of the present invention and/or other construct through engineering approaches; And be to have the multivalence (for example polyspecific comprises bispecific) of effector function in conjunction with the expression construct of the nucleotide sequence of albumen or its fragment or variant or the method for other constructs about comprise coding by recombinant technique.

Being used in the situation of gene therapy, under the control of suitable promoter, decide and look character (for example terminal differentiation or the active division after the mitosis of the principal host of institute cell on the character (for example type of aforesaid promoter) of construct; For example but expression construct is maintained episome or is integrated in the host cell gene group) and decide, various construct of the present invention (comprising that the multivalence (for example polyspecific) with effector function is in conjunction with albumen) in fact can be expressed in any host cell, comprises host cell in the body.

The suitable cloning vector and the expression vector that are used for protokaryon and eucaryon host are described in such as people such as Sambrook, Molecular Cloning:A Laboratory Manual, and the 2nd edition, Cold Spring Harbor, NY is in (1989). Exemplary clone/expression vector includes, but is not limited to cloning vector, shuttle vector and expression construct, and it can or be suitable for increasing, shifting and/or express any nucleic acid medium of wherein contained polynucleotide as known in the art based on plasmid, phasmid (phagemid), phasmid (phasmid), clay, virus, artificial chromosome. As described herein, in a preferred embodiment of the invention, in the mammalian cell of nucleic acid transfection of the present invention, conversion or transduction, carry out recombinant expressed. Also referring to for example Machida, CA., " Viral Vectors for Gene Therapy:Methods and Protocols (viral vectors of gene therapy: method and experimental program) "; Wolff, JA, " Gene Therapeutics:Methods and Applications of Direct Gene Transfer (gene therapy: the methods and applications of direct gene transfer) " (Birkhauser 1994); Stein, U and Walther, W (volume), " Gene Therapy of Cancer:Methods and Protocols (gene therapy of cancer: method and experimental implementation) " (Humana Press 2000); Robbins, PD (volume), " Gene Therapy Protocols (gene therapy scheme) " Humana Press 1997); Morgan, JR (volume), " Gene Therapy Protocols (gene therapy scheme) " (Humana Press 2002); Meager, A (volume), " Gene Therapy Technologies, Applications and Regulations:From Laboratory to Clinic (gene therapy technology, application and adjusting: the laboratory is to clinical) " (John Wiley ﹠ Sons Inc.1999); MacHida, CA and Constant, JG, " Viral Vectors for Gene Therapy:Methods and Protocols (viral vectors of gene therapy: method and experimental implementation) " (Humana Press 2002); " New Methods Of Gene Therapy For Genetic Metabolic Diseases NIH Guide (the gene therapy new method NIH guide that is used for Inherited Metabolic Disorders) " the 22nd volume, the 35th phase, on October 1st, 1993. Also referring to United States Patent (USP) the 6th, 384, No. 210, the 6th, 384, No. 203, the 6th, 384, No. 202, the 6th, 384, No. 018, the 6th, 383, No. 814, the 6th, 383, No. 811, the 6th, 383, No. 795, the 6th, 383, No. 794, the 6th, 383, No. 785, the 6th, 383, No. 753, the 6th, 383, No. 746, the 6th, 383, No. 743, the 6th, 383, No. 738, the 6th, 383, No. 737, the 6th, 383, No. 733, the 6th, 383, No. 522, the 6th, 383, No. 512, the 6th, 383, No. 481, the 6th, 383, No. 478, the 6th, 383, No. 138, the 6th, 380, No. 382, the 6th, 380, No. 371, the 6th, 380, No. 369, the 6th, 380, No. 362, the 6th, 380, No. 170, the 6th, 380, No. 169, the 6th, 379, No. 967 and the 6th, 379, No. 966.

Expression construct normally is derived from plasmid vector.A kind of preferred construct is that (CA), it has nucleotide sequence, polyadenylation signal and the T7 promotor site of coding Ampicillin Trihydrate (ampicillin) resistant gene to modified pNASS carrier for Clontech, Palo Alto.Other suitable mammalian expression vectors are known (referring to people such as for example Ausubel, 1995; People such as Sambrook, the same; Also referring to for example Invitrogen, San Diego, the catalogue of CA; Novagen, Madison, WI; Pharmacia, Piscataway, NJ).For the output of the multivalent binding proteins that promotes to have effector function increases (described output is to use suitable selective agent (for example Rheumatrex) back because of due to the gene amplification), at present can be under suitable adjustable control preparation comprise the preferred construct of Tetrahydrofolate dehydrogenase (DHFR) encoding sequence.

Usually, recombinant expression vector will comprise the mark selected of replication orgin and permissive host cell transformation as mentioned above, and be derived from gene through high expression level directly to transcribe the promotor of downstream configurations sequence.Carrier is connected with the operability of polynucleotide of the present invention to produce clones construct or expression construct.Exemplary clone/expression construct contains at least one the expression controlling elements that is connected with polynucleotide operability of the present invention, for example promotor.Also contain other in carrier of the present invention and the clone/expression construct and express controlling elements, such as enhanser, factor-specific binding site, terminator and ribosome bind site.The structural sequence of the allos of polynucleotide of the present invention is that the phase assembles with translation initiation sequence and terminator sequence in due course.Therefore, for example, can be used as the multivalent binding proteins coding nucleic acid that is provided herein and to be used for expressing this proteic recombinant expression construct body and being included in any one of various expression vector establishment bodies at host cell.In some preferred embodiment, described construct is to be included in the preparation of vivo medicine-feeding.Described carrier and construct comprise chromosomal DNA sequence, nonchromosomal DNA sequence and synthetic DNA sequence, for example derivative of SV40; Bacterial plasmid; Phage DNA; Yeast plasmid; Be derived from the carrier of the combination of plasmid and phage DNA, viral DNA (lacking sex reversal record virus) such as cowpox, adenovirus, fowlpox virus and pseudorabies or as described below duplicating.Yet, can use any other carrier to prepare the recombinant expression construct body, and in preferred embodiments, this carrier is reproducible and can survive among the host.

Suitable dna sequence dna for example can be inserted in the carrier by various programs.Generally speaking, dna sequence dna is inserted in the suitable restriction endonuclease cleavage site by program as known in the art.Contain be used to clone, the standard technique of DNA separation, amplification and purifying, be used to relate to the standard technique and the various isolation technique of the enzymatic reaction of dna ligase, archaeal dna polymerase, restriction endonuclease and similar enzyme thereof.The multiple standards technical description is in people (1993 Current Protocols in Molecular Biology (the existing experimental program of molecular biology), Greene Publ.Assoc.Inc.﹠amp such as for example Ausubel; John Wiley ﹠amp; Sons, Inc., Boston, MA); People such as Sambrook (1989 Molecular Cloning (molecular cloning), the 2nd edition, Cold Spring Harbor Laboratory, Plainview, NY); People such as Maniatis (1982Molecular Cloning (molecular cloning), Cold Spring Harbor Laboratory, Plainview, NY); Glover (volume) (1985 DNA Cloning (dna clone) I and II volume, IRL Press, Oxford, UK); Hames and Higgins (volume), (1985 Nucleic AcidHybridization (nucleic acid hybridization), IRL Press, Oxford, UK); And in other documents.

Make the dna sequence dna in the expression vector functionally be connected with guiding mRNA synthetic with at least one suitable expression control sequenc (for example composition promotor or modulability promotor).The representative example of described expression control sequenc comprises aforesaid eukaryotic cell or its viral promotor.Promoter region can be selected from any gene of wanting that uses CAT (CAT) carrier or have other carriers that can select mark.Eukaryotic promoter comprise the quick early promoter of CMV, HSV thymidine kinase, in early days reach SV40 in late period, be derived from retroviral LTR, and mouse metallothionein(MT)-I.Selecting suitable carrier and promotor is to belong in those of ordinary skills' the horizontal extent, and describe the preparation of some particularly preferred recombinant expression construct body herein, described recombinant expression construct body comprises at least one promotor or modulability promotor of functionally being connected with the nucleic acid of coding albumen of the present invention or polypeptide.

The DNA that transcribes coding albumen of the present invention and polypeptide by higher eucaryotic cells can strengthen by enhancer sequence is inserted in the carrier.Example comprises the sub-enhanser of SV40 enhanser (bp100 to 270), cytomegalovirus early promoter that is positioned at the replication orgin nearside, the polyoma enhanser and the adenovirus enhanser of replication orgin nearside.

Also contain the gene therapy of using nucleic acid of the present invention (comprise the displacement defective gene or novel gene is added into strategy in cell and/or the tissue), and it is being researched and developed for being used for the treatment of cancer, adjusting Metabolic disorder and being used for the immunotherapy field.Gene therapy of the present invention comprises uses the of the present invention various constructs that have or do not have separate carrier or delivery vehicle or construct for treatment described disease, illness and/or state herein.Described construct also can be used as the vaccine that is used for the treatment of or prevents described disease, illness and/or state herein.Thereby dna vaccination for example utilizes the polynucleotide of coding immunogenic protein and nucleic acid determiner to defend pathogenic agent or tumour cell with stimulating immune system.Described strategy can stimulate acquired character or innate immunity, or can relate to the modification of immunologic function via cytokine-expressing.The vivo gene therapy be usually directed to genetic material directly inject in patient or the animal body with treatment, prevent or improve disease or with the symptom of disease-related.Vaccine and immunomodulatory are the general therapy.For therapy in the organizing specific gonosome,, be preferably gene location and send and/or express/targeted system such as those therapies that are intended to treat cancer.The several genes therapy carrier of targeting specific tissue is known in this area, and has developed the program of complete targeting specific tissue, for example uses the technology based on conduit, and all described contents all are covered by herein.

Also contain stripped gene therapy herein, and described method relate to remove, heredity is modified, amplification and give self cell of individuality (for example human patients) again.Example comprises that the heredity of the bone marrow transplantation that is used for cancer therapy or lymph progenitor cell modifies.Stripped gene therapy preferably is applicable to handle and is easy to get and can survives in cells in culture (such as blood or skin cells) during the transgenosis process.

The gene therapy carrier that is suitable for comprises adenovirus carrier, lentiviral vectors, adeno-associated virus (AAV) carrier, herpes simplex virus (HSV) carrier and retroviral vector.Gene therapy also can use " naked DNA ", based on the delivering method of liposome, carry out based on delivering method (comprising the DNA that is connected with positively charged lipid), electroporation and the trajectory projective method of lipid.

In some embodiment (including but not limited to the gene therapy embodiment), carrier can be virus vector, such as retroviral vector.People such as Miller, 1989 BioTechniques7:980; Coffin and Varmus, 1996 Retroviruses, Cold Spring HarborLaboratory Press, NY.For example, the retroviral plasmid vector retrovirus of originating includes but not limited to Moloney (Moloney) murine leukemia virus, spleen necrosis virus, retrovirus (such as Lloyd's (Rous) sarcoma virus), Ha Wei (Harvey) sarcoma virus, avian leukosis viruses, gibbon ape leukemia virus, HIV (human immunodeficiency virus), adenovirus, bone marrow proliferative sarcoma virus and mammary tumor virus.

Retrovirus is reproducible and can be integrated in RNA viruses in the genome of host cell via the DNA intermediate.This DNA intermediate or provirus can stably be integrated in the host cell DNA.According to certain embodiments of the present invention, expression construct can comprise wherein to incorporate into has the coding alien gene of foreign protein and the retrovirus of improper retrovirus RNA.When retrovirus RNA enter in the host cell, when take place infecting simultaneously, alien gene also is introduced in the cell, and then can be integrated in the host cell DNA, it is the part of reverse transcription virus gene group seemingly.In the host, express this external gene and cause expressing foreign protein.

Develop the most of retroviral vector system that is used for gene therapy and be based on the mouse retrovirus.Described retrovirus exists with two kinds of forms: be called the cell free virus of virus particle or be integrated in provirus in the host cell DNA.The virus of virus particle form contains: retroviral structural and enzymatic albumen (comprising reversed transcriptive enzyme), virus genomic two RNA copy and contain the part of the source cell plasma membrane of viral envelope glycoprotein.The reverse transcription virus gene group is organized as four main districts: long terminal repetition (LTR), its contain promising transcription initiation and stop necessary cis-acting elements and be positioned at 5 of encoding gene ' with 3 '; Gene with three encoding gag, pol and env.These three gene gag, pol and env encode respectively inner virus structure, enzymatic albumen (such as intergrase) and envelope glycoprotein (called after gp70 and p15e), it gives the uncertain function of virus with infectivity and host range specificity and " R " peptide.

For with use the relevant security consideration of retrovirus (comprise and be used for expression construct), the clone and the preparing carriers sexual cell of having developed independent packing are.In brief, this method is to utilize the purposes of two kinds of components (retroviral vector and package cell line (PCL)).Retroviral vector contains long terminal repetition (LTR), foreign DNA and packaging sequence (y) to be transferred.This retroviral vector because of the gene of coding structure and envelope protein is not included in can't be in the vector gene group by autosynthesis.PCL contains the proteic gene of encoding gag, pol and env, but does not contain packaging signal " y ".Therefore, but PCL only self forms empty virus particle.In this general method, retroviral vector is to be introduced among the PCL, thereby forms preparing carriers sexual cell system (VCL).This VCL makes the virus particle of the alien gene group that only contains retroviral vector, and therefore formerly is regarded as safe retroviral vector and uses for therapeutic.

" retroviral vector construct body " is meant the molectron of the expression that can guide the one or more sequences paid close attention to or gene (such as the nucleotide sequence of coding multivalent binding proteins) in a preferred embodiment of the invention.In brief, the retroviral vector construct body must comprise 5 ' LTR, tRNA binding site, packaging signal, second chain DNA synthetic starting point and the 3 ' LTR.Can comprise multiple heterologous sequence in the vector construction body, comprise the sequence of proteins encoded for example (for example cytotoxic protein, disease-related antigen, immune accessory molecule or sub stituent because of) or sequence (for example as rnase or antisense sequences) itself as molecule.

Retroviral vector construct body of the present invention can be easy to come construction by multiple retrovirus, described retrovirus for example comprises that B, C and D type retrovirus and foamy virus and slow virus are (referring to for example RNA Tumor Viruses (RNA tumour virus), the 2nd edition, ColdSpring Harbor Laboratory, 1985).Described retrovirus is easily available from depository or preservation center, such as American type culture collection (" ATCC "; Rockville Maryland), or uses common available technology to be separated by known source and get.Known disclosure that is provided herein and standard recombinant technology, can be easy to utilize any above-mentioned retrovirus with assembling or make up retroviral vector construct body of the present invention, packing cell or producer's cell (people such as Sambrook for example, Molecular Cloning:A Laboratory Manual (molecular cloning: lab guide), the 2nd edition, Cold Spring Harbor Laboratory Press, 1989; Kunkle, 1985 Proc.Natl.Acad.Sci. (USA) 82:488).

Be applicable to that the promotor in the virus vector can include but not limited to usually: retrovirus LTR; The SV40 promotor; And human cytomegalovirus (CMV) promotor (is described in people such as Miller, among the 1989 Biotechniques 7:980-990) or other any promotors (cellularity promotor for example, such as the eukaryotic cell promotor, include but not limited to histone, polIII and beta-actin promotor).Spendable other viral promotors include but not limited to adenovirus promoter, thymidine kinase (TK) promotor and B19 small virus promotor.For those skilled in the art, the selection of suitable promotor will be apparent via contained herein instruction, and optional inherent regulation promotor or aforesaid promotor.

Use the transducible package cell line of retroviral plasmid vector to form producer's clone.Can include but not limited to as Miller through the example of the packing cell of transfection, Human GeneTherapy (people's gene treatment), PE501, the PA317 described in the 1:5-14 (1990), ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψ CRE, ψ CRIP, GP+E-86, GP+envAm12 and DAN clone.Carrier can be via any way as known in the art packing cell of transduceing.Described mode includes but not limited to electroporation, uses liposome and CaPO ₄Precipitation.In a selection, retroviral plasmid vector can be encapsulated in the liposome, or with the lipid coupling, and then give the host.

Producer's clone produces infectious retroviral vector particle, and described particle comprises that coding has the nucleotide sequence of the multivalent binding proteins of effector function.Described retroviral vector particle then can be used for external or the interior transduction of body eukaryotic cell.To express the nucleotide sequence of this albumen of coding or polypeptide through the eukaryotic cell of transduction.Can include but not limited to lymphocyte and reticuloendothelial cell, keratinocyte, endotheliocyte and the bronchial epithelial cell of embryonic stem cell and hemopoietic stem cell, liver cell, fibroblast, circulation peripheral blood lymphocytes and polymorphonuclear cell (comprising myelomonocyte), lymphocyte, myoblast, tissue macrophages, dendritic cell, Kupffer cell (Kupffer cell), lymphoglandula and spleen through the eukaryotic cell of transduction.

Host cell

Another aspect of the present invention provides a kind of host cell through any polynucleotide of the present invention or clone/expression construct conversion or transfection, or contains the host cell of any polynucleotide of the present invention or clone/expression construct.Polynucleotide and clone/expression construct can be utilized any method as known in the art (comprising conversion, transfection and transduction) and be introduced in the suitable cell.Host cell comprises the cell of the individuality of experience isolated cells therapy (comprising the gene therapy that for example exsomatizes).The eukaryotic host cell that an aspect of of the present present invention contained has polynucleotide of the present invention, when carrier or albumen, except that the cell that comprises intrasubject (for example cell of human patients self), also comprise the VERO cell, HeLa cell (HeLa cell), Chinese hamster ovary (CHO) clone (the modified Chinese hamster ovary celI that comprises the glycosylation pattern that to modify expressed multivalence binding molecule, referring to laid-open U.S. Patents application the 2003/0115614th A1 number, it is incorporated herein with way of reference), COS cell (such as COS-7), W138, BHK, HepG2,3T3, RIN, MDCK, A549, PC12, K562, the HEK293 cell, the HepG2 cell, the N cell, the 3T3 cell, frugiperda cell (for example Sf9 cell) is coveted on the meadow, brewing yeast cell and any other eukaryotic cell of expressing and randomly separating albumen of the present invention or peptide that is applicable to as known in the art.Also contain prokaryotic cell prokaryocyte, include but not limited to intestinal bacteria (Escherichia coli), Bacillus subtilus (Bacillus subtilis), salmonella typhimurium (Salmonella typhimurium), streptomycete (Streptomycete) or any prokaryotic cell prokaryocyte that is suitable for expressing and randomly separating albumen of the present invention or peptide as known in the art.In prokaryotic cell prokaryocyte protein isolate or peptide, especially contain and to use as known in the art being used for from the technology of inclusion body extracting protein.By herein instruction as can be known, suitably host's selection is in those skilled in the art's technology category.

The through engineering approaches host cell can be incubated in the conventional substratum, and this substratum is modified to be fit to the activation promotor, to select transformant or amplification specific gene.The culture condition of the particular host cell through selecting to be used to express such as temperature, pH value and conditions of similarity thereof, is conspicuous for general those skilled in the art.Also can use various mammalian cell culture systems to come express recombinant protein.The example of mammalian expression system comprises that the COS-7 system of monkey kidney fibroblast is (by Gluzman, 1981 Cell 23:175 describe) and can express other clones of consistency carrier, for example C127,3T3, CHO, hela cell line and bhk cell are.Mammalian expression vector will comprise replication orgin, suitable promotor and optional enhanser, and any essential ribosome bind site, polyadenylation site, donor splicing site and acceptor site, transcription termination sequence and 5 ' flank non-transcribed sequence, for example as herein about as described in the preparation of multivalent binding proteins expression construct.Be derived from the dna sequence dna of SV40 montage and polyadenylation site and can be used for the non-transcribed genetic elements that provides required.Construct imports in the host cell and can realize by several different methods well known to those skilled in the art, described method includes but not limited to calcium phosphate transfection method, DEAE-dextran mediation infection protocol or electroporation people such as (, 1986 Basic Methods in Molecular Biology (molecular biology basic skills)) Davis.

In one embodiment, host cell is transduceed by the recombinant virus construct that guides albumen of the present invention or polypeptide expression.Host cell through transduction produces the virion that contains through expressed proteins or polypeptide, and this is the part that is derived from the host cell membrane that merges through virion between the virus budding period through expressed proteins or polypeptide.

Composition pharmaceutically

In certain embodiments, composition of the present invention (such as comprising the coding multivalent binding proteins or the composition of this proteic polynucleotide as described herein) be suitable for be enough to allow coded albumen in host cell in body or under the condition of vivoexpression and time administration to be used for gene therapy and homoeopathy thereof.Described composition can be configured to the composition pharmaceutically according to the well-known process administration.Composition pharmaceutically comprises expression product and pharmaceutically acceptable carrier, vehicle or the thinner of one or more recombinant expression construct body and/or described construct usually.Described carrier should be nontoxic to the receptor under used dosage and concentration.For based on the preparation of nucleic acid or for the preparation that comprises expression product of the present invention, can by intracutaneous for example, subcutaneous, intramuscular or intravenous route or by as known in the art under one group of given environment suitable any approach, per kilogram of body weight gives about 0.01 μ g extremely about 100mg.Preferred dose for example is extremely about 1mg/kg of about 1 μ g/kg, is preferably about 5 μ g/kg especially to about 200 μ g/kg.

It will be apparent to those skilled in the art that administration number of times and frequency will decide on host's reaction.The pharmaceutically acceptable carrier that is suitable for therapeutic use has been well known in the drug technique, and be described in for example Remingtons Pharmaceutical Sciences (Lei Mingdun pharmaceutical science), among the Mack Publishing Co. (A.R.Gennaro compiles, 1985).For example, can use Sterile Saline and phosphate buffered saline (PBS) under the physiology pH value.Can provide sanitas, stablizer, dyestuff and analogue thereof in the composition pharmaceutically.For example, can add the ester of Sodium Benzoate, Sorbic Acid and P-hydroxybenzoic acid as sanitas.The same, at the 1449th page.In addition, can use antioxidant and suspension agent.The same.Compound of the present invention can free alkali or salt form use, two kinds of forms all are considered as belonging to category of the present invention.

Contain one or more nucleic acid construct of the present invention or can be and allow that composition gives any form of patient corresponding to proteic composition pharmaceutically by the coded product of described nucleic acid construct.For example, composition can be the form of solid, liquid or gas (aerosol).Typical route of administration includes but not limited in per os, part, parenteral (for example through the hypogloeeis or through cheek), hypogloeeis, rectum, vagina and the nose.The term parenteral comprises subcutaneous injection, intravenously, intramuscular as used herein, breastbone is interior, cavernous body is interior, sheath is interior, duct is interior, urethra is interior injects or infusion techniques.Composition pharmaceutically can be prepared so that wherein contained active ingredient can be by biological utilisation after giving the patient with said composition.The composition that gives the patient can be taked the form of one or more dose unit, and wherein for example lozenge can be single dose unit, and the container of one or more compound of the present invention of aerosol form can have most dose units.

For oral administration, can there be vehicle and/or tackiness agent.Example is sucrose, kaolin, glycerine, amylodextrin, sodium alginate, carboxymethyl cellulose and ethyl cellulose.Can exist toner and/or seasonings.Can use the dressing shell.

Composition can be liquid form, for example elixir, syrup, solution, emulsion or suspension.Lift two examples, liquid can or pass through injected delivery for oral administration.When being intended to oral administration, preferred composition also contains one in sweeting agent, sanitas, dyestuff/tinting material and the sweetener or many persons except that containing one or more integrated structure domain-immunoglobulin fusion constructs or expression product.In the composition that is intended to by drug administration by injection, can comprise one in interfacial agent, sanitas, wetting agent, dispersion agent, suspension agent, buffer reagent, stablizer and the isotonic agent or many persons.

Pharmaceutically composition of liquid (no matter solution, form of suspension or other similar type) can comprise one in the following adjuvant or many persons as used herein: sterile diluent, such as water for injection, salt brine solution (being preferably physiological saline), Ringer's solution (Ringer ' s solution), isotonicity sodium-chlor, non-volatility oils (such as the synthetic direactive glyceride that can be used as solvent or suspension medium or two glyceryl ester, polyoxyethylene glycol, glycerine, propylene glycol or other solvents); Antiseptic-germicide is such as benzylalcohol or methyl p-hydroxybenzoate; Antioxidant is such as xitix or sodium bisulfite; Sequestrant is such as ethylenediamine tetraacetic acid (EDTA); Buffer reagent such as acetate, Citrate trianion or phosphoric acid salt, and is used to adjust the tensile medicament, such as sodium-chlor or glucose.Parenteral administration can be packaged in the peace bottle of being made by glass or plastics, deserted syringe or the multiple dose vials.Physiological saline is preferred adjuvant.Injectable composition pharmaceutically is preferably aseptic.

Also need in the preparation to comprise other components,, include but not limited to aluminium salt, water-in-oil emulsion, biodegradability oil vehicle, O/w emulsion, biodegradability microcapsule and liposome such as delivery vehicle.The example that is used for the immunostimulating material (adjuvant) of described vehicle comprises N-ethanoyl muramyl-L-L-Ala-D-isoglutamine (MDP), lipopolysaccharide (LPS), dextran, IL-12, GM-CSF, IFN-and IL-15.

Although can use the known any suitable carrier of those of ordinary skills in the composition pharmaceutically of the present invention, whether bearer type will be looked mode of administration and need continue to discharge and change.For administered parenterally (such as subcutaneous injection), the carrier preferred package is moisture, salt solution, alcohol, fat, wax or buffer reagent.For oral administration, can use above-mentioned any carrier or solid carrier, such as mannitol, lactose, starch, Magnesium Stearate, soluble saccharin, talcum powder, Mierocrystalline cellulose, glucose, sucrose and magnesiumcarbonate.Biodegradability microsphere (for example poly(lactic acid) galactoside) also can be used as the carrier of composition pharmaceutically of the present invention.Suitable biodegradability microsphere is for example to be disclosed in the United States Patent (USP) the 4th, 897, No. 268 and the 5th, 075, No. 109.Thus, microsphere is preferably greater than about 25 microns.

Composition pharmaceutically also can contain thinner (such as buffer reagent), antioxidant (such as xitix), lower molecular weight (being less than about 10 residues) polypeptide, albumen, amino acid, carbohydrate (for example glucose, sucrose or dextrin), sequestrant (for example EDTA), gsh and other stablizers and vehicle.Neutral buffered saline inclusive NAND specific serum albumin blended salt solution is exemplary suitable thinner.Preferably, use suitable vehicle solution (for example sucrose) product to be formulated as lyophilized products as thinner.

(comprise the technology that is disclosed in laid-open U.S. Patents application the 2006/0008415th A1 number according to technology as known in the art, it is incorporated herein with way of reference), composition pharmaceutically of the present invention also comprises stabilize proteins and stabilising liq pharmaceutical preparation.Described technology comprises proteic derivatize, and wherein this albumen comprises the thiol group with N-ethanoyl-L-halfcystine, N-ethyl-maleimide or halfcystine coupling.

As mentioned above, the present invention includes the composition of the nucleic acid molecule of the multivalent binding proteins that can send encodes has effector function.Described composition comprises recombinant viral vector, for example retrovirus (referring to WO 90/07936, WO 91/02805, WO 93/25234, WO 93/25698 and WO 94/03622), adenovirus (referring to Berkner, 1988 Biotechniques 6:616-627; People such as Li, 1993 Hum.Gene Ther.4:403-409; People such as Vincent, people such as Nat.Genet.5:130-134 and Kolls, 1994 Proc.Natl.Acad.Sci.USA 91:215-219), poxvirus is (referring to United States Patent (USP) the 4th, 769, No. 330, United States Patent (USP) the 5th, 017, No. 487 and WO89/01973)), with polycation type molecule compound recombinant expression construct body nucleic acid molecule (referring to WO 93/03709) and with liposome bonded nucleic acid (referring to people such as Wang, 1987 Proc.Natl.Acad.Sci.USA 84:7851).In certain embodiments, DNA can be connected with the adenovirus of deactivation or inactivation (referring to people such as Curiel, 1992 Hum.Gene Ther.3:147-154; People such as Cotton, 1992 Proc.Natl.Acad.Sci.USA 89:6094).Other suitable compositions comprise the DNA-part (referring to people such as Wu, 1989J.Biol.Chem.264:16985-16987) and lipid-DNA make up (referring to people such as Felgner, 1989 Proc.Natl.Acad.Sci.USA 84:7413-7417).

Except that the guide body internal program, also can use stripped program, wherein cell is removed, modifies from host's (for example individual, such as human patients) and place same or another host animal.Obviously, in the environment that exsomatizes, can use above-mentioned any composition that construct of the present invention (albumen/polypeptide or its nucleic acid of encoding) is introduced in the histocyte.The experimental program of virus, physics and chemical acquisition method is well known in this area.

The formation of antibody

By means of repeatedly subcutaneous or peritoneal injection antigenic peptide or its fragment and adjuvant, usually can in animal (for example rabbit, hamster, goat, sheep, horse, pig, rat, gerbil jird, cavy, mouse or any other suitable Mammals, and other nonmammalian kinds), produce polyclonal antibody at antigenic peptide.Adjuvant includes but not limited to Fu Shi complete or Freund (complete or incomplete Freund ' s adjuvant), mineral substance gel (such as aluminium hydroxide) and surfactant, such as lysolecithin, general stream Buddhist nun gram (pluronic) polyvalent alcohol, polyanion, peptide, fat liquor and dinitrophenol(DNP).BCG (bacille Calmette-Guerin vaccine (bacilli Calmette-Guerin)) and CBP also are potential suitable adjuvant.It is applicable to making antigenic peptide and having immunogenic carrier proteins and combine in the species for the treatment of immunity; Typical carriers comprises keyhole spiral shell hemocyanin, serum albumin, bovine thyroglobulin or Trypsin inhibitor SBTI.In addition, can use aggregating agent prepared therefrom to react with enhancing immunity such as alum.After the immunity, with the animal bloodletting and utilize routine techniques to measure anti--antigenic peptide antibody titer of serum.Polyclonal antibody can be used for serum, in serum, detect polyclonal antibody, for example maybe can utilize the antigen affinity chromatography polyclonal antibody purifying in serum.

Can utilize any method to prepare at the monoclonal antibody of antigenic peptide by the generation of the continuous cell line in culture antibody molecule.For example, monoclonal antibody can be by as people such as Kohler, Nature 256:495[1975] described in hybridoma method; Human B-quadroma technology (people such as Kosbor, Immunol Today 4:72,1983; People such as Cote, Proc Natl AcadSci 80:2026-2030,1983) and EBV-hybridoma technology (people such as Cole, MonoclonalAntibodies and Cancer Therapy, Alan R Liss Inc, New York N.Y., the 77-96 page or leaf, (1985)) prepare.

When using hybridoma technology, can use myeloma cell line.Preferably, be applicable to that the clone in the fusion program that produces hybridoma does not produce endogenous antibody, have high fusion efficiencies, and show enzymatic defect, thereby it can not be grown in only supporting some selective medium that institute's fused cell of wanting (hybridoma) is grown.For example, be mouse as if animal through immunity, then can use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 41, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG1.7 and S194/5XX0 Bul; If rat then can be used R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; And U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 all can use in conjunction with cytogamy.

In an alternate embodiment, human antibodies can via phage show the library prepare (people such as Hoogenboom, J.Mol.Biol.227:381[1991]; People such as Marks, J.Mol.Biol.222:581; Also referring to United States Patent (USP) the 5th, 885, No. 793).Described method is simulated immunoselection via following steps: show whole antibody on the filobactivirus surface, and select phage according to it with selected antigenic the combination subsequently.A kind of this technology is that the PCT that is described in people's such as Adams name application applies in PCT/US98/17364 number, and its description utilizes this method to separate the high-affinity and the functional short effect antibody of MPL-acceptor and msk-acceptor.In the method, the complete list of human antibody gene can form (people such as Mullinax, Proc.Natl.Acad.Sci. (USA) 87:8095-8099[1990]) by the human V gene of the natural rearrangement of clone in the described peripheral blood lymphocyte before freely.

Perhaps, complete whole synthetic human heavy chain can by with each human VH section with the D section of random nucleotide together with human J section makes up by form (people such as Hoogenboom, J.Mol.Biol.227:381-388[1992]) without the V constant gene segment C of resetting.Equally, all light chains can make up (people such as Griffiths, EMBO J.13:3245-3260[1994]) by each human V section and J section are merged.The Nucleotide of complete antibody (that is heavy chain and light chain) of will encoding is connected to strand Fv fragment, and the Nucleotide of this polynucleotide with the less important coat protein of coding filobactivirus is engaged.When this fusion rotein was expressed on phage surface, the polynucleotide of coding specific antibody can be selected to differentiate by using immobilized antigen.

Except that the typical method that forms polyclone and monoclonal antibody, also contain any method that is used to form any known antibodies form.It is external to remove polyclone and monoclonal anti, and antibody formation also comprises chimeric antibody, humanized antibody, CDR-grafted antibody and antibody fragment and varient.

The varient of specific-binding agent and derivative

In one embodiment, provide the insertion varient, wherein one or more amino-acid residue is augmented the specific-binding agent aminoacid sequence.Insertion can be positioned at proteic arbitrary end or two ends, maybe can be arranged in the inner area of specific-binding agent aminoacid sequence.Varient product of the present invention also comprises ripe specific-binding agent product, that is leader sequence or signal sequence removes and gained albumen has the specific-binding agent product of other n terminal residues wherein.Other n terminal residues can be derived from another kind of albumen can comprise that maybe one or more is because of being derived from the residue that specific proteins can't be differentiated.Be encompassed in the polypeptide (the Met-1-multivalence binding peptide that for example has effector function) that position-1 has other methionine residues, also be included in the polypeptide of the present invention (Met-2-Lys-1-multivalent binding proteins) that position-2 and position-1 has other methionine(Met)s and lysine residue with effector function.Varient with polypeptide of the present invention of other Met, Met-Lys or Lys residue (or generally speaking being one or more alkaline residue) is particularly useful for enhancing and produces recombinant protein in bacterial host cell.

The present invention also comprises the specific polypeptide of the present invention with other amino-acid residues because of using specific expressed system to produce.For example, use to express as the commercial carrier of the polypeptide of wanting of the part of glutathione-S-transferase (GST) fusion product can the GST component and the polypeptide of wanting be provided at the polypeptide of wanting that position-1 has other glycine residues after cutting.Also be encompassed in the varient that produces via expression in other carrier systems, comprise that Histidine mark wherein incorporates those varients in the aminoacid sequence at the C-terminal of sequence and/or N-terminal usually.

In another aspect, the invention provides the deletion mutation body, one or more amino-acid residue in the polypeptide wherein of the present invention is removed.Disappearance can realize at an end or two ends of polypeptide, or realize via removing of one or more residue in the aminoacid sequence.The deletion mutation body must comprise all fragments of polypeptide of the present invention.

Antibody fragment is meant the polypeptide of sequence to small part that has corresponding in the immunoglobulin variable domain sequence.Fragment for example can form by enzymatic lysis or the chemical cracking polypeptide corresponding to full length antibody.Other binding fragments comprise by synthetic technology or those fragments of forming by recombinant DNA technology (recombinant plasmid that contains the nucleotide sequence of encoding part antibody variable region such as expression).Preferred polypeptide fragment presents the peculiar or specific immunological characteristic of target as described herein.Fragment of the present invention with the immunological characteristic of wanting can be by knowing in this area and any method of conventional practice prepares.

In aspect another, the invention provides multivalence with effector function replacement varient in conjunction with polypeptide.Replace that varient comprises that one or more amino-acid residue in the aminoacid sequence wherein is removed and through substituting residue metathetical those polypeptides.In certain embodiments, described being substituted in is conservative in nature; Be the replacement of non-conservation yet also the present invention includes.Amino acid can according to physical property and to secondary and three grades of protein structures be used for classify.In the art, the conservative property replacement can be regarded as another amino acid that a kind of aminoacid replacement has similar characteristics.Enumerate exemplary conservative property in the Table A and replace (referring to 97/09433, the 10 page of WO, on March 13rd, 1997 open (PCT/GB96/02197 submitted on June 9th, 96)), be right after as follows.

Table A

Perhaps, as cited in table B, conservative amino acid can be as Lehninger, [Biochemistry, second edition; Worth Publishers, Inc.NY:NY (1975), 71-77 page or leaf] described in divide into groups, be right after as follows.

Table, B:

Conservative property replaces II

Side chain feature amino acid

Nonpolar (hydrophobicity)

A. aliphatic: ALIVP

B. aromatics: FW

C. sulfur-bearing: M

D. border: G

Neutral-polarity

A. hydroxyl: STY

B. acid amides: NQ

C. sulfhedryl: C

D. border: G

Positively charged (alkalescence) KRH

Electronegative (acidity) DE

The present invention also provides the derivative of specific-binding agent polypeptide.Derivative comprises the specific-binding agent polypeptide of the modification insertion, disappearance or the replacement that has except that amino-acid residue.Preferably, described modification has covalency character, and for example comprises the chemical bonding with polymkeric substance, lipid, other organic moiety and inorganic part.Derivative of the present invention can be produced to increase the circulating half-life of specific-binding agent polypeptide, maybe can be designed to improve the ability of polypeptide target to want cell, tissue or organ.

The present invention further comprises as United States Patent (USP) the 4th, 640 No. 835, the 4th, 496, No. 689, the 4th, 301, No. 144, the 4th, 670, No. 417, the 4th, 791, No. 192 and the 4th, described in 179, No. 337, through covalent modification or derivatize to comprise the multivalent binding proteins with effector function of one or more water-soluble polymers connector (such as polyoxyethylene glycol, polyoxyethylene glycol or polypropylene glycol).Other suitable polymers as known in the art comprise mono methoxy polyethylene glycol, dextran, Mierocrystalline cellulose and other polymkeric substance based on carbohydrate, poly-(N-vinyl pyrrole pyridine ketone)-polyoxyethylene glycol, propylene glycol homopolymer, polyoxytrimethylene/ethylene oxide copolymer, polyoxyethylene polyvalent alcohol (for example glycerine) and polyvinyl alcohol, and described mixture of polymers.Especially be preferably polyoxyethylene glycol (PEG) derivatize albumen.Water-soluble polymers can be bonded to specific position, for example is bonded to the N-terminal of albumen of the present invention and polypeptide, or is connected at random with one or more side chain of polypeptide.The purposes that PEG is used for the improved treatment ability is to be described in No. the 6th, 133,426, the United States Patent (USP) giving people such as Gonzales.

The target spot of immunoglobulin (Ig) mutagenesis

Some strategy is available for controlling the natural characteristics of antigen specific immune sphaeroprotein (for example antibody), and described strategy is invalid to the binding molecule based on NIg.The good example of for example supporting molecule based on antibody to be better than described surrogate in the described strategy is, regulate the affinity of antibody to its target in vivo via the affinity maturation, it is to utilize the somatic hypermutation of immunoglobulin gene to produce the antibody that affinity increases with the immune response progress.In addition, the structure of change immunoglobulin (Ig) and the recombinant technology of immunoglobulin domain and structural domain have been developed.Therefore, can prepare specifying antigen to show the polypeptide that is derived from antibody of affinity, and knownly in this area multiplely be used to differentiate and purifying or separate the purification scheme and the monitoring screen of described polypeptide through changing.Use described known technology, can obtain antigen is shown the affinity that reduces or increase, the polypeptide that comprises the binding domains that is derived from antibody.The strategy of polypeptide variants that is used for producing the affinity of display change comprises: the DNA to encoding antibody uses site-directed mutagenesis or random mutagenesis to change the existing amino acid of albumen, then be the screening step, this step will change the antibody variation body of (for example, increasing or the affinity of minimizing with respect to not modified parental antibody or with reference to antibody) reclaim to show through design.

In the mutagenesis strategy for a change affinity the amino-acid residue of normal target be those residues in the light chain of antibody and complementarity-determining region of variable region of heavy chain (CDR) or the super variable region.Steric other amino acid that carry out the interactional residue of materialization and influence described residue with antigen are contained in described district.Yet, also proved that the antigen binding characteristic of the amino acid antagonist in the CDR district variable domains framework region in addition has substantial role, and can be through target to control described characteristic.Referring to Hudson, P.J.Curr.Opin.Biotech., 9:395-402 (1999) and reference wherein.

Littler and more effective screening library of antibody variation body can be by preparing site restriction random mutagenesis or the site-directed mutagenesis corresponding to the zone of tending to " super sudden change " during somatocyte affinity ripening process among the CDR.Referring to people such as Chowdhury, Nature Biotech., 17:568-572 (1999) and reference wherein.The known type that limits the DNA element in super mutational site in this way comprises repetition in the same way and oppositely repetition, some consensus sequence, secondary structure and palindromic sequence.Total dna sequence dna comprises four base sequences purine-G-pyrimidine-A/T (that is A or G-G-C or T-A or T) and Serine codon AGY (wherein Y can be C or T).

Therefore, another aspect of the invention is one group of mutagenesis strategy that is used for modified antibodies to the affinity of its target.Described strategy comprises the mutagenesis of the whole variable region of heavy chain and/or light chain, the only interior mutagenesis, the mutagenesis of framework region or any combination (" mutagenesis " can be random mutagenesis or site-directed mutagenesis in the present context) of described method that have super mutational site of mutagenesis, CDR in CDR district.Solve described antibody and antibody via technology well known by persons skilled in the art (such as the x-ray crystal analysis method): thus the structure of ligand complex can be finished clearly describing of CDR district reached discriminating to the residue of the binding site that comprises antibody.Based on known to those skilled in the art to the whole bag of tricks of the analysis of described antibody crystals structure and sign and can be used for district near CDR.The example of described common method comprises Kabat, Chothia, AbM and contact definition.

The Kabat definition is based on the sequence mutability and is the most frequently used definition in prediction CDR district.People such as Johnson, Nucleic Acids Research, 28:214-8 (2000).The Chothia definition is based on the position in structural ring district.(people such as Chothia, J.Mol.Biol., 196:901-17[1986]; People such as Chothia, Nature, 342:877-83[1989]).The AbM definition is to take into account between Kabat and Chothia definition.AbM be Oxford University's group of molecules (Oxford MolecularGroup) a whole set of program that is used for the antibody structure modeling of being produced (people such as Martin, Proc.Natl.Acad.Sci (USA) 86:9268-9272[1989]; People such as Rees, ABMTM (computer program that is used for the antibody variable region modeling), Oxford, UK; Oxford Molecular, Ltd.).The AbM package is to utilize the combination of knowledge data base and the method that starts anew by the tertiary structure modeling of primary sequence antagonist.Introduce another kind of definition recently, be called the contact definition.Referring to people such as MacCallum, J.Mol.Biol., 5:732-45 (1996).This definition is based on the analysis to obtainable compound crystal structure.

According to convention, the CDR structural domain in the heavy chain is commonly referred to H1, H2 and H3, and the order number consecutively that moves to C-terminal according to N-terminal.The order number consecutively that CDR district in the light chain is commonly referred to L1, L2 and L3 and moves to C-terminal according to N-terminal.

CDR-H1 is long for about 10 to 12 residues and according to Chothia and AbM definition, starts from the 4th residue behind the Cys usually, or definition starts from thereafter the 5th residue usually according to Kabat.Be generally Trp after the H1, be generally Trp-Val, and can be Trp-Ile or Trp-Ala.According to the AbM definition, the length of H1 is about 10 to 12 residues, and the Chothia definition does not comprise 4 last residues.

According to Kabat and AbM definition, CDR-H2 starts from the 15th residue behind the H1 end usually.Residue before the H2 is generally Leu-Glu-Trp-Ile-Gly, but can have multiple variation.Be generally aminoacid sequence Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala after the H2.According to the Kabat definition, the length of H2 is about 16 to 19 residues, and wherein AbM definition prediction length is generally 9 to 12 residues.

CDR-H3 starts from the 33rd residue behind the H2 end usually, and usually after aminoacid sequence Cys-Ala-Arg.Be generally amino acid Gly after the H3.The scope of H3 length is 3 to 25 residues.

CDR-L1 starts from about residue 24 and usually usually after Cys.Residue after the CDR-L1 is generally Trp and begins with person one of in the following sequence usually: Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln or Trp-Tyr-Leu.The length of CDR-L1 is about 10 to 17 residues.

CDR-L2 starts from about 16 residues behind the L1 end.It is usually after following residue: Ile-Tyr, Val-Tyr, Ile-Lys or Ile-Phe.The length of CDR-L2 is about 7 residues.

CDR-L3 start from usually behind the L2 end the 33rd residue and usually after Cys.Be generally aminoacid sequence Phe-Gly-XXX-Gly after the L3.The length of L3 is about 7 to 11 residues.

The whole bag of tricks that is used for modified antibodies has been described in this area, comprise for example preparation method of humanized antibody, wherein the sequence of the sequence of Humanized immunoglobulin variable region of heavy chain framework and donor immunity sphaeroprotein variable region of heavy chain framework has 65% to 95% consistence.Each humanization immunoglobulin chain is except that comprising CDR, usually also comprise amino acid from donor immunity sphaeroprotein framework, described amino acid for example can interact realizing binding affinity with CDR, such as one or more amino acid that is close to CDR in the donor immunity sphaeroprotein or about 3 dusts (angstrom) with those interior amino acid (as being predicted) by the molecule modeling.Heavy chain and light chain can be separately by using any or all standards in the various localization criterias to design.When being combined into complete antibody, Humanized immunoglobulin has non-immunogenic substantially and antigen (such as the albumen that contains epi-position or other compounds) is kept the affinity identical substantially with the donor immunity sphaeroprotein in the mankind.

In one embodiment, description has similar binding specificity with parental antibody, but has the preparation method of the antibody and the antibody fragment of enhanced human characteristic.Humanized antibody is to replace method and comprise by the chain that utilizes phage display technology for example the specific non-human antibody's of antigen tool that paid close attention to the heavy chain or the polypeptide of variable region of light chain are obtained, then with itself and all human complementary (light chain or heavy chain) chain variable regions combinations.Discriminating is right to the specific hybridization of being paid close attention to of antigen tool, and with the human chain of selected centering and all human complementary variable domains (heavy chain or light chain) combinations.In another embodiment, with among the CDR among non-human antibody's component and the CDR from whole integral parts combinations of human antibodies.Select crossbred from the dimeric gained of antibody polypeptides library, and it can be used in the second humanization replacement step; Perhaps, if this crossbred has had enough human characteristic and had therapeutic value, then get rid of this second step.The modifying method that strengthens human characteristic is known in this area.

Another embodiment is a kind of method that is prepared as follows humanized antibody: replace corresponding human cdr amino acid sequence and/or replace corresponding human FR aminoacid sequence with the FR aminoacid sequence with the cdr amino acid sequence.

Another embodiment is provided for differentiating the method for amino-acid residue that can be modified in the antibody variable territory, and described method does not weaken the natural affinity of antigen binding domains in its immunogenic while with respect to the allos species of minimizing; And the described method that is suitable for giving the modified antibody variable region of allos species of preparation.

Can be designed to reach the antigen binding affinity that increases or reduce to the modification of immunoglobulin (Ig) (such as antibody) and/or to reduce antibody by any method as known in the art in the intravital immunogenicity of receptor and/or to regulate the effect level of activity.In one approach, thus humanized antibody can be modified to get rid of glycosylation site and strengthened antibody the affinity of its isogeneic (people such as Co, Mol.Immunol.30:1361-1367[1993]).The technology that reaches " facial ornament/resurfacing " such as " reconstruct ", " super chimeric " has prepared the humanized antibody with bigger treatment potential.People such as Vaswami, Annals of Allergy, Asthma ， ﹠amp; Immunol 81:105 (1998); People such as Roguska, Prot.Engineer.9:895-904 (1996)].Also referring to United States Patent (USP) the 6th, 072, No. 035, it describes the antibody reconstructing method.Although described technology weakens antibody mediated immunity originality by the number that reduces external residue, after the antibody repeat administration, it can't stop anti-genotype reaction and the reaction of anti-allotype.The alternative method that is used to reduce immunogenic described method is to be described in people such as Gilliland, among J.Immunol.62 (6): the 3663-71 (1999).

Under many circumstances, humanized antibody causes antigen binding capacity to descend.Therefore, preferably make humanized antibody " reverse mutation " to comprise existing amino-acid residue in one or more initial (the most normal is rodent) antibody, to attempt to recover the binding affinity of antibody.Referring to people such as for example Saldanha, Mol.Immunol.36:709-19 (1999).

The glycosylation that has proved immunoglobulin (Ig) can influence effector function, structural stability and from antibody producing cells excretory speed (referring to people such as Leatherbarrow, Mol.Immunol.22:407 (1985), the document is incorporated herein with way of reference).Cause the carbohydrate group of described characteristic to be connected with the constant region of antibody usually.For example, at C _H2Asn297 place in the structural domain can impel the whole abilities of IgG complement activation dependent cell dissolved (people such as Tao, J.Immunol.143:2595 (1989)) with the IgG glycosylation.At C _H3Asn 402 places in the structural domain for example can facilitate the IgM glycosylation the suitable assembling and the cell lysis activity (people such as Muraoka, J.Immunol.142:695 (1989)) of antibody.C at IgA antibody _H1And C _H3Position 162 in the structural domain and 419 places remove glycosylation site and cause in the cell degraded and to excretory at least 90% restraining effect people such as (, Wall, Mol.Cell.Biol.8:4197 (1988)) Taylor.Therefore, molecule of the present invention comprises the immunoglobulin (Ig) that changes through the sudden change mode, and described immunoglobulin (Ig) is because of the glycosylation pattern of display change of undergoing mutation of the specificity residue in the constant subprovince for example, thereby changes effector function.Referring to people such as Co, Mol.Immunol.30:1361-1367 (1993), people such as Jacquemon, J.Thromb.Haemost.4:1047-1055 (2006), people such as Schuster, Cancer Res.65:7934-7941 (2005) reaches people such as Warnock, Biotechnol Bioeng.92:831-842 (2005), each document is incorporated herein with way of reference.

The present invention also comprises the multivalence binding molecule with at least one binding domains, this binding domains has at least 80%, preferred 90% or 95% or 99% consistence and has the residue that at least one is different from this immune globulin variable region with known immunoglobulin variable domain sequence on sequence, and the residue that is wherein changed increases glycosylation site, changes the position of one or more glycosylation site or preferably remove glycosylation site with respect to immune globulin variable region.In certain embodiments, this variation removes the N-connection glycosylation site in the immune globulin variable region framework or removes and is present in the immunoglobulin heavy chain variable region framework, (use people such as Co at about amino-acid residue 65 to about amino-acid residue 85, J.Immunol.148:1149, the numbering of (1992) regulation) N-in the zone of scope connects glycosylation site.

Contain any method that is used to prepare with respect to the multivalence binding molecule of the glycosylation pattern of immunoglobulin (Ig) canonical sequence display change as known in the art.For example, can utilize any technology in the multiple genetic technique to change one or more specific residue.Perhaps, be used to the glycosylation pattern that the host cell for preparing can change with preparation through through engineering approaches.For example, a kind of method as known in the art provides the glycosylation of change with the non-fucosylated varient form of separable type that strengthens ADCC.Described varient is because of producing at the host cell expression that contains the oligosaccharides modifying enzyme.Perhaps, contain the Potelligent technology of BioWa/Kyowa Hakko to reduce the Fucose content in the glycosylation molecule of the present invention.In a kind of currently known methods, provide via producing the CHO host cell that is used for the recombination immunoglobulin preparation that the GDP-Fucose comes the glycosylation pattern in modified immunoglobulin FC district.This technology can be used for modifying the glycosylation pattern of the constant subprovince of multivalence binding molecule of the present invention.

Except that the binding characteristic of modifying binding domains (such as the binding domains of immunoglobulin (Ig)), with except that such as the humanized modification, the present invention also comprises by the residue that causes effector function (such as the effector function of constant subprovince) being changed or effector function is regulated in sudden change.Described modification can utilize any technology as known in the art to realize, such as people such as Presta, and the method that is disclosed among the Biochem.Soc.Trans.30:487-490 (2001), the document is incorporated herein with way of reference.Exemplary methods will comprise that the experimental programs that the people disclosed such as using Presta is to modify known the influence corresponding to the bonded specificity residue in one or more constant subprovince of FC γ RI, FC γ RII, FC γ RIII, FC α R and FC ε R.

In another approach, Xencor XmAb technology can be used for making the constant subprovince through engineering approaches corresponding to the FC structural domain to kill effector function to strengthen cell.Referring to people such as Lazar, Proc.Natl.Acad.Sci. (USA) 103 (11): 4005-4010 (2006), the document is incorporated herein with way of reference.For example, utilize this method to form and make F _CEffector function is killed thereby strengthen cell in the optimized constant subprovince of γ R specificity and associativity.

Preparation has the multivalent binding proteins of effector function

Can use the multiple expression vector/host system that contains and express multivalent binding proteins of the present invention (having effector function).Described system includes but not limited to microorganism, such as the bacterium that transforms through recombinant phage, plasmid, clay or other expression vectors; Yeast through yeast expression or shuttle vectors conversion; Insect cell system through virus expression carrier (for example baculovirus) infection; Through virus expression carrier (cauliflower mosaic virus (cauliflower mosaic virus for example; CaMV); Tobacco mosaic virus (TMV) (tobacco mosaic virus; TMV)) transfection or through bacillary expression vector (for example Ti or pBR322 plasmid) plant transformed cell system; Or zooblast system.The mammalian cell that is applicable to the preparation of reorganization multivalent binding proteins includes but not limited to: VERO cell, HeLa cell, Chinese hamster ovary (CHO) clone, COS cell (such as COS-7), W138, BHK, HepG2,3T3, RIN, MDCK, A549, PC12, K562 and HEK293 cell.The following herein exemplary experiment scheme of describing recombinant expressed multivalent binding proteins.

Expression vector can comprise transcriptional units, and this transcriptional units comprises following each person's combination: (1) has one or more genetic elements of regulating effect in genetic expression, for example promotor, enhanser or factor-specific binding site; (2) encoding transcription becomes mRNA and translates into the structure or the sequence of proteic wedding agent; And (3) suitable transcription initiation and terminator sequence.The structural unit of desiring to be used for yeast or eukaryotic expression system preferably includes the proteic leader sequence that host cell can exocytosis be translated.Perhaps, as if express recombinant multivalent binding proteins under the situation of no leading sequence or transit sequence, then it can comprise the N-terminal methionine residues.This residue subsequently can be from expressed recombinant protein cracking or not from its cracking so that final multivalent binding proteins to be provided.

For example, can use and be purchased expression system (pichia expression system (PichiaExpression System) (Invitrogen, San Diego, CA)) for example is according to manufacturer specification recombinant expressed multivalent binding proteins in yeast.Before this system also depends on-former-α sequence is with the guiding secretion, but transcribing of inset is to be driven by alcohol oxidase (AOX1) promotor under methanol induction.Secreted multivalence binding peptide is purifying in the yeast growth substratum by the following method, for example by being used for from method bacillary and mammalian cell supernatant liquor purified peptide.

Perhaps, the cDNA of coding multivalence binding peptide can be cloned into rhabdovirus expression vector pVL1393 (PharMingen, San Diego, CA) in.This carrier can use to infect greedy frugiperda cell in meadow and the preparation recombinant protein in the proteic substratum of no SF9 according to manufacturer specification (PharMingen).Multivalent binding proteins can use heparin-agarose post (Pharmacia, Piscataway, NJ) purifying and concentrated in substratum.The insect system (such as the SF9 system) that is used for protein expression is well known to those skilled in the art.In a kind of this system, Autographa californica multicapsid nucleopolyhedrosisvirus NPV (Autographa californica nuclear polyhedrosis virus; AcNPV) can be used as the carrier of coveting in the frugiperda cell on the meadow or in cabbage looper larva (Trichoplusialarvae), expressing alien gene.Multivalence binding peptide encoding sequence can be cloned into virus nonessential region (such as polyhedron gene) and under the control of polyhedrin promotor, settle.The successful insertion of multivalence binding peptide will make polyhedron gene have nonactive and produce the recombinant virus that lacks coat protein.Recombinant virus can be used for infecting the greedy frugiperda cell in meadow of expression of peptides or cabbage looper larva (people such as Smith, J Virol 46:584,1983; People such as Engelhard, Proc Nat Acad Sci (USA) 91:3224-7,1994).

In another embodiment, the dna sequence dna of coding multivalence binding peptide can and be cloned in the suitable carrier by pcr amplification, for example pGEX-3X (Pharmacia, Piscataway, NJ).The pGEX carrier is designed to prepare the fusion rotein that comprises glutathione-S-transferase (GST) by this vector encoded, and the multivalent binding proteins by the dna fragmentation coding in the cloning site of this carrier of insertion.Can form and for example comprise the suitably PCR primer of cracking site.Only be used for promoting expression if multivalent binding proteins merges part, or need not the connector as the peptide of being paid close attention to, the multivalent binding proteins syzygy of then recombinating can be cut from the GST of fusion rotein portion.PGEX-3X/ multivalence binding peptide construct is transformed in the intestinal bacteria XL-1Blue cell (Stratagene, La Jolla CA), and indivedual transformant are separated and cultivation.To and can use automatic sequencer that it is carried out the part order-checking from the plasmid DNA purifying of indivedual transformant and have the multivalent binding proteins coding nucleic acid inset of being wanted with suitable orientation with proof.

Can be used as the fusion multivalent binding proteins that insoluble inclusion body prepares in bacterium can following purifying in addition.By centrifugal collection host cell; In 0.15M NaCl, 10mM Tris (pH8), 1mM EDTA, wash; And at room temperature use 0.1mg/ml N,O-Diacetylmuramidase (Sigma ChemicalCo.) to handle 15 minutes.Make the lysate clarification by ultrasonic treatment, and cell debris is passed through to precipitate with centrifugal 10 minutes of 12,000 times of gravity.Can be in 50mM Tris (pH8) and 10mM EDTA with the precipitation resuspending that contains the multivalent binding proteins syzygy, through 50% glycerine layering, and with centrifugal 30 minutes of 6000 times of gravity.Can be with the precipitation resuspending in not containing Mg ⁺⁺And Ca ⁺⁺Standard phosphate buffered saline solution (PBS) in.The multivalent binding proteins syzygy can be further purified people such as () Sambrook by being deposited in the sex change sds page of resuspending separated.Gel is soaked among the 0.4M KCl manifesting albumen, with its excision and in the gel electrophoresis buffer reagent that lacks SDS electroelution.If GST/ multivalence binding peptide fusion rotein is to prepare in bacterium with the form of soluble proteins, then it can use GST purifying module (Pharmacia Biotech) to come purifying.

Preferably make the multivalent binding proteins syzygy stand digestion so that GST from multivalence binding peptide of the present invention cracking.Can (20-40 μ g fusion rotein, 20-30 unit's human prothrombin (4000U/mg (Sigma) is in 0.5ml PBS) be at room temperature hatched 16 to 48 hours and were loaded on the sex change SDS-PAGE gel with reaction product isolated with the digestion reaction thing.Gel can be soaked in 0.4M

Among the KCl to manifest protein band.(Applied Biosystems Model 473A, Foster City CA) proves by amino acid sequence analysis can to use automatic sequencer corresponding to the characteristic of protein band of the multivalence binding peptide with expection molecular weight.Perhaps, can be by described peptide being carried out HPLC and/or mass spectroscopy proves its characteristic.

Perhaps, the dna sequence dna of coding multivalence binding peptide can be cloned into contain and want promotor to reach in the plasmid that randomly contains leader sequence (referring to people such as for example Better, Science, 240:1041-43,1988) to some extent.The sequence of this construct can prove by automatic sequencing.Then can use bacterium is carried out CaCl ₂Hatch and standard program that heat-shocked is handled people such as () Sambrook, plasmid is converted in the suitable coli strain (such as bacterial strain MC1061).Bacterium through transforming can be incubated in the LB substratum, and this LB culture medium supplemented has the Gepcillin (carbenicillin) or the candidate of other suitable forms as known in the art, and comes the proteic generation of abduction delivering in the suitable culture medium by being incubated at.If exist, then leader sequence can be realized the secretion of multivalence binding peptide and cracking between the secretory phase.Secreted recombinant protein can pass through method purifying in bacteria culture medium of the following stated herein.

The mammalian hosts system that is used for express recombinant protein has been well known to those skilled in the art and has been vote.Can select to have the expressed albumen of processing or produce the host cell system of the certain capabilities of some posttranslational modification that is used to provide protein-active.The described modification of polypeptide includes but not limited to: acetylize, carboxylated, glycosylation, phosphorylation, lipidization and acidylate.Different host cells (such as CHO, sea draw, MDCK, 293, WI38 and similar host cell thereof) have and be specific to described translation afterwards active cell mechanism and feature mechanism and can be selected to guarantee that foreign protein is carried out correct modification and processing.

Cell transformed preferably can be used for the protein production of extended high rate rate, and thereby needs stably express.Described cell was cultivated described cell 1-2 days after preferably containing at least one carrier that can select the mark and the expression cassette of wanting conversion in enrichment medium, be transferred to selective medium afterwards.Can select mark to be designed to give the candidate resistance and its existence makes the cell of successful expression foreign protein to grow and to reclaim.The resistance group of stable transformed cells can utilize the tissue culture technique that is suitable for this cell to breed.

Can use the multiple choices system to reclaim cell transformed to be used to produce recombinant protein.Described selective system includes but not limited to lay respectively at HSV thymidine kinase, hypoxanthine-guanine phosphoribosyl transferase and the adenine phosphoribosyltransferase gene in tk-cell, hgprt-cell or the aprt-cell.Also can utilize the selection basis of metabolic antagonist resistance: the dhfr that gives the Rheumatrex resistance as following each person; Give the gpt of mycophenolic acid resistance; Give aminoglycoside G418 resistance and give the neo of grand (chlorsulfuron) resistance of chlorine sulphur; And give the hygro of hygromycin resistance.Applicatory other can select gene to comprise trpB, and it makes cell can use indoles to substitute tryptophane; Or hisD, it makes cell can use histidinol alternate sets propylhomoserin.For differentiating that transformant provides the mark of range estimation indication to comprise anthocyanidin, beta-Glucuronidase and substrate GUS thereof, reaches luciferase and its substrate luciferin.

Protein purification

The protein purification technology is well known to those skilled in the art.Described technology is included in the roughing out of a certain rank with polypeptide and non-polypeptide fraction.With multivalence in conjunction with polypeptide and at least a other albumen sepn, but the polypeptide that purifying is paid close attention to but need utilize chromatographic technique and electrophoretic technique to be further purified to reach partially or completely purifying (or purifying is a uniformity) usually.The analytical procedure that is particularly suited for preparing pure multivalence binding peptide is ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis and isoelectric focusing method.Especially effectively the peptide purification method is fast protein liquid chromatography method and HPLC.

Some aspect of the present invention is about the purifying of coded multivalent binding proteins or peptide and purifying substantially in specific embodiments.As used herein term " multivalent binding proteins of purifying or peptide " desire refer to can with other component composition isolated, wherein multivalent binding proteins or peptide are purified to any degree with respect to its natural obtainable state.Therefore, the multivalent binding proteins of purifying or peptide also are meant non-existent multivalent binding proteins of institute or peptide in its naturally occurring environment.

Usually, " purifying " is meant that keep its expressed bioactive multivalent binding proteins composition a kind of the separation to remove various other components and said composition substantially.If use term " purifying substantially ", then this title is meant the multivalent binding proteins composition, wherein multivalent binding proteins or peptide form the main component of composition, such as in composition, constitute albumen weight about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, more than about 99% or 99%.

Those skilled in the art will be known the various methods that are used for the purification degrees of quantitative multivalent binding proteins according to the disclosure.Described method comprises the specific binding activity of for example measuring active ingredient or analyzes by SDS/PAGE assesses in the component multivalence in conjunction with the amount of polypeptide.The preferred method that is used to assess multivalent binding proteins component purity for calculate this component in conjunction with active, with its with original extract combine specific activity, thereby and calculating purification degrees (assessing by " purifying multiple " herein).Be used to represent that the effective unit in conjunction with active amount decides on the particular assay technology that is selected to purifying certainly, and no matter whether expressed multivalent binding proteins or peptide shows detectable in conjunction with active.

The various technology that are applicable to the multivalent binding proteins purifying are well known to those skilled in the art.Described technology for example comprises: through the precipitation of ammonium sulfate, PEG, antibody and analogue thereof or by thermally denature, centrifugal then; Chromatographic step is such as ion-exchange, gel-filtration, reversed phase chromatography, hydroxyapatite and affinity chromatography; Isoelectrofocusing; Gel electrophoresis; And the combination of described technology and other technologies.As in this area usually known to, think that the order that carries out various purification steps can change, or some step can be omitted and still can yet be regarded as and prepares the appropriate method of the multivalent binding proteins of purifying substantially.

Generally not needing always provides multivalent binding proteins with purified state.In fact, expection can be used in certain embodiments and not be the multivalent binding proteins of purifying substantially.Partial purification can realize by being used in combination less purification step, or by using the multi-form of identical conventional purification scheme to realize.For example, should be appreciated that and use cation exchange column chromatography that the HPLC device carried out usually than the bigger purifying of constructed generation that uses the low pressure chromatographic system.The method that shows relatively low purification degrees has advantage aspect active aspect the recovery total of multivalent binding proteins product or in the combination of keeping expressed multivalent binding proteins.

The migration of known peptide can change under the different condition of SDS/PAGE, noticeable change sometimes (people such as Capaldi, Biochem.Biophys.Res.Comm., 76:425,1977).Therefore should be appreciated that under different deposition conditions, the apparent molecular weight of purifying or partially purified multivalent binding proteins expression product can change.

The effector cell

At the target cell induction for example ADCC, ADCP (antibody dependent cellular phagolysis) and fellow's thereof effector cell comprise that human leucocyte, scavenger cell, monocyte, activation neutrophil, active natural kill and wound (NK) cell and eosinophil.The effector cell expresses F _Cα R (CD89), Fc γ RI, Fc γ RII, Fc γ RIII and/or F _Cε R1 and for example comprise monocyte and the activation neutrophil.Found that for example the expression of Fc γ RI can be raised by interferon-gamma (IFN-γ).This enhanced is expressed the cellular cytoxicity activity that can strengthen monocyte and neutrophil defence target cell.Therefore, the effector cell can be through (IFN-γ) or other cytokines (for example TNF-α or β, group's stimulating factor, IL-2) activation, with increase cell surface before multivalence albumen of the present invention contact on the existing of Fc γ RI.

Multivalence albumen of the present invention provides the antibody mediated effect function, such as the cytotoxicity (ADCC) of antibody dependent effector cell mediation, for being used to defend the target cell.Have effector function multivalence albumen can such as herein instruction give separately, or with effector cell's coupling after give, thereby form " activating effect cell "." activating effect cell " is effector cell as defined herein, and it is connected with also as defined herein the multivalence albumen with effector function, so that the effector cell with target function effectively was provided before administration.

The activating effect cell can cell on physiology the form of suspension in the acceptable solution by vivo medicine-feeding.The cell number that is given is about 10 ⁸-10 ⁹On the individual order of magnitude, but look therapeutic purpose and variation to some extent.Generally speaking, this amount will be enough to obtain the effector cell and reach the effector cell function that the degree of wanting is provided in this position in the location at target cell place, such as passing through ADCC and/or phagocytotic cell killing effect.Acceptable solution is intended to comprise and can makes the stable any carrier soln of target effector cell that gives in the body on the term physiology as used herein, comprises for example salt solution and aqueous buffer solution, solvent, antiseptic-germicide and anti-mycotic agent, isotonic agent and analogue thereof.

Therefore, other aspects of the present invention provide a kind of in individuality the method at cell induction specific antibody effector function (such as ADCC), this method comprises to individuality and gives multivalence albumen (or coding nucleic acid) or the activating effect cell in the acceptable substratum on physiology.As known in the art, route of administration can change and suitable route of administration will be by those skilled in the art based on the consideration of case specificity variable and conventional procedure is judged.

Acellular effect

Acellular effect is also provided by multivalent molecule of the present invention, for example by providing CDC functional.Complement system is immune biological chemistry cascade, and it helps exotic (such as pathogenic agent) is removed in organism.It is to be derived from a lot of little plasma proteinss, and described plasma proteins operate together is induced the cytolysis of target cell by the plasma membrane that destroys the target cell.Complement system is formed by the solubility more than 35 and in conjunction with the albumen of cell, and wherein 12 directly are involved in the complement path.Described albumen has activity in causing three kinds of biological chemistry paths of complement system activatory: classical complement path, substituting complement path and mannose binding lectin path.Antibody (especially IgG1 antibody-like) also can " combination " complement.Described path has obtained detail knowledge in the art and has no longer repeated herein, but it should be noted that CDC does not depend on the interaction of binding molecule and immune cell (for example B cell).Also it should be noted that complement system regulates by Complement Regulatory Protein.Described albumen is present in the blood plasma with the concentration that is higher than complement proteins.Complement Regulatory Protein is to be present on self cell surface, provides to prevent that self cell is subjected to the mechanism of complement proteins target.The expection complement system works in having some kinds of diseases of immune component, such as Ba-Xi Er Shi (Barraquer-Simon) syndrome, A Zihai Mo's disease (Alzheimer ' s disease), asthma, lupus erythematosus disease, various forms of sacroiliitis, autoimmunity heart trouble and multiple sclerosis.Lack terminal path and make individual easy infection autoimmune disorders and transmissible disease (especially meningitis).

Disease, illness and state

The invention provides the multivalent binding proteins with effector function and varient and derivative, and this binding events is applicable to treatment, prevention or improvement and the relevant symptom of disease, illness or pathologic state (preferably making human painful disease, illness or pathologic state) in conjunction with one or more binding partner.In the preferred embodiment of described method, have conjugated protein the making of the multivalence (and polyspecific) of effector function and have target the cell of (such as the tumour-specific cell surface marker) combines with effector cell's (such as cell of showed cell cytotoxic activity in the immunity system).In other embodiments, polyspecific, multivalent binding proteins with effector function combine with effector cell's (such as immune cytotoxic cell) to guarantee correct target specifically in conjunction with two kinds of different disease specific cell surface markers, illness specific cell surface marker or state specific cell surface marker.In addition, can use multivalent binding proteins to induce or enhancement antigen activity or inhibition antigenic activity with effector function.Multivalent binding proteins with effector function also is suitable for combination treatment and palliative treatment.

On the one hand, the invention provides and be applicable to that treatment or prevention are the disease of feature and the composition and the method for state with the antigenic activity amount relevant with cell unusually.Described disease comprises cancer and other excess proliferative states, such as hyperplasia, psoriasis, contact dermatitis, immunity illness and infertility.The multiple cancer that comprises noumenal tumour and leukemia is subject to composition disclosed herein and method effect.Medicable cancer types includes but not limited to: the gland cancer of breast, prostate gland and colon; The pulmonary branches tracheocarcinoma of form of ownership; Encephaloid; Melanoma; Hepatoma; Neuroblastoma; Papilloma; Picked-up of ammonia precursor and decarboxylation glucagonoma (apudoma); Choristoma; Branchioma; Malignant carcinoid syndrome; Carcinoid heart disease; And cancer (for example walker's cancer (Walker), rodent cancer, basosquamous cell carcinoma, Bu-Pi Er Shi (Brown-Pearce) family name cancer, mammary gland tuberculation, Ai Lixishi (Ehrlich) knurl, Krebs 2 cancers, Merkel (merkel) cell carcinoma, cement tumour, nonsmall-cell lung cancer, oat-cell carcinoma, papillary cancer, inocarcinoma, bronchiolar carcinoma, bronchogenic carcinoma, squamous cell carcinoma and transsitional cell carcinoma).Medicable other cancer types comprise: the histocyte illness; Leukemia; Malignant histiocytosis; Hodgkin's (the disease of Hodgkin ' s); Little immunoproliferation disease; The non-Hodgkin lymphomas; Plasmoma; The reticuloendothelial cell hyperplasia disease; Melanoma; Chondroblastoma; Chondroma; Chondrosarcoma; Fibroma; Fibrosarcoma; Giant cell tumor; Histiocytoma; Lipoma; Liposarcoma; Mesothelioma; Myxoma; Myxosarcoma; Osteoma; Osteosarcoma; Chordoma; Craniopharyngioma; Dysgerminoma; Progonoma; Mesenchymoma; Mesonephroma; Myosarcoma; Ameloblastoma; Cementoma; Odontoma; Teratoma; Thymoma; Trophoblastic tumor.In addition, also contain the following cancer types that is subject to therapeutic action: adenoma; Cholangioma; Cholesteatoma; Cylindroma; Cystadenocarcinoma; Cystadenoma; Grain guided cell knurl; Gynandroblastomal; Hepatoma; Syringoadenoma; Islet cell tumor; Lai Shi (Leydig) glucagonoma; Papilloma; Sai Shi (sertoli) glucagonoma; Theca cell tumor; Leiomyoma; Leiomyosarcoma; Myoblastoma; Myomata; Myosarcoma; Rhabdomyoma; Rhabdosarcoma; Ependymoma; Ganglioneuroma; Neurospongioma; Medulloblastoma; Meningioma; Schwannoma; Neuroblastoma; Neuroepithelioma; Neurofibroma; Neuroma; Chromaffinoma; Receptoma.Medicable cancer types also includes but not limited to: angiokeratoma; Blood vessel lymph sample hyperplasia occurs together and has a liking for Yihong blood cell increase disease; Sclerosing hemangioma; Angiomatosis; Glomus tumor; Hemangioendothelioma; Vascular tumor; Hemangiopericytoma; Angiosarcoma; Lymphangioma; Lymphangiomyoma; Lymphangiosarcoma; Pinealoma; Sarcocarcinoma; Chondrosarcoma; Cystosarcoma phyllodes; Fibrosarcoma; Angiosarcoma; Leiomyosarcoma; Leukosarcoma; Liposarcoma; Lymphangiosarcoma; Myosarcoma; Myxosarcoma; The ovarian cancer knurl; Rhabdosarcoma; Sarcoma; Anything superfluous or useless; Neurofibroma; And cervical dysplasia.The present invention further provides composition and the method for cell wherein for the treatment of that be applicable to because of other states of antigenic abnormal expression high become immortalization or hyper-proliferative.

The various excess proliferative illnesss that are subject to composition of the present invention and method effect are illustrated as B-cell cancer, comprise B-cell lymphoma (such as various forms of lymphogranulomatosises, non-Hodgkin lymphomas (NHL) or central nervous system lymphoma), leukemia (such as acute lymphoblastic leukemia (ALL), lymphocytic leukemia (CLL), hairy cell leukemia and chronic myoblast leukemia) and myelomatosis (such as multiple myeloma).Other B cell cancers comprise small lymphocytic lymphoma, B-cell PL, the lymph-plasma cell lymphoma, the splenic marginal zone lymphoma, plasma cell myeloma, bone isolatism plasmocyte sarcoma, the outer plasmoma of bone, the joint outer edge area B-cell lymphoma of mucosa associated lymphoid tissue (MALT), joint marginarium B-cell lymphoma, follicular lymphoma, lymphoma mantle cell, the big B-cell lymphoma of diffustivity, the big B-cell lymphoma of mediastinum (thymus gland), big B-cell lymphoma in the blood vessel, lymphoma primary effusion, Bai Jiteshi (lymphoma/the leukemia of Burkitt ' s), the B-cell proliferation of uncertain malignant potential tumour, lymphomatoid granulomatosis and transplanting back Lymphoid tissue proliferative disorders.

To produce autoantibody is that the illness of feature is considered as autoimmune disorders usually.Autoimmune disorders includes but not limited to: sacroiliitis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, arthritic psoriasis, psoriasis, dermatitis, polymyositis/dermatomyositis, inclusion body myositis, inflammatory myositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, crest syndrome (CREST syndrome), the reaction that inflammatory bowel is followed, Crow engler (the disease of Crohn ' s), ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerule ephritis, the supersensitivity state, eczema, asthma, relate to the state that the T cell infiltrates and chronic inflammatory reacts, atherosclerosis, autoimmunity myocarditis, leukocyte adhesion deficiency disease, systemic lupus erythematosus disease (SLE), subacute cutaneous lupus erythematosis disease, discoid lupus disease, lupus osteomyelitis, lupus brain inflammation, teenager's outbreak type diabetes, multiple sclerosis, allergic encephalomyelitis, optic neuromyelitis, rheumatic fever, SC (Sydenham ' schorea), with by the irritated relevant immune response of the acute and delayed of cytokine and T-cell mediated, tuberculosis, sarcoidosis, granulomatosis (comprises Wei Genashi (Wegener ' s) granuloma disease and Qiu-Shi Er Shi (Churg-Strauss) disease), agranulocytosis, vasculitis (comprises anaphylaxis vasculitis/vasculitis, ANCA and rheumatoid vasculitis), aplastic anemia, Dai-Bu Er Shi (Diamond Blackfan) anaemia, immune hemolysis anaemia (comprising autoimmune hemolytic anemia (AIHA)), pernicious anemia, simple red blood corpuscle underdevelopment (PRCA), the blood coagulation factor VIII deficiency disease, haemophilia A, the autoimmunity neutropenia, pancytopenia, leukopenia, relate to the disease that white cell oozes out, central nervous system (CNS) inflammatory conditions, multiple organ injury's syndrome, myasthenia gravis, the disease of antigen-antibody complex mediation, the anti-GBM disease, antiphospholipid antibody syndrome, the supersensitivity neuritis, Bei Qieteshi (Behcet) disease, the triumphant plucked instrument Man (syndrome of Castleman ' s), Goodpasture (Goodpasture) syndrome, Yi-Lan Er Shi (Lambert-Eaton) Eaton-Lambert myasthenic syndrome, Lei Nuoshi (the syndrome of Reynaud ' s), repair the Gram (syndrome of Sjorgen ' s), Shi Difen-strong Sen Shi (Stevens-Johnson) syndrome, the solid organ transplantation rejection, graft versus host disease (GVHD), bullous pemphigoid, pemphigus, the autoimmunity polyendocrinopathy, the seronegativity SpA, Lai Teershi (the disease of Reiter ' s), whole body splinting syndrome, giant cell arteritis, immunity plyability ephritis, IgA nephropathy, the neuropathy of IgM polyneuropathy or IgM mediation, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), Heng Nuo-permitted Lan Shi (Henoch-Schonlein) purpura, the autoimmunity thrombocytopenia, the autoimmune disorders of testis and ovary (comprising autoimmunity testitis and ovaritis), the primary subthyroidism; The autoimmunity endocrinopathy comprises that autoimmunity thyroiditis, chronic thyroiditis (Hashimoto (Hashimoto ' s) thyroiditis), subacute thyroiditis, specially send out property subthyroidism, A Disenshi (Addison ' s) is sick, Ge Leishi (Grave ' s) disease, autoimmunity polyadenous syndrome (or polyadenous incretopathy syndrome), type i diabetes (also being called insulin-dependent diabetes (IDDM)) and the seat Han Shi (syndrome of Sheehan ' s); Auto immune hepatitis, lymph interstitial pneumonia (HIV), occurring together property of bronchiolitis obliterans (Nonimplantation) NSIP, Ji Lan-Ba Leishi (Guillain-Barr é) syndrome, great vessels inflammation (comprising polymyalgia rheumatica and giant cells (high iS-One (Takayasu ' s)) arteritis), the medium vessels inflammation (comprises Chuan Qishi (Kawasaki ' s) disease and polyarteritis nodosa), polyarteritis nodosa (PAN), ankylosing spondylosis, uncle Jie Shi (Berger ' s) sick (IgA nephropathy), carry out sex pilus spheroid ephritis fast, primary biliary cirrhosis, celiac disease (amylan enteropathy), cryoglobulinemia, the cryoglobulinemia hepatitis that occurs together, amyotrophic lateral sclerosis (ALS), coronary artery disease, familial Mediterranean fever, the many vasculitises of microscopically, the Gen Shi of the section (syndrome of Cogan ' s), prestige Scott-Aldrich Er Shi (Whiskott-Aldrich) syndrome and obstruction thromboangiitis.

Rheumatoid arthritis (RA) is the chronic disease of feature for the inflammation with the joint, and it causes swelling, pain and afunction.The patient who suffers from RA for a long time shows that usually carrying out property joint is damaged, lopsided, anergy and even early death.Except that RA; generally speaking inflammatory diseases, illness and state all are subject to the effect of treatment, prevention or the improvement of the symptom relevant with the inflammation process (for example heating, pain, swelling, hyperemia); and composition of the present invention and method are of value to treatment, prevent or improve unusual or unusual inflammatory process, comprise RA.

Crohn disease and relative disease ulcerative colitis are for belonging to the disease group's who is called inflammatory bowel (IBD) two class principal diseases.Crohn disease is for causing the chronic disease of digestive tube or stomach and intestine (GI) road inflammation.Although it can involve the GI any zone of oral cavity to anus, its most normal small intestine and/or colon of influencing.In ulcerative colitis, involving of GI only limits to colon.The antigenic antibody of neutrophil is resisted in being characterized as of Crohn disease, that is " examining all anti-NAs " (pANCA); And the antibody of anti-yeast saccharomyces cerevisiae, that is " anti-yeast saccharomyces cerevisiae antibody " (ASCA).A lot of patients of ulcerative colitis have pANCA antibody in its blood, but do not have ASCA antibody; And a lot of Crohn disease patients show ASCA antibody and do not show pANCA antibody.A kind of method of assessment Crohn disease is for using Crohn disease activity index (CDAI), and this activity index is based on 18 kinds of collected predictive variables of doctor and keeps the score.CDAI value below 150 and 150 is relevant with static disease; Value indicative of active disease more than 150, and the value more than 450 is regarded as extremely serious disease [people such as Best, " Development of aCrohn ' s disease activity index (development of Crohn disease activity index). " Gastroenterology 70:439-444 (1976)].Yet, beginning from research, some investigator uses 200 to 250 " subjective value " to keep the score as health.

Systemic lupus erythematosus disease (SLE) is an autoimmune disorders, and it is because of due to the blood vessel generation repeatability in the multiple organ that comprises kidney, skin and the joint damage.In suffering from the patient of SLE, unsuitable interaction causes producing the autoantibody of attack cells nuclear between T cell and the B cell.Usually consistently think that autoantibody causes SLE, therefore exhausting the cytophyletic novel therapy of B (making immunity system to restore because new B cell is formed by precursor cell) is the lasting benefited hope that provides of SLE patient.

Multiple sclerosis (MS) also is an autoimmune disorders.It is characterized by the inflammation of central nervous system and the destruction of myelin, make the intravital neurocyte fibrous insulation of brain, spinal cord and body.Even, think that extensively autoimmunity T cell is the pathogenetic perpetrator of this disease although the cause of disease of MS is unknown.Yet, have high-load antibody in MS patient's the celiolymph, and the B cell response that some theoretical prophesy causes antibody to produce plays an important role for this disease of mediation.

The autoimmunity thyropathy is because of produce to stimulate Tiroidina to cause hyperthyroidism (lattice RD) or destroy due to the autoantibody that Tiroidina causes hypothyroidism (struma lymphomatosa).Thyroid stimulation be because of autoantibody in conjunction with and activation thyrotropic hormone (TSH) acceptor due to.Thyroid destruction is because of due to the reaction of autoantibody and other thyroid antigens.

Comprised by other diseases, illness and the state of composition of the present invention and the benefit effect that method provided and repair Gram syndrome, this syndrome is the autoimmune disorders of feature for the destruction that produces the body of gland of moisture with health.In addition, immunologic thrombocytopenic purpura (ITP) be because of autoantibody in conjunction with thrombocyte and cause due to it destroys, and this state is suitable for using material of the present invention and method.Myasthenia gravis (MG) (a kind of unable chronic autoimmunity neuromuscular illness of random muscle group that causes, it is characterized by autoantibody and be combined in the acetylcholine receptor that express in the myoneural junction place) for having the disease of the symptom that can use composition of the present invention and method treatment, and expection the present invention will be of value to and treat and/or prevent MG.In addition, use composition of the present invention and method, expection Lloyd's (Rous) sarcoma virus infects and can be subjected to the treatment of at least a symptom or the effect of improvement.

Another aspect of the present invention is to use material of the present invention and method to prevent and/or treat any hyper-proliferative sexual state of skin, comprises psoriasis and contact dermatitis or other excess proliferative diseases.The psoriasic autoimmune inflammation of skin and also relevant that is characterized as with sacroiliitis (under 30% situation) and scleroderma, inflammatory bowel (comprising Crohn disease and ulcerative colitis).Proved that the patient that suffers from psoriasis and contact dermatitis has the antigenic activity that increases (people such as Ogoshi, J.Inv.Dermatol., 110:818-23[1998]) at described intralesional.The polyspecific multivalent binding proteins can directly be delivered to the cell of for example expressing the antigenic intralesional of high-content with immune cytotoxic cell.Multivalence (for example polyspecific) is conjugated protein can be given or give near focus by any approach in described herein various route of administration of use and the other administration route well known to those skilled in the art through subcutaneous.

Also contain the treatment of the special property sent out inflammatory myopathy (IIM) (comprising dermatomyositis (DM) and polymyositis (PM)).Inflammatory myopathy can utilize multiple classification schemes to be classified.Mi Leshi classification (Miller ' s classification schema) (Miller, Rheum Dis Clin North Am.20:811-826,1994) is differentiated 2 kinds of spy's property sent out inflammatory myopathies (IIM): polymyositis (PM) and dermatomyositis (DM).

PM-DM for involve muscle and (under the DM situation) skin chronic, make weak inflammatory diseases.Described illness is rare illness, according to reports, in the U.S., annual morbidity be annual 0.6 to 3.2 example among annual about 5 to 10 examples and per 1,000,000 children among per 1,000,000 adults (Targoff, Curr Probl Dermatol.1991,3:131-180).Special send out the property inflammatory myopathy and significantly sickness rate and mortality ratio are relevant, noticed reach among the ill adult half once suffered from major injury (people such as Gottdiener, Am J Cardiol.1978,41:1141-49).Miller (Rheum DisClin North Am.1994,20:811-826 and Arthritis and Allied Conditions (sacroiliitis and have off status), the 75th chapter, Koopman and Moreland compile, Lippincott Williams and Wilkins, 2005) enumerate the standard that five classes are used to diagnose IIM, that is, the special property sent out inflammatory myopathy standard (IIMC) assessment, comprise myasthenia, the examination of living tissue sign of muscle deterioration, the serum content of muscle relevant enzyme raises, the electromagnetism triad of myopathy, the sign of fash in the dermatomyositis, and the sign that also comprises autoantibody is as second standard.

IIM correlation factor (comprising muscle relevant enzyme and autoantibody) includes but not limited to that creatine kinase (CK), serum lactic dehydrogenase, zymohexase, proteins C reactive, aspartic transaminase (AST), alanine aminotransferase (ALT) and anti-nuclear autoantibody (ANA), myositis specific antibody (MSA) reach at the antibody that can extract nuclear antigen.

The preferred autoimmune disorders that is subject to method effect of the present invention comprises Crohn disease, Ji Lan-Ba Lei Cotard (GBS; Also be called acute inflammation demyelination polyneuropathy, acute spy's property sent out polyradiculoneuritis, acute spy's property sent out polyneuritis and blue De Lishi (Landry ' s) ascending paralysis), lupus erythematosus disease, multiple sclerosis, myasthenia gravis, optic neuritis, psoriasis, rheumatoid arthritis, hyperthyroidism (for example lattice RD), subthyroidism (for example Hashimoto's disease), Ao Deshi (the thyroiditis (being similar to the thyroiditis of Hashimoto's disease) of Ord ' s), diabetes (1 type), aplastic anemia, conjunctivo-urethro-synovial syndrome, auto immune hepatitis, primary biliary cirrhosis, antiphospholipid antibody syndrome (APS), opsoclonus-myoclonic syndrome (OMS), temporal arteritis (also being called " giant cell arteritis "), acute disseminated encephalomyelitis (ADEM), Goodpasture, Wei Genashi granuloma disease, coeliac disease, pemphigus, the dog polyarthritis, warm autoimmune hemolytic anemia.In addition, the present invention is contained and is used for the treatment of or the method for the symptom of improvement and following disease-related: endometriosis, interstitial cystitis, neuromyotonia, scleroderma, leukodermia, vulvitis, cause card Ge Shi (Chagas ') disease, sarcoidosis, chronic fatigue syndrome and the autonomic nerve obstacle of expansion type heart trouble (megalocardia).

Think that complement system works in having a lot of diseases of immune component, such as A Zihai Mo's disease, asthma, lupus erythematosus disease, various forms of sacroiliitis, autoimmunity heart trouble and multiple sclerosis, all described diseases are all as the disease that is subject to therapeutic action, illness or state or utilize the amendatory symptom of method of the present invention and be covered by among the present invention.

Decide by one or more specific effect functions that multivalence strand binding molecule shows, be preferably some constant subprovince.For example, for complement activation, IgG (IgG1, IgG2 or IgG3) and IgM are preferred; For opsonization and toxin neutralizing effect, the IgG of any hypotype is all preferred; For the pathogenic agent combination, IgA is preferred; And for for the parasitic combination of worm, IgE is preferred.

For example, there is F on the human leucocyte as the constant region of the identification IgG antibody of three kinds of dissimilar Fc γ acceptors _CR, it can be distinguished according to structural performance and functional performance and according to the antigenic structure that the CD monoclonal antibody is detected.It is called Fc γ RI, Fc γ RII and Fc γ RIII and on leukocytic (overlapping) subclass through differential expression.

FcgRI (CD64) (high-affinity receptor through expressing on monocyte, scavenger cell, neutrophil, bone marrow precursor and dendritic cell) comprises isotype 1a and 1b.FcgRI has high-affinity to monosomy IgG 1 and IgG3.Its affinity to IgG4 is low about 10 times, the IgG2 of its debond simultaneously.FcgRI does not show genetic polymorphism.

Fc γ RII (CD32) (comprising isotype lla, llb1, llb2, llb3 and llc) is the human Fc gamma R type the most widely that distributes, and it is expressed on the blood leucocyte of most of type and is expressed on youth's lattice Han Shi (Langerhans) cell, dendritic cell and the thrombocyte.Fc γ RII is only in conjunction with the low affinity receptor of the accumulative IgG of institute.Only Fc γ R class can be in conjunction with IgG2.Fc γ RIIa shows genetic polymorphism, produces two kinds of different allotypes respectively: Fc γ Rlla-H131 and Fc γ Rlla-R131.This functional polymorphism is attributable to the single amino acid difference at 131 places, position: Histidine (H) or arginine (R) residue, this is most important for the IgG combination.As if Fc γ Rlla be easy in conjunction with IgG and IgG3 and debond IgG4.Fc γ Rlla-H131 has much higher affinity than Fc γ Rlla-R131 allotype to compound IgG2.

Fc γ RIII (CD16) has two kinds of isotypes or allelotype, its both all can be in conjunction with IgG1 and IgG3.The Fc γ RIIa that IgG is had medium affinity is expressed on scavenger cell, monocyte, NK cell (NK) cell and the T cell subclass.Fc γ RIIIb is that it is optionally expressed on neutrophil to the acceptor of the low affinity of IgG tool.The high mobility acceptor can carry out effective cooperation with other membrane receptors.Dimeric research proves for myelomatosis IgG, only IgG1 and IgG3 in conjunction with Fc γ RIIIb (with low affinity in conjunction with), and do not find the combination of IgG2 and IgG4.Fc γ RIIIb has codominance diallele polymorphism, described allotype called after NA1 (neutrophil antigen) and NA2.

Another aspect of the present invention be to use material of the present invention and method by treatment, prevent or the effect that alleviates infection is resisted because of the infection due to any infectious agent in the multiple infectious agent.Multivalence polyspecific binding molecule of the present invention is designed to raise efficiently and effectively the organic immunity system of host is derived from external organism, external cell, adventitious viruses or external lifeless object with opposing infection.For example, the polyspecific binding molecule can have one specifically in conjunction with the binding domains of the target on the infectious agent and another specifically conjugated antigen be the binding domains of the target (such as CD40, CD80, CD86, DC-SIGN, DEC-205, CD83 and fellow thereof) on the delivery cell.Perhaps, each binding domains of multivalence binding molecule all can be specifically in conjunction with infectious agent, thereby more effectively and infectious agent.In addition, the present invention is contained specifically in conjunction with the target on the infectious agent and in conjunction with the polyspecific multivalence binding molecule of the relevant binding partner of acellular, described polyspecific multivalence binding molecule can be in conjunction with the effector function of polyspecific binding molecule effectively treatment or prevention because of the infection due to the infectious agent.

The infectious cell that the present invention is contained comprises any known infectious cell, includes but not limited to various bacteriums (pathogenicity bo intestinal bacteria (E.coli) for example, salmonella typhimurium (S.typhimurium), Pseudomonas aeruginosa (P.aeruginosa), Bacillus anthracis (B.anthracis), Clostridium botulinum (C.botulinum), refractory clostridium (C.difficile), clostridium perfringens (C.perfringens), Hp (H.pylori), vibrio cholerae (V.cholerae) and class bacteroid thereof), mycobacterium, mycoplasma, fungi (comprising yeast and mould) and parasite (comprise primary Piroplasmea (Protozoa), Trematoda (Trematoda), the any known parasitics member of merozoic cestode guiding principle (Cestoda) and nematoda (Nematoda)) any person in.Infectious virus includes but not limited to eucaryon virus (for example adenovirus (adenovirus), bunyavirus (bunyavirus), simplexvirus (herpesvirus), papovavirus (papovavirus), paramyxovirus (paramyxovirus), picornavirus (picornavirus), poxvirus (poxvirus), arc reovirus virus (reovirus), retrovirus (retrovirus) and similar virus thereof) and phage.Exotic comprises that (no matter mode of entrance and no matter whether have wanton injury) enters the object in the organism (preferred human).In vogue day by day in view of many resistances infectious agent (for example bacterium) (the especially pathogenic agent of hospital infection), therefore material of the present invention and method can provide a kind of methods of treatment to strengthen the difficulty that is caused to avoid antibiotics resistance.

Relevant with infectious agent and be subject to the disease of material disclosed herein and method (preventative or therapeutic) treatment, state or illness include but not limited to anthrax, aspergillosis, bacterial meningitis, bacterial pneumonia (for example Chlamydia pneumoniae), blastomycosis, sausage poisoning (botulism), brucellosis (brucellosis), moniliosis (candidiasis), cholera, coccidioidomycosis of brain, torulosis, diarrheagenic E. coli, enterohemorrhagic Escherichia coli or enterotoxigenic Escherichia coli, diphtheria, glanders, histoplasmosis, veteran's disease, leprosy, listeriosis (listeriosis), nocardiasis (nocardiosis), Whooping cough, salmonellosis (salmonellosis), scarlet fever, sporotrichosis, Strep throat, toxic shock syndrome, traveler diarrhea and typhoid fever.

Other aspects of the present invention and details will be apparent by following examples, described embodiment be intended to the explanation and unrestricted.Embodiment 1 describes the regroup clone of heavy chain immunoglobulin and variable region of light chain.Embodiment 2 describes the structure of little module immune drug.Embodiment 3 describes the structure of the former flask of the multivalent binding proteins that is used to have effector function.Embodiment 4 describes the combination of this initial prototype molecule and the research of expression.Embodiment 5 describes the structure of the substituting construct that is derived from this initial prototype molecule, and the sequence that wherein is connected the subarea between EFD and the BD2 changes on length and sequence.In addition, described alternative form, wherein the orientation in V district also changes in the binding domains 2.Embodiment 6 describes subsequently described combination and functional study with substituting construct of variation connexon form, thereby identifies the cutting that connects the subarea in some described derivative forms; And for addressing this problem the novel sequence variant of being researched and developed.Embodiment 7 describes the structure of the preferred alternate embodiment of polyspecific multivalence fusion rotein (wherein BD1 and BD2 all combine the antigen (CD20 and CD37) on the same cell type) or another kind of polyspecific fusion rotein (wherein sign an undertaking for BD2 and close specific antigen and changed into human CD3 but not CD28).Embodiment 8 describes combination and the functional study of being carried out with CD20-hIgG-CD37 polyspecific construct.Embodiment 9 describes combination and the functional study of being carried out with CD20-hIgG-CD3 multivalence fusion protein construct.Embodiment 10 discloses the multivalence binding molecule with connexon, and described connexon is based on the given zone of immunoglobulin superfamily member's ectodomain.Embodiment 11 discloses the mensuration of differentiating binding domains, and described binding domains is expected in the multivalence binding molecule can effectively reach at least a useful effect, and this effect is through differentiating relevant with described molecule (for example disease treatment).

Embodiment 1

The clone of heavy chain immunoglobulin and variable region of light chain

Can use any method as known in the art to bring out antibody at set antigen target.In addition, can use any method as known in the art to clone light chain immunoglobulin and/or variable region of heavy chain, and the constant subprovince of one or more antibody.Following method provides exemplary cloning process.

A. the separation of total RNA

Be clone's heavy chain immunoglobulin and variable region of light chain or constant subprovince, from the hybridoma of secreting suitable antibody, separate total RNA.Will be available from the cell (2 * 10 of hybridoma cell line ⁷) wash 1 time with PBS, and in 12 * 75mm round bottom polypropylene tube (No. the 2059th, Falcon), precipitate via centrifugal.In each pipe, add TRIzol ^TMTotal RNA separation agent (GibcoBRL, Life Technologies, catalog number (Cat.No.) 15596-018 number) (8ml) and via repeating is drawn cytolysis.At room temperature lysate is hatched 5 minutes, add 1.6ml (0.2 volume) chloroform and 15 seconds of concuss afterwards.After at room temperature leaving standstill 3 minutes, with lysate in 4 ℃ of pre-cooled Beckman JA-17 turners with 9, centrifugal 15 minutes of 000rpm is so that separate water with organic phase.To mix in the new pipe of upper aqueous phase (about 4.8ml) immigration and with the 4ml Virahol light and slowly.After at room temperature hatching 10 minutes, by in 4 ℃ of JA-17 turners with 9,000rpm made RNA precipitation in centrifugal 11 minutes, with ice-cold 75% washing with alcohol of RNA precipitation with 8ml, and in the JA-17 turner, coming redeposition in centrifugal 7 minutes under 4 ℃ with 7,000 * rpm.Washing with alcohol liquid is decanted, and RNA is precipitated air-dry 10 minutes.RNA is precipitated the ddH that resuspending is handled through diethylpyrocarbonate (DEPC) in 150 μ l ₂Among the O, the ddH that every 1ml handles through DEPC ₂Contain 1 μ l ribonuclease inhibitor (No. the 799017th, catalog number (Cat.No.) among the O; Boehringer Mannheim/Roche).With described precipitation by light and slow absorption resuspending and under 55 ℃, hatching 20 minutes.By measuring OD through the dilution aliquots containig _260nm(1.0OD _260nmUnit=40 μ g/mlRNA) comes the RNA sample is carried out quantitatively.

The rapid amplifying of B.cDNA end

Carry out the end of 5 ' RACE with amplification heavy chain and variable region of light chain or constant subprovince.According to manufacturer specification, use 5 ' RACE system (LifeTechnologies, catalog number (Cat.No.) 18374-058 number) of the terminal test kit of rapid amplifying cDNA 2.0 editions.Use the oligonucleotide Oligo 5.1 editions (Molecular Biology Insights, Cascade CO) that designs program that degeneracy 5 ' RACE Oligonucleolide primers is designed to constant region coupling with for example two classes mouse immuning ball protein heavy chain (IgG1 and IgG2b) commonly used.Primer also is designed to the constant region coupling with mouse IgG κ light chain.This is the light chain immunoglobulin of unique classification, therefore need not degeneracy in design of primers.The sequence of primer is as follows:

Title sequence SEQ ID NO.

Heavy chain GSP1

5′AGGTGCTGGAGGGGACAGTCACTGAGCTGC3′ 7

Nested heavy chain

5′GTCACWGTCACTGRCTCAGGGAARTAGC3′ 8

(W=A or T; R=A or G)

Light chain GSP1

5′GGGTGCTGCTCATGCTGTAGGTGCTGTCTTTGC3′ 9

Nested light chain 5 ' CAAGAAGCACACGACTG

AGGCACCTCCAGATG3′ 10

5 ' Race simplified style anchor primer

5′GGCCACGCGTCGACTAGTACGG

GNNGGGNNGGGNNG3′ 11

Be amplification mouse immuning ball protein heavy chain component, carry out reverse transcriptase reaction in 0.2ml thin-walled PCR pipe, the heavy chain GSP1 primer (SEQID NO:7), 4 μ g that this PCR pipe contains 2.5 picomole (pmole) cloned (for example cloning 4A5 or clone 4B5) the isolating total RNA of institute and the 12 μ l ddH through the DEPC processing from suitable hybridoma ₂O.Equally, for mouse light chain component, carry out reverse transcriptase reaction in 0.2ml thin-walled PCR pipe, light chain GSP1 primer (SEQ ID NO:9), 4 μ g that this PCR pipe contains 2.5 picomole clone total RNA of (for example cloning 4A5 or clone 4B5) and the ddH that 12 μ l handle through DEPC available from suitable hybridoma ₂O.

(MJ research Inc., Waltham react in MA) at PTC-100 programmable thermo cycler.Mixture hatched under 70 ℃ 10 minutes so that RNA sex change and then wet cooled on ice 1 minute.Described pipe is brief centrifugal so that the pipe lid is collected moisture content certainly.Subsequently, following component is added in the reaction: the 10 x PCR buffer reagents (200mM Tris-HCl (pH8.4), 500mM KCl) of 2.5 μ l, the 25mM MgCl of 2.5 μ l ₂, the 10mM dNTP mixture of 1 μ l and the 0.1M DTT of 2.5 μ l.After each pipe being mixed, place 42 ℃ PTC-100 thermo cycler to last 1 minute described pipe so that mixture is warm in advance by light and slow absorption.Subsequently, with the SuperScript of 1 μ l (200 units) ^TMII reversed transcriptive enzyme (Gibco-BRL; Catalog number (Cat.No.) 18089-011 number) is added in each pipe, mixes, and under 42 ℃, hatch 45 minutes by drawing light and slowly.Make reaction cycle to 70 ℃ last 15 minutes, and then be circulated to 37 ℃ with termination reaction.Then Yeast Nucleic Acid enzyme mixture (1 μ l) is added in each reaction tubes, mixes light and slowly, and under 37 ℃, hatch 30 minutes.

The first chain cDNA that reverse transcriptase reaction produced is separated rotating filter cartridge (Gibco-BRL) according to manufacturer specification purifying in addition with GlassMAX DNA.In each first chain reaction, add the 6M NaI binding soln of 120 μ l.Then cDNA/NaI solution is transferred in the GlassMAX rotating filter cartridge and with 13,000 times of gravity centrifugal 20 seconds.The filter cylinder insert carefully removed and abandons the mobile part in the pipe.Then rotating filter cartridge is put back in the blank pipe and with 0.4ml cold (4 ℃) 1 * lavation buffer solution and be added in each rotating filter cartridge.With described pipe with 13,000 times of centrifugal 20 seconds of gravity and abandon mobile part.With this washing step triplicate again.Then with 0.4ml cold (4 ℃) 70% washing with alcohol 4 times of GlassMAX filter cylinder.After the mobile part that abandons last 70% ethanol washing lotion, put back to filter cylinder in the pipe and with centrifugal again 1 minute of 13,000 times of gravity so that make the filter cylinder complete drying.Then the rotating filter cartridge insert is transferred to fresh sample recovery tube, wherein the ddH that 50 μ l are handled through 65 ℃ of (preheating) DEPC ₂O is added in each rotating filter cartridge fast.With filter cylinder with 13,000 times of gravity centrifugal 30 seconds with wash-out cDNA.

C. terminal deoxynucleotidyl transferase (TdT) tailing

For each first chain cDNA sample, following component is added in the 0.2ml thin-walled PCR pipe: the ddH that 6.5 μ l handle through DEPC ₂The 2mM dCTP of 5 * tailing buffer reagent of O, 5.0 μ l, 2.5 μ l and 10 μ l are through the suitable cDNA sample of GlassMAX purifying.Each 24 μ l reactant hatched under 94 ℃ 2-3 minute in thermo cycler so that the DNA sex change, and wet cooled on ice 1 minute.Inclusion by simple centrifugal collection tube.Subsequently, 1 μ l terminal deoxynucleotidyl transferase (TdT) is added in each pipe.Described pipe is mixed via light and slow absorption and in the PTC-100 thermo cycler, under 37 ℃, hatched 10 minutes.After hatching in these 10 minutes, TdT is lasted 10 minutes and with its hot deactivation by being circulated to 65 ℃.With reactant in cooled on ice and will be stored under-20 ℃ through the first chain cDNA of TdT tailing.

D. through the PCR of the first chain cDNA of dC tailing

Carry out dual multiple pcr amplification (each first chain cDNA sample through the dC tailing carries out twice independently PCR reaction) in 50 μ l volumes, this 50 μ l volume contains: any one among the 5 ' RACE simplified style anchor primer of 200 μ M dNTP, 0.4 μ M (SEQ ID NO:11) and the nested heavy chain GSP2 of 0.4 μ M (SEQ ID NO:8) or the nested light chain GSP2 (SEQ ID NO:10), 10mM Tris-HCl (pH8.3), 1.5mM MgCl ₂, 50mM KCl, 5 μ l the Expand through dC tailing cDNA and 5 units ^TMThe Hi-Fi archaeal dna polymerase (Roche/BoehringerMannheim GmbH, Germany).Adopt " successively decreasing/increase progressively " annealing temperature experimental program, in PTC-100 programmable thermal cycler (MJ Research Inc.) with following condition amplification PCR reactant: initial 95 ℃ of 40 seconds of sex change, following 5 circulations: 94 ℃ following 20 seconds, 61 ℃-2 ℃/circulated for 20 seconds, 72 ℃ of 40 seconds+1 second/circulations; Follow following 5 circulations: 94 ℃ following 25 seconds, 53 ℃+1 ℃/circulation 20 seconds, 72 ℃ 46 seconds+1 second/circulation; Follow following 20 circulations: 94 ℃ following 25 seconds, 55 ℃ 20 seconds, 72 ℃ 51 seconds+1 second/circulation; And under 72 ℃, hatched 5 minutes at last.

E.TOPO TA-clone

(QIAGEN Inc., Chatsworth CA) carry out gel-purified with gained PCR product through 1.0% sepharose, and use TOPO TA to use QIAQuick gel-purified system

Test kit (Invitrogen, San Diego, CA, catalog number (Cat.No.) K4550-40 number) is cloned into pCR2.1 with its TA-, and according to manufacturer specification it is converted in the intestinal bacteria TOP10F ' cell (Invitrogen).According to manufacturer specification, differentiate clone by blue/white screening method with inset, wherein the white clone is considered as positive colony.The culture that 3.5ml is contained the liquid Luria meat soup (LB) of 50 μ g/ml Ampicillin Trihydrates is inoculated with white colony, and cultivates (about 16 hours) overnight down in 37 ℃ down with the 225rpm concussion.

Use qiagen plasmid micropreparation test kit (QIAGEN Plasmid MiniprepKit, QIAGEN Inc., No. the 12125th, catalog number (Cat.No.)), according to the plasmid DNA in the manufacturer specification purifying culture.Plasmid DNA is suspended in the 1 x TE buffer reagent (pH8.0) of 34 μ l and then as previously mentioned, by two deoxyribonucleotide order-checkings of fluorescent and automatic detection method, use ABI Big Dye Terminator 3.1 reagent positive colony to be checked order, and use ABI 3100 dna sequencing instrument to analyze with the extent of dilution of 1:4-1:8.Used sequencing primer comprise T7 (5 ' GTAATACGACTCACTATAGG3 '; SEQ ID NO:12) and the reverse (5 ' CAGGAAACAGCTATGACC3 ' of M13; SEQ ID NO:13) primer.Sequencing result will prove that described clone is corresponding to mouse IgG sequence.

F. utilize the synthetic of overlapping oligonucleotide extension PCR from tau gene

This method relates to uses overlapping Oligonucleolide primers and PCR, adopts the mixture of high-fidelity DNA polymerase or polysaccharase, synthetic immunoglobulin V-district or other genes.Originate in the middle part of V-region sequence, design 40 to 50 base primerses, so that make 20 to 30 bases of growing chain amplification with either direction, and in abutting connection with minimum overlapping 20 bases of primer.Each PCR step needs two primers, and one cause antisense strand (forward primer or adopted primer is arranged) and causes sense strand (reverse primer or antisense primer), to form the double-stranded PCR product of growth.During design of primers, the nucleotide sequence that can change final product is to form the Restriction Enzyme site, destroy existing Restriction Enzyme site, add and flexibly connect son, change, disappearance or insert the base that changes aminoacid sequence, the whole dna sequence dna of optimization is synthetic and meet for expection and be used to express the organic codon service regulations of synthetic gene to strengthen primer.

With primer involutory and the dilution so that first pair is 5 μ M, each is up to 80 μ M to having the concentration more than 2 times thereafter.The 1 μ L amplification of using Platinum PCR SuperMix-High Fidelity (Invitrogen, San Diego, CA, catalog number (Cat.No.) 12532-016 number) in 50 μ L PCR reaction, will be got each from described primer mixture.Be initially at 94 ℃ of following sex change after 2 minutes, carry out 30 circulations of PCR with following recycle scheme: 94 ℃ following 20 seconds, 60 ℃ following 10 seconds and 68 ℃ are following 15 seconds.Use Qiaquick PCR purification column (QiagenInc., No. the 28704th, catalog number (Cat.No.)) purified pcr product to remove excessive primer and enzyme.Then utilize the as above definite PCR condition of describing of institute (the exception part was 30 seconds for each being circulated to 68 ℃ time lengthening), the primer that seemingly dilutes with next category is to increasing this PCR product again.With gained PCR product through aforesaid primer and enzyme purifying once more, through the TOPO-TA clone, and as above E part in definite description of institute check order.

Embodiment 2

The structure of little module immune drug (SMIP)

Form with strand reorganization (mouse/people) scFv makes up the polyspecific multivalent binding proteins with effector function that contains binding domains 1, with its called after 2H7 (VL-connexon-VH).ScFv2H7 is for discerning the little module immune drug (SMIP) of CD20 specifically.Binding domains is based on the human CD20 antibody sequence that can openly obtain, for GenBank accession number for the VH be M17953 and for VL the GenBank accession number be M17954.CD20-specificity SMIP is described in the U.S. Patent Publication of owning together 2003/133939,2003/0118592 and 2005/0136049, and described openly is to be incorporated herein in full with way of reference.Separate the fifteen amino acid connexon of the peptide connexon of VL and VH for the following sequence of coding: Asp-Gly ₃Ser-(Gly ₄Ser) ₂Binding domains 1 is to be positioned at the protein-bonded N-end of polyspecific, and the terminal N-with constant subprovince of the C-of this structural domain is terminal directly to be connected, and hinge, C are contained in this constant subprovince _H2Structural domain and C _H3Structural domain (being orientated) with amino to carboxyl.Constant subprovince is to be derived from IgG1 antibody, and it is to separate by pcr amplification IgG 1 from human PBMC.The modification of hinge area is passed through: replace existing three Cys residues in the wild-type IgG 1 hinge arrangement territory with three Ser residues, this hinge area is by fifteen amino acid sequence: EPKSCDKTHTCPPCP (SEQ ID NO:14; Indicate with runic through Ser residue metathetical Cys residue for these three) coding.In alternate embodiment, hinge area is modified at one or more halfcystine place, so that form SSS and CSC type hinge.In addition, last proline(Pro) is replaced by Serine sometimes, and halfcystine replaces.

C _H3The C-of structural domain is terminal covalently bound with a series of alternative connexon structural domains, described connexon structural domain and place constant subprovince C-terminal and the N-terminal of binding domains 2 between.Folding characteristic on BD2 is decided, the preferred multivalent binding proteins with effector function will have described connexon one so that constant subprovince and binding domains 2 separately, although this connexon is not the necessary component of the present composition.For some specificity multivalent molecule, this connexon may be important for the separation of structural domain, and for other persons, this connexon may be accessory.This connexon and the terminal (V that is connected of the N-of the scFv 2E12 that discerns CD28 specifically _H-connexon-V _L).Making the VH structural domain and the isolating connexon of VL structural domain of the scFv 2E12 part of multivalence binding molecule is 20 amino acid connexon (Gly ₄Ser) ₄, but not insert in standard (Gy between the V structural domain of scFv usually ₄Ser) ₃Connexon.Through observation, longer connexon can be improved the binding characteristic of the 2e12 scFv of VH-VL orientation.

Constructed polyspecific multivalence binding molecule contains binding domains 1, and this binding molecule comprises: from the 2E12 leader peptide sequences of the amino acid/11-23 of SEQ ID NO:171; Be shown in the human CD20 variable region of light chain of 2H7 mouse-anti of the position 24 of SEQ ID NO:171; Start from the Asp-Gly of the residue 130 of SEQ ID NO:171 ₃-Ser-(Gly ₄Ser) ₂Connexon; The human CD20 variable region of heavy chain of 2H7 mouse-anti, wherein the amino acid at residue 11 places of the variable domains of VH is substituted by Serine (VHL11S) by leucine, and this binding molecule has single serine residue (that is VTVS at heavy chain district end, wherein canonical sequence is VTVSS) (Genbank accession number M17953), and insert between two binding domains BD1 (2H7) and the BD2 (2E12) person and be the constant subprovince of human IgG1, it comprises the modified hinge area that comprises " CSC " or " SSS " sequence, and wild-type C _H2And C _H3Structural domain.Nucleotide and aminoacid sequence with multivalent binding proteins of effector function show with SEQ ID NO:228 and 229 respectively for the CSC form, and show with SEQ ID NO:170 and 171 respectively for the SSS form.

The formation of stable expression cell line is passed through: via electroporation will not cut or linearizing recombinant expression plasmid transfection to Chinese hamster ovary cell (CHO DG44 cell), in containing the substratum of Rheumatrex, select then.The a large amount of cultures and the main aperture that produce the multivalent binding proteins of maximum amount are increased in the Rheumatrex of incremental change, and clone the culture of adaptation subsequently by restricted dilution.In bio-reactor or ripple bag, (Excell 302 available from the serum free medium of JRH Biosciences in use, catalog number (Cat.No.) 14324-1000M number, be supplemented with 4mM glutamine (Invitrogen, 25030-081), Sodium.alpha.-ketopropionate (Invitrogen 11360-070, dilute 1 times), non-essential amino acid (Invitrogen, 11140-050,1 times of final dilution), penicillin-Streptomycin sulphate 100IU/ml (Invitrogen, 15140-122) and the Recombulin of 1 μ g/mL (Invitrogen, 97-503311)) cultivate to produce multivalent binding proteins through transfection CHO cell.The CHO basic medium of other serum-frees (such as CD-CHO and analogue thereof) also can be used for preparation.

By the albumin A affinity chromatography, from consuming CHO culture supernatants purified fusion protein.Use a series of chromatographies and filtration step (comprising that virus reduces strainer) purifying multivalent binding proteins.Cell culture supernatant liquid is filtered, then on GE Healthcare XK 16/40 post, stand the albumin A agarose affinity chromatography.After the protein binding post, post is washed with dPBS, then with 1.0M NaCl, 20mM sodium phosphate (pH6.0) washing, and then with 25mMNaCl, 25mN NaOAc (pH5.0) washing, to remove non-specific binding albumen.With 100mM glycine (Sigma) (pH3.5) with institute's bonded albumen from the post wash-out, and (pH6.0) make the pH value reach 5.0 with 0.5M2-(N-morpholinyl) ethane sulfonic acid (MES).The albumen sample concentration to 25mg/mL, is prepared against the GPC purifying.Use GE healthcare XK post and Superdex 200 preparation scale (GE healthcare), on GE Healthcare AKTA Explorer 100Air device, carry out size exclusion chromatography.

Then material is concentrated and prepare with 20mM sodium phosphate and 240mM sucrose, gained pH value is 6.0.Composition is filtered, be packed in the sterile vials with various concentration afterwards, this amount on institute's recover materials is decided.

Embodiment 3

Make up scorpion shape developed by molecule box

To contain the Synthetic 2 H7 scFv (anti-CD 20 that is connected with constant subprovince as described in example 2 above; SEQ ID NO:1) nucleic acid called after TRU-015.Use the synthetic scFv 2E12 of the TRU-015 nucleic acid and the little module immune drug of encoding (anti--CD28VL-VH; SEQ IDNO:3) and synthetic scFv 2E12 (anti--CD28VH-VL; SEQ ID NO:5) nucleic acid is as the template that is used for the various components of pcr amplification scorpion shape molecule box.The template of binding domains 1 and constant subprovince or skeleton provide by TRU-015 (nucleic acid of the scFv 2H7 (anti-CD 20) that coding is connected with constant subprovince), and this template is construction in expression vector pD18.Above-mentioned containing has two kinds of orientation (V _L-V _HAnd V _H-V _L) in the nucleic acid of scFv 2E12 of arbitrary orientation provide the coding region for binding domains 2.

The TRU 015 SSS hinge C that is used for BD2/ connexon inset _H2C _H3

Use contains the Synthetic 2 H7 scFv IgG1 type of SSS hinge, forms scorpion shape molecule box by serving as the template of adding the EcoRI site to replace existing terminator codon and XbaI site.Use primer 9 (SEQ ID NO:23; Referring to table 1) and primer 87 (SEQ ID NO:40; Referring to table 1) and Platinum PCR high-fidelity mixture (Invitrogen), by this molecule of pcr amplification.With gained 1.5Kbp fragment purification and be cloned among the carrier pCR2.1-TOPO (Invitrogen), and be transformed among the coli strain TOP10 (Invitrogen) the validating DNA sequence.

Table 1

SEQ

Numbering title sequence 5 '-3 ' ID NO

The PCR primer

GCGATAAAGCTTGCCGCCATGGAA

1 hVK3L-F3H3 GCACCAGCGCAGCTTCTCTTCC 15

ACCAGCGCAGCTTCTCTTCCTCCTG

2 hVK3L-F2 CTACTCTGGCTCCCAGATACCACCG 16

GGCTCCCAGATACCACCGGTCAAAT

3 hVK3L-F1-2H7VL TGTTCTCTCCCAGTCTCCAG 17

GCGATAGCTAGCCAGGCTTATCTAC

4 2H7VH-NheF AGCAGTCTGG 18

GCGATAGCTAGCCCCACCTCCTCCA

5 G4S-NheR GATCCACCACCGCCCGAG 19

GCGTACTCGAGGAGACGGTGACCGT

6 015VH-XhoR GGTCCCTGTG 20

GCAGTCTCGAGCGAGCCCAAATCTTG

7 G1H-C-XHO TGACAAAACTC 21

GCAGTCTCGAGCGAGCCCAAATCTTC

8 G1H-S-XHO TGACAAAACTC 22

GCGTGAGAATTCTTACCCGGAGACAGG

9 CH3R-EcoR1 GAGAGGCTC 23

GCGACGTCTAGAGTCATTTACCCGGAG

10 G1-XBA-R ACAGG 24

AATTATGGTGGCGGTGGCTCGGGCGGT

11 G4SLinkR1-S GGTGGATCTGGAGGAGGTGGGAGTGGG 25

AATTCCCACTCCCACCTCCTCCAGATCCA

12 G4SLinkR1-AS CCACCGCCCGAGCCACCGCCACCAT 26

GCGTGTCTAGATTAACGTTTGATTTCCAG

13 2E12VLXbaR CTTGGTG 27

GCGATGAATTCTGACATTGTGCTCACCCA

14 2E12VLR1F ATCTCC 28

GCGATGAATTCTCAGGTGCAGCTGAAGGA

15 2E12VHR1F GTCAG 29

GCGAGTCTAGATTAAGAGGAGACGGTGAC

16 2E12VHXbaR TGAGGTTC 30

17 2e12VHdXbaF1 GGGTCTGGAGTGGCTGGGAATGATATG 31

18 2e12VHdXbaR1 ATTCCCAGCCACTCCAGACCCTTTCCTG 32

19 IgBsrG1F GAGAACCACAGGTGTACACCCTG 33

20 IgBsrG1R GCAGGGTGTACACCTGTGGTTCTCG 34

SEQ

Numbering title sequence 5 '-3 ' ID NO

Sequencing primer

82 M13R CAGGAAACAGCTATGAC 35

83 M13F GTAAAACGACGGCCAGTG 36

84 T7 GTAATACGACTCACTATAGG 37

85 pD18F-17 AACTAGAGAACCCACTG 38

86 pD18F-20 GCTAACTAGAGAACCCACTG 39

87 pD18F-1 ATACGACTCACTATAGGG 40

88 pD18R-s GCTCTAGCATTTAGGTGAC 41

89 CH3seqF1 CATGAGGCTCTGCACAAC 42

90C H3seqF2 CCTCTACAGCAAGCTCAC 43

91 CH3seqR1 GGTTCTTGGTCAGCTCATC 44

92 CH3seqR2 GTGAGCTTGCTGTAGAGG 45

Table 1: Oligonucleolide primers is used to make up CD20-CD28 scorpion shape molecule box.Primer is divided into 2 groups: PCR group and order-checking group.The PCR primer is to be used to make up described box, and sequencing primer is the dna sequence dna that is used to prove all middle constructs and final construct.

N2H7 V _KWith human V _K3The leader sequence syzygy

Use the primer 3 and 5 in the table 1, utilize the PCR mutagenesis of oligonucleotide guiding AgeI (ACCGGT) restriction site to be introduced 5 ' end of the coding region of TRU 015 VK, Nhe I (GCTAGC) restriction site is introduced 3 ' end of the coding region of (G4S) 3 connexons.Last 6 amino acid of human VK3 leading (gb:X01668) because primer 3 is also encoded, therefore use the primer 1,2 and 5 in the table 1, utilize overlapping PCR to increase leading N-end sequence (comprising consistence Kozak box and HinDIII (AAGCTT) restriction site) in regular turn.

N2H7 IgG1 SSS hinge-C _H2C _H3Construct

Use primer 4 and 6 (to be respectively SEQ ID NO:18 and 20; Table 1) TRU-015V that increases again _H, wherein make the V of NheI site 5 ' and TRU-015 _KMerge and make Xho I (5 '-CTCGAG-3 ') site at 3 ' end and IgG1 hinge-C _H2C _H3Structural domain engages.Equally, the primer 8 in the use table 1 and 9 amplification IgG1 hinge-C _H2-C _H3The district, thus 5 ' XhoI site introduced, will have 3 ' end now and replace for the clone, and the destruction terminator codon is so that can translate the binding domains 2 that CH3 structural domain downstream is connected with EcoRI (5 '-GAATTC-3 ') site.This scorpion shape molecule box type is different from the box of above-mentioned prefix for " n ".

Except that above-mentioned multivalent binding proteins, albumen of the present invention can have corresponding to the binding domains of the single variable region of immunoglobulin (Ig) (binding domains 1 or 2, or both).The exemplary embodiment of this aspect of the present invention will comprise the V corresponding to camellid antibody _HThe binding domains of structural domain, or can in conjunction with antigenic other species antibody of target through single modification or not modified V district, although contain any single variable domains that is applicable in the albumen of the present invention.

2E12 VL-VH and VH-VL construct

Be the 2E12 scFv that preparation is compatible with described box, the overlapping Oligonucleolide primers 17 and 18 in the palpus use table 1 destroys inner Xba I (5 '-TCTAGA-3 ') site.Use these two primers and primer to the increase binding domains of two opposed orientation of the combination of 14/16 (VL-VH) or 13/15 (VH-VL), so that it carries EcoRI site and XbaI site respectively at its 5 ' and 3 ' end.Also encode the place ahead, Xba I site next-door neighbour's terminator codon (TAA) of primer 13 and 16.

2H7 SSS IgG1 2e12 LH/HL construct

Effector domain-binding domains 2 connexons add (STD connexon-STD1 and STD2)

With complementary primer 11 in the table 1 and 12 combinations, be heated to 70 ℃, and slowly cool to room temperature so that this two chain is bonding.Utilize manufacturers's experimental program, the T4 polynucleotide kinase (Roche) that uses the 1X with 1mM ATP to connect in the buffer reagent (Roche) adds 5 ' phosphate.Then use T4 dna ligase (Roche) that the double-stranded connexon of gained is engaged to and be positioned at IgG1C _H3In the EcoRI site between the terminal coding region and the top of binding domains 2.Existence and C to the EcoRI site of gained DNA construct screening connexon-BD2 joint _H3The existence of the nucleotide sequence GAATTA of-connexon joint.Then suitable STD1 connexon construct is digested with EcoRI again, and repeat the connexon connection has the connexon of being made up of the Lx1 sequence of two (STD2) consistence iteration with generation molecule.As above screening DNA construct once more.

Embodiment 4

Expression study

The nucleic acid that above-mentioned coding is had the multivalent binding proteins of effector function carries out expression study.With the nucleic acid of coding multivalent binding proteins through transient transfection to the COS cell and will maintain through cells transfected allow knowing under the condition that heterologous gene expressed in described cell.As mentioned above, use PEI or DEAE-dextran with the DNA transient transfection to the COS cell (people such as PEI=Boussif O., PNAS 92:7297-7301, (1995), the document is incorporated herein with way of reference; People such as Pollard H., JBC 273:7507-7511, (1998), the document is incorporated herein with way of reference).Each novel molecule is carried out repeatedly independent transfection so that measure the average expression degree of each new form.If by the PEI transfection, then the COS cell is coated in the 60mm tissue culturing plate in the DMEM/10% FBS substratum and hatch overnight so that it covered with about 90% in transfection the same day.Substratum is changed to do not contain antibiotic serum-free DMEM and hatched 4 hours.Transfection media (4 milliliters/plate) contains and has the serum-free DMEM that 50 μ g PEI and 10-20 μ g pay close attention to the DNA plasmid.Transfection media is mixed by eddy current, at room temperature hatched 15 minutes, and after extracting existing substratum out, be added in the plate.Culture hatched regather supernatant liquor after 3-7 days.By the protein expression of SDS-PAGE, Western blotting mensuration culture supernatants, verify combination and utilize various mensuration (comprising the experiment of ADCC, CDC and coculture) to come measurement function by flow cytometry.

SDS-PAGE analyzes and western blot analysis

The albumen aliquots containig that is contained proteic thick culture supernatants of 8 μ g (common 30 microlitres/hole) or purifying by every hole prepares sample, and 2X Tris-glycine SDS buffer reagent (Invitrogen) is added into 1 times of ultimate density.(CA) electrophoresis is to provide MW size criteria for Invitrogen, Carlsbad to 10 microlitre SeeBlue marks.(Invitrogen, San Diego stand the SDS-PAGE analysis on CA) in 4-20%Novex Tris-glycine gels in conjunction with (fusions) protein variants to make multivalence.95 ℃ down heating under reduction or non-reduced condition, use Novex Tris-glycine SDS sample buffer reagent (2X) sample loading after 3 minutes, electrophoresis 60 minutes under 175V then.Use 1X Novex Tris-glycine SDS electrophoresis buffer reagent (Invitrogen) to carry out electrophoresis.

Behind the electrophoresis, (Ellard, Seattle WA) are transferred to pvdf membrane with albumen and last 1 hour under 100mAmp to use the half dry type electroblotting device.Western comprises the following three kinds of buffer reagents that are present on the saturated Whatman filter paper for transferring buffered dose: contain 36.34g/ and rise Tris (pH10.4) and 20% methyl alcohol for No. 1; Contain 3.02g/ for No. 2 and rise Tris (pH10.4) and 20% methyl alcohol; Containing 3.03g/ for No. 3 rises Tris (pH9.4), 5.25g/ and rises epsilon-amino caproic acid and 20% methyl alcohol.Under agitation, blocking-up in the bovine lacto transfer technique optimizer (=5% skimming milk) of film in PBS is overnight.(the Fc specificity Caltag) one arises from and hatches one hour in the bovine lacto transfer technique optimizer, then washing 3 times in PBS-0.5% Tween 20, each 15 minutes with the anti-IgG of HRP bonded goat of cytolemma and 5 μ g/ml.Wet film was hatched 1 minute with ECL solution, be exposed to the X-omat film then and lasted for 20 seconds.Fig. 2 is presented at albumen electrophoretic western blotting under non-reduced condition expressed in the COS cell culture supernatant liquid (30 microlitres/hole).Swimming lane is with mark 1-9 indication and contain following sample: swimming lane 1 (cuts off=See Blue mark kDa indication trace side.Swimming lane 2=2H7-sssIgG P238S/P331S-STD1-2e12 VLVH; Swimming lane 3=2H7-sssIgGP238S/P331S-STD1-2e12 VHVL; Swimming lane 4=2H7-sssIgG P238S/P331S-STD2-2e12 VLVH; Swimming lane 5=2H7-sssIgG P238S/P331S-STD2-2e12VHVL; Swimming lane 6=2e12 VLVH SMIP; Swimming lane 7=2e12 VHVL SMIP; Swimming lane 8=2H7 SMIP.2H7 in the described construct is always V _LV _HOrientation, sssIgG indication is positioned at the characteristic (as shown in Figure 5) of the hinge/connexon of connexon position 1, and the P238S/P331S indication sports IgG 1 pattern of mutant (shown in the 2nd aa) and is present in wild-type IgG l C from wild-type (shown in an aa) _H2And C _H3Amino acid position in the structural domain, the STD1 indication is positioned at 20 amino acid (18+ restriction site) connexons (as shown in Figure 5) of connexon position 2, and the STD2 indication is positioned at 38 amino acid (36+ restriction site) connexons (as shown in Figure 6) of connexon position 2.

In conjunction with research

Carry out in conjunction with the dual specific binding characteristic of research with assessment CD20/CD28 polyspecific multivalence binding peptide.Initially, the WIL2-S cell is added into 96 orifice plates and centrifugal for precipitating the shape cell.On entire plate, use twice volumetry (20 μ g/ml titration to 0.16 μ g/ml), the CD20/CD28 purifying protein is added in the plate of inoculation.Also the twice serial dilution (the TRU-015 concentration range is 20 μ g/ml to 0.16 μ g/ml) with TRU-015 (source of binding domains 1) purifying protein is added in the plate hole of inoculation.The background contrast is served as in not protein-contg hole.

To contain proteic inoculation plate hatched on ice one hour.Subsequently, with 200 μ l1% FBS (in PBS) washing once with described hole.Then in each hole, add and hatched again on ice one hour through the anti-human antibodies of FITC (FcSp) labelled goat and with described plate with 1:100.Then described plate is washed once with 200 μ l, 1% FBS (in PBS), and with described cell resuspending in 200 μ l, 1% FBS and pass through facs analysis.

Anti-for assessing-CD28 peptide 2E12 V _HV _LBinding characteristic, the Chinese hamster ovary celI that will express CD28 is by applying in the indivedual holes that are inoculated in culture plate.Then use twice dilution scheme (concentration range is 20 μ g/ml to 0.16 μ g/ml) that the CD20/CD28 purifying protein is added in indivedual holes.Reuse twice dilution scheme (that is 20 μ g/ml to 0.16 μ g/ml), 2E12IgG-VHVL SMIP purifying protein is added in indivedual inoculations hole.Albumen is not accepted so that the background contrast to be provided in a hole.Then described plate was hatched on ice one hour, with 200 μ l1%FBS (in PBS) washing once, and in each hole, add with 1:100 through FITC (Fc Sp, CalTag, Burlingame, CA) the anti-human antibodies of labelled goat.Again with described plate in hatching one hour on ice and using 200 μ l, 1% FBS (in PBS) washing subsequently once.After described cell resuspending is in 200 μ l, 1% FBS, carry out facs analysis.The result shows that the multivalent binding proteins with the terminal CD20 binding domains 1 of N-is in conjunction with CD20; Has N-V _H-V _LThose albumen of the terminal CD28 binding domains 2 of C-of-C orientation are also in conjunction with CD28.

Flow cytometry (FACS) shows that expressed albumen can be in conjunction with CD20 (referring to Fig. 3) that is presented on the WIL-2S cell and the CD28 (with reference to figure 3) that can present in conjunction with Chinese hamster ovary celI, thereby proof BD1 or BD2 can have the antigenic function of binding specificity target.Each data presentation shown on the figure of Fig. 3, different multivalence have associativity in conjunction with (fusion) proteic serial dilution in indicated titration scope.Use the data indication that described initial construction body obtained, under equivalent concentration, the multivalence with binding domains 2 types (using the 2e12 of VHVL orientation) is orientated than VLVH that form is more preferably expressed in conjunction with (fusions) albumen and more preferably in conjunction with CD28.

Fig. 4 shows and to use available from each the diagrammatic representation in conjunction with result of study that purified albumen carried out in described transfection body/construct.This figure shows the overview that combines of albumen and the WIL-2S cell of expressing CD20, thereby proves this multivalent molecule in conjunction with CD20, and the single specificity SMIP under same concentrations.The top figure of Fig. 5 and base map show the overview of BD2 specificity (2e12=CD28) in conjunction with the CD28 Chinese hamster ovary celI.For the combining of binding domains 2 and CD28, the orientation in V district influences the combination of 2e12.2H7-sss-hIgG-STD1-2e12 multivalent binding proteins with 2e12 of VH-VL (HL) orientation shows being equivalent to the degree combination of single specificity SMIP, and the 2e12LH molecule shows the poor efficiency combination under same concentrations.

Embodiment 5

The structure of the various connexon forms of multivalence fusion rotein

This embodiment describes the structure of different connexon forms listed in the form shown in Figure 6.

C _H3The structure of-BD2 connexon H1 to H7

For probing into C _H3-BD2 connexon length and expression and the bonded formed scorpion shape molecule influence, and contrived experiment is compared like construct with the bigger category with different connexons will have molecule 2H7sssIgG1-Lx1-2e12HL now.Use 2H7sssIgG1-Lx1-2e12HL as template, listed primer carries out a series of PCR reactions in the use oligonucleotide table 2, is 0 to 16 connexon that amino acid does not wait thereby form length.Described connexon can be configured to nucleic acid fragment, and described nucleic acid fragment is crossed over the C of BsrGI site _H3The coding region is to the nucleic acid end of the coding connexon-BD2 contact of EcoRI site.

Table 2

SEQ ID

Numbering title sequence 5 '-3 ' NO.

The PCR primer

GCGATAGAATTCCCAGATCCACCACCGCCCGA

1 L1-11R GCCACCGCCACCATAATTC 46

GCGATAGAATTCCCAGAGCCACCGCCACCATA

2 L1-6R ATTC 47

GCGTATGAATTCCCCGAGCCACCGCCACCCTTA

3 L3R CCCGGAGACAGG 48

GCGTATGAATTCCCAGATCCACCACCGCCCGAG

4 L4R CCACCGCCACCCTTAC 49

GCGTATGAATTCCCGCTGCCTCCTCCCCCAGATC

5 L5R CACCACCGCC 50

6 IgBsrG1F GAGAACCACAGGTGTACACCCTG 51

GCGATAGAATTCGGACAAGGTGGACACCCCTTAC

7 L-CPPCPR CCGGAGACAGGGAGAG52

Table 2: the sequence that is used to form the primer of CH3-BD2 connexon varient

Fig. 6 illustrates multivalence in conjunction with (fusion) proteic schematic structure and show the orientation in the V district of each binding domains, is present in the sequence (only listing the Cys residue) of connexon position 1 and is positioned at the sequence and the identifier of connexon of the connexon position 2 of molecule.

Embodiment 6

Combination and functional study at the varient connexon form of 2H7-IgG-2e12 prototype multivalence fusion rotein

This embodiment shows to a series of expression of " prototype " 2H7-sssIgG-Hx-2e12 VHVL construct that has various connexons (H1-H7) in the connexon position 2 with in conjunction with the result who studies.Described in above embodiment, use the albumin A affinity chromatography, extensive COS transient transfection by molecule and purifying are expressed each in the described albumen.Then make purified albumen stand to analyze, comprise SDS-PAGE, Western blotting, bonded analyzed and researched and bioactive functional assays by flow cytometry.

The relatively combination research of different B D2 orientation

Carry out combination research described in above embodiment, the exception part is to use the material through the albumin A purifying, and uses combination (fusion) albumen of constant basis that is 0.72 μ g/ml for each varient of being studied.Fig. 7 shows that each connexon varient of comparison and 2e12 are orientated the histogram of varient for the binding characteristic of CD20 and CD28 target cell.H1-H6 is meant the construct of the 2e12 with H1-H6 connexon and VH-VL orientation.L1-L6 is meant the construct of the 2e12 with H1-H6 connexon and VL-VH orientation.Data show, the specific binding domains 2 of 2e12 tool is being compared when existing with (sample L1-L6) the more effectively combination when existing of LH orientation with HL orientation (sample H1-H6).Shown in the following set of diagrams, find that the influence of connexon length is complicated, complicated part is that the molecule with longer connexon contains some single specificity cracking molecule, and described cracking molecule lacks the CD28 binding specificity at C-terminal.Carry out other experiments to determine the combination of selected connexon, the results are shown among Figure 10,12 and 13.

The SDS-PAGE of the H1-H7 connexon varient of purifying analyzes

Branches such as albumen by purifying prepare sample, and 8 μ g albumen are contained in every hole, and add 2X Tris-glycine SDS buffer reagent (Invitrogen) to the 1X ultimate density.For reduced form sample/gel, the buffer reagent that then 10X reduced adds Tris-glycine SDS buffer reagent and is added into together in the sample to 1X.(CA) electrophoresis is to provide MW size criteria for Invitrogen, Carlsbad to 10 μ l SeeBlue marks.(Invitrogen, San Diego carry out the SDS-PAGE analysis on CA) in 4-20% Novex Tris-glycine gels in conjunction with (fusions) protein variants to make multivalence.After 3 minutes, under reduction or non-reduced condition, use Novex Tris-glycine SDS sample buffer reagent (2X) sample loading 95 ℃ of heating, then 175V electrophoresis 60 minutes.Use 1X Novex Tris-glycine SDS that buffer reagent (Invitrogen) is carried out electrophoresis.Behind the electrophoresis, under agitation gel dyeed 30 minutes in Ku Masi (Coomassie) SDS PAGE R-250 staining agent, and decoloured at least one hour.Fig. 8 shows that [2H7-sss-hIgG P238S/P331S-Hx-2e12VHVL] multivalence is in conjunction with (fusions) protein variants and the non-reduced and reductive Ku Masi of the TRU-015 of sample and the 2e12 HLSMIP gel that dyes in contrast.When connexon length increases, increase in the electrophoretic proteic amount of about SMIP size (or 52kDa).The increase of protein content is corresponding to the top reduction of protein content in the electrophoretic band of about 90kDa in this band.Gels data show full-length molecule or contiguous connexon place's cracking, forms a molecule that lacks the BD2 district.Less BD2 fragment does not exist, and shows that the nucleotide sequence in (1) connexon sequence may form a hidden splice site, and this is removed in the rna transcription thing of montage than small segment; Or (2) albumen in full-length polypeptide translation after the cracking of proteolysis mode, the BD2 fragment instability that this is less, that is be vulnerable to proteolytic treatment.The Western blotting analysis of some described molecules shows that described albumen all contains CD20 BD1 sequence, but this less band lacks CD28 BD2 reactivity.On gel quav or trace, all do not observe less band with " naked " scFv size (25-27kDa) migration, show in the sample not have this less peptide fragment.

Western blot analysis BD1 and BD2 combine with 2H7 specificity Fab or CD28mIg's

Fig. 9 shows the result that western blot analysis 2H7-sss-hIgG-H6 multivalence is compared with each single specificity SMIP in conjunction with (fusion) albumen.

Under non-reduced condition, carry out electrophoresis, and before loading, do not make the sample boiling.Behind the electrophoresis, (Ellard, Seattle WA) are transferred to pvdf membrane with albumen and last 1 hour under 100mAmp to use the half dry type electroblotting device.Under agitation, film blocking-up in bovine lacto transfer technique optimizer (5% skimming milk among the PBS) is overnight.Fig. 9 A: in bovine lacto transfer technique optimizer, film was hatched one hour with the AbyD02429.2 (at the Fab of 2H7 antibody) of 5 μ g/mL, then in PBS-0.5%Tween 20, wash 3 times each 5 minutes.Then film was hatched in 6X His-HRP one hour with the concentration of 0.5 μ g/ml.Trace is washed three times each 15 minutes in PBST.Wet film was hatched 1 minute with ECL solution, be exposed to the X-omat film then and lasted for 20 seconds.

Fig. 9 B: (Ancell, Bayport MN) are hatched together, then washing three times in PBS-0.5% Tween 20, each 15 minutes with the CD28Ig of 10 μ g/ml in bovine lacto transfer technique optimizer with film.Then (CalTag, Burlingame CA) are hatched in bovine lacto transfer technique optimizer with the goat anti-mouse HRP binding substances of 1:3000 with film.With film washing three times, each 15 minutes, then in ECL solution, hatched 1 minute, be exposed to the X-omat film then and lasted for 20 seconds.Western blot analysis result indication, CD28 binding domains are to exist with multivalence " monomer " component (this component moves under about 90kDa) existence and with the form of higher category.Do not observe the band migration in position for single SMIP or the expection of naked scFv size fragment.When using the anti-genotype Fab of CD20, detect the fragment of SMIP size, illustrate to have the CD28 scFv BD2 peptide fragment partly that contains (2H7-sss-hIgG) and lack fusion rotein.

Combination research to selected connexon

Figure 10 shows the result in conjunction with research that purified 2H7-sss-hIgG-Hx-2e12 fusion rotein is carried out.Carry out in conjunction with the dual specific binding characteristic of research with assessment CD20/CD28 polyspecific binding peptide.Utilize routine techniques to apply the WIL2-S cell when initial.Using twice volumetry (20 μ g/ml to 0.16 μ g/ml) the CD20/CD28 purifying protein to be added in the plate of inoculation on the entire plate.Also the twice serial dilution (concentration range of TRU-015 is 20 μ g/ml to 0.16 μ g/ml) with TRU-015 (source of binding domains 1) purifying protein is added in the plate hole of inoculation.The background contrast is served as in not protein-contg hole.

To contain proteic through the inoculation plate hatched on ice one hour.Subsequently, with 200 μ l, 1% FBS (in PBS) washing once with described hole.Then in each hole, add and hatched again on ice one hour through the anti-human antibodies of FITC (FcSp) labelled goat and with described plate with 1:100.Then described plate is washed once with 200 μ l, 1% FBS (in PBS), and with described cell resuspending in 200 μ l, 1% FBS and pass through facs analysis.

Anti-for assessing-CD28 peptide 2E12 V _HV _LBinding characteristic, the Chinese hamster ovary celI that will express CD28 is by applying in the indivedual holes that are inoculated in culture plate.Then use twice dilution scheme (concentration range is 20 μ g/ml to 0.16 μ g/ml) that the CD20/CD28 purifying protein is added in indivedual holes.Reuse twice dilution scheme (that is 20 μ g/ml to 0.16 μ g/ml), 2E12IgGvHvL SMIP purifying protein is added in indivedual inoculations hole.A hole does not receive albumen so that the background contrast to be provided.Then described plate was hatched on ice one hour, with 200 μ l 1%FBS (in PBS) washing once, and in each hole, add with 1:100 through the anti-human antibodies of FITC (Fc Sp) labelled goat.Again described plate was hatched on ice one hour and use 200 μ l1% FBS (in PBS) washing once subsequently.With after described cell resuspending is in 200 μ l, 1% FBS, carry out facs analysis.Flow cytometry (FACS) show the CD20 (referring to Figure 10 A) that on expressed proteins can be in conjunction with the WIL-2S cell, presented and can be in conjunction with Chinese hamster ovary celI on the CD28 (with reference to figure 10B) that presented, thereby show that BD1 or BD2 can have the antigenic function of binding specificity target.In addition, do not find that used connexon (H1-H6) is to having remarkably influenced in conjunction with the antigenic avidity of target.

SEC separates multivalence in conjunction with (fusion) albumen.On GE Healthcare XK 16/40 tubing string, by albumin A agarose affinity chromatography method purifying combination (fusion) albumen in cell culture supernatant liquid., to tubing string, tubing string is washed with dPBS in protein binding, then with 1.0M NaCl, 20mM sodium phosphate (pH6.0) washing, and then with 25mM NaCl, 25mN NaOAc (pH5.0) washing, to remove non-specific binding albumen.(pH3.5) with institute's bonded albumen wash-out in tubing string, and (pH6.0) make its pH value reach 5.0 with 100mM glycine (Sigma) with 0.5M 2-(N-morpholinyl) ethane sulfonic acid (MES).Utilize routine techniques with the albumen sample concentration to 25mg/ml, in order to the GPC purifying.Use GE healthcare XK tubing string and Superdex 200 preparation scale (GE healthcare), on GE Healthcare AKTA Explorer 100Air device, carry out size exclusion chromatography (SEC).

The form that Figure 12 shows is summarized different from the isolating result of (fusion) proteic SEC.Increase with connexon length, the mixture of molecule also increases in the solution, makes to be difficult to by HPLC from peak (or POI) that the higher category isolated in form is paid close attention to.As if the H7 connexon can be split as this most of mixture the more form of homogeneous in solution, so that soluble form is most of with single POI form migration under about 172kDa.

Other are in conjunction with research

Multivalence to smaller subset is more carried out second series experiment (referring to Figure 12 and Figure 13) in conjunction with (fusions) albumen, this time comparison connexon H3, H6 and H7.Data show, and compare in conjunction with CD20, and it is more obvious to descend in conjunction with the degree of CD28, but when connexon length increases, both are decline slightly all.In addition, data presentation H7 connexon all shows the combination of top to two kinds of antigens.Use is through the albumin A purifying, but obtains described data without the multivalence that SEC is further purified in conjunction with (fusion) albumen, so can have the molecule of various ways in the solution.As if the result also indicates, and clipped form is poorer than the stability of actual multivalence polypeptide, because binding curve does not reflect a large amount of single specificity forms of existing connexon H6 in the solution fully.

Prove the polyspecific combination of single molecule

Carry out substituting in conjunction with measuring (referring to Figure 13), wherein use the specific reagent of CD28 BD2 tool is detected combining of CD20 and WIL-2S cell surface, thereby show that the BD1 on same polyspecific combination (fusion) albumen can take place to combine (with reference to Figure 12) with two targets are antigenic with BD2 simultaneously.This measures the proteic polyspecific binding characteristic of proof.

Embodiment 7

Has polyspecific combination (fusion) the proteic structure that substitutes specific BD2

Except that prototype CD20-CD28 polyspecific binding molecule, can prepare other two kinds of forms with 2 districts of substituting binding domains (comprising CD37 binding domains and CD3 binding domains).Also can prepare and have about the molecule of [2H7-sss-IgG-Hx/STDx-2e12HL] polyspecific in conjunction with the described some connexon structural domains of (fusion) albumen.Described other polyspecifics are described below in conjunction with the structure of (fusion) molecule.

Anti--CD37 binding domains construct

Table 3

Numbering title sequence SEQ ID NO.

ACTGCTGCAGCTGGACCGCGCT

23 G281LH-NheR AGCTCCGCCGCCACCCGAC 53

GGCGGAGCTAGCGCGGTCCAGC

24 G281LH-NheF TGCAGCAGTCTGGACCTG 54

GCGATCACCGGTGACATCCAGAT

25 G281-LH-LPinF GACTCAGTCTCCAG 55

GCGATACTCGAGGAGACGGTGAC

26 G281-LH-HXhoR TGAGGTTCCTTGAC 56

GCGATCGAATTCAGACATCCAGAT

27 G281-LH-LEcoF GACTCAGTCTCCAG 57

GCGATTCTAGATTAGGAAGAGACG

28 G281-LH-HXbaR GTGACTGAGGTTCCTTGAC 58

GCGATAACCGGTGCGGTCCAGCTG

29 G281-HL-HF CAGCAGTCTGGAC 59

GACCCACCACCGCCCGAGCCACCG

CCACCAGAAGAGACGGTGACTGAGG 60

30 G281-HL-HR3 TTC

ACTCCCGCCTCCTCCTGATCCGCCG

31 G281-HL-HR2 CCACCCGACCCACCACCGCCCGAG 61

GAGTCATCTGGATGTCGCTAGCACTC

32 G281-HL-HNheR CCGCCTCCTCCTGATC 62

ATCAGGAGGAGGCGGGAGTGCTAGC

33 G281-HL-LNheF GACATCCAGATGACTCAGTC 63

GCGATACTCGAGCCTTTGATCTCCAG

34 G281-HL-LXhoR TTCGGTGCCTC 64

GCGATATCTAGACTCAACCTTTGATCT

35 G281-HL-LXbaR CCAGTTCGGTGCCTC 65

GCGATAGAATTCGCGGTCCAGCTGCA

36 G281-HL-EcoF GCAGTCTGGAC 66

Table 3: the G28-1 that is used to form SMIP molecule and scorpion molecule is anti--Oligonucleolide primers of CD37 binding domains.

Use the primer in the Table X above, G28-1scFv (SEQ ID NO:102) is converted into G28-1LH SMIP by PCR.With primer 23 and 25 and 10ng G28-1 scFv combination, use Platinum PCR Supermix Hi-Fidelity PCR mixing tank (Invitrogen, Carlsbad, CA) VK (30 following circulations: lasted for 20 seconds under 94 ℃, lasted for 15 seconds under 58 ℃, lasted for 15 seconds under 68 ℃) that in ABI 9700 thermo cyclers, increases.This PCR product has restriction site PinAI (AgeI) and has NheI at the end of scFv (G4S) 3 connexons at the 5 ' end of VK.By in PCR running, with condition that above VK is identical under with primer 24 and 26 and 10ng G28-1scFv make up and change VH similarly.This PCR product has restriction site NheI and has XhoI at 3 ' end at the 5 ' end of VH.Because important overlapping primer 23 and 24 of being engineered to of sequence identity, therefore with 5 times of VK and VH dilutions, then with the 1:1 ratio use flank primer 25 and 26 be added into PCR and by with 68 ℃ of time length by extending to for 45 seconds 15 seconds and as above increase total length scFv.This PCR product represents whole G28-1 scFv as the PinAI-XhoI fragment, and can come purifying to remove excessive primer, enzyme and salt by MinElute tubing string (Qiagen) purifying.Eluate is being digested 4 hours until finishing with PinAI (Invitrogen) and XhoI (Roche) under 37 ℃ in the 1X H buffer reagent (Roche) of 50 μ L volumes.Then will be through PCR product electrophoresis in 1% sepharose of digestion, fragment is removed and uses buffer reagent QG repurity on the MinElute tubing string in gel, and follow intermittent the mixing that gel-buffer mixture is hatched 10 minutes with the dissolving agarose under 50 ℃, remove back PCR as primer afterwards and on tubing string, carry out purifying.3 μ L are connected buffer reagent (Promega with 1 μ L through the pD18-n2H7sssIgG1 SMIP and the 5 μ L 2X LigaFast of PinAI-XhoI digestion through the G28-1LH of PinAI-XhoI digestion, Madison, WI) and 1 μ L T4 dna ligase (Roche) be combined into 10 μ L reactants, thorough mixing and at room temperature hatching 10 minutes.Then utilize manufacturers's scheme that 3 these connectors of μ L are transformed into competence TOP 10 (Invitrogen).Be coated on described transformant on the LB agar plate with 100 μ g/ml Gepcillins (Teknova) and under 37 ℃, hatch overnight.Cultivate after 18 hours, selection bacterium colony and being inoculated among the 1mlT-Broth (Teknova) that contains 100 μ g/ml Gepcillins in deep hole type 96 orifice plates, and in 37 ℃ of concussion thermostat containers, cultivate overnight.Cultivate after 18 to 24 hours, use QIAprep 96 Turbo test kits (Qiagen) to go up DNA isolation in each culture overnight at BioRobot8000 (Qiagen).Then get 10 μ L from each clone, the reaction volume with 15 μ L in 1X B buffer reagent digests with HindIII and XhoI Restriction Enzyme.Will (Invitrogen CA) goes up electrophoresis for the restriction site analysis at 1% agarose E-gel through the DNA of digestion.The segmental clone of the HindIII-XhoI that contains suitable size is carried out the sequence checking.Hold and make (G4S) 4 connexons to be terminated at the Nhe I site of G28-1VH by using primer 29,30,31 and 32 in the Table X above that the PinAI site is placed in 5 ', make up G28-1HL SMIP in a similar manner.Use the primer 33 and 34 in the Table X to change VK, so that the NheI site is introduced the 5 ' end of VK and XhoI is introduced 3 ' end by PCR.Then described PCR is as above made up and with flank primer 29 and 34 amplifications to produce the complete G28-1scFv DNA of VH-VL orientation, just like being cloned into G28-1LH SMIP in pD18-(n2H7) the sssIgG1 SMIP that PinAI-XhoI digests.

2H7sssIgG1-STD1-G28-1 LH/HL construct

Use G28-1LH and G28-1HL SMIP as template, resist-the CD37 binding domains by PCR change LH and HL, so that its flank restriction site is compatible with scorpion shape molecule box.Use primer 27 (LH) or 36 (HL) that the 5 ' end of each scFv is introduced in the EcoRI site, and use primer 28 (LH) or 35 (HL) that 3 ' end is introduced in terminator codon/XbaI site.With the gained dna clone in the pD18-2H7sssIgG-STD1 of EcoRI-XbaI digestion.

The 2H7sssIgG1-Hx-G28-1HL construct

With 2H7sssIgG1-Hx-2e12HL DNA with BsrGI and EcoRI and by the C-end of IgG1 be connected molecular 325bp fragment and digest.By using BsrGI-EcoRI to remove the STD1 connexon described thing is replaced with the equivalence district among the 2H7sssIgG1-STD1-G19-4HL, and it is replaced with the corresponding connexon among the 2H7sssIgG1-Hx-2e12HL clone.

Anti--CD3 binding domains construct

Table 4

SEQ ID

Numbering title sequence NO.

GCGTATGAACCGGTGACATCCAGAT

37 194-LH-LF1 GACACAGACTACATC 67

ATCCAGATGACACAGACTACATCCTC

38 194-LF2 CCTGTCTGCCTCTCTGGGAGACAG 68

GTCTGCCTCTCTGGGAGACAGAGTCA

39 194-LF3 CCATCAGTTGCAGGGCAAGTCAGGAC 69

GTTGCAGGGCAAGTCAGGACATTCGC

40 194-LF4 AATTATTTAAACTGGTATCAGCAG 70

ATTTAAACTGGTATCAGCAGAAACCAG

41 194-LF5 ATGGAACTGTTAAACTCCTGATC 71

GAACTGTTAAACTCCTGATCTACTACA

42 194-LF6 CATCAAGATTACACTCAGGAGTC 72

CAAGATTACACTCAGGAGTCCCATCAA

43 194-LF7 GGTTCAGTGGCAGTGGGTCTGGAAC 73

CAGGTTGGCAATGGTGAGAGAATAATC

44 194-LR7 TGTTCCAGACCCACTGCCACTGAAC 74

GCAAAAGTAAGTGGCAATATCTTCTGGT

45 194-LR6 TGCAGGTTGGCAATGGTGAGAG 75

GAACGTCCACGGAAGCGTATTACCC

46 194-LR5 TGTTGGCAAAAGTAAGTGGCAATATC 76

CGTTTGGTTACCAGTTTGGTGCCTCCAC

47 194-LR4 CGAACGTCCACGGAAGCGTATTAC 77

ACCACCGCCCGAGCCACCGCCACC

48 194-LR3 CCGTTTGGTTACCAGTTTGGTG 78

GCTAGCGCTCCCACCTCCTCCAGATCCA

49 194-LR2 CCACCGCCCGAGCCACCGCCAC 79

GTTGCAGCTGGACCTCGCTAGCGCT

50 194-LH-LR1 CCCACCTCCTCCAGATC 80

GATCTGGAGGAGGTGGGAGCGCTAGC

51 194-LH-HF1 GAGGTCCAGCTGCAACAGTCTGGACCTG 81

AGCTGCAACAGTCTGGACCTGAACT

52 194-HF2 GGTGAAGCCTGGAGCTTCAATGAAG 82

AGCCTGGAGCTTCAATGAAGATTTCC

53 194-HF3 TGCAAGGCCTCTGGTTACTCATTC 83

GCAAGGCCTCTGGTTACTCATTCACT

54 194-HF4 GGCTACATCGTGAACTGGCTGAAGCAG 84

ATCGTGAACTGGCTGAAGCAGAGCC

55 194-HF5 ATGGAAAGAACCTTGAGTGGATTGGAC 85

GAACCTTGAGTGGATTGGACTTATTA

56 194-HF6 ATCCATACAAAGGTCTTACTACCTAC 86

AATGTGGCCTTGCCCTTGAATTTCTG

57 194-HR6 GTTGTAGGTAGTAAGACCTTTGTATG 87

CATGTAGGCTGTGCTGGATGACTTGT

58 194-HR5 CTACAGTTAATGTGGCCTTGCCCTTG 88

ACTGCAGAGTCTTCAGATGTCAGACTG

59 194-HR4 AGGAGCTCCATGTAGGCTGTGCTGGATG 89

ACCATAGTACCCAGATCTTGCACAG

60 194-HR3 TAATAGACTGCAGAGTCTTCAGATGTC 90

GCGCCCCAGACATCGAAGTACCAGTC

61 194-HR2 CGAGTCACCATAGTACCCAGATCTTG 91

GCGAATACTCGAGGAGACGGTGACCG

62 194-LH-HR1 TGGTCCCTGCGCCCCAGACATCGAAG 92

GCGTATGAACCGGTGAGGTCCAGC

631 94-HL-HF1 TGCAACAGTCTGGACCTG 93

ACCGCCACCAGAGGAGACGGTGACCGT

641 94-HL-HR1 GGTCCCTGCGCCCCAGACATCGAAGTAC 94

ACCTCCTCCAGATCCACCACCGCCCG

651 94-HL-HR0 AGCCACCGCCACCAGAGGAGACGGTG 95

GCGGGGGAGGTGGCAGTGCTAGCGA

661 94-HL-LF1 CATCCAGATGACACAGACTACATC 96

GCGAATACTCGAGCGTTTGGTTACCA

671 94-HL-LR3Xho GTTTGGTG 97

GCGATATCTAGATTACCGTTTGGTTAC

68 194-HL-LR3Xba CAGTTTGGTG 98

GCGTATGAGAATTCAGAGGTCCAGCTG

69 194-HL-HF1R1 CAACAGTCTGGACCTG 99

GCGTATGAGAATTCTGACATCCAGA

70 194-LH-LF1R1 TGACACAGACTACATC 100

GCGTATCTAGATTAGGAGACGGTGACC

71 194-LH-HR1Xba GTGGTCCCTGCGCCCCAGACATCGAAG 101

Table 4: the oligonucleotide that is used to form the anti--CD3 binding domains of G19-4scFv sequence.

Synthesize the G19-4 binding domains by prolonging overlapping Oligonucleolide primers as mentioned above.Light chain PCR carries out with two steps, is initiated with the primer 43/44,42/45,41/46 and 40/47 that concentration is respectively 5 μ M, 10 μ M, 20 μ M and 40 μ M and makes up in Platinum PCR SupermixHi-Fidelity and carry out 30 following circulations: lasted for 20 seconds under 94 ℃, lasted for 10 seconds under 60 ℃, lasted for 15 seconds under 68 ℃.The PCR condition that 1 μ L gained PCR product utilization is identical (the exception part is for increasing to for 25 seconds with 68 ℃ of time length) is used primer mixture (for the HL orientation) amplification again of primer mixture (for the LH orientation) or 66/67 (the 40 μ M) of 39/48 (10 μ M), 38/49 (20 μ M) and 37/50 (40 μ M).VK with LH orientation 5 ' end by PinAI in conjunction with and at 3 ' end by the NheI combination, and the VK with HL orientation 5 ' end by NheI in conjunction with and at 3 ' end by the XhoI combination.

Be synthetic heavy chain, by primer 56/57,55/58,54/59 and 53/60 combination being prepared the primer mixture with concentration same as above in a PCR step.In the 2nd PCR, utilize the PCR condition identical to come amplimer 52/61 (20 μ M) and 51/62 (50 μ M) with 1 μ l from a PCR with the 2nd PCR of light chain, have NheI and have the LH orientation of XhoI at 3 ' end to be formed on 5 ' end.In the 2nd PCR, with primer 52/61 (10 μ M), 63/64 (20 μ M), 63 (20 μ M)/65 (40 μ M) and 63 (20 μ M)/5 (80 μ M) with from the 1 μ L of last PCR combination, to be formed on the heavy chain that 5 ' end has PinAI and has the HL orientation of NheI at 3 ' end.As above-mentioned construct, with overlapping being designed to the NheI site of abundance is the primer at center, so that heavy chain by will having LH orientation and light chain PCR combination and increase to synthesize G19-4LH again, and by HL PCR being made up and increasing to synthesize G19-4HL again with primer 63 and 67 with flank primer 37 and 62.

Total length G19-4LH/HL PCR product is separated excision and with aforesaid Qiagen MinElute tubing string purifying in gel by agarose gel electrophoresis.Then described DNA is cloned among the pCR2.1 (Invitrogen) through TOPO, is transformed into TOP10, and bacterium colony is at first passed through the screening of EcoRI clip size, then by the dna sequencing screening.Then G19-4LH/HL is cloned among the pD18-IgG1 so that express in mammalian cell via PinAI-XhoI.

The 2H7sssIgG1-STD1-G19-4LH/HL construct

Use G19-4LH and G19-4HL SMIP as template, resist-the CD3 binding domains by PCR change LH and HL, so that its flank restriction site is compatible with scorpion shape molecule box.Use primer 27 (LH) or 36 (HL) that the 5 ' end of each scFv is introduced in the EcoRI site, and use primer 28 (LH) or 35 (HL) that 3 ' end is introduced in terminator codon/XbaI site.With the gained dna clone in the pD18-2H7sssIgG-STD1 of EcoRI-XbaI digestion.

The 2H7sssIgG1-Hx-G19-4HL construct

With 2H7sssIgG1-Hx-2e12HL DNA with BsrGI and EcoRI and by the C-end of IgG1 be connected molecular 325bp fragment and digest.By using BsrGI-EcoRI to remove the STD1 connexon described thing is replaced with the equivalence district among the 2H7sssIgG1-STD1-G19-4HL, and it is replaced with the corresponding connexon among the 2H7sssIgG1-Hx-2e12HL clone.

Consider to may be obvious that behind the various multivalent binding proteins disclosed herein the feature of the described molecule that easily is combined to form molecule of the present invention.This feature comprises binding domains 1, constant subprovince (comprising hinge or hinge spline structure territory), connexon structural domain and binding domains 2.Intrinsic modularized in the conjugated protein design of described novelty makes those skilled in the art directly to control dna sequence dna at any N-end of module and/or C-end wanted, and compares with parent's molecule that it is originated through revising or the functional novel molecule of enhanced to form demonstration so that this sequence can be inserted almost any position.For example, contain binding domains 1 or the binding domains 2 of any binding domains of the member who is derived from immunoglobulin superfamily as molecule of the present invention.Described institute deutero-binding domains comprises: have with immunoglobulin superfamily member's sequence one to one aminoacid sequence and even the structural domain of coding polynucleotide sequence, and preferably have the varient and the derivative of 80%, 90%, 95%, 99% or 99.5% sequence identity with the immunoglobulin superfamily member.Described binding domains (1 and 2) preferably via as herein other the place described connexons with different sequences and length be connected with other modules of molecule of the present invention, its restricted condition is reached necessary any space of functional tertiary structure and flexibility for described connexon is enough to provide molecule.Another module of multivalent binding proteins is a hinge area, and it not only can be corresponding to immunoglobulin superfamily member's hinge area, and can be its varient, such as described " CSC " or " SSS " hinge area herein.In addition, constant subprovince comprises proteic module of the present invention, and it can be corresponding to the subprovince of immunoglobulin superfamily member's constant region, as hinge-C _H2-C _H3The structure of constant subprovince is illustrated.Also contain the varient and the derivative of constant subprovince, it preferably has the aminoacid sequence that has 80%, 90%, 95%, 99% or 99.5% sequence identity with the immunoglobulin superfamily member.

Exemplary primary structure with described characterization of molecules is shown in the table 5, its disclosure binding domains 1 and 2 polynucleotide and homologous amino acid sequence, and the primary structure of constant subprovince (comprising hinge or hinge spline structure territory) and can insert connexon between the N-end in terminal and binding domains 2 districts of the C-of the constant subprovince of multivalent binding proteins for example.Other examples of molecule of the present invention comprise above-mentioned feature, and wherein for example any one in binding domains 1 and the binding domains 2 or both comprise the V that is derived from the immunoglobulin superfamily member _LOr V _LSpline structure territory and be derived from the identical or different member's of immunoglobulin superfamily V _HOr V _HThe structural domain in spline structure territory, described structural domain can be separated by the connexon of as disclosed herein any connexon representative.The V that is oriented to of containing BD1 in the wherein said structural domain and/or BD2 _L-V _HOr V _H-V _LMolecule.The more complete representation of primary structure with various features of multivalence binding molecule of the present invention is to be present in the appended form of disclosure ending.The present invention further comprises the polynucleotide of the described molecule of encoding.

Table 5

Binding domains	Nucleotide sequence	Aminoacid sequence	SEQ IDNOS. (aminoacid sequence)
				2H7 LH	atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggacaaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccccatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggagttttaacccacccacgttcggtgctgggaccaagctggagctgaaagatggcggtggctcgggcggtggtggatctggaggaggtgggagctctcaggcttatctacagcagtctggggctgagtcggtgaggcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaagcagacacctagacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctacaatcagaagttcaagggcaaggccacactgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaagactctgcggtctatttctgtgcaagagtggtgtactatagtaactcttactggtacttcgatgtctggggcacagggaccacggtcaccgtctct	mdfqvqifsfllisasvimsrgqivlsqspailsaspgekvtmtcrasssvsymhwyqqkpgsspkpwiyapsnlasgvparfsgsgsgtsysltisrveaedaatyycqqwsfnpptfgagtklelkdgggsggggsggggssqaylqqsgaesvrpgasvkmsckasgytftsynmhwvkqtprqglewigaiypgngdtsynqkfkgkatltvdkssstaymqlssltsedsavyfcarvvyysnsywyfdvwgtgttvtvs	1(2)

Binding domains	Nucleotide sequence	Aminoacid sequence	SEQ IDNOS. (aminoacid sequence)
				2e12 LH	atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtcgacattgtgctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgctagcaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagactttagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccatggacgttcggtggaggcaccaagctggaaatcaaacggggtggcggtggatccggcggaggtgggtcgggtggcggcggatctcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactatcgatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgcccgagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctct	MDFQVQIFSFLLISASVIMSRGVDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRGGGGSGGGGSGGGGSQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSS	3(4)
2e12 HL	atggattttcaagtgcagattttcagcttcctgctaatcagtgcttcagtcataatgtccagaggagtccaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatcacatgcaccgtctcagggttctcattaaccggctatggtgtaaactgggttcgccagcctccaggaaagggtctggagtggctgggaatgatatggggtgatggaagcacagactataattcagctctcaaatccagactatcgatcaccaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccagatactactgtgcccgagatggttatagtaactttcattactatgttatggactactggggtcaaggaacctcagtcaccgtctcctctgggggtggaggctctggtggcggtggatccggcggaggtgggtcgggtggcggcggatctgacattgt	MDFQVQIFSFLLISASVIMSRGVQVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKR	5(6)

Binding domains	Nucleotide sequence	Aminoacid sequence	SEQ IDNOS. (aminoacid sequence)
					gctcacccaatctccagcttctttggctgtgtctctaggtcagagagccaccatctcctgcagagccagtgaaagtgttgaatattatgtcacaagtttaatgcagtggtaccaacagaaaccaggacagccacccaaactcctcatctctgctgctagcaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagactttagcctcaacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagcaaagtaggaaggttccatggacgttcggtggaggcaccaagctggaaatcaaacgt
G28-1LH	accggtgacatccagatgactcagtctccagcctccctatctgcatctgtgggagagactgtcaccatcacatgtcgaacaagtgaaaatgtttacagttatttggcttggtatcagcagaaacagggaaaatctcctcagctcctggtctcttttgcaaaaaccttagcagaaggtgtgccatcaaggttcagtggcagtggatcaggcacacagttttctctgaagatcagcagcctgcagcctgaagattctggaagttatttctgtcaacatcattccgataatccgtggacgttcggtggaggcaccgaactggagatcaaaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgctagcgcagtccagctgcagcagtctggacctgagctggaaaagcctggcgcttcagtgaagatttcctgcaaggcttctggttactcattcactggctacaatatgaactgggtgaagcagaataatggaaagagccttgagtggattggaaatattgatccttattatggtggtactacctacaaccggaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcaagagtctgacatctgaggactctgcagtctattactgtgcaagatcggtcggccctatggactactggggtcaaggaacctcagtcaccgtctcgag	DIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLLVSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDNPWTFGGGTELEIKGGGGSGGGGSGGGGSASAVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLEWIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARSVGPMDYWGQGTSVTVS	102(103)
				G28-1HL	accggtgaggtccagctgcaacagtctggacctgaactggtgaagcctggagcttcaatgaagatttcctgcaaggcctctggttactcattcactggctacatcgtgaactggctgaagcagagccatggaaagaaccttgagtggattggacttattaatccatacaaaggtcttactacctacaaccagaaattcaagggcaaggccacattaactgtagacaagtcatccagcacagcctacatggagctcctcagtctgacat	EVQLQQSGPELVKPGASMKISCKASGYSFTGYIVNWLKQSHGKNLEWIGLINPYKGLTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSASDIQMTQTTSSLSASLGDRVTISCRASQDIR	104(105)

Binding domains	Nucleotide sequence	Aminoacid sequence	SEQ IDNOS. (aminoacid sequence)
					ctgaagactctgcagtctattactgtgcaagatctgggtactatggtgactcggactggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctctggtggcggtggctcgggcggtggtggatctggaggaggtgggagcgggggaggtggcagtgctagcgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattcgcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattgccaacctgcaaccagaagatattgccacttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcaccaaactggtaaccaaacgctcgag	NYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIANLQPEDIATYFCQQGNTLPWTFGGGTKLVTKRS
G19-4LH	accggtgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattcgcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattgccaacctgcaaccagaagatattgccacttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcaccaaactggtaaccaaacggggtggcggtggctcgggcggtggtggatctggaggaggtgggagcgctagcgaggtccagctgcaacagtctggacctgaactggtgaagcctggagcttcaatgaagatttcctgcaaggcctctggttactcattcactggctacatcgtgaactggctgaagcagagccatggaaagaaccttgagtggattggacttattaatccatacaaaggtcttactacctacaaccagaaattcaagggcaaggccacattaactgtagacaagtcatccagcacagcctacatggagctcctcagtctgacatctgaagactctgcagtctattactgtgcaagatctgggtactatggtgactcggactggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctcgag	DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIANLQPEDIATYFCQQGNTLPWTFGGGTKLVTKRGGGGSGGGGSGGGGSASEVQLQQSGPELVKPGASMKISCKASGYSFTGYIVNWLKQSHGKNLEWIGLINPYKGLTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVSS	106(107)
				G19-4HL	accggtgaggtccagctgcaacagtctggacctgaactggtgaagcctggagcttcaatgaagattt	EVQLQQSGPELVKPGASMKISCKASGYSFTGYIVNWLKQSHGK	108(109)

Binding domains	Nucleotide sequence	Aminoacid sequence	SEQ IDNOS. (aminoacid sequence)
					cctgcaaggcctctggttactcattcactggctacatcgtgaactggctgaagcagagccatggaaagaaccttgagtggattggacttattaatccatacaaaggtcttactacctacaaccagaaattcaagggcaaggccacattaactgtagacaagtcatccagcacagcctacatggagctcctcagtctgacatctgaagactctgcagtctattactgtgcaagatctgggtactatggtgactcggactggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctctggtggcggtggctcgggcggtggtggatctggaggaggtgggagcgctagcgacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattcgcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattgccaacctgcaaccagaagatattgccacttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcaccaaactggtaaccaaacgctcgag	NLEWIGLINPYKGLTTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSASDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIANLQPEDIATYFCQQGNTLPWTFGGGTKLVTKRS

Hinge area	Nucleotide sequence	Aminoacid sequence	SEQ ID NO. (aminoacid sequence)
				sss(s)-hIgG1	gagcccaaatcttctgacaaaactcacacatctccaccgagctca	EPKSSDKTHTSPPSS	230(231)
csc(s)-hIgG1	gagcccaaatcttgtgacaaaactcacacatctccaccgtgctca	EPKSCDKTHTSPPCS	232(233)
				ssc(s)-hIgG1	gagcccaaatcttctgacaaaactcacacatctccaccgtgctca	EPKSSDKTHTSPPCS	110(111)
scc(s)-hIgG1	gagcccaaatcttctgacaaaactcacacatgtccaccgtgctca	EPKSSDKTHTCPPCS	112(113)
				css(s)-hIgG1	gagcccaaatcttgtgacaaaactcacacatctccaccgagctca	EPKSCDKTHTSPPSS	114(115)
scs(s)-hIgG1	gagcccaaatcttgtgacaaaactcacacatgtccaccgagctca	EPKSSDKTHTCPPSS	116(117)
				ccc(s)-hIgG1	gagcccaaatcttgtgacaaaactcacacatgtccaccgtgctca	EPKSCDKTHTSPPCS	118(119)
ccc(p)-	Gagcccaaatcttgtgacaaaactcacacatgt	EPKSCDKTHTSPPCP	120(121)

hIgG1	ccaccgtgccca
				sss(p)-hIgG1	gagcccaaatcttctgacaaaactcacacatctccaccgagccca	EPKSSDKTHTSPPSP	122(123)
csc(p)-hIgG1	gagcccaaatcttgtgacaaaactcacacatctccaccgtgccca	EPKSCDKTHTSPPCP	124(125)
				ssc(p)-hIgG1	gagcccaaatcttctgacaaaactcacacatctccaccgtgccca	EPKSSDKTHTSPPCP	126(127)
scc(p)-hIgG1	gagcccaaatcttctgacaaaactcacacatgtccaccgtgccca	EPKSSDKTHTCPPCP	128(129)
				css(p)-hIgG1	gagcccaaatcttgtgacaaaactcacacatctccaccgagccca	EPKSCDKTHTSPPSP	130(131)
scs(p)-hIgG1	gagcccaaatcttgtgacaaaactcacacatgtccaccgagccca	EPKSSDKTHTCPPSP	132(133)
				scppcp	agttgtccaccgtgccca	SCPPCP	134(135)
EFD	Nucleotide sequence	Aminoacid sequence	Recognition sequence symbol (aminoacid sequence)
				hIgG1(P238S)C H2C H3	gcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga	APELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	142(143)
hIgG1(P331S)C H2C H3	gcacctgaactcctgggtggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgt	APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELT	144(145)

	cctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga	KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
				hIgG1(P238S/P331S)C H2C H3	gcacctgaactcctgggtggatcgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaacaatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga	APELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	146(147)
Connexon	Nucleotide sequence	Aminoacid sequence	The recognition sequence symbol
				STD1	aattatggtggcggtggctcgggcggtggtggatctggaggaggtgggagtgggaattct	NYGGGGSGGGGSGGGGSGNS	148(149)
STD2	aattatggtggcggtggctcgggcggtggtggatctggaggaggtgggagtgggaattatggtggcggtggctcgggcggtggtggatctggaggaggtgggagtgggaattct	NYGGGGSGGGGSGGGGSGNYGGGGSGGGGSGGGGSGNS	150(151)
				H1	aattct	NS	152(153)
H2	ggtggcggtggctcggggaattct	GGGGSGNS	154(155)

H3	aattatggtggcggtggctctgggaattct	NYGGGGSGNS	156(157)
				H4	ggtggcggtggctcgggcggtggtggatctgggaattct	GGGGSGGGGSGNS	158(159)
H5	aattatggtggcggtggctcgggcggtggtggatctgggaattct	NYGGGGSGGGGSGNS	160(161)
				H6	ggtggcggtggctcgggcggtggtggatctgggggaggaggcagcgggaattct	GGGGSGGGGSGGGGSGNS	162(163)
H7	gggtgtccaccttgtccgaattct	GCPPCPNS	164(165)
				(G4S)3	ggtggcggtggatccggcggaggtgggtcgggtggcggcggatct	GGGSGGGSGGGS	166(167)
(G4S)4	ggtggcggtggctcgggcggtggtggatctggaggaggtgggagcgggggaggtggcagt	GGGSGGGSGGGSGGGGS	168(169)

Table 5: primary structure (polynucleotide and homologous amino acid sequence) with exemplary feature of multivalence binding molecule

Embodiment 8

To substituting the combination and the functional study of polyspecific fusion rotein

Each of above-mentioned other multivalence binding molecules is carried out combining with above-mentioned prototype CD20-IgG-CD28 polyspecific the parallel laboratory test of the experiment of (fusion) molecule.Generally speaking, the data that described other molecules obtained are similar with the result who is observed for the prototype molecule.Some of described experiment be remarkable result be disclosed in hereinafter.Figure 14 shows the result of the blocking-up research that one in the novel molecule carried out, and wherein BD1 and BD2 all combine the target antigen (being CD20 and CD37 in the case) on same cell or the cell type.This polyspecific multivalence combination (fusion) albumen is through being designed to have the binding domains 1 (2H7 in conjunction with CD20; VLVH orientation) and in conjunction with the binding domains 2 (G28-1VL-VH (LH) or VH-VL (HL)) of CD37.Carry out described experiment and be intended to prove proteic polyspecific characteristic.

Blocking-up research: with Ramos or BJAB B Lymphoblastoid (2.5x10 ⁵) arising from the dyeing substratum (PBS) preincubate in 96 hole V-arrangement base plates with mouse-anti-CD20 (25 μ g/ml) antibody or mouse-anti-CD37 (10 μ g/ml) antibody one with 2% mice serum, the substratum that will dye together or separately places in the dark and lasts 45 minutes on ice.Blocking antibody with cell preincubate 10 minutes at room temperature, is added polyspecific in conjunction with (fusion) albumen with prescribed concentration scope (being generally 0.02 μ g/ml to 10 μ g/ml) afterwards, and in the dark in hatching again 45 minutes on ice.With cell washing 2 times in the dyeing substratum, and in the dyeing substratum in that (Burlingame, CA) the anti-IgG of FITC goat (1:100) was hatched one hour together, to detect polyspecific combining in conjunction with (fusion) albumen and cell with Caltag on ice.Then (Cleveland Ohio) fixes for No. the 19943rd, catalog number (Cat.No.), USB with PBS washing 2 times and with 1% trioxymethylene with cell.(BD Biosciences, San Jose is CA) by the flow cytometry cell to use FACsCalibur instrument and CellQuest software.Each data set is described 2H7-sss-hIgG-STD1-G28-1HL fusion rotein combining in the presence of CD20, CD37 or the two blocking antibody of CD20 and CD37.Though this experiment is used the existence of one, two blocking antibodies in the cracking connexon to get rid of fully with polyspecific and combined (fusion) proteic combination, this proves that most of molecules have combined function to CD20 and CD37.The data class of in figure A and figure B, two kinds of clone Ramos and BJAB being tested seemingly, wherein the CD20 blocking antibody more effectively reduces (fusion) the protein bound degree of being observed that combines with polyspecific than CD37 blocking antibody.

ADCC measures

Figure 15 shows the result of the ADCC mensuration that CD20-CD37 polyspecific combination (fusion) albumen is carried out.Use BJAB lymphoblast sample B cell to carry out ADCC mensuration as target and human PBMC action effect cell.Under 37 ℃, in IMDM/10% FBS, with bjab cell with 500 μ Ci/ml ⁵¹Cr Sodium chromate (250 μ Ci/ μ g) mark 2 hours.To in RPMI.10% FBS, wash three times through the cell of mark and with 4 * 10 ⁵Individual cells/ml resuspending is in RPMI.The heparinization human whole blood is available from anonymous laboratory donor, and separates PBMC via lymphocyte separating medium (LSM, ICN Biomedical) gradient by fractional separation.Results buffy coat (Buffy coats) and in RPMI/10% FBS washed twice, afterwards with 5 * 10 ⁶The ultimate density resuspending of individual cells/ml is in RPMI/10% FBS.Before being used for subsequent measurements, use the hemocytometer pair cell to count by trypan blue exclusion method.The reagent sample is added into for 4 times in the RPMI substratum with 10% FBS with ultimate density, and to three parts of 10 times of serial dilutions of each reagent preparation.Then described reagent is added in the 96 hole U type base plates until indicated ultimate density with 50 microlitres/hole.Will through ⁵¹The bjab cell of Cr mark is with 50 microlitres/hole (2 * 10 ⁴Individual cells/well) is added in the plate.Follow PBMC with 100 microlitres/hole (5 * 10 ⁵Individual cells/well) be added in the plate, make effector (PBMC): the final ratio of target (BJAB) is 25:1.Effector and target are added in the simple substratum to measure the background kill ratio.Will through ⁵¹The cell of Cr mark is added in the simple substratum to measure ⁵¹The spontaneous release rate of Cr and be added into and have 5% NP40 (No. the 28324th, catalog number (Cat.No.), Pierce, Rockford, in substratum IL) to measure ⁵¹The maximum release rate of Cr.Triplicate reacts in the hole of 96 orifice plates.Polyspecific is added in the hole with the ultimate density (as shown in the figure) in 0.01 μ g/ml to the 10 μ g/ml scope in conjunction with (fusion) albumen.Each data set is described different polyspecifics combination (fusion) albumen or the corresponding single specificity SMIP in the described titration scope.Results and the counting before, make be reflected under 37 ℃, at 5% CO ₂In carried out 6 hours.Then will be transferred to from the 25 μ l supernatant liquors in each hole Luma Plate 96 (No. the 6006633rd, catalog number (Cat.No.), Perkin Elmer, Boston, Mass) in and at room temperature dry overnight.On PackardTopCounNXT, measure the CPM that is discharged.Calculate specificity and kill percentage by deducting (this mean value of cpm{ three increments of sample }-the spontaneous release rate of cpm)/(the spontaneous release rate of the maximum release rate-cpm of cpm) x100.Killing the relative protein concentration of % with specificity maps to data.Described digital proof polyspecific can mediate the ADCC activity of the antigenic cell of the opposing one or more targets of expression and the SMIP single specificity to CD20 and/or CD37 in conjunction with (fusions) albumen, but does not show the increase of this effector function degree.

Co-culture experiments

Figure 16 demonstration is conceived to this type polyspecific in conjunction with the designed result of experiment of (fusion) proteic other characteristics, wherein by through two institute's bonded surface receptor signal conduction/combinations, two binding domainss of resisting targets are expressed on same cell or cell type can produce synergistic effect.Use is measured described isolating PBMC about ADCC as mentioned and is carried out co-culture experiments.With described PBMC with 2x10 ⁶Individual cells/ml, with the final volume resuspending in 500 microlitres/hole in substratum, and single culture, or use H7 connexon [2H7-sss-IgG-H7-G28-1HL] to hatch in conjunction with (fusion) proteic single specificity SMIP with CD20, CD37, CD20+CD37 or polyspecific.Add each test agent with the ultimate density of 20 μ g/ml.Cultivate after 24 hours, the % that does not observe B cell in the culture has actual variance; Yet, when described cell stands flow cytometry, visible cell agglomerate in the FWD X 90 dyeing patterns that contain polyspecific combination (fusion) proteic culture, this explanation is expressed the antigenic B cell of these two targets and is participated in homotype adhesion.Cultivate after 72 hours, polyspecific makes existing nearly all B necrocytosis in conjunction with (fusion) albumen.The combination of two kinds of single specificity SMIP also acutely reduces the percentage of B cell, but does not see that for the polyspecific binding molecule this amount descends.Described data show, the binding domains of CD20 and the binding domains of CD37 all are added on the same polyspecific molecule, can make to produce the homotype adhesion between the B cell, and also can make CD20 combine same cell with CD37 antigen.Although be not wishing to be bound by theory, the synergistic effect of removing the target cell be attributable to (1) via binding domains 1 and 2 in conjunction with the same cell type; And/or (2) in the PBMC culture, and the effector function structural domain of multivalence binding molecule (constant subprovince) interacts with monocyte or other cell types, makes and kills delay.This kinetics speed of killing effect is unhappy, and finishing needs to illustrate that it can be second-order effect more than 24 hours, observes described effect again after needing to produce cytokine or other molecules.

Apoptosis is measured

Figure 17 is shown as and probes into the designed result of experiment of cell death inducing after using [2H7-sss-hIgG-H7-G28-1HL] polyspecific multivalence combination (fusion) albumen or single specificity CD20 and/or CD37SMIPS to reach mutual combined treatment B clone separately.Under 37 ℃, at 5% CO ₂In, in Iscoves (Gibco) perfect medium, with Ramos cell (figure A with 10% FBS and 5 μ g/ml, 10 μ g/ml or 20 μ g/ml fusion roteins; ATCC CRL-1596 number) and the Daudi cell (figure B; ATCC CCL-213 number) with 3 * 10 ⁵Individual cells/ml is hatched (24 hours) overnight.If carry out combination experiment with single specificity SMIP, then described albumen is to use with following concentration: TRU-015 (at the SMIP of CD20)=10 μ g/ml, G28-1LH (at the SMIP of CD37)=5 μ g/ml.Perhaps, the TRU-015 of 20 μ g/ml and the G28-1LH of 10 μ g/ml are made up.Then use BD Pharmingen apoptosis test regent box (No. the 556547th, catalog number (Cat.No.)) with cell with the dyeing of annexin V-FITC (AnnexinV-FITC) and propidium iodide and according to test kit explanation handle.Cell is rotated light and slowly, under room temperature, hatched 15 minutes in the dark, and in 400 μ l binding buffer agent, dilute, analyze afterwards.On FACsCalibur (Becton Dickinson) instrument that uses Cell Quest software (Becton Dickinson), pass through the flow cytometry sample.Data show with the percentage of the describing annexin V/propidium iodide positive cell histogram with respect to the relation of handling type.Obviously, compare with single specificity reagent when using (even), polyspecific all can be induced the apoptosis of remarkable higher degree to two kinds of clones in conjunction with (fusion) albumen.This enhanced functional activity reflection BD1 and BD2 (to CD20 and CD37 tool specificity) the coordination binding interactions of acceptor on the target cell.

Embodiment 9

The 2H7-hIgG-G19-4 polyspecific is in conjunction with (fusion) proteic combination and functional performance

This embodiment describes the combination and the functional performance of 2H7-hIgG-G19-4 polyspecific fusion rotein.The structure of described molecule is described among the embodiment 7.Expression and purifying are described in above embodiment.

As carry out the combination experiment as described for above molecule, the exception part is to be used to measure CD3 bonded target cell for expressing the Jurkat cell of CD3 in its surface.With reference to Figure 18, wherein top figure shows the purifying protein that uses by 20 μ g/ml serial dilution to 0.05 μ g/ml, the binding curve that is obtained in conjunction with the Jurkat cell about CD20-CD3 polyspecific molecule.As if compare with the LH orientation, the G19-4 with HL orientation combines more excellent with the antigenic specificity of CD3.Figure below shows the binding curve that is obtained about BD1 (binding domains of identification CD20).All molecules are all in conjunction with well and with approximate being equivalent to combine with the degree of the monospecific SMIP of CD20 tool.

ADCC measures

For data shown among Figure 19, described in above-mentioned embodiment, carry out ADCC and measure.In the case, fusion rotein is the combination of 2H7-hIgG-G19-4 varient or single specificity SMIP (to the specific 2H7 of CD20 tool) or antibody (to the specific G19-4 of CD3 tool).In addition, for the shown data of the figure below among Figure 19, before using, pass through to use MACS (Miltenyi Biotec, Auburn, CA) the magnetic beads exhaustion method of tubing string tripping device and NK cell-specific CD16 magnetic micro-beads grain (catalog number (Cat.No.) 130-045-701 number) exhausts the NK cell in PBMC.Shown digital proof among two figure, whether all CD20-hIgG-CD3 polyspecific molecules all mediate ADCC, and exhaust with the NK cell or whether total PBMC is used for measuring irrelevant.For the combination of TRU 015 or G19-4 and TRU015, the culture that only contains the NK cell can mediate ADCC.Although G19-4 can in conjunction with the NK T cell of expressing CD3 and shown in first measure in the described cell of activation, in the arbitrary mensuration at the BJAB target, G19-4 all operates not good, it does not express CD3.The polyspecific of being observed in figure below may be via opposite with the antigenic BJAB target of expression CD20 in conjunction with (fusion) proteic killing effect, mediates by come activating T cell group's cytotoxicity in conjunction with CD3.In used concentration range, this kills active dosage for molecule and seems relative insensitivity, and even under the concentration of 0.01 μ g/ml, still obviously be different from other molecules of being tested.

Embodiment 10

The multivalence binding molecule

Other embodiments comprise the connexon structural domain that is derived from immunoglobulin (Ig).More particularly, the source sequence of described connexon is the following sequence that obtains: relatively be present between other members' the V-spline structure territory of immunoglobulin superfamily or be present in district between V-spline structure territory and the C-spline structure territory.Because described sequence is expressed as the part of the extracellular domain of cell surface receptor usually, expect that therefore it is more stable and also should not have immunogenicity for proteolytic cleavage.Expection uncomfortable as multivalence in conjunction with (fusions) proteic connexon, be expressed sequence type on the surface expression member of-Ig superfamily but be present in a kind of sequence type in the insertion district between C-spline structure territory and the membrane spaning domain.Through observing many described molecules have soluble form and, illustrating that described sequence more is subject to the cracking influence than all the other molecules in described insertion district's cracking near cytolemma.

As described herein, above-mentioned connexon is inserted among the single specificity SMIP, inserts between binding domains and the effector function structural domain, or insert multivalence combination (fusion) proteic two kinds of possibility connexon positions one in.

The complete sequence table that is disclosed among the application that encloses, and this sequence table is incorporated herein in full with way of reference.Colour coding indicates the sequence of various districts in specific polynucleotide and the polypeptide or structural domain to be applicable to correspondence district or structural domain in the sequence of differentiating any molecule disclosed herein.

Embodiment 11

The screening matrix that is used for the scorpion shape molecule candidate of target B-cell

Foreword

As the method for the combination of differentiating the paired monoclonal antibody binding domains that produces suitable and effective multivalence binding molecule or the scorpion shape molecule of resisting target colony probably, in the anti-B clone composite substrate of the various non-Hodgkin lymphomas of representative, the monoclonal antibody of a series of opposing B cell antigens is tested.By guaranteeing known or expection might be pursued more all being measured in conjunction with the institute of the antibody of institute's concerns cell, can use set cell type, the two-dimentional matrix of use antibody is guided research and design.Knownly claim the monoclonal antibody of the multiple B cell antigen of opposing of (CD) to be recorded in left column according to its cluster name.Some described antibody (naming according to the one or more antigens of its specificity bonded) (that is CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and CL II (II class MHC)) is hatched with antigen positive target cell separately or with other member's combinations of this monoclonal antibody group.The variable domains that contains described antibody is as the binding domains in the exemplary embodiment of multivalence binding molecule.Those skilled in the art utilize understanding and the conventional procedure to this area, can in the database that for example can openly obtain, differentiate suitable antibody sequence (nucleic acid coding sequence and aminoacid sequence), to form suitable antibodies or its fragment (for example by clone, PCR, method of peptide synthesis and fellow thereof) and to use described compound to make up the multivalence binding molecule based on hybridization.The source that obtains the exemplary antibody of binding domains as described herein is provided in the table 6.Usually will use the clone or the synthesis strategy in the CDR district that can obtain antibody chain, keep specificity any antibody, its fragment or derivatives thereof in conjunction with the antigenic ability of target but still contain.

In more detail, be standard in this area from the heavy chain of hybridoma clonal antibody and/or variable region of light chain.Do not need to know the sequence of the variable region of paying close attention to so that utilize conventional clone technology to obtain this district.Referring to people such as for example Gilliland, Tissue Antigens 47 (1): 1-20 (1996).For preparation comprises the single chain polypeptide of the variable region that can discern mouse or human leucocyte antigen, design a kind of method that is used for quick clone and expression, this method can be at generation functional protein in two to three weeks of hybridoma isolation of RNA.The variable region is by the poly-G tailing first chain cDNA, then by preposition poly-C anchor primer and the constant region sequence is had specific reverse primer grappling PCT clone.Two kinds of primers all contain the flank restriction endonuclease site that is useful in the insertion pUC19.Be formed for separating mouse, hamster and rat V _LAnd V _HThe PCR primer sets of gene.Measuring specificity V _LWith V _HBehind the right consensus sequence, ((Gly usually encodes to get involved the peptide connexon by coding ₄Ser) ₃) DNA make V _LWith V _HGene connects, and with V _L-connexon-VH box gene is transferred in the pCDM8 mammalian expression vector.The construct transfection is reclaimed sFv to the COS cell and from the conditioned medium supernatant liquor.This method successfully is used for forming anti-human CD2, CD3, CD4, CD8, CD28, CD40, the functional sFv of CD45 and the functional sFv of anti-mouse CD3 and gp39 from the hybridoma that produces mouse, rat or hamster antibody.Originally, sFv is expressed as the hinge-C with IgG 1 _H2-C _H3The fusion rotein of structural domain is beneficial to use anti-IgG reagent of goat or albumin A to carry out fast characterizing and purifying.The active also available little peptide of sFv (for example mark) is expressed or is expressed with the anury form.Express the cell signaling activity enhancing of CD3 (G19-4) sFv anury formal proof and disclose the potential that sFv has activated receptor.

Perhaps, can following direct realization to the discriminating of the one-level aminoacid sequence of the variable domains of monoclonal antibody: for example limit the proteolyzing of antibody, for example use the Edman degradation method then or carry out the order-checking of N-terminal peptide by the fracture mass spectroscopy.N-end sequencing method has been well known in the art.Behind the one-level aminoacid sequence of measuring variable domains,, assemble the cDNA of this sequence of coding then by the scFv forming method by synthetic property nucleic acid synthetic method (for example PCR).Described must or the preferred nucleic acid control method be standard in this area.

Fragment, derivative and the analogue of also containing aforesaid antibody are as suitable binding domains.In addition, contain above-mentioned any constant subprovince, comprise the constant subprovince that comprises above-mentioned any hinge area.In addition, the multivalence strand binding molecule described in this embodiment can comprise described any or all of connexon herein.

Initially make monoclonal antibody be exposed to cell and then use goat anti-mouse second step antibody linked (the 2nd step).Randomly, can before making cell and antibody contacts, make antibody linked (for example antibody linked) by in solution, making.As another alternative method, can followingly make monoclonal antibody crosslinked with solid phase: by on the plastic bottom layer that is adsorbed to the tissue culture hole or by means of the goat anti-mouse antibody that is adsorbed to plastics " holding back " on these plastics, then by assess for example growth interruption or cell survival based on the mensuration of orifice plate.

The phosphatidyl Serine reverses the accepted index of the outside cell surface of plasma membrane so far for short apoptosis incident via the kytoplasm side of cytolemma.Advance to apoptosis and cause the cell membrane integrity forfeiture, this can insert entering of dyestuff (for example propidium iodide (PI)) by the cell opacity and detect., after monoclonal antibody alone or in combination, carry out dual short apoptosis and measure in cellular exposure, and treated cell mass is marked with regard to positive annexin V (ANN) of cell surface and/or PI inclusion aspect.

Fractional analysis in the annexin V combination/propidium iodide

Cell and cell culture condition.Experiment is intended to detect the crosslinked influence to four kinds of human B-clones of two kinds of different monoclonal antibodies that makes the expressed target of opposing.Measure the influence of pair cell system by measuring exposure back ANN and/or the painted degree of PI.Under 37 ℃, at 5%CO ₂In, in having the Iscoves of 10%FBS (Gibco) perfect medium, human B clone BJAB, Ramos (ATCC#CRL-1596), Daudi (ATCC#CCL-213) and DHL-4 (DSMZ#ACC495) were hatched 24 hours.Before the research, make cell density maintain 2 * 10 ⁵To 8 * 10 ⁵Individual cells/ml and survival rate are common〉95%.

With 2 * 10 ⁵Each competitive list clonal antibody of the cell density of individual cells/ml and 2 μ g/ml experimentizes via the matrix of resisting the B-cell antigen.Each competitive list clonal antibody is to add separately with 2 μ g/ml, or also individually adds with each matrix monoclonal antibody combination with 2 μ g/ml.Table 6 is listed the catalog number (Cat.No.) and the source of monoclonal antibody used in the described experiment.For making described monoclonal antibody for crosslinked in the solution, then with the concentration ratio (goat anti-mouse antibody: each monoclonal antibody) be added in each hole of goat anti-mouse IgG (Jackson Labs catalog number (Cat.No.) 115-001-008 number) with 2:1, for example, the Kong Zhongke that only has a kind of monoclonal antibody (2 μ g/ml) is added with the goat anti-mouse antibody that ultimate density is 4 μ g/ml, and has the goat anti-mouse antibody that competitive list clonal antibody (2 μ g/ml) and the Kong Zhongke from the monoclonal antibody (2 μ g/ml) of matrix are added with 8 μ g/ml.

Cell under 37 ℃ in 5% CO ₂In hatch 24 hours after, use BD Pharmingen annexin V-FITC apoptosis test regent box I (#556547), with it with annexin V-FITC and propidium iodide dyeing.In brief, with cell with cold PBS washed twice and with 1 * 10 ⁶Individual cells/ml resuspending is in " binding buffer agent ".Then the cell of 100 microlitres in the binding buffer agent dyeed with 5 μ l annexin V-FITC and 5 μ l propidium iodides.Cell is mixed light and slowly and at room temperature in dark, hatched 15 minutes.Then 400 microlitre binding buffer agent are added in each sample.Then go up sample readings and use Cell Quest software (Becton Dickinson) are analyzed at FACsCalibur (Becton Dickinson).

Table 6

Title Catalog number (Cat.No.) Supplier

Anti--CD19 #C2269-74 US Biological (Swampscott, MA)

Anti-CD 20 #169-820 Ancell Corp (Bayport, MN)

Anti--CD21 #170-820 Ancell Corp (Bayport, MN)

Anti--CD22 #171-820 Ancell Corp (Bayport, MN)

Anti--CD23 #172-820 Ancell Corp (Bayport, MN)

Anti--CD30 #179-820 Ancell Corp (Bayport, MN)

Anti--CD37 #186-820 Ancell Corp (Bayport, MN)

Anti-CD 40 #300-820 Ancell Corp (Bayport, MN)

Anti--CD70 #222-820 Ancell Corp (Bayport, MN)

Title Catalog number (Cat.No.) Supplier

Anti--CD72 #C2428-41B1 US Biological (Swampscott, MA)

Anti--CD79a #235-820 Ancell Corp (Bayport, MN)

Anti--CD79b #301-820 Ancell Corp (Bayport, MN)

Anti--CD80 #110-820 Ancell Corp (Bayport, MN)

Anti--CD81 #302-820 Ancell Corp (Bayport, MN)

Anti--CD86 #307-820 Ancell Corp (Bayport, MN)

Anti--CLII DR, DQ, DP #131-820 Ancell Corp (Bayport, MN)

Table 6: the antibody and the source thereof of employed opposing B cell antigen in this research.

Cross-linking antibody (for example goat anti-mouse antibody) is added into makes cellular sensitivity strengthen among the independent monoclonal antibody A, illustrate with constructed multivalence binding molecule or the scorpion shape molecule of two binding domainss of discerning same antigen effectively to strengthen cellular sensitivity.Be not wishing to be bound by theory, this susceptibility enhancing is attributable to antigen aggregation and the conduction of modified signal.TNF receptor family member for example needs the homotype multimerization so that signal transduction, and can promote this interaction at the scorpion shape molecule that each end of molecule has an equivalent binding domains.The gathering of CD40 and the conduction of signal subsequently are the example of this phenomenon at the B cell system.

As shown in Figure 20,21 and 22, add different antigenic monoclonal antibody A of opposing and monoclonal antibody B and can produce the short apoptosis effect of addition, or in some combination, produce short apoptosis effect greater than addition (that is collaborative) to treated cell.In Figure 20, for example, the combination of monoclonal antibody of anti-CD 20 and other B cell antigens of opposing all can make cellular sensitivity obtain in various degree enhancing.Yet, some combination is (such as the combination of anti-CD 20 with anti--CD19, or the combination of anti-CD 20 and anti--CD21) produces short apoptosis effect, illustrate that multivalence binding molecule or the scorpion shape molecule be made up of described binding domains can remove the B cell of conversion especially effectively greater than addition.With reference to Figure 20, when being exposed to independent anti-CD 20 antibodies, the per-cent that shows the short active cell of apoptosis is about 33% (corresponding to the vertical bar shaped post of " 20 ", that is anti-CD 20 antibodies); Be exposed to anti--during CD19 antibody, the per-cent of short apoptosis cell is about 12% (among Figure 20 corresponding to the vertical bar shaped post of " 19 ", that is anti--CD19 antibody); And be exposed to anti-CD 20 with anti--during CD19 antibody, the per-cent of short apoptosis cell is the about 73% horizontal bar shaped post of (among the Figure 20 corresponding to " 19 ").After being exposed to two kinds of antibody, short apoptosis cell is 73%, significantly greater than the effect summation 45% (33%+12%) that each independent antibody produced, illustrates that anti--CD19 and anti-CD 20 antibodies are to producing synergistic effect.The multivalence binding molecule that is suitable for comprise wherein two binding domainss can to the B-cell behavior produce the molecule of additive effect and wherein two binding domainss can produce the multivalence binding molecule of synergistic effect to the B-cell behavior.In certain embodiments, a binding domains will not have detectable influence to measured cell behavior parameter, in the wherein paired binding domains each is facilitated the active different aspect of multivalence binding molecule, (for example binding domains A is in conjunction with target cell and promotion apoptosis such as polyspecific multivalence binding molecule, and binding domains B is in conjunction with the solubility therapeutical agent, such as cytotoxin).Design on the multivalence binding molecule is decided, two binding domainss can be uncorrelated to the type (addition, collaborative or retarding effect) of the combined effect of target cell generation, this is because one in the binding domains can be to acellular (for example solubility) binding partner tool specificity or pair cell dependency binding partner tool specificity, and really not so to different cell types.

Produce addition, exemplary binding domains pairing (as shown in Figure 20-23) collaborative or retarding effect is apparent by table 7 and table 8.Table 7 provides the quantitative data of the per-cent of the cell that dyeing is positive about ANN and/or PI that is extracted in each figure of Figure 20-23.The numerical value that table 8 provides the data of utilizing table 7 to calculate, whether right interaction produces addition, collaborative or retarding effect based on the given antibody of described numerical value decidable, and and for example the per-cent of the cell that is positive according to ANN and/or PI dyeing is assessed.

Table 7

Title	Anti-CD	20	Anti--CD79b	Anti--CL II	Anti--CD22
						Anti--CD19	13/73 *	18/76/66	14/47/46	12/11
Anti-CD 20	33/NA	42/94/92	33/71/76	28/33
					Anti--CD21	14/75	22/50/76	18/24/40	11/11
Anti--CD22	8/55	12/39/33	12/19/17	10/12
					Anti--CD23	8/41	12/63/55	14/22/17	10/12
Anti--CD30	8/38	14/72/61	12/56/61	10/11
					Anti--CD37	15/45	19/92/86	20/60/62	19/20
Anti-CD 40	10/48	12/44/30	13/21/28	14/13
					Anti--CD70	9/40	12/56/39	15/21/15	10/10
Anti--CD72	NA	16/60/64	30/78/63	17/17

Title	Anti-CD	20	Anti--CD79b	Anti--CL II	Anti--CD22
						Anti--CD79a	21/66	43/42/50	28/55/51	14/14
Anti--CD79b	46/88	70/70/68	45/80/76	26/16
					Anti--CD80	7/41	14/35/30	15/19/17	11/11
Anti--CD81	14/65	25/86/83	25/54/43	19/20
					Anti--CD86	7/38	16/58/42	15/24/18	14/11
Anti--CL II	53/77	52/96/98	47/52/43	72/57

^*Numerical value in the 2-4 row of table 7 reflects the height of the histogram among Figure 20-22 respectively, the height of the vertical bar shaped post of first numeric representation of each cell, and second value is represented the height of horizontal bar shaped post, and the height of third value (if existence) reflection strokes and dots post.In the 5th row, the height of the twill post among the height of first numerical value reflection solid post and second value reflection Figure 23.

Table 8

Title	Anti-CD 20	Anti--CD79b	Anti--CL II	Anti--CD22
					Anti--CD19	S：13+33＝46 *	A：18+56＝74A：18+43＝61	S：14+26＝40S：14+18＝32	I：12+10＝22
Anti-CD 20	NA	A：42+56＝98A：42+43＝85	S：33+26＝59S：33+18＝51	A/I：28+10＝38
					Anti--CD21	S：14+33＝47	I：22+56＝78S：22+43＝65	I：18+26＝44A：18+18＝36	I：11+10＝21
Anti--CD22	S：8+33＝41	I：12+56＝68I：12+43＝55	I：12+26＝38I：12+18＝30	NA
					Anti--CD23	A：8+33＝41	A：12+56＝68A：12+43＝55	I：14+26＝40I：14+18＝32	I：10+10＝20
Anti--CD30	A：8+33＝41	A：14+56＝70A：14+43＝57	S：12+26＝38S：12+18＝30	I：10+10＝20
					Anti--CD37	A：15+33＝48	S：19+56＝75S：19+43＝62	S：20+26＝46S：20+18＝38	I：19+10＝29
Anti-CD 40	A/S：10+33＝43	I：12+56＝68I：12+43＝55	I：13+26＝39A：13+18＝31	I：14+10＝24
					Anti--CD70	A：9+33＝42	I：12+56＝68I：12+43＝55	I：15+26＝41I：15+18＝33	I：10+10＝20
Anti--CD72	NA	I：16+56＝72A：16+43＝59	S：30+26＝56S：30+18＝48	I：17+10＝27

Title	Anti-CD	20	Anti--CD79b	Anti--CL II	Anti--CD22
						Anti--CD79a	S：21+33＝54	I：43+56＝99I：43+43＝86	A：28+26＝54A：28+18＝46	I：14+10＝24
Anti--CD79b	S：46+33＝79	NA	S：45+26＝71S：45+18＝63	I：26+10＝36
					Anti--CD80	A：7+33＝40	I：14+56＝70I：14+43＝57	I：15+26＝41I：15+18＝33	I：11+10＝21
Anti--CD81	S：14+33＝47	A：25+56＝81S：25+43＝68	A：25+26＝51A：25+18＝43	I：19+10＝29
					Anti--CD86	A：7+33＝40	I：16+56＝72I：16+43＝59	I：15+26＝41I：15+18＝33	I：14+11＝25
Anti--CL II	I：53+33＝86	A：52+56＝108A：52+43＝95	NA	I：72+10＝82

" A " means and observes " addition " effect;

" S " means and observes " working in coordination with " effect;

" I " means and observes " inhibition " effect;

^*Schematic equation: A+B=C, wherein " A " is because of ANN and/or the PI positive cell percentage of matrix antibody due to separately, " B " is that (Figure 20 is anti--ANN and/or PI positive cell percentage CD22) due to for anti--CD79b, Figure 22 for anti--CLII and Figure 23 for anti-CD 20, Figure 21, and " C " is for estimating additive effect because of common antibody.(referring to quantitative data corresponding in the above table 7) with Figure 20-23.If there are two equations in the cell, then go up the result that the common antibody gained of higher indication concentration is used in the equation reflection; The result of the common antibody gained of low indication concentration is used in following equation reflection.

In certain embodiments, two binding domainss interact with inhibition, addition or cooperative mode in sensitization (or desensibilization) target cell (such as the B cell).Figure 23 shows and will resist-protection or retarding effect that CD22 antibody and strong short apoptosis monoclonal antibody (such as anti--CD79b antibody or anti--MHC II class (that is anti--CL II) antibody) combination are produced.For example, Figure 23 and table 7 show, anti--CD22 antibody induction is no more than about 10% cell and shows that the short apoptosis behavior solid post of (among the Figure 23 corresponding to " 22 ") and anti--CD79b induce about 26% the short apoptosis cell solid post of (among the Figure 23 corresponding to " CD79b ") separately.Yet only about 16% the short apoptosis cell twill post of (among the Figure 23 corresponding to " 79b ") is induced in anti--CD22 and the combination of anti--CD79b.Therefore, the short apoptosis cell of the antibody induction 16% that is made up, it is less than the summation 38% of anti--CD22 (12%) and anti--independent effect that produces of CD79b (26%).Utilize this method check Figure 23 and/or table 7-8 to disclose, anti--CD22 antibody and comprise the polyspecific multivalence binding molecule of anti--CD22 binding domains via prolongation will obtain the inhibition population effect with following each antibody (or corresponding binding domains) when independently being used in combination: anti--CD19, anti-CD 20, anti--CD21, anti--CD23, anti--CD30, anti--CD37, anti-CD 40, anti--CD70, anti--CD72, anti--CD79a, anti--CD79b, anti--CD80, anti--CD81, resist-CD86 and resisting-MHC II antibody-like/binding domains.

Be not wishing to be bound by theory, described data can be made description below: the polyspecific multivalence binding molecule that anti--CD22 antibody is described or comprises anti--CD22 binding domains can prevent or relax any antibody mediated effect of above just having listed.More generally, comprise the polyspecific multivalence binding molecule of anti--CD22 binding domains with any one effect that interacts and produced in inhibition and CD19, CD20, CD21, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and the MHC II quasi-molecule.In Figure 23 and table 8 as seen, anti--CD22 antibody and will work the active effect that suppresses or relax any antibody/binding domains of identification B-cell surface marker (such as T cell differentiation antigen) via prolonging binding domains (comprising anti--CD22 binding domains).Expection multivalence binding molecule (comprising polyspecific multivalence binding molecule) is applicable in the improvement treatment plan of various diseases, wherein needs to weaken or control the activity of binding domains.

Except that producing inhibition, addition or the synergistic combination effect via combining of cell surface part usually with two binding domainss of target cell interaction, experimental result disclosed herein proves, given binding domains be to can providing dissimilar combined effects according to the relative concentration of two binding domainss, thereby strengthens versatility of the present invention.For example, table 8 discloses, and anti--CD21 and anti--CD79b interact with suppressor mode when the test concentrations of anti--CD79b is higher, but when the test concentrations of anti--CD79b was hanged down, these two kinds of antibody interacted with cooperative mode.Although some embodiment will be used the multivalence binding molecule of single type, that is comprise the monospecific multivalence binding molecule of for example single CD21 binding domains and single CD79b binding domains, but the present invention includes the mixture of multivalence binding molecule, described mixture will make that can adjust relative binding domains concentration is wanted effect to reach, such as inhibition, addition or synergistic effect.In addition, method of the present invention comprises single multivalence binding molecule and another kind of binding molecule (such as the conventional antibody molecule) is used in combination to adjust or the relative concentration of the structural domain of optimizing integration.Those skilled in the art can utilize standard technique (for example by two groups of experimental serial dilution matrix of design, each binding domains uses a group) to measure the suitable relative concentration of binding domains.

Be not wishing to be bound by theory, should be appreciated that, the surface expression of second part on the same cell type can be induced or regulate to the combination of a part, thereby or its surperficial environment that can change second part change itself and specific binding molecules (such as antibody or multivalence binding molecule) bonded susceptibility.

Although herein to use B clone and antigen to be example, the described method of the effective multivalence binding molecule of optimum determining (that is scorpion shape molecule) is applicable to other diseases background and target cell colony, comprise other normal cells, its abnormal cells counterpart, comprise chronic stimulation hematopoietic cell, cancer cells and cells infected.

In the method for the direct effect that the screening monoclonal antibody makes up, also can use other signals conduction phenotypes (such as Ca ²⁺Move; Tyrosine phosphorylation is regulated; Caspase (caspase) activation; The NF-KB activation; Cytokine, somatomedin or chemokine processing; Or genetic expression (for example in reporter gene system)).

So that one-level is antibody linked and the alternative of simulation multivalence binding molecule or scorpion shape molecular structure, can use other molecules (comprising solubility Fc acceptor, albumin A, complement component (comprising C1q), mannose binding lectin, the bead that contains reagent or linking agent or matrix, difunctionality chemistry linking agent and plastics sorbent material) of the Fc part of binding antibody to make the identical or different antigenic multiple monoclonal antibody of opposing crosslinked as using secondary antibody.

Embodiment 12

Multivalent binding proteins with structure of effector function or scorpion shape molecule

The general schematic structure of scorpion shape polypeptide is H2N-binding domains 1-scorpion shape connexon-constant subprovince-binding domains 2.Scorpion shape molecule also can have the hinge sample district of the N-end that is placed in binding domains 1, is generally the peptide district that is derived from the antibody hinge.In some scorpion shape molecule embodiment, binding domains 1 and binding domains 2 are to be derived from immune globulin binding structural domain separately, for example are derived from V _LAnd V _HV _LWith V _HUsually connect through connexon.Experiment is intended to proof, scorpion shape polypeptide can have the binding domains that is different from immune globulin binding structural domain, comprise the Ig binding domains that scorpion shape molecule binding domains is originated, difference is aminoacid sequence difference, causes with respect to source Ig binding domains usually less than 5% and preferably less than 1% sequence divergence.

Usually, sequence difference causes single amino acid to change, such as replacement.The optimum position that described amino acid changes is arranged in one or more district of pairing scorpion shape molecule binding domains, or shows and Ig complementarity-determining region (CDR) at least 80% of the Ig binding domains that this scorpion shape molecule binding domains is originated and preferred 85% or 90% sequence identity.Provide further guidance by peptide relatively in conjunction with the model of identical target (such as CD20).With respect to CD20, the epi-position collection of illustrative plates discloses, can discern Ala-Asn-Pro-Ser (ANPS) motif of CD20 in conjunction with the 2H7 antibody of CD20, and expect and also can discern this motif in conjunction with the scorpion shape molecule of CD20.Go into by the aminoacid sequence due in the formed bag in scorpion shape molecule binding domains district (corresponding to Ig CDR) to change the functional wedding agent that is contemplated to CD20 with the ANPS motif is buried.Model research also discloses, corresponding to CDR3 (V _L), CDR1-3 (V _H) scorpion shape district contact CD20, and expection is kept or facilitate the variation of described contact can produce scorpion shape molecule in conjunction with CD20.

Except that the interaction that promotes scorpion shape molecule and its target, contain in the scorpion shape molecule binding domains promotion corresponding to Ig V _LAnd V _HInteractional sequence variation (with respect to homology Ig binding domains sequence) between the scorpion shape molecule binding domains district of structural domain.For example, corresponding to V _LThe scorpion shape molecular regime in conjunction with CD20 in, sequence SYIV can change by replace Val (V33) with amino acid (such as His), produces sequence SYIH.This changes expection and can improve corresponding to V _LAnd V _HInteraction between the scorpion shape molecular regime of structural domain.In addition, be expected at corresponding to V _HThe terminal interpolation of the N-of the scorpion shape molecular regime of-CDR3 residue will change the orientation of this scorpion shape molecular regime, and this may influence it in conjunction with feature, because V _HThe terminal Ser of the N-of-CDR3 contacts with CD20.Conventional determining discloses those orientations and can make in conjunction with the feature generation and will change.Also expection is corresponding to V _H-CDR2 and/or V _HSudden change in the scorpion shape molecular regime of-CDR3 will form with the potential novelty of target (such as CD20) and contact.For example, based on model research, expection replaces corresponding to V _HWhat any will change scorpion shape molecule in the mode that easily stands conventional determining among Y105 in the district of-CDR3 and the W106 (being present among the sequence NSYW) has modified scorpion shape molecule in conjunction with feature in conjunction with feature to be used to differentiate.In addition for example, expection will influence combination corresponding to the change of the sequence (such as the Trp among the sequence C QQW (W)) of the scorpion shape molecule binding domains of Ig VL-CDR3.Usually, for target is shown those scorpion shape molecules that strengthen affinity, screening is corresponding to the change in the scorpion shape molecular regime of Ig CDR.

Model structure based on humanization CD20 scFv binding domains 20-4, based on the public information (people such as Du of CD20 extracellular loop structurally associated, J Biol.Chem.282 (20): 15073-80 (2007)), and based on the CD20 that is discerned by mouse 2H7 antibody (it is the source of the CDR of humanization 20-4scFv binding domains) in conjunction with epi-position, in the CDR district of 2Lm20-4x2Lm20-4 scorpion shape molecule, carry out through engineering approaches sudden change, so that improve itself and CD20 bonded affinity.At first, design can influence the more effective bonded sudden change of 20-4CDR configuration and promotion and CD20 extracellular loop.Secondly, design can provide the drawing-in system of novel molecular interphase interaction to change between 2Lm20-4x2Lm20-4 scorpion shape molecule and its target.Described sudden change comprises: VL CDR1 V33H (that is with His replace Val at 33 places, position of the CDR1 in VL district), VL CDR3 W90Y, VH CDR2 D57E, VH CDR3 insert V, VH CDR3 Y101K, VH CDR3 N103G, VH CDR3 N104G and VH CDR3 Y105D behind residue S99.Because expection can produce synergistic effect with some described sudden change combination, therefore design 11 kinds of mutant with difference sudden change combination as shown in table 9 (the residue font-weight of introducing by sudden change and underline).

Table 9

V L CDR1	V L CDR3	V H CDR2	V H CDR3
				RASSSVSYI H	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SVYYSNYWYFDL
RASSSVSYI H	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SVYY GGYWYFDL
				RASSSVSYI H	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SYYSNS DWYFDL
RASSSVSYI H	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SYYS GGDWYFDL
				RASSSVSYIV	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SY KSNSYWYFDL
RASSSVSYIV	QQWSFNPPT	AIYPGNG ETSYNQKFKG	SYYSNSYWYFDL
				RASSSVSYIV	QQ YSFNPPT	AIYPGNGDTSYNQKFKG	SYYSNSYWYFDL
RASSSVSYI H	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SY KSNS DWYFDL
				RASSSVSYI H	QQWSFNPPT	AIYPGNG ETSYNQKFKG	SYYSNS DWYFDL
RASSSVSYI H	QQ YSFNPPT	AIYPGNGDTSYNQKFKG	SYYSNS DWYFDL
				RASSSVSYI H	QQ YSFNPPT	AIYPGNG ETSYNQKFKG	SY KS GGDWYFDL

Use the primer of the sequence area of coding change, will suddenly change by PCR mutagenesis and introduce in the binding domains of CD20xCD20 scorpion shape molecule (2Lm20-4x2Lm20-4).After the sequence checking, the segmental dna fragmentation of 2Lm20-4scFv that coding is had a corresponding sudden change is cloned in the conventional expression vector of coding region of the constant subprovince of containing scorpion shape molecule, thereby produces the polynucleotide of the global DNA sequence that contains novel 2Lm20-4x2Lm20-4 scorpion shape molecule.Varient with 2Lm20-4x2Lm20-4 scorpion shape molecule of CDR sudden change is to produce by expressing in moment COS cell system, and via albumin A and size exclusion (SEC) purification by chromatography.Use elementary B-cell and WIL2-S B-lymphoma cell line, assess the binding characteristic of 2Lm20-4x2Lm20-4 scorpion shape molecular variant by facs analysis.

Also used similar approach to form other mutant to optimize the CD20 binding domains.The CD20SMIP of called after TRU015 serves as the substrate that forms mutant, unless and opposite explanation, otherwise all structural domains are human structural domain.Find that following mutant contains suitable functional CD20 binding domains.The Q (single-letter amino acid coding) that replaces S is contained at 27 places, position of the CDR1 of 018008 molecule in VL, and contain the S and 102 places, the position in the CDR3 of VH that replace T and contain the L that replaces V at 28 places, position in the CDR1 of VH.According to the modularized design of scorpion shape molecule, following part scorpion shape connexon sequence corresponding to the CCCP sequence in the IgG1 hinge is made up with VL that suddenlys change and VH independently: CSCS, SCCS and SCCP.018009 molecule contains the Q that replaces S at 27 places, position of the CDR1 of VL, contains the S that replaces T at 28 places, position of the CDR1 of VH, and in the CDR3 of VH, and the S that replaces V is contained at 96 places in the position, the L that replaces V is contained and the 95 places disappearance V in the position in 102 places in the position.Use in 018009 above-mentioned with 018008 in the identical scorpion shape connexon subsequence found in the employed scorpion shape connexon.018010 molecule is in the CDR1 of VL, in the position 27 places contain the Q that replaces S, in the position 33 places have the I that replaces M and in the position 34 places contain the V that replaces H, and contain the S that replaces T, and contain the L that replaces V at 102 places, position of the CDR3 of VH at 28 places, position of the CDR1 of VH.Scorpion shape connexon by CSCS and the definition of SCCS subsequence is applicable to 018010.018011 in the CDR1 of the CDR1 of VL and VH, contain with as identical sudden change as described in 018010, and in the CDR3 of VH, in the position 95 places disappearance V, in the position 96 places contain the S that replaces V and in the position 102 places contain the L that replaces V.Scorpion shape connexon by CSCS, SCCS and the definition of SCCP subsequence is used for 018011 molecule.018014VL is the mouse VL that does not suddenly change, and wherein human VH contains the variation that S replaces the variation of T and contains L replacement V at 102 places of CDR3 at 28 places of CDR1.018015 also contains not the mouse VL and the human VH of sudden change, and this mankind VH contains the variation that S replaces T at 28 places of CDR1, and in CDR3, contains the S that replaces V at the disappearance V of 95 places, at 96 places and contains the L that replaces V at 102 places.27 places of 2Lm5 molecule in the CDR1 of VL have the Q that replaces S; In the CDR1 of VH, have F that replaces Y and the S that has replacement T at 30 places at 27 places; And in the CDR3 of VH, have S that replaces V and the L that has replacement V at 102 places at the disappearance V of 95 places, at 96 places.Be used for independently by the scorpion shape connexon of CSCS, SCCS and SCCP definition 018014 and 018015 each.2Lm5-1 is identical with 2Lm5, and the exception part is that 2Lm5-1 does not have sudden change in the CDR1 of VH, and only uses the scorpion shape connexon by the definition of CSSS subsequence.2Lm6-1 has the sudden change of 2Lm5 and in the CDR3 of VL, has T that replaces S and the S that has replacement F at 93 places at 92 places, and only uses the scorpion shape connexon by the definition of CSSS subsequence.Sporting among the 2Lm16 only above to the sudden change among the CDR3 of the listed VH of 2Lm5-1.Scorpion shape connexon by subsequence CSCS, SCCS and SCCP definition is used for 2Lm16 independently.27 places of 2Lm16-1 in the CDR1 of VL have the Q that replaces S; And in the CDR3 of VL, have T that replaces S and the S that has replacement F at 93 places at 92 places; And among the CDR3 of VH, have S that replaces V and the L that has replacement V at 102 places at the disappearance V of 95 places, at 96 places; Only use scorpion shape connexon by the definition of CSSS subsequence.2Lm19-3 has the Q of replacement S, has I that replaces M and the V that has replacement H at 34 places at 33 places, and have the sudden change among the CDR3 of the listed VH of 2Lm16-1 in the CDR1 of VL at 27 places.Scorpion shape connexon by subsequence CSCS, SCCS and SCCP definition is used for 2Lm19-3 independently.The 2Lm20-4 molecule contains the I that replaces M and contains the V that replaces H at 34 places, and contain the sudden change among the CDR3 of the listed VH of 2Lm16-1 in the CDR1 of VL at 33 places.For 2Lm5-1,2Lm6-1,2Lm16,2Lm16-1,2Lm19-3 and 2Lm20-4, also there is the S that replaces L at 11 places, position of the framework region of VH.Scorpion shape connexon by CSCS, SCCS and the definition of SCCP subsequence is used for 2Lm20-4 independently.At last, there is the S that replaces P in 331 places in the position in the following mutant: wherein scorpion shape connexon is by 018008 of the CSCS definition; Wherein each scorpion shape connexon is by 018009 of CSCS and SCCP definition; Wherein scorpion shape connexon is by 018010 of the CSCS definition; Wherein scorpion shape connexon is by 018011 of the SCCP definition; Wherein scorpion shape connexon is by 018014 of the CSCS definition; Wherein scorpion shape connexon is by 018015 of the CSCS definition; Wherein scorpion shape connexon is by the 2Lm16 of any one definition among CSCS, SCCS and the SCCP; Wherein scorpion shape connexon is by the 2Lm19-3 of CSCS or SCCP definition; And wherein scorpion shape connexon by the 2Lm20-4 of CSCS or SCCP definition.

In addition, contain two districts making binding domains (such as in the scorpion shape molecule binding domains corresponding to Ig V _LAnd V _HThe district) variation of the connexon length that connects.For example, the terminal Asp expection of C-of wherein finding to remove connexon between structural domain can influence scorpion shape molecule in conjunction with feature, as replacing Asp with Gly.

Also contain the scorpion shape connexon that has for the hinge area of Ig through prolonging (insert C-terminal with constant subprovince between and between N-end and the binding domains 2) scorpion shape molecule, wherein amino-acid residue is added between the terminal and any halfcystine (corresponding to the Ig hinge cysteine) of C-in the scorpion shape molecule, and wherein this scorpion shape molecule halfcystine can form interchain disulfide bond.The scorpion shape molecule that contains described feature has made up and in hereinafter characterizing.

Expression, stability and the curative effect of scorpion shape molecule have been made great efforts to improve via the binding domains 2 covalently bound scorpion shape connexons that optimization is settled constant subprovince and C-terminal.The prototype scorpion shape molecule that is used to optimize research contain make N-terminal be derived from IgG1 C _H2And C _H3The anti-CD 20 scFV (binding domains 1) that merges of constant subprovince and the two anti-CD 20 scFv that make the constant therewith subprovince fusion of C-end.The expection of this scorpion shape molecule (as immunoglobulin molecules) via constant region (or subprovince) in conjunction with the peptide chain formation homodimer mixture that is connected by disulfide linkage.For obtaining the high expression level degree that its CD20 target is had the tetravalence stable molecule of high-affinity, the scorpion shape connexon between the constant subprovince and second binding domains must satisfy following consideration.At first, sterically hindered (the having a scFv fragment on each of two scorpion shape molecule monomers) between the homology binding domains that is carried by two scFv fragments should minimize the native conformation that is beneficial to keep each binding domains.Next, but the configuration of binding domains and orientation should make each structural domain producibility combination and each binding domains combine with its target high-affinity.The 3rd, scorpion shape connexon itself should have proteolytic enzyme tolerance and non-immunogenic relatively.

In exemplary CD20xCD20 scorpion shape molecule construction body S0129, C _H3C-terminal be connected via 2H7 scorpion shape connexon (a kind of be derived from and corresponding to the segmental peptide of the natural human hinge sequence of IgG1) with two anti-CD 20 scFV structural domains.2H7 scorpion shape connexon serves as the basis of design studies, and design studies is used and to be intended to the area of computer aided model in conjunction with feature improveing the expression of scorpion shape molecule and improve expressed molecule.

For analyzing 2H7 scorpion shape connexon, use Insight II software that 3 dimension structures of the dimeric forms of IgG 1 hinge are carried out modeling.Has V _H-V _LThe crystalline structure of the anti-CD 20 scFV of orientation is elected to be reference configuration (the RCSB albumen database clauses and subclauses coding: 1A14) of 20-4 binding domains.In complete IgG1, hinge makes C _H1The C-end and the C of structural domain _H2The N-of structural domain is terminal to be connected, and the configuration of each structural domain should make that the hinge cysteine residue can be in pairs to form homodimer.In exemplary scorpion shape molecule, the 2H7 connexon that is derived from hinge makes and is derived from IgG1 C _H3The C-in the scorpion shape molecular structure territory of structural domain terminal be derived from IgG1 V _H2The N-of this part of the scorpion shape molecule binding domains 2 of structural domain is terminal to be connected.Utilize V _H-V _LThe three-dimensional model structure of scFV, the optimum distance between the C-end of expection 2H7 connexon is subjected to the influence of following three kinds of Considerations.The first, must keep hinge stability, and stability can support by Dimerized (for example homodimerization), this means hinge cysteine must can be paired in the presence of two folding binding domainss.The second, two binding domains (for example scFV) must satisfy 2H7 connexon C-end and not produce the steric hindrance interference so that albumen can suitably fold.The 3rd, the CDR of each binding domains should be able to face same direction (as in natural antibody), because each binding domains of prototype scorpion shape molecule can be in conjunction with the lip-deep contiguous acceptor of same cell (CD20).For described consideration, the distance between two N-ends of scFv is contemplated to about 28

In dimer scorpion shape molecular form, the distance between the C-end of the 2H7 connexon of Design Theory is contemplated to about 16

Be contemplated to the required distance of usefulness of optimizing scorpion shape molecule for satisfying, at least 3 amino acid of the terminal expansion of the C-of 2H7 connexon.This expansion expection makes and can form disulfide linkage between 2H7 connexon cysteine residues, thereby makes the terminal binding domains 2 of C-can suitably correctly fold and facilitate the correct orientation of CDR.In addition, in complete IgG1, owing to have C between hinge and the binding domains _H1And V _L1Therefore structural domain, the distance between the binding domains that carried of two chains further increases and expection can further promote the crosslinked of the lip-deep contiguous acceptor of same cell.In view of above-mentioned consideration, design one group of connexon (table 10) with different lengths.For immunogenicity is minimized, use C _H2The terminal existing natural residue (Ala-Pro-Glu-Leu or APEL) of the N-of structural domain, the C-end that is added into scorpion shape connexon by sequence prolongs 2H7 scorpion shape connexon.Longer construct contains one or more known (Gly4Ser) connexon unit with proteolytic enzyme tolerance and flexibility.

C in constant subprovince _H3Contain CD20 * CD20 scorpion shape molecule construction body through the scorpion shape connexon of expansion between the terminal scFv binding domains of structural domain and C-and be utilize the PCR mutagenesis to make up and by subclone to conventional mammalian expression vector.Can analyze of the influence of connexon length by express in the experiment more secreted proteic output in the moment of using COS or HEK293 cell, or the pulse-chase of the methionine(Met)/halfcystine by western blot analysis method or use [35] S-mark is researched and analysed albumen and is synthesized and gather and analyze the influence of connexon length to CD20 * CD20 scorpion shape developed by molecule to CD20 * CD20 scorpion shape developed by molecule.

Table 10

The construct numbering	Scorpion shape connexon core (2H7) sequence	Sequence spreading	Scorpion shape connexon sequence through expansion
				1	GCPPCPNS	APEL	GCPPCPNSAPEL
2	GCPPCPNS	APELGGGGS	GCPPCPNSAPELGGGGS
				3	GCPPCPNS	APELGGGGSGGGGS	GCPPCPNSAPELGGGGSGGGGS
4	GCPPCPNS	APELGGGGSGGGGSGGGGS	GCPPCPNSAPELGGGGSGGGGSGGGGS

Also contain glycosylation scorpion shape molecule, and in the present context, contain can be in the presence of the carbohydrate modifying agent host cell of culture expression scorpion shape molecule, the carbohydrate modifying agent is defined as the small molecules organic compound that preferably has less than 1000 daltonian molecular weight in this article, it can suppress to take place such as connecting the carbohydrate ripening period at proteic N as the interpolation of the sugar of the part of the carbohydrate that is connected to polypeptide, the activity of related enzyme in removing or modifying.Glycosyl turns to the complex process that betides in endoplasmic reticulum (" core glycosylation ") and gorky (Golgi) body (" terminal saccharideization ").Various Glycosylases and/or mannoside enzyme inhibitors provide following one or more to want effect: the glycosylation pattern that strengthens ADCC activity, enhancing Fc receptors bind and change.Exemplary inhibitor comprises (but being not limited to) castanospermine (castanospermine) and suppresses alpha-Mannosidase (kifunensine).The effect of scorpion shape molecule is expressed in the existence that is disclosed at least a this inhibitor in following examples down.

Embodiment 13

Scorpion shape molecule protein is expressed degree and sign

Measure scorpion shape molecule protein expression degree and characterize expressed albumen and can produce product with practical benefit with the design of proof albumen.Utilize routine techniques, express monospecific CD20 * CD20 scorpion shape molecule and dual specific CD20 * CD37 scorpion shape molecule in the CHO DG44 cell in nutrient solution.

The stably express of the benchmark degree in the observation CHO DG44 cell that CD20 * CD20 scorpion shape molecule S0129 (21m20-4x21m20-4) is cultivated in the presence of various supplement feeds, as shown in Figure 34.All substratum all contain the 50nM Rheumatrex, and this concentration can be kept the copy number of the polynucleotide of coding scorpion shape molecule.Polynucleotide contains the coding region of scorpion shape molecule protein, this coding region without the codon optimization in CHO DG44 cell, expressing.Use the pD18 carrier that polynucleotide is introduced in the cell.By Figure 34 obviously as seen, obtain the expression degree of about 7-46 μ g/ml.

Also be determined at the expression degree behind the polynucleotide of amplification coding dual specific CD20xCD37 scorpion shape molecule.Use pD18 carrier cloning CD20 * CD37 scorpion shape molecule encoding district, and plasmid is introduced in the CHO DG44 cell.Use dhfr-Rheumatrex technology as known in the art to realize the amplification of coding polynucleotide, wherein use the MTX that increases concentration to select the increase copy number of dihydrofolate reductase gene (dhfr), thereby the close-connected polynucleotide of being paid close attention to is increased jointly.Figure 35 shows that the stably express degree that observes dual specific CD20 * CD37 scorpion shape molecule is generally about 22-118 μ g/ml.Under different condition (comprising the Rheumatrex concentration that is used to increase), observe change of production, but those skilled in the art easily optimize described variation.Also make described other various scorpion shape molecules stand expression analysis herein in CHO and/or COS cell, analytical results is provided in the following table in 11.Described result's proof utilizes routine techniques and conventional amplification optimisation technique can obtain the scorpion shape molecule protein of high yield.

Expressed albumen is also analyzed by SDS-PAGE and is characterized, to assess the molecular weight of expressed proteic homology degree and integrity and checking monomeric peptide.The reduction and non-reduced condition under to denaturing polyacrylamide gel (4-20%Tris glycine) electrophoresis.Shown result is disclosed under the reductive condition among Figure 36, has the 2Lm20-4 SCC SMIP of expection monomer molecule amount and each the single protein band among the S1000 (CD20 (21m20-4) xCD20 (21m20-4) monospecific scorpion shape molecule S0126).Described data declaration SMIP and scorpion shape molecule easily stand purifying with complete form.Under non-reduced condition, it is consistent with the monomer SMIP of desired size to observe the trace peptide, and wherein most of albumen occurs with the single clear band form consistent with dimeric structure.Under described non-reduced condition, monospecific scorpion shape molecule protein shows the single clear band with molecular weight consistent with dimeric structure.SMIP is consistent with the dimeric structure and the monomer whose structure of scorpion shape molecule, and it contains hinge sample scorpion shape connexon separately, and this connexon contains at least one can participate in the halfcystine that disulfide linkage forms.

Also assess scorpion shape connexon to the expression of scorpion shape molecule and the influence of integrity, and the results are shown in the table 12.This tabular goes out the scorpion shape connexon varient of monospecific CD20xCD20 (2Lm20-4x2Lm20-4) S0129 scorpion shape molecule and CD20xCD28 S0033 scorpion shape molecule (2H7sccpIgG1-H7-2e12), and its integrity is expressed degree as single chain molecule and its moment with respect to parent scorpion shape molecule S0129 that optionally has H7 connexon (being set at 100%) or S0033 in the COS cell.Table 13 provides assessment to incorporate the data of the scorpion shape connexon varient gained in CD20 * CD20 scorpion shape molecule into, and at the class likelihood data of CD20 * CD28 scorpion shape molecule.Table 13 provides assessment to contain the data of the S0129 varient gained of scorpion shape connexon, and described scorpion shape connexon is not the hinge sample connexon that contains at least one halfcystine that can form disulfide linkage; On the contrary, the scorpion shape connexon in the described molecule is to be derived from II type C-lectin stem district.Apparent by data shown in the table 13, in express measuring in moment, hinge sample scorpion shape connexon can combine with the scorpion shape molecule with the degree expression that is higher or lower than not modified parent scorpion shape connexon.In addition, some connexon varients shows than the not modified bigger proteolytic cleavage resistance of parent's connexon, this be related to all or nearly all through expressed proteins.The data presentation of table 13 present with the slightly different scorpion shape molecule that combines feature of the scorpion shape molecule that contains hinge sample scorpion shape connexon in have non-hinge sample connexon, such as the connexon in the stem district that is derived from II type C-lectin.In addition, the shown effector function (ADCC) of scorpion shape molecule that contains non-hinge sample scorpion shape connexon is equal to or surpasses and the relevant ADCC of scorpion shape molecule with hinge sample scorpion shape connexon.

Table 11

The connexon title	Upstream (CH3) sequence	S0129 (2Lm20-4 x 2Lm 20-4) connexon varient-aminoacid sequence 1	Based on	#AAs	Express COS 2	Cracking 3	Express CHO 2
								H7	QKSLSLSPGK	GCPPCPNS	H7	18	100	-	100
H16	QKSLSLSPGK	LSVKADFLTPSIGNS	CD80-	25	174	+
								H18	QKSLSLSPGK	LSVLANFSQPEIGNS	CD86	25	165	++
H19	QKSLSLSPGK	LSVLANFSQPEISCPPCPNA	CD86+H7	30	161	+	109
								H26	QKSLSLSPGK	RIHQMNSELSVLANS	CD86	25	170	++
H32	QKSLSLSPGK	RIHLNVSERPFPPNS	CD22	25	184	++
								H47	QKSLSLSPG	LSVKADFLTPSIGNS	H16	24	141	-	206

H48	QKSLSLSPG	KADFLTPSIGNS	H16	21	137	-
								H50	Q	LSVLANFSQPEIGNS	H18	16	21	-
H51	QKS	LSVLANFSQPEIGNS	H18	18	110	-
								H52	QKSLSLSPG	SQPEIVPISNS	H18	20	95	-
H53	QKSLSL	SQPEIVPISCPPCPNS	H19	26	96	-
								H54	Q	SVLANFSQPEISCPPCPNS	H19	21	72	+/-
H55	OKSLSLSPG	RIHQMNSELSVLANS	H26	24	118	+
								H56	QKSLSLSPG	QMNSELSVLANS	H26	21	130	-	163
H57	QKSLSLSPG	VSERPFPPNS	H32	19	118	-
								H58	QKSLSLSPG	KPFFTCGSADTCPNS	CD72	24	103	-
H59	CKSLS	KPFFTCGSADTCPNS	CD72	20	94	-

¹NFS is the consistent motif of glycosylation

²With respect to SO129-H7 (%), moment expresses in COS (6W plate) or CHO (single flask)

³The split product of being observed by the SDS-PAGE/ silver staining:-=do not have, +=fuzzy band; ++=master tape, ++ +=50% cracking)

Table 12

Table 13

Albumen	Describe	Output (μ g purifying protein/ml fill-in)	POI% (M.wt that represents with Kd is according to MALS)	The improvement of S0129wt POI	In conjunction with Ramos	ADCC measures	The sequence of scorpion shape connexon
								S0129wt	The H7 connexon	1.6	67(167)	-	-	-	GCPPC
S0129-CD69	CD69 stem district	2.9	66(167)	1.8	Be weaker than S0129wt	*Be better than S0129wtPOI slightly	QYNCPGQYTFSM
								S0129-CD72	CD72 brachymemma stem district	2.0	69(165)	1.2	Be similar to S0129wt	*Be better than S0129wtPOI slightly	PFFTCGSADTC
S0129-CD94	CD94 stem district	2.9	67(171)	1.8	Be similar to S0129wt	*Be better than S0129wtPOI slightly	EPAFTPGPNIELQKDSDC
								S0129-NKG2A	NKG2A stem district	2.5	93(170)	2.2	Be better than S0129wt slightly	Be similar to S0129wtPOI	QRHNNSSLNTRTQKARHC
S0129-NKG2D	NKG2D stem district	1.9	70(166)	1.2	Be similar to S0129wt	*Be better than S0129wtPOI slightly	NSLFNQEVQIPLTESYC

Described in above embodiment, contain by in the nutrient solution that contains the carbohydrate modifying agent, expressing and prepare scorpion shape molecule.In exemplary embodiment, it is about 200 μ M (corresponding to about 37.8 μ g/mL) that castanospermine (MW189.21) is added in the substratum until ultimate density, or concentration range reaches up to about 300,275,250,225,200,175,150,125,100,75,60 or 50 μ g/mL greater than about 10,20,30,40,50,60,70,80,90,100,110,120,130,140 or 150 μ M.For example, the scope that contains 10-50 μ M or 50-200 μ M or 50-300 μ M or 100-300 μ M or 150-250 μ M.In other exemplary embodiment, it is that about 200 μ M (corresponding to about 32.6 μ g DMJ/mL) or concentration range are greater than about 10,20,30,40,50,60,70,80,90,100,110,120,130,140 or 150 μ M, and up to about 300,275,250,225,200,175,150,125,100,75,60 or 50 μ g/mL that DMJ (for example DMJ-HCl (MW199.6)) is added in the substratum until ultimate density.For example, the scope that contains 10-50 μ M or 50-200 μ M or 50-300 μ M or 100-300 μ M or 150-250 μ M.In other exemplary embodiment, to suppress that alpha-Mannosidase (MW232.2) is added in the substratum until ultimate density is that about 10 μ M (corresponding to about 2.3 μ g/mL) or concentration range are greater than about 0.5,1,2,3,4,5,6,7,8,9 or 10 μ M, and up to about 50,45,40,35,30,25,20,19,18,17,16,15,14,13,12 or 11 μ M.For example, the scope that contains 1-10 μ M or 1-25 μ M or 1-50 μ M or 5-10 μ M or 5-25 μ M or 5-15 μ M.

In an experiment, express monospecific CD20xCD20 scorpion shape molecule (S0129) with the cell that is incubated in 200 μ M castanospermines (S0129 CS200) or 10 μ M (excessive) the inhibition alpha-Mannosidases (S0129 KF 10), and measure of combination or the dyeing of expressed scorpion shape molecule, as shown in Figure 42 to the WIL2S cell.In addition, in relatively in conjunction with research, the binding ratio of glycosylation S0129 scorpion shape molecule and CD16 (FC γ RIII) not glycosylation S0129 scorpion shape molecule above about three times.

In another research, probe into the ADCC mediation property killing effect of humanization CD20xCD20 scorpion shape molecule (S0129) to the BJABB-cell.Result shown in Figure 43 confirms that scorpion shape molecule causes producing significantly stronger ADCC mediation property BJAB B-necrocytosis when expressing in institute's cultured cells in the presence of castanospermine or inhibition alpha-Mannosidase under the scorpion shape molecule of given concentration exposes.

Embodiment 14

The combination of scorpion shape molecule

A. structural domain spacing

The binding domains that dual specific scorpion shape molecule can utilize the terminal and C-end of the N-that is positioned at molecule to and at least two targets of combination simultaneously.In the case, for the cell surface target, said composition is can be with described target crosslinked or impel collaborative convergence on the described target entity.It will be apparent to those skilled in the art that multiple receptor system is crosslinked and activate through this, produce the signal induction that impels cell phenotype to change.Designing composition disclosed herein partly is intended to make this signal conduction maximization and controls the phenotype that is produced.

In scorpion shape molecule architecture design, should be appreciated that and consider the approximate dimension of each structural domain in the scorpion shape molecular composition and structural domain that angular region between with structural domain is represented between flexible desired value.For the scorpion shape molecule that uses scFv binding domains (for for the binding domains 1 and 2 (BD1 and BD2)), the terminal hinge (H1) of IgG1 N-and described H7PIMS connexon herein, the binding domains maximum of the binding domains of N-end and C-end can be separated by approximately

And I is separated by approximately

The binding domains maximum of N-end can be separated by approximately

And I is separated by approximately

(people such as Deisenhofer, 1976, Hoppe-Seyler ' s Z.Physiol.Chem.Bd.357, S.435-445; People such as Gregory, 1987, Mol.Immunol.24 (8): 821-9.; People such as Poljak, 1973, Proc.Natl.Acad.Sci., 1973,70:3305-3310; People such as Bongini, 2004, Proc.Natl.Acad.Sci.101:6466-6471; People such as Kienberger, 2004, EMBO Reports, 5:579-583, each document is incorporated herein with way of reference).Partly select described size can make in scorpion shape molecule institute bonded receptor complex, acceptor-acceptor distance is less than about 50

, this is because can be (people such as Paar, 2002 of the best less than the distance of this distance for the peak signal conduction speech of some acceptor oligomer, J.Immunol., 169:856-864, the document is incorporated herein with way of reference), make simultaneously and can incorporate the required F of effector function into _CStructure.

The binding domains of the terminal and C-end of the N-of scorpion shape molecule is through being designed to flexible structure to promote the target combination and to allow geometrical dimension scope in conjunction with target.Those skilled in the art also will understand, the flexibility between the terminal binding domains of N-end or C-(being respectively BD1 and BD2) and the binding domains and the F of molecule _CMinimum and maximum distance between flexibility between the structural domain and BD1 and/or the BD2 institute bonded acceptor can be for example by selecting the terminal hinge arrangement territory (H1) of N-and being modified by the structural simulation terminal localized scorpion shape connexon structural domain of more close C-(H2).For example, the hinge sample C of the hinge arrangement territory of IgG1, IgG2, IgG3, IgG4, IgE, IgA2, synthetic hinge and IgM _H2The flexibility of structural domain showed different and different lengths.The optimal selection that it will be apparent to those skilled in the art that H1 and scorpion shape connexon (H2) will be decided on following factor: scorpion shape molecule is designed interaction receptor system with it, and scorpion shape molecule is in conjunction with institute's inductive desired signal conduction phenotype.

In certain embodiments, scorpion shape molecule has scorpion shape connexon (H2), and this connexon is the hinge sample connexon corresponding to Ig hinge (such as the IgG1 hinge).Described embodiment comprises the scorpion shape molecule of the aminoacid sequence with scorpion shape molecule hinge, this aminoacid sequence for respect to for example H7 sequence or wild-type IgG1 hinge sequence by the sequence of the terminal expansion of N-.The exemplary scorpion shape connexon of this type will have by H ₂N-APEL (x) _y-CO ₂H carries out the H7 hinge sequence of the terminal expansion of N-, and wherein x is that Gly4Ser connexon unit and y are 0 to 3 numerical value.Use two kinds of scorpion shape molecules (dual specific CD20xCD28 scorpion shape molecule and monospecific CD20xCD20 scorpion shape molecule) to study of the influence of scorpion shape connexon illustratively to scorpion shape stability of molecule.For in these two kinds of scorpion shape molecular designing each, insert various scorpion shape connexons.Especially, the key distinction of scorpion shape connexon H16 and H17 is that H17 has the sequence of H16, and the sequence of H7 is appended hereto the C-end, and the key distinction of scorpion shape connexon H18 and H19 is that in forming H19, the sequence of H7 is appended hereto the C-end of H18 similarly.For in two kinds of scorpion shape molecular backbone chains (20 * 28 and 20 * 20) each, in above-mentioned four kinds of scorpion shape connexons each is inserted appropriate location.In the COS cell, obtain the moment of described construct is expressed, and apply the scorpion shape molecule protein purifying that will be present on the hole (Pierce SEIZE IP test kit) in the culture supernatants at albumin A/G-.On the SDS-PAGE gel, separate purified albumen and manifest by silver staining.Check Figure 44 discloses, and another H7 sequence in the scorpion shape connexon can increase the stability of all types of scorpion shape connexons and all types of scorpion shape molecule proteins.In other words, the C-end that H7 is attached to H16 or H18 can increase the stability of scorpion shape molecule, and no matter whether this scorpion shape molecule is CD20xCD28 or CD20xCD20, and this observation is all set up.Target in conjunction with aspect, the scorpion shape molecule protein with CD20 * CD20 framework shows and parent's monospecific humanization CD20 * similar binding characteristic of CD20 scorpion shape molecule S0129, as shown in Figure 45.

Yet, except that above-mentioned embodiment, need prevent that institute's bonded acceptor is closer to each other in about 50

With interior and have a mind to form time peak signal (people such as Paar, J.Immunol., 169:856-864).In the case, expection selects that shorter and flexible littler H1 and scorpion shape connexon (H2) are suitable than above-mentioned those connexons.

Uniform distances considers to be applicable to the scorpion shape connexon of non-hinge sample.Described scorpion shape connexon for example is the peptide class of aminoacid sequence in stem district with C-lectin.The exemplary scorpion shape hinge that comprises C-lectin stem district is the scorpion shape hinge that is derived from CD72 stem district, CD94 stem district and NKG2A stem district.The scorpion shape molecule that contains described scorpion shape hinge is through making up and expressing, characterizing aspect cracked susceptibility and purifying adaptability.Data presentation is in table 14.

Table 14

The connexon title	G 4S is codon optimized 1	The end of CH3	S0129 scorpion shape connexon varient aminoacid sequence	Based on following connexon sequence	Express (%S0129) 2	Cracking 3	Experiment table purifying %POI
								H7	N	K	GCPPCPNS	H7	100	-	70
H60	Y(17)	K	GCPPCPNS	H7	114	-	ND
								H61	Y(15)	K	GCPPCPNS	H7	90	-	66

H62	N	G	QRHNNSSLNTRTQKARHCPNS	NKG2A stem district	129	-	89
								H63	Y(17)	G	QRIINNSSLNTRTQKARHCPNS	NKG2A stem district	100	-	85
H64	Y(15)	G	QRHNNSSLNTRTQKARHCPNS	NKG2A stem district	81	-	93
								H65	N	G	EPAFTPGPNIELQKDSDCPNS	CD94 stem district	133	-	66
H66	Y(17)	G	EPAFTPGPNIELQKDSDCPNS	CD94 stem district	200	-	64
								H67	Y(15)	G	EPAFTPGPNIELQKDSDCPNS	CD94 stem district	129	-	65
H68	N	G	RTRYLQVSQQLQQTNRVLEVTNSSLRQQLRLKITQLGQSAEDLQGSRRELAQSQEALQVEQRAHQAAEGQLQACQADRQKTKETLQSEEQQRRALEQKLSNMENRLKPFFTCGSADT	The complete stem of CD72 district	110	-	75

¹Gly ₄Ser connexon codon optimized has (17) restriction site or do not have (15) restriction site

²Be evaluated at expression among the COS based on the protein recovery of experiment table purifying

³By the purified one or more split products that albumen observed of the blue dyeing of SDS-PAGE/ storehouse Maas

The combination of b.N-end and the terminal binding domains of C-

N-is terminal to be combined with C-end structure territory participation target cell

The combination (TRU015+SMIP016) of CD20SMIP (TRU015), CD37SMIP (SMIP016), CD20 and CD37SMIPS and the target cell binding ability of CD20xCD37 dual specific scorpion shape molecule (015x016) are to block separately with the bonded ability of relevant target (CD37 or CD20) specificity competition bonded antibody and assess by measuring described molecule.Competitive antibody optionally be through the FITC mark monoclonal anti-CD37 antibody or through the mono-clonal anti-CD 20 antibodies of PE mark.Ramos B-cell provides target.

PBS (#100-113, Gemini Bio-Products, WestSacramento, CA) the Ramos B-cell (1.2 * 10 in (dyeing medium) that will have 5% mice serum ⁷Individual cells/ml) is added in the 96 hole V-arrangement base plates (25 microlitres/hole).Various SMIP and scorpion shape molecule are diluted to 75 μ g/ml in dyeing medium, and 4 times are diluted to the concentration shown in Figure 38.Diluted compound is added in the cell of institute's coated plate (except that the medium that is used for control wells separately).Cell was hatched 10 minutes with described compound, and then the FITC with 5 μ g/ml resists-CD37 antibody (#186-040, Ancell, Bayport, MN) and the PE anti-CD 20 antibodies (#555623 of 3 μ g/ml, BD Pharmingen, San Jose, CA) (pure) is added in the 25 μ l dyeing mediums in the hole together.With described cell in the dark in hatching 45 minutes on ice and then with PBS washing 2.5 times.(Cleveland OH) fixes for #19943 1LT, USB Corp, and then (BD Biosciences, San Jose CA) go up running at FACs Calibur with 1% trioxymethylene with cell.With Cell Quest software (BD Biosciences, San Jose, CA) analytical data.Result shown in Figure 38 confirms, all SMIP, SMIP combination and the scorpion shape molecule that contains the CD20 binding site all successfully are bonded to Ramos B-cell (last figure) with anti-CD 20 antibodies competition through the PE mark; All SMIP, SMIP combination and the scorpion shape molecule that contains the CD37 binding site all successfully are bonded to Ramos B-cell (figure below) with anti--CD37 antibody competition through the FITC mark.Therefore, dual specific CD20 * CD37 scorpion shape molecule is proved to be for the target on the B-cell and has the terminal and terminal binding site of C-of exercisable N-.

C. cell surface persistence

Bonded SMIP of institute and scorpion shape molecule (monospecific and dual specific) the cell surface persistence on the B-cell surface discloses scorpion shape molecular ratio SMIP and shows stronger cell surface persistence.With 6 * 10 in the dyeing medium (2.5% lowlenthal serum among the PBS, 2.5% mice serum) ⁶The Ramos B-cell (3 * 10 of individual cells/ml ⁵Individual cells/well) is added in the 96 hole V-arrangement base plates.In dyeing medium, prepare the test agent of twice ultimate density by 5 times of serial dilution 500nM parent materials, and then be added in the Ramos B-cell with 1:1.In addition, also apply control media.With cell in the dark in hatching 45 minutes on ice.Then plate is washed 3.5 times with cold PBS.Then (Carlsbad CA) makes an addition in the dyeing medium for #H10501, Caltag/Invitrogen with the second reagent FITC goat Anti-Human class IgG with the 1:100 extent of dilution.With cell in the dark in hatching 30 minutes on ice.Then cell is washed 2.5 times by the cold PBS of centrifugal usefulness, (Cleveland OH) fixes for #199431LT, USB Corp, and then (BD Biosciences, San Jose CA) go up running at FACs Calibur with 1% trioxymethylene solution.With CellQuest software (BD Biosciences, San Jose, CA) analytical data.Data analysis the results are shown among Figure 37, and this figure shows some kinds of SMIP (monospecific CD20 * CD20 scorpion shape molecule and dual specific CD20 * CD37 scorpion shape molecule) and its target combining on Ramos B cell.

To have Ramos B-cell (7 x 10 ⁵Individual cells/ml) two pipes were hatched on ice 30 minutes, each (respectively being 25 μ g/ml, in the Iscoves medium with 10%FBS) in two kinds of compounds (that is humanization CD20 (2Lm20-4) SMIP and humanization CD20 * CD20 (2Lm20-4 x 2Lm20-4) scorpion shape molecule) studied.Incubation period, wash two pipes 3 times by centrifugal when finishing.Then with the cell (in 150 μ l Iscoves media) of a pipe with 2 * 10 ⁵Individual cells/well is coated in the 96 hole flat undersides, then a plate is placed 37 ℃ of thermostat containers and another plate is hatched on ice.With second pipe respectively organize the cell resuspending in cold PBS (dyeing medium) with 2% mice serum and 1% sodiumazide, and with 2 * 10 ⁵Individual cells/well is coated in the 96 hole V-arrangement base plates so that through second antibody (that is the anti-IgG of FITC goat (#H10501, Caltag/Invitrogen, Carlsbad, CA)) rapid dyeing.Make an addition in dyeing medium with the final extent of dilution of 1:100 second antibody and make cell on ice in the dark in dyeing 30 minutes.Then (Cleveland OH) fixes for #199431LT, USB Corp with cold PBS washing 2.5 times and with 1% trioxymethylene with cell.

Specified time point in Figure 39 is gathered in the crops sample in 96 hole flat undersides, hatch down or on ice at 37 ℃, and place 96 hole V-arrangement base plates (2 * 10 ⁵Individual cells/well) in.With cell once with the washing of cold dyeing look medium, resuspending, and the final extent of dilution of second antibody with 1:100 made an addition in the dyeing medium.With described cell in the dark in hatching 30 minutes on ice.Then cell is washed 2.5 times in cold PBS, and fix with 1% trioxymethylene subsequently by centrifugal.(BD Biosciences, San Jose CA) go up running, and with CellQuest software (BD Biosciences, San Jose, CA) analytical data at FACS Calibur with sample.Result shown among Figure 39 proves that SMIP and scorpion shape molecule continue at least six hours with combining of B-cell surface, and wherein the degree that continues of monospecific hu CD20xCD20 (2Lm20-4x2Lm20-4) scorpion shape molecular ratio huCD20 (2Lm20-4) SMIP is bigger.

Embodiment 15

The direct cell killing effect of monospecific and dual specific scorpion shape molecule

Experiment is intended to assess monospecific and dual specific scorpion shape molecule directly kills the ability of lymphoma cell, that is does not relate to the ability that ADCC or CDC kill described cell.Especially, make Su-DHL-6 and DoHH2 lymphoma cell line stand monospecific scorpion shape molecule (that is CD20 * CD20 scorpion shape molecule or CD37 * CD37 scorpion shape molecule) individually, or stand dual specific CD20 * CD37 scorpion shape molecule.

Utilize routine techniques to set up the nutrient solution of Su-DHL-6, DoHH2, Rec-1 and WSU-NHL lymphoma cell, and then make some described nutrient solution individually be exposed to monospecific CD20SMIP, monospecific scorpion shape molecule (CD20 * CD20 or CD37 * CD37) or dual specific scorpion shape molecule (CD20 * CD37 or CD19 * CD37).Cellular exposure is to carry out under the crosslinked condition not causing in SMIP or scorpion shape molecule.Cell and described molecule were kept in touch 96 hours, afterwards as known in the art, measure its growth by detecting ATP.The cell killing effect that apparent CD20SMIP and CD20xCD20 monospecific scorpion shape molecule are produced in Figure 24 and table 15.The cell of apparent CD37 * CD37 monospecific scorpion shape molecule kills ability in Figure 25 and table 15, in Figure 26 and table 15 apparent CD20 * CD37 dual specific scorpion shape molecule kill the ability of lymphoma cell and in Figure 27 and table 15 apparent CD19 * CD37 dual specific scorpion shape molecule kill the ability of lymphoma cell.With the data combination of three independent experiments, and each point is represented mean+/-standard error (SEM).Symbol as table 15 is annotated, the IC in the table 15 ₅₀Value determines by the curve among Figure 24, Figure 25 and Figure 26, and is defined as to compare with undressed culture and produces 50% inhibiting concentration.Digital proof in described figure and the form, scorpion shape molecule are killed the effectiveness of described clone and are compared to go out greatly more than 10 times with the free SMIP that uses the identical combination structural domain.

Table 15

^*Data source is from Figure 24.

^*Data source is from Figure 25.

^{* *}Data source is from Figure 26.

^{* * *}Data source is from Figure 27.

In following cell, carry out other experiments of humanization CD20xCD20 scorpion shape molecule S0129: Su-DHL-4, Su-DHL-6, DoHH2, Rec-1 and WSU-NHL cell.The results are shown among Figure 46 and Figure 47.The data that provided among the described figure are expanded above-mentioned conclusion, and its proof scorpion shape molecule has the ability of directly killing various clones.

Above-mentioned conclusion may extend to other monospecifics and dual specific scorpion shape molecule, and various scorpion shape molecule proofs have the ability of directly killing the B cell.Make the DoHH2B-cells in vitro be exposed to monospecific CD20 * CD20 scorpion shape molecule, monospecific CD37 * CD37 scorpion shape molecule or dual specific CD20 * CD37 scorpion shape molecule.Shown result proves that dual specific scorpion shape molecule has the kill ratio curve that form is different from monospecific scorpion shape molecule among Figure 48.

In the presence of 70nM CD20xCD20 scorpion shape molecule (S0129), CD20xCD37 scorpion shape molecule or CD37xCD37 scorpion shape molecule, cultivate the Su-DHL-6 cell and also in external environment, produce direct B-cell killing effect (Figure 49).Equally, the Su-DHL-6 cellular exposure is in dual specific CD19xCD37 scorpion shape molecule or be exposed to

Produce direct cell killing effect, and dual specific scorpion shape molecule is than showing lethality under the low dosage, such as among Figure 50 announcement.

By making the DHL-4 cellular exposure, can provide another proof of direct cell killing effect in identification four kinds of CD20 monospecific scorpion shape molecule independently.Two kinds of patterns of CD20xCD20 scorpion shape molecule are designed to merge the crossbred of two 20-4 binding domainss (20-4x20-4 and S0129) and other two kinds of merging 011 and 20-4 binding domains.All four kinds of two kinds of CD20 * CD20 scorpion shape molecular designing through independently structure and purified pattern ((20-4 * 20-4 and S0129) and crossbred (011 * 20-4 and 011 * 20-4 Δ Asp)) are effectively killed the DHL-4 cell with direct mode.For this research, with 1 μ g/ml specify albumen with the DHL-4 cell through extracorporeal treatment 24 hours.Then cell is used annexin V and propidium iodide (necrocytosis early stage and late period mark) dyeing respectively, and passed through FACS quantization cell group.As strengthen with the dyeing shown in the black post proved that shown result proves the ability of directly killing of each CD20 * CD20 construct among Figure 51.In addition, described result proves, with respect to the scorpion shape molecule based on 20-4x20-4, crossbred 011x20-4 albumen shows slightly for direct cell killing effect and strengthen, although described scorpion shape molecule monospecific identification CD20 separately.In experimental group out of the ordinary, measure the dose-response of four kinds of independent scorpion shape molecule construction bodies by facs analysis annexin V staining cell group and propidium iodide staining cell group.Result shown in Figure 52 proves, in handle the necrocytosis that the DHL-4 cells are caused with various independent scorpion shape molecule construction bodies, and the dose-response increase.

Embodiment 16

The attach feature (ADCC and CDC) that scorpion shape molecule is mediated

A. the cytotoxicity of scorpion shape molecule dependent cell

Experiment is intended to judge whether scorpion shape molecule mediates the killing effect of BJAB B lymphoma cell.Through observation, BJAB B lymphoma cell can be killed by CD20 and/or CD37 scorpion shape molecule.

When initial, under 37 ℃, in having the Iscoves medium of 10% FBS with BJABB-cell (1 * 10 ⁷Individual/milliliter) with 500 μ Ci/ml ⁵¹The Cr Sodium chromate (#CJS1, AmershamBiosciences, Piscataway, NJ) mark is 2 hours.Then will be loaded with ⁵¹The BJAB B cell of Cr washs 3 times in the RPMI medium with 10% FBS and with 4 * 10 ⁵Individual/milliliter resuspending is in RPMI.By the lymphocyte separating medium (#50494, MP Biomedicals, Aurora, Oh) centrifugal, the peripheral blood monocyte (PBMC) of separating experiment chamber donor from the heparinization whole blood is with RPMI medium washing 2 times and with 5 * 10 ⁶Individual/milliliter resuspending is in the RPMI with 10%FBS.The reagent sample is added in the RPMI medium with 10%FBS with 4 times of ultimate densities, and to three parts of 10 times of serial dilutions of each reagent preparation.Then described reagent is added in the 96 hole U type base plates until specified ultimate density with 50 microlitres/hole.Then will through ⁵¹The bjab cell of Cr mark is with 50 microlitres/hole (2 * 10 ⁴Individual cells/well) is added in the plate.Follow PBMC with 100 microlitres/hole (5 * 10 ⁵Individual cells/well) be added in the plate, make effector (PBMC): the final ratio of target (BJAB) is 25:1.Effector and target are added in the independent medium to measure the background kill ratio.Will through ⁵¹The BJAB of Cr mark is added in the independent medium to measure ⁵¹The spontaneous release rate of Cr and be added into and have 5% NP40 (#28324, Pierce, Rockford, in medium I11) to measure ⁵¹The maximum release rate of Cr.With described plate under 37 ℃ in 5% CO ₂In hatched 6 hours.Then get from each hole 50 μ l (25 μ l are also for suitable) supernatant liquor be transferred to LumaPlate-96 (#6006633, Perkin Elmer, Boston, Mass) in and at room temperature dry overnight.

After the drying, on Packard TopCount-NXT, quantize radioactive emission with cpm.Sample value is three increments mean values originally.Use following equation to calculate specificity and kill per-cent: kill %=((sample-spontaneous release rate)/(maximum release rate-spontaneous release rate)) * 100.Curve display BJAB B cell among Figure 30 kills by monospecific scorpion shape molecule CD20 * CD20 and CD37 * CD37.BJAB B cell is also killed in the combination of CD20SMIP and CD37SMIP.Described result proves that scorpion shape molecule shows the cytotoxicity of scorpion shape molecule dependent cell and expects that this functionally provides by the constant subprovince that the active scorpion shape of ADCC molecule is provided.

B. the effect of scorpion shape molecule in CDC

Experiment also proves that scorpion shape molecule has CDC (CDC) activity.As described below and as shown in Figure 31, experiment relates to makes Ramos B-cellular exposure in CD19 and/or CD37SMIP and scorpion shape molecule.

Experiment is initially 5 * 10 in the Iscoves medium (no FBS) of 50 μ l ⁵To 2.5 * 10 ⁵Individual Ramos B-cell is added in the hole of 96 hole V-arrangement base plates.(or independent Iscoves) is added in the hole with the appointment ultimate density at twice with 50 μ l with the test compounds among the Iscoves.Cell and reagent were hatched under 37 ℃ 45 minutes.With cell with the Iscoves of no FBS washing 2.5 times and with the Iscoves with human serum of prescribed concentration resuspending in 96 orifice plates (#A113, Quidel, San Diego, CA) in.Then cell was hatched under 37 ℃ 90 minutes.With cell by centrifugal wash and resuspending in the cold PBS of 125 μ l.Then with cell transfer to FAC bunch of pipe (#4410, CoStar, Corning, NY) and add propidium iodide (#P-16063, Molecular Probes, Eugene, PBS OR) that 125 μ l have 5 μ g/ml.Cell is at room temperature hatched 15 minutes with propidium iodide in dark, and then be placed on ice, quantize, and on FACsCalibur, analyze through CellQuest software (Becton Dickinson).Result shown among Figure 31 proves that CD19SMIP (but not CD37SMIP) shows the CDC activity, and the combination of these two kinds of SMIP shows and the independent roughly the same CDC level of activity of CD19SMIP.Yet CD19xCD37 scorpion shape molecule shows than the remarkable stronger CDC activity of independent SMIP or SMIP combination, and this explanation scorpion shape molecule framework can provide than other molecular designing CDC greatly.

The ADCC/CDC activity of c.CD20 * CD20 monospecific scorpion shape molecule

To three kinds of different CD20xCD20 monospecific scorpion shape molecules and suitably contrast molecular testing ADCC and CDC functional.Utilize routine techniques to measure ADCC, and the results are shown among Figure 53.ADCC activity by apparent remarkable (but non-unanimity) relevant among the figure with each CD20 that is tested * CD20 monospecific scorpion shape molecule.

Be assessment CDC, under 37 ℃ with Ramos B-cell sample (4 * 10 ⁵) hatched 3.5 hours with each CD20 * CD20 scorpion shape molecule (0,0.5,5,50 and 500nM) and serum (10%).Assess necrocytosis by 7-AAD dyeing and facs analysis.The results are shown among Figure 54, this figure discloses scorpion shape molecule and shows some CDC activity.In similar experiment, under 37 ℃ with Ramos B-cell sample (4 * 10 ⁵) hatched 2 hours with CD20 * CD20 scorpion shape molecule protein (5,50,100nM) and serum (10%).Hatch with cell washing 2 times and with anti-human C1qFITC antibody.Assess the bonded C1q of institute by facs analysis, and the results are shown among Figure 55.Result shown among described result and Figure 54 is consistent, illustrate that each CD20xCD20 monospecific scorpion shape molecule and some CDC are active relevant, but activity is weaker than the activity relevant with CD20SMIP.

D. scorpion shape molecule and F _CThe interaction of γ RIII

ELISA studies show that under the non-existent situation of target cell, scorpion shape molecule is with the low Fc γ RIII (CD16) of enhancing degree combination (low affinity isotype or allelotype).Initially utilize routine techniques, apply elisa plate with low affinity or high-affinity CD16mIgG.Assess this fixedly fusion rotein catch the ability of CD20SMIP or CD20 * CD20 monospecific scorpion shape molecule.Detect bonded SMIP of institute and scorpion shape molecule with the anti-IgG of goat (HRP) secondary antibody, and measure average candle power intensity (MFI).PBS (negative control thing) shows with single-point separately.The results are shown among Figure 32 A among (merge by CD16 high-affinity isotype and catch) and Figure 32 B (merge by the low affinity isotype of CD16 and catch).By apparent to the consideration of Figure 32 A and Figure 32 B, CD20SMIP and the combine enhancing of CD20 * CD20 monospecific scorpion shape molecule demonstration with high-affinity and low affinity CD16 isotype syzygy, wherein CD20 * CD20 scorpion shape molecule shows and hangs down combining with protein concentration of affinity isotype syzygy and increase and significantly enhancing.

Also be evaluated at the target cell and have combining of scorpion shape molecule and Fc γ RIII isotype down.Data presentation, in the presence of the target cell, scorpion shape molecule strengthens with low affinity isotype of Fc γ RIII (CD16) or combining with protein concentration of allelotype and high-affinity isotype or allelotype.

In experimentizing, allowing under the positive target cell of SMIP or scorpion shape molecule and the CD20 bonded condition, make the positive target cellular exposure of CD20 in CD20SMIP or CD20 * CD20 monospecific scorpion shape molecule.Subsequently, make carry SMIP or scorpion shape molecule the target cellular exposure in CD16 high-affinity or low affinity isotype through mouse IgFc mark.Then will add the immobilization CD16 that is connected with mouse IgFc with mark through the anti-mouse Fc of labelled goat as secondary antibody.Then (BD Biosciences, San Jose utilize the Flow cytometry cell on CA), and with Cell Quest software (BD Biosciences, San Jose, CA) analysis at FACs Calibur.As shown in Figure 33, in the presence of the target cell, increase each concentration of CD20SMIP and CD20 * CD20 monospecific scorpion shape molecule and can make the enhancing that combine with the CD16 isotype, it is more remarkable that wherein the enhancing of the associativity of CD20 * CD20 scorpion shape molecule is compared to the being seen associativity enhancing of CD20SMIP.

Embodiment 17

Scorpion shape molecule is to the cell cycle effect of target lymphoma cell

By making lymphoma cell be exposed to the cell cycle effect that SMIP, monospecific scorpion shape molecule and dual specific scorpion shape molecule are assessed scorpion shape molecule.More particularly, with DoHH2 lymphoma cell (0.5 * 10 ⁶) handled 24 hours with 0.4nM Rituximab (rituximab), CD20 * CD37 scorpion shape molecule, TRU-015 (CD20SMIP)+SMIP-016 combination (respectively being 0.2nM), 100nMSMIP-016 or 100nM CD37 * CD37 scorpion shape molecule.In 96 hours growth-inhibiting are measured, the IC that described concentration shows ₅₀Value is than the IC of scorpion shape molecule ₅₀Value goes out about 10 times (referring to Figure 24-27) greatly.Under 37 ℃, with culture with 10 μ M BrdU (bromine uracil deoxyriboside) marks 20 minutes.After fixing, cell is used anti--BrdU-FITC antibody staining and used the propidium iodide counterstain.Numerical value among Figure 28 be 4 parts of copy type cultures of 2-3 independent experiment mean value+/-SD.Analyze all sampled datas simultaneously and utilize BrdU and PI merges point curve and makes up demonstration.Graphic representation proves, the main effects that scorpion shape molecule is handled is for to the exhausting of the cell that is in the S-phase, and G ₀/ G ₁Increase at interval.

Embodiment 18

The physiological effect of scorpion shape molecule

A. plastosome current potential

Institute's announcement in measuring as JC-1, CD20 * CD20 scorpion shape molecule is induced the mitochondrial membrane potential decline of DHL4B-cell.JC-1 for the cationic carbon cyanine dye that shows mitochondrial voltage-dependent and gather (from Molecular Probes's The JC-1 flow cytometry is measured test kit).Compare with plasma membrane, JC-1 has more specificity to mitochondrial membrane, and is used to measure the variation of mitochondrial membrane potential.Mitochondrial gather to be offset by the fluorescent of green (529nm) to red (590nm) indicate.

In experimentizing, initially with DHL-4B-cell (5 * 10 ⁵Individual cells/ml) be incubated in 24 orifice plates and cultivate in the thermostat container in normal structure, under 37 ℃ in 5% CO ₂Middle CD20 * CD20 scorpion shape molecule with 1 μ g/ml, Rituximab, IgG control antibodies or 5 μ M star spore rhzomorphs (staurosporine) were handled 24 hours.Add JC-1 dyestuff (10 μ l/ml, 2 μ M ultimate densities) and under 37 ℃, cell was hatched 30 minutes again.Cell is gathered in the crops by centrifugal (1200rpm, 5 minutes), used 1ml PBS washing and resuspending in 500 μ l PBS.(FACSCalibur BD) excites via 488nM and 530nM and 585nM emission spectral filter comes analysis of cells by flow cytometry.For the representative scatter diagram shown in Figure 56, measuring red fluorescent on the Y-axis and on X-axis, measuring green fluorescent.As with positive control CCCP (carbonyl cyanide 3-chlorobenzene hydrazone) (a kind of known mitochondrial membrane potential upset agent) finding, the depolarize of mitochondrial membrane is to measure as weakening of red fluorescent.Have reaction for checking JC-1 changes membrane potential, with the DHL-4B-cell at 37 ℃, 5%CO ₂Use the CCCP (50 μ M and 250 μ M) of two kinds of concentration to handle down 5 minutes.Another kind of positive control is for handling the cell with cell death inducing with wide spectrum kinase inhibitor star spore rhzomorph.Result shown in Figure 56 is 10, the 000 point curve figure that count, and wherein red fluorescent is depicted on the Y-axis and green fluorescent is depicted on the X-axis.Generality histogram with cell per-cent of upsetting mitochondrial membrane potential (is upset MMP: black post) be shown among Figure 56.Described result proves, handles the reduction that can cause the mitochondrial membrane potential relevant with necrocytosis with 20-4 * 20-4 scorpion shape molecule or 011 * 20-4 scorpion shape molecule.

B. calcium current amount

Utilize Ca ⁺⁺Mobile (common trait of cell signaling) analyzes the influence of scorpion shape molecule pair cell signal transduction path as measuring.The SU-DHL-6 lymphoma cell is handled with calcium 4 dye markers and with the test molecule of hereinafter being differentiated.The pair cell reading lasted for 20 seconds with mensuration background fluorescent, and then added SMIP/ scorpion shape molecule (first dotted line among Figure 28), and measured fluorescent until 600 seconds.When 600 seconds, add anti-human F (ab) ' 2 of 8 times of excessive crosslinked goats, and measure 300 seconds of fluorescent again.Figure among Figure 28 (A) shows, compares (blue line) with unprovoked cell, by the result that combination obtained (red line) of CD20 SMIP and CD37 SMIP; By result's (black line) that CD20 * CD37 dual specific scorpion shape molecule is obtained.Among the figure B in Figure 28, the result who handles cell (red line) with independent CD20 SMIP causes Ca ⁺⁺Flow phenomenon, but this is also strong like that not as the signal (black line) that monospecific CD20 * CD20 scorpion shape molecule is produced.The Ca of Figure 28 ⁺⁺Flow graph is represented available from the fluorescent through three holes of the scorpion shape molecule of equimolar amount and SMIP/SMIP combined treatment.

C. caspase 3,7 and 9

Such as increase by the dyeing of annexin V and propidium iodide and mitochondrial membrane potential decline proof, the ability of directly killing the B-cell in conjunction with the scorpion shape molecule of CD20 causes in conjunction with the further research to other apoptosis correlation effects of B-cell of the scorpion shape molecule of CD20.The method that is adopted is the DHL-4B-cell that is exposed to CD20 * CD20 scorpion shape molecule or suitable contrast to be carried out Apo1 measure.Apo1 measures the synthetic peptide substrates that is based on caspase 3 and 7.Measure component can available from Promega (

Homology caspase-3/7 is measured).Caspase mediation discharges fluorescent rhodamine 110 marks through the peptide Z-DEVD-of mark rhodamine 110 (Rhodamine 110) cracking, uses 485nm to excite to detect with 530nm it is measured.

In experiment, with 100 μ l DHL-4B-cells (1 * 10 ⁶Individual cells/ml) is coated in the flat tissue culturing plate in black 96 holes and cultivates in the thermostat container, at 37 ℃, 5%CO in normal structure ₂Use CD20 * CD20 scorpion shape molecule, Rituximab, IgG control antibodies or the 5 μ M star spore rhzomorphs of 1 μ g/ml to handle down 24 or 48 hours.(star spore rhzomorph is a small molecules wide spectrum kinases inhibitor, is known in the art it and is classical apoptotic effective inductor of various kinds of cell type).After 24 or 48 hours, 100 times of 100 μ l dilution substrates are added in each hole, go up at board-like oscillator (300rpm) and mixed one minute light and slowly and at room temperature hatched two hours.Use 485nM to excite and measure fluorescent with 527nM emission spectral filter (Fluoroskan Ascent FL, Thermo Labsystems).Behind the graphic presentation that shows among Figure 57 24 hours and 48 hours (for the star spore rhzomorph, only 24 hours), the average candle power intensity of three processing+/-standard deviation.Described result proves, directly do not kill the B-cell via relating to caspase 3/7 activatory apoptotic pathways in conjunction with the scorpion shape molecule of CD20.

The result who is obtained in the Apo-1 mensuration verifies that by the western blot analysis method described western blot analysis method is through being designed to the cracking that detection causes the short caspase cracking of caspase activatory or detects PARP (poly-(ADP-ribose) polysaccharase) (a kind of known passing through activates caspase 3 and cracked albumen).Make the DHL-4B-cellular exposure last 4,24 or 48 hours, and cell lysate is separated and utilizes routine techniques to carry out the Western blotting analysis through SDS-PAGE in scorpion shape molecule or contrast in conjunction with CD20.Figure 58 result displayed is the form of western blotting set.Bottom three place's western blottings are to use anti-caspase antibody to detect reflection proteolysis activatory caspase change of molecular weight.For caspase 3,7 and 9, do not exist caspase to be subjected to any CD20-binding molecule activatory sign.Star spore rhzomorph serves as the positive control of mensuration and to the short caspase cracking of each the induced activity caspase in caspase 3,7 and 9.The 4th western blotting shown in Figure 58 shows, not cracking of PARP (a kind of substrate of known activation caspase 3), and this is with to fail to activate caspase 3 in conjunction with the scorpion shape molecule of CD20 consistent.All described result of experiment are all consistent, illustrates that it not is key character by in conjunction with the direct cell killing effect of the scorpion shape molecule inductive DHL-4B-of the institute cell of CD20 that caspase 3 activates.

In addition, the time is studied continuously and is intended to measure the effect of CD20 conjugated protein (comprising CD20 * CD20 scorpion shape molecule) to caspase 3.With DoHH2 or Su-DHL-6B-cell and 10nM CD20 conjugated protein (S0129 scorpion shape molecule, 2Lm20-4SMIP or

)+/-solubility CD16 Ig (40nM), independent solubility CD16 Ig or medium are hatched together.With cell with 3 * 10 ⁵Individual/hole/300 microlitres are incubated among the complete RPMI with 10% FBS and results when 4 hours, 24 hours or 72 hours.The sample of 72 hours time points is coated in the 500 μ l test agents.Cell is washed with PBS, and (San Jose CA) dyes with intracellular reactive caspase-3 for No. the 55048th, catalog number (Cat.No.), BDPharmingen then to use BD Pharmingen caspase 3 (activity form), mAB apoptosis test kit: FITC.In brief, carry out 2 washings again with cold PBS after, cell suspension was hatched 20 minutes in cold cytofix/cytoperm solution and on ice.Then come washed cell by centrifugal, suction, and at room temperature use Perm/Wash buffer reagent washed twice.Then at room temperature in dark, sample was dyeed 30 minutes with the anti-caspase 3 of 20 μ l FITC-in the Perm-Wash buffer reagent of 100 μ l.Then sample is used Perm-Wash buffer reagent washed twice, and resuspending is in the Perm-Wash buffer reagent of 500 μ l.Then the cell transfer that will be washed is to the FACs pipe, and (BD Biosciences, San Jose CA) go up running, and with Cell Quest software (BD Biosciences, San Jose, CA) analysis at FACs Calibur.The results are shown in the table 16.

Table 16

All described result of experiment are all consistent, illustrate CD16 not in the presence of, limited to the activation of caspase 3, this is not to mean caspase 3 to activate the key character of serving as reasons in conjunction with the direct cell killing effect of scorpion shape molecule institute's inductive of CD20.

The d.DNA fracture

Induce classical apoptosis signal transduction path finally to cause concentrating of chromosomal DNA and fracture degraded.For whether the scorpion shape molecule of measuring in conjunction with CD20 directly kills the B-cell via classical Apoptosis Mechanism, make cellular exposure after scorpion shape molecule or contrast in conjunction with CD20, check the state of B-cell chromosome DNA.When initial, with the CD20 binding molecule (that is monospecific CD20 * CD20 (the scorpion shape molecule of 2Lm20-4 * 2Lm20-4), CD20 * CD20 (011 * 2Lm20-4) scorpion shape molecule or Rituximab) or with contrast extracorporeal treatment DHL-4B-cell last 4 hours, 24 hours or 48 hours.Subsequently, with cytolysis and utilize routine techniques with the chromosomal DNA purifying.Then chromosomal DNA is carried out fractional separation by gel electrophoresis.Gel electrophoresis figure shown in Figure 59 shows the shortage of dna break, proves by the caused necrocytosis of scorpion shape molecule in conjunction with CD20 to be mediated by classical apoptotic pathways.The cell of handling through star spore rhzomorph is used as positive control in described mensuration.

The e.SYK phosphorylation

SYK is that the phosphoric acid with some phosphorylation sites is regulated albumen, and it suppresses son as transcribing.SYK is arranged in nucleus, but can relocate in cytolemma fast after the activation.For activation, SYK must keep its nucleus positioning sequence.Activation SYK has the effect and the SYK that suppress breast cancer tumor and passes through short apoptosis signal (crosslinked such as ionizing rays, BCR connection and MHC II class) activation.In addition, proved that SYK can influence PLC-γ and Ca ⁺⁺Path.Via described observation, research is in conjunction with the ability of the scorpion shape molecules influence SYK of CD20.

Make the DHL-6B-cellular exposure last 0,5,7 or 15 hour and make cytolysis in dual specific CD20 * CD37 scorpion shape molecule.Molten born of the same parents' thing immunoprecipitation is separated immunoprecipitate by gel electrophoresis, and the results are shown among Figure 60 with anti-phosphorylated tyrosine antibody or with anti--SYK antibody.Figure 60 is apparent by check, and dual specific CD20 * CD37 scorpion shape molecule is failed to bring out the phosphorylation of SYK, thereby failed its activation.Consistent with above-mentioned research to caspase activation and chromosomal DNA fracture, as if do not utilize classical apoptotic pathways (such as the caspase dependent pathway) directly to kill the B-cell in conjunction with the scorpion shape molecule of CD20.Although be not wishing to be bound by theory, but expection is directly to kill the B-cell via caspase independence approach and SYK independence approach in conjunction with the scorpion shape molecule of CD20, this approach does not have significant chromosomal DNA fracture characteristic, does not have significant chromosomal DNA fracture characteristic in the identical time period of taking place at least to rupture during caspase dependent cell apoptosis.

Embodiment 19

Scorpion shape molecular application

A. the activity in vivo of scorpion shape molecule

The activity of scorpion shape molecule also utilizes mouse model to assess.Measurement for scorpion shape molecule activity in vivo relates to the serum-concentration that gives 10-300 μ g scorpion shape molecule and measure this scorpion shape molecule subsequently according to chronological order.The results are shown among Figure 40 of described research, it is shown as in two kinds of dual specific scorpion shape molecules to mouse three all pharmacokinetic studies (that is S0033, CD20 * CD27 scorpion shape molecule and CD20 * CD37 scorpion shape molecule) the serum-concentration curve of each.Data presentation among Figure 40, after the administration, the serum content of each falls after rise to the baseline content in these two kinds of dual specific scorpion shape molecules, need expend at least 500 hours.Therefore, scorpion shape molecule demonstration serum stability and recyclability, persistent body-internal-circulation transformation period.

Also assess the interior effect of body of scorpion shape molecule.Contrast at SMIP and to use aggressive Ramos heteroplastic transplantation model in the parallel laboratory test of historic immunoglobulin (Ig) contrast.The survivorship curve that provided among Figure 41 discloses and gives 10 μ g dual specific scorpion shape molecules survival is had negligible influence, but the administration of 100-300 μ g has important positive effect to the survival of the mouse that carries Ramos xenotransplantation body.

B. combination treatment

Expection scorpion shape molecule will be applicable to prevention, treatment or improve the symptom of the influence mankind, other Mammalss and other organic various states.For example, expection in conjunction with the scorpion shape molecule of CD20 applicable to treatment or prevention is excessive with the B-cell or relevant multiple disease unusually.In fact, any disease that easily stands treatment (relate to and exhaust the B-cell) all can stand treatment in conjunction with the scorpion shape molecule of CD20.In addition, scorpion shape molecule (for example in conjunction with CD20 scorpion shape molecule) can be used from the combination treatment with other treatment agent one.For the feasibility of various combination treatments is described, give Su-DHL-6B-cell with monospecific CD20 * CD20 scorpion shape molecule (S0129) and hydroxyl daunoblastin (doxorubicin), vincristine(VCR) (vincristine) or rapamycin (rapamycin) combination.The hydroxyl daunoblastin is a topoisomerase II type toxic agent, and it disturbs DNA biochemical mechanism and belongs to drug categories at anticancer therapy.Rapamycin (Sirolimus) is a macrolide antibiotic, and its arrestin synthetic is initial and suppress immunity system, is applicable to organ transplantation and can be used as antiproliferative to cooperate the coronary artery inner support to use to suppress or prevention of restenosis.Vincristine(VCR) is for suppressing the vinca alkaloids that tubule formed and be used for the treatment of cancer.

Experimental result shown in Figure 61 is to be shown as the combinatorial index value that respectively is combined in the effect extent and scope.The interaction of monospecific CD20 * CD20 scorpion shape molecule S0129 and various kinds of drug has nothing in common with each other, but with

(RTXN) curve form is similar.The being seen effect of hydroxyl daunoblastin by high density can reflect that trend unit price bonded changes.Described data declaration can be used in combination with other multiple therapeutical agents and described combination to those skilled in the art will be apparent after understanding the disclosure in conjunction with the scorpion shape molecule of CD20.

Variation with Structural Theme of the multivalence binding molecule of effector function or scorpion shape molecule to those skilled in the art will be apparent after looking back the disclosure, and described variation structure belongs to category of the present invention.

Sequence table

＜110〉Tang Pusen etc.

＜120〉has the single-chain multivalent binding proteins of effector function

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<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G1HC-Xho

<400>21

<210>22

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G1H-S-Xho

<400>22

<210>23

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>CH3REcoR1

<400>23

<210>24

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G1XBZ-R

<400>24

<210>25

<211>54

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G4SLinkR1-S

<400>25

<210>26

<211>54

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G4SLinkR1-AS

<400>26

<210>27

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>2E12VLXbaR

<400>27

<210>28

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>2E12VLR1F

<400>28

<210>29

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>2E12VHR1F

<400>29

<210>30

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>2E12VHXbaR

<400>30

<210>31

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>2e12VHdXbaF1

<400>31

<210>32

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>2e12VHdXbaR1

<400>32

<210>33

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>IgBsrG1F

<400>33

<210>34

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>IgBsrG1R

<400>34

<210>35

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>M13R

<400>35

<210>36

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>M13F

<400>36

<210>37

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>T7

<400>37

<210>38

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>pD18F-17

<400>38

<210>39

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>pD18F-20

<400>39

<210>40

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>pD18F-1

<400>40

<210>41

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>pD18R-s

<400>41

<210>42

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>CH3seqF1

<400>42

<210>43

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>CH3seqF2

<400>43

<210>44

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>CH3seqR1

<400>44

<210>45

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>CH3seqR2

<400>45

<210>46

<211>51

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>L1-11R

<400>46

<210>47

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>L1-6R

<400>47

<210>48

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>L3R

<400>48

<210>49

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>L4R

<400>49

<210>50

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>L5R

<400>50

<210>51

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>IgBsrG1F

<400>51

<210>52

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>L-CPPCPR

<400>52

<210>53

<211>41

<212>DNA

＜213〉artificial sequence

<220>

<223>G281LH-NheR

<220>

＜223〉synthetic primer

<400>53

<210>54

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281LH-NheF

<400>54

<210>55

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281-LH-LPinF

<400>55

<210>56

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281-LH-HXhoR

<400>56

<210>57

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281-LH-LEcoF

<400>57

<210>58

<211>43

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281-LH-HXbaR

<400>58

<210>59

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281-HL-HF

<400>59

<210>60

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281-HL-HR3

<400>60

<210>61

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281-HL-HR2

<400>61

<210>62

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281-HL-HNheR

<400>62

<210>63

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281-HL-LNheF

<400>63

<210>64

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281-HL-LXhoR

<400>64

<210>65

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281-HL-LXhoR

<400>65

<210>66

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>G281-HL-EcoF

<400>66

<210>67

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LH-LF1

<400>67

<210>68

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LF2

<400>68

<210>69

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LF3

<400>69

<210>70

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LF4

<400>70

<210>71

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LF5

<400>71

<210>72

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LF6

<400>72

<210>73

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LF7

<400>73

<210>74

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LR7

<400>74

<210>75

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LR6

<400>75

<210>76

<211>51

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LR5

<400>76

<210>77

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LR4

<400>77

<210>78

<211>46

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LR3

<400>78

<210>79

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LR2

<400>79

<210>80

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LH-LR1

<400>80

<210>81

<211>54

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LH-HF1

<400>81

<210>82

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HF2

<400>82

<210>83

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HF3

<400>83

<210>84

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HF4

<400>84

<210>85

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HF5

<400>85

<210>86

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HF6

<400>86

<210>87

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HR6

<400>87

<210>88

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HR5

<400>88

<210>89

<211>55

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HR4

<400>89

<210>90

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HR3

<400>90

<210>91

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HR2

<400>91

<210>92

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LH-HR1

<400>92

<210>93

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HL-HF1

<400>93

<210>94

<211>55

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HL-HR1

<400>94

<210>95

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HL-HRO

<400>95

<210>96

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HL-LF1

<400>96

<210>97

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HL-LR3Xho

<400>97

<210>98

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HL-LR3Xba

<400>98

<210>99

<211>43

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-HL-HF1R1

<400>99

<210>100

<211>41

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LH-LF1R1

<400>100

<210>101

<211>54

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>194-LH-HR1Xba

<400>101

<210>102

<211>725

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>G28-1VLVH(DNA)

<400>102

<210>103

<211>239

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>G-28-1VLVH(AA)

<220>

<221>misc_feature

<222>(1)..(107)

<223>VL

<220>

<221>misc_feature

<222>(108)..(124)

＜223〉connexon

<220>

<221>misc_feature

<222>(125)..(239)

<223>VH

<400>103

<210>104

<211>767

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>G28-1 VHVL(DNA)

<400>104

<210>105

<211>253

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>G28-1 VHVL(AA)

<220>

<221>misc_feature

<222>(1)..(121)

<223>VH

<220>

<221>misc_feature

<222>(122)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(253)

<223>VL

<400>105

<210>106

<211>749

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>G19-4 VLVH(DNA)

<400>106

<210>107

<211>247

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>G-19-4 VLVH(AA)

<220>

<221>misc_feature

<222>(1)..(108)

<223>VL

<220>

<221>misc_feature

<222>(109)..(125)

＜223〉connexon

<220>

<221>misc_feature

<222>(126)..(247)

<223>VH

<400>107

<210>108

<211>752

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>G19-4 VHVL(DNA)

<400>108

<210>109

<211>248

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>G19-4 VHVL(AA)

<220>

<221>misc_feature

<222>(1)..(122)

<223>VH

<220>

<221>misc_feature

<222>(123)..(139)

＜223〉connexon

<220>

<221>misc_feature

<222>(140)..(248)

<223>VL

<400>109

<210>110

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<221>misc_feature

<223>ssc(s)-hIgG1(DNA)

<400>110

<210>111

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<221>misc_feature

<223>ssc(s)-hIgG1(AA)

<400>111

<210>112

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<221>misc_feature

<223>scc(s)-hIgG1(DNA)

<400>112

<210>113

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<221>misc_feature

<223>scc(s)-hIgG1(AA)

<400>113

<210>114

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<221>misc_feature

<223>css(s)-hIgG1(DNA)

<400>114

<210>115

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<221>misc_feature

<223>css(s)-hIgG1(AA)

<400>115

<210>116

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<221>misc_feature

<223>scs(s)-hIgG1(DNA)

<400>116

<210>117

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<221>misc_feature

<223>scs(s)-hIgG1(AA)

<400>117

<210>118

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>118

<210>119

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>119

<210>120

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>120

<210>121

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>121

<210>122

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>122

<210>123

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>123

<210>124

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<221>misc_feature

<223>csc(p)-hIgG1(DNA)

<400>124

<210>125

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<221>misc_feature

<223>csc(p)-hIgG1(AA)

<400>125

<210>126

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<221>misc_feature

<223>ssc(p)-hIgG1(DNA)

<400>126

<210>127

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<221>misc_feature

<223>ssc(p)-hIgG1(AA)

<400>127

<210>128

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<221>misc_feature

<223>scc(p)-hIgG1(DNA)

<400>128

<210>129

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<221>misc_feature

<223>scc(p)-hIgG1(AA)

<400>129

<210>130

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<221>misc_feature

<223>css(p)-hIgG1(DNA)

<400>130

<210>131

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<221>misc_feature

<223>css(p)-hIgG1(AA)

<400>131

<210>132

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<221>misc_feature

<223>ssc(p)-hIgG1(DNA)

<400>132

<210>133

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<221>misc_feature

<223>ssc(p)-hIgG1(DNA)

<400>133

<210>134

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<223>ssc(p)(DNA)

<220>

＜223〉synthetic primer

<400>134

<210>135

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>135

<210>136

<211>7

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>136

<210>137

<211>2

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>137

<210>138

<211>7

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<400>138

<210>139

<211>2

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>139

<210>140

<211>654

<212>DNA

＜213〉homo sapiens

<220>

＜223〉hIgG1 wild-type

<400>140

<210>141

<211>217

<212>PRT

＜213〉homo sapiens

<220>

＜223〉hIgG1 wild-type

<400>141

<210>142

<211>654

<212>DNA

＜213〉homo sapiens

<220>

<223>hIgG1(P238S)

<400>142

<210>143

<211>217

<212>PRT

＜213〉homo sapiens

<220>

<223>hIgG1(P238S)

<400>143

<210>144

<211>654

<212>DNA

＜213〉homo sapiens

<220>

<223>hIgG1(P331S)

<400>144

<210>145

<211>217

<212>PRT

＜213〉homo sapiens

<220>

<223>hIgG1(P331S)

<400>145

<210>146

<211>654

<212>DNA

＜213〉homo sapiens

<220>

<223>hIgG1(P238S/P331S)

<400>146

<210>147

<211>217

<212>PRT

＜213〉homo sapiens

<220>

<223>hIgG1(P238S/P331S)

<400>147

<210>148

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>STD1(DNA)

<400>148

<210>149

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>STD1(AA)

<400>149

<210>150

<211>114

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

<223>STD2(DNA)

<400>150

<210>151

<211>38

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>STD2(AA)

<400>151

<210>152

<211>6

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

＜223〉connexon H1 (PN)

<400>152

<210>153

<211>2

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H1 (AA)

<400>153

<210>154

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

＜223〉connexon H2 (PN)

<400>154

<210>155

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

＜223〉connexon H2 (AA)

<400>155

<210>156

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

＜223〉connexon H3 (PN)

<400>156

<210>157

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H3 (AA)

<400>157

<210>158

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

＜223〉connexon H4 (PN)

<400>158

<210>159

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H4 (AA)

<400>159

<210>160

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

＜223〉connexon H5 (PN)

<400>160

<210>161

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H5 (AA)

<400>161

<210>162

<211>54

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

＜223〉connexon H6 (PN)

<400>162

<210>163

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H6 (AA)

<400>163

<210>164

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

＜223〉connexon H7 (PN)

<400>164

<210>165

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H7 (AA)

<400>165

<210>166

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

＜223〉connexon (G4S) 3

<400>166

<210>167

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon (G4S) 3

<400>167

<210>168

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic primer

<220>

＜223〉connexon (G4S) 4

<400>168

<210>169

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon (G4S) 4

<400>169

<210>170

<211>2337

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-STD1-2e12HL(DNA)

<400>170

<210>171

<211>772

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>2H7sssIgG1-STD1-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(281)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(519)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(520)..(640)

<223>VH2

<220>

<221>misc_feature

<222>(641)..(660)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(661)..(772)

<223>VL2

<400>171

<210>172

<211>772

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1 (P238S/P331S)-STD1-2e12IIL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(519)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(520)..(640)

<223>VH2

<220>

<221>misc_feature

<222>(641)..(660)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(661)..(772)

<223>VL2

<400>172

<210>173

<211>2322

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-STD1-2e12LH(DNA)

<400>173

<210>174

<211>767

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1-STD1-2e12LH (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(519)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(520)..(631)

<223>VL2

<220>

<221>misc_feature

<222>(632)..(646)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(647)..(767)

<223>VH2

<400>174

<210>175

<211>767

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1 (P238S/P331S)-STD1-2e12LH (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(519)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(520)..(631)

<223>VL2

<220>

<221>misc_feature

<222>(632)..(646)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(647)..(767)

<223>VH2

<400>175

<210>176

<211>2376

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-STD2-2e12LH(DNA)

<400>176

<210>177

<211>785

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1-STD2-2e12LH (w/2e12 leader sequence)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(129)

<223>VL

<220>

<221>misc_feature

<222>(130)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(537)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(538)..(649)

<223>VL2

<220>

<221>misc_feature

<222>(650)..(664)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(665)..(785)

<223>VH2

<400>177

<210>178

<211>785

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1 (P238S/P331S)-STD2-2e12LH (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(281)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(537)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(538)..(649)

<223>VL2

<220>

<221>misc_feature

<222>(650)..(664)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(665)..(785)

<223>VH2

<400>178

<210>179

<211>2391

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-STD2-2e12HL(DNA)

<400>179

<210>180

<211>790

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1-STD2-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(537)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(538)..(658)

<223>VH2

<220>

<221>misc_feature

<222>(659)..(678)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(679)..(790)

<223>VL2

<400>180

<210>181

<211>790

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1 (P238S/P331S)-STD2-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(537)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(538)..(658)

<223>VH2

<220>

<221>misc_feature

<222>(659)..(678)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(679)..(790)

<223>VL2

<400>181

<210>182

<211>2283

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-H1-2e12HL(DNA)

<400>182

<210>183

<211>754

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1-H1-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(281)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(501)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(502)..(622)

<223>VH2

<220>

<221>misc_feature

<222>(623)..(642)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(643)..(754)

<223>VL2

<400>183

<210>184

<211>2301

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-H2-2e12HL(DNA)

<400>184

<210>185

<211>760

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1-H2-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(127)

<223>VL

<220>

<221>misc_feature

<222>(128)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(507)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(508)..(628)

<223>VH2

<220>

<221>misc_feature

<222>(629)..(648)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(649)..(760)

<223>VL2

<400>185

<210>186

<211>2307

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-H3-2e12HL(DNA)

<400>186

<210>187

<211>762

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1-H3-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(509)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(510)..(630)

<223>VH2

<220>

<221>misc_feature

<222>(631)..(650)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(651)..(762)

<223>VL2

<400>187

<210>188

<211>2316

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-H4-2e12HL(DNA)

<400>188

<210>189

<211>765

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>2H7sssIgG1-H4-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(512)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(513)..(633)

<223>VH2

<220>

<221>misc_feature

<222>(634)..(653)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(654)..(765)

<223>VL2

<400>189

<210>190

<211>2322

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-H5-2e12HL(DNA)

<400>190

<210>191

<211>767

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1-H5-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(514)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(515)..(635)

<223>VH2

<220>

<221>misc_feature

<222>(636)..(655)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(656)..(767)

<223>VL2

<400>191

<210>192

<211>2331

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-H6-2e12HL(DNA)

<400>192

<210>193

<211>770

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1-H6-2e12HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(517)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(518)..(638)

<223>VH2

<220>

<221>misc_feature

<222>(639)..(658)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(659)..(770)

<223>VL2

<400>193

<210>194

<211>2301

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7ssc_IgG1-H7-2e12HL(DNA)

<400>194

<210>195

<211>760

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7ssc_IgG1-H7-2e12HL (w/2e12 connexon) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(507)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(508)..(628)

<223>VH2

<220>

<221>misc_feature

<222>(629)..(648)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(649)..(760)

<223>VL2

<400>195

<210>196

<211>2283

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-H7-G194HL(DNA)

<400>196

<210>197

<211>754

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1-H7-G194HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH1

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(507)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(508)..(629)

<223>VH2

<220>

<221>misc_feature

<222>(630)..(646)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(647)..(754)

<223>VL2

<400>197

<210>198

<211>2298

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-H7-G281HL(DNA)

<400>198

<210>199

<211>759

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7sssIgG1-H7-G281HL (w/2e12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL1

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH1

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(507)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(508)..(629)

<223>VH2

<220>

<221>misc_feature

<222>(630)..(651)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(652)..(759)

<223>VL2

<400>199

<210>200

<211>1533

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2e12-sss-IgG1HL SMIP(DNA)

<400>200

<210>201

<211>510

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>2e12-sss-IgG1HL SMIP(AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(24)..(144)

<223>VH

<220>

<221>misc_feature

<222>(145)..(164)

＜223〉connexon

<220>

<221>misc_feature

<222>(165)..(276)

<223>VL

<220>

<221>misc_feature

<222>(279)..(293)

＜223〉hinge

<400>201

<210>202

<211>1518

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2e12-sss-IgG1LH SMIP(DNA)

<400>202

<210>203

<211>505

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>2e12-sss-IgG1LH SMIP(AA)

<220>

<221>misc_feature

<222>(1)..(23)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(24)..(135)

<223>VL

<220>

<221>misc_feature

<222>(136)..(150)

＜223〉connexon

<220>

<221>misc_feature

<222>(151)..(271)

<223>VH

<220>

<221>misc_feature

<222>(274)..(288)

＜223〉hinge

<400>203

<210>204

<211>1498

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>G28-1LH SMIP(DNA)

<400>204

<210>205

<211>492

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>G28-1LH SMIP(AA)

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(21)..(127)

<223>VL

<220>

<221>misc_feature

<222>(128)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(260)

<223>VH

<220>

<221>misc_feature

<222>(261)..(275)

＜223〉hinge

<400>205

<210>206

<211>1522

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>G28-1HL SMIP(DNA)

<400>206

<210>207

<211>500

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>G28-1HL SMIP(AA)

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(21)..(136)

<223>VH

<220>

<221>misc_feature

<222>(137)..(158)

＜223〉connexon

<220>

<221>misc_feature

<222>(159)..(266)

<223>VL

<220>

<221>misc_feature

<222>(268)..(283)

＜223〉hinge

<400>207

<210>208

<211>1522

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>G19-4LH SMIP(DNA)

<400>208

<210>209

<211>500

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>G19-4LH SMIP(AA)

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(21)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(145)

＜223〉connexon

<220>

<221>misc_feature

<222>(146)..(267)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<400>209

<210>210

<211>1525

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>G19-4HL SMIP(DNA)

<400>210

<210>211

<211>501

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>G19-4HLSMIP(AA)

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(21)..(142)

<223>VH

<220>

<221>misc_feature

<222>(143)..(159)

＜223〉connexon

<220>

<221>misc_feature

<222>(160)..(267)

<223>VL

<220>

<221>misc_feature

<222>(270)..(284)

＜223〉hinge

<400>211

<210>212

<211>2328

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>n2H7sssIgG1-STD1-2e12HL(DNA)

<400>212

<210>213

<211>769

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>n2H7sssIgG1-STD1-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(21)..(127)

<223>VL

<220>

<221>misc_feature

<222>(128)..(142)

＜223〉connexon

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

＜223〉hinge

<220>

<221>misc_feature

<222>(497)..(516)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(517)..(637)

<223>VH2

<220>

<221>misc_feature

<222>(638)..(657)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(658)..(769)

<223>VL2

<400>213

<210>214

<211>2313

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>n2H7sssIgG1-STD2-2e12LH(DNA)

<400>214

<210>215

<211>764

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>n2H7sssIgG1-STD2-2e12LH(AA)

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

＜223〉connexon

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

＜223〉hinge

<220>

<221>misc_feature

<222>(497)..(516)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(517)..(628)

<223>VL2

<220>

<221>misc_feature

<222>(629)..(643)

＜223〉connexon

<220>

<221>misc_feature

<222>(644)..(764)

<223>VH2

<400>215

<210>216

<211>2048

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>n2H7sssIgG1-H1-2e12HL(DNA)

<400>216

<210>217

<211>751

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>n2H7sssIgG1-H1-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

＜223〉connexon

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

＜223〉hinge

<220>

<221>misc_feature

<222>(497)..(498)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(499)..(619)

<223>VH2

<220>

<221>misc_feature

<222>(620)..(639)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(640)..(751)

<223>VL2

<400>217

<210>218

<211>2292

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>n2H7sssIgG1-H2-2e12HL(DNA)

<400>218

<210>219

<211>757

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>n2H7sssIgG1-H2-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

＜223〉connexon

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

＜223〉hinge

<220>

<221>misc_feature

<222>(497)..(504)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(505)..(625)

<223>VH2

<220>

<221>misc_feature

<222>(626)..(645)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(646)..(757)

<223>VL2

<400>219

<210>220

<211>2298

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>n2H7sssIgG1-H3-2e12HL(DNA)

<400>220

<210>221

<211>759

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>n2H7sssIgG1-H3-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

＜223〉connexon

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

＜223〉hinge

<220>

<221>misc_feature

<222>(497)..(506)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(507)..(627)

<223>VH2

<220>

<221>misc_feature

<222>(628)..(647)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(648)..(759)

<223>VL2

<400>221

<210>222

<211>2307

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>n2H7sssIgG1-H4-2e12HL(DNA)

<400>222

<210>223

<211>762

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>n2H7sssIgG1-H4-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

＜223〉connexon

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

＜223〉hinge

<220>

<221>misc_feature

<222>(497)..(509)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(510)..(630)

<223>VH2

<220>

<221>misc_feature

<222>(631)..(650)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(651)..(762)

<223>VL2

<400>223

<210>224

<211>2313

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>n2H7sssIgG1-H5-2e12HL(DNA)

<400>224

<210>225

<211>764

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>n2H7sssIgG1-H5-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

＜223〉connexon

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

＜223〉hinge

<220>

<221>misc_feature

<222>(497)..(511)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(512)..(632)

<223>VH2

<220>

<221>misc_feature

<222>(633)..(652)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(653)..(764)

<223>VL2

<400>225

<210>226

<211>2322

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>n2H7sssIgG1-H6-2e12HL(DNA)

<400>226

<210>227

<211>767

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>n2H7sssIgG1-H6-2e12HL(AA)

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(21)..(126)

<223>VL

<220>

<221>misc_feature

<222>(127)..(142)

＜223〉connexon

<220>

<221>misc_feature

<222>(143)..(264)

<223>VH

<220>

<221>misc_feature

<222>(265)..(279)

＜223〉hinge

<220>

<221>misc_feature

<222>(497)..(514)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(515)..(635)

<223>VH2

<220>

<221>misc_feature

<222>(636)..(655)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(656)..(767)

<223>VL2

<400>227

<210>228

<211>2337

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>2H7sssIgG1-H7-G281HL(DNA)

<400>228

<210>229

<211>772

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉2H7cscIgG1-STD1-2e12HL (w/2E12 leader sequence) (AA)

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉leader sequence

<220>

<221>misc_feature

<222>(23)..(128)

<223>VL

<220>

<221>misc_feature

<222>(129)..(144)

＜223〉connexon

<220>

<221>misc_feature

<222>(145)..(265)

<223>VH

<220>

<221>misc_feature

<222>(268)..(282)

＜223〉hinge

<220>

<221>misc_feature

<222>(500)..(519)

＜223〉EFD-BD2 connexon

<220>

<221>misc_feature

<222>(520)..(640)

<223>VH2

<220>

<221>misc_feature

<222>(641)..(660)

＜223〉connexon 2

<220>

<221>misc_feature

<222>(661)..(772)

<223>VL2

<400>229

<210>230

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>scs(s)-hIgG1(DNA)

<400>230

<210>231

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>scs(s)-hIgG1(AA)

<400>231

<210>232

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

<223>ccc(s)(DNA)

<400>232

<210>233

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

<223>ccc(s)(AA)

<400>233

<210>234

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H8 (PN)

<400>234

<210>235

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>235

<210>236

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H9 (PN)

<400>236

<210>237

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H9 (AA)

<400>237

<210>238

<400>238

000

<210>239

<400>239

000

<210>240

<211>51

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H11 (PN)

<400>240

<210>241

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H11 (AA)

<400>241

<210>242

<211>51

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H12 (PN)

<400>242

<210>243

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H12 (AA)

<400>243

<210>244

<211>51

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H13 (PN)

<400>244

<210>245

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H13 (AA)

<400>245

<210>246

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H15 (PN)

<400>246

<210>247

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H15 (AA)

<400>247

<210>248

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H16 (PN)

<400>248

<210>249

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H16 (AA)

<400>249

<210>250

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H17 (PN)

<400>250

<210>251

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H17 (AA)

<400>251

<210>252

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H18 (PN)

<400>252

<210>253

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H18 (AA)

<400>253

<210>254

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H19 (PN)

<400>254

<210>255

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H19 (AA)

<400>255

<210>256

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H20 (PN)

<400>256

<210>257

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H20 (AA)

<400>257

<210>258

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉connexon H21 (PN)

<400>258

<210>259

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H21 (AA)

<400>259

<210>260

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic polypeptide

<220>

＜223〉connexon H22 (PN)

<400>260

<210>261

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H22 (AA)

<400>261

<210>262

<211>63

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H23 (PN)

<400>262

<210>263

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H23 (AA)

<400>263

<210>264

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H24 (DNA)

<400>264

<210>265

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H24 (AA)

<400>265

<210>266

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H25 (PN)

<400>266

<210>267

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H25 (AA)

<400>267

<210>268

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H26 (PN)

<400>268

<210>269

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H26 (AA)

<400>269

<210>270

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H27 (PN)

<400>270

<210>271

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H27 (AA)

<400>271

<210>272

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H28 (PN)

<400>272

<210>273

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H28 (AA)

<400>273

<210>274

<211>63

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H29 (PN)

<400>274

<210>275

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H29 (AA)

<400>275

<210>276

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H30 (PN)

<400>276

<210>277

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H30 (AA)

<400>277

<210>278

<211>63

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H31 (PN)

<400>278

<210>279

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H31 (AA)

<400>279

<210>280

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H32 (PN)

<400>280

<210>281

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H32 (AA)

<400>281

<210>282

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H33 (PN)

<400>282

<210>283

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H33 (AA)

<400>283

<210>284

<400>284

<210>285

<400>285

<210>286

<400>286

<210>287

<400>287

<210>288

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H36 (PN)

<400>288

<210>289

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H36 (AA)

<400>289

<210>290

<400>290

<210>291

<400>291

<210>292

<400>292

<210>293

<400>293

<210>294

<400>294

<210>295

<400>295

<210>296

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H40 (PN)

<400>296

<210>297

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H40 (AA)

<400>297

<210>298

<400>298

<210>299

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<221>misc_feature

＜223〉connexon H41

<400>299

<210>300

<400>300

<210>301

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H42 (AA)

<400>301

<210>302

<400>302

<210>303

<400>303

<210>304

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H44 (PN)

<400>304

<210>305

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H44 (AA)

<400>305

<210>306

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H45 (PN)

<400>306

<210>307

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H45 (AA)

<400>307

<210>308

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉synthesized polynucleotide

<220>

＜223〉connexon H46

<400>308

<210>309

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H46

<400>309

<210>310

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H47

<400>310

<210>311

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H48

<400>311

<210>312

<400>312

<210>313

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H50

<400>313

<210>314

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H51

<400>314

<210>315

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H52

<400>315

<210>316

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H53

<400>316

<210>317

<211>19

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H54

<400>317

<210>318

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H55

<400>318

<210>319

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H56

<400>319

<210>320

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H57

<400>320

<210>321

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H58

<400>321

<210>322

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H59

<400>322

<210>323

<211>14

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H60

<400>323

<210>324

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H61

<400>324

<210>325

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H62

<400>325

<210>326

<211>19

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H63

<400>326

<210>327

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<400>327

<210>328

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H65

<400>328

<210>329

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H66

<400>329

<210>330

<211>21

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H67

<400>330

<210>331

<211>118

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉connexon H68

<400>331

<210>332

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti--CD-20 VL CDR1

<400>332

<210>333

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti--CD-20 VL CDR1

<400>333

<210>334

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti--CD-20 VL CDR3

<400>334

<210>335

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti--CD-20 VL CDR3

<400>335

<210>336

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti--CD-20 VH CDR2

<400>336

<210>337

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti--CD-20 VH CDR2

<400>337

<210>338

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti-CD 20 VH CDR3

<400>338

<210>339

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti-CD 20 VH CDR3

<400>339

<210>340

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti-CD 20 VH CDR3

<400>340

<210>341

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti-CD 20 VH CDR3

<400>341

<210>342

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti-CD 20 VH CDR3

<400>342

<210>343

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti-CD 20 VH CDR3

<400>343

<210>344

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti--CD-20 VH CDR3

<400>344

<210>345

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti-CD 20 VH CDR3

<400>345

<210>346

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉scorpion shape connexon core (2H7) sequence

<400>346

<210>347

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉sequence spreading

<400>347

<210>348

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉sequence spreading

<400>348

<210>349

<211>14

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉sequence spreading

<400>349

<210>350

<211>19

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉sequence spreading

<400>350

<210>351

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉the scorpion shape connexon through expanding

<400>351

<210>352

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the scorpion shape connexon through expanding

<400>352

<210>353

<211>22

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉the scorpion shape connexon through expanding

<400>353

<210>354

<211>27

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉the scorpion shape connexon through expanding

<400>354

<210>355

<211>51

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>VL CDR1-X-VL CDR3-X-VH CDR2-X-VH CDR3

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉the amino acid scope of Xaa=between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉the amino acid scope of Xaa=between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

＜223〉the amino acid scope of Xaa=between VH CDR2 and VH CDR3

<400>355

<210>356

<211>51

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉the amino acid scope of Xaa=between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉the amino acid scope of Xaa=between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

＜223〉the amino acid scope of Xaa=between VH CDR2 and VH CDR3

<400>356

<210>357

<211>51

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉the amino acid scope of Xaa=between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉the amino acid scope of Xaa=between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

＜223〉the amino acid scope of Xaa=between VH CDR2 and VH CDR3

<400>357

<210>358

<211>51

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉the amino acid scope of Xaa=between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉the amino acid scope of Xaa=between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

＜223〉the amino acid scope of Xaa=between VH CDR2 and VH CDR3

<400>358

<210>359

<211>51

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉the amino acid scope of Xaa=between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉the amino acid scope of Xaa=between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

＜223〉the amino acid scope of Xaa=between VH CDR2 and VH CDR3

<400>359

<210>360

<211>51

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉the amino acid scope of Xaa=between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉the amino acid scope of Xaa=between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

＜223〉the amino acid scope of Xaa=between VH CDR2 and VH CDR3

<400>360

<210>361

<211>51

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉the amino acid scope of Xaa=between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉the amino acid scope of Xaa=between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

＜223〉the amino acid scope of Xaa=between VH CDR2 and VH CDR3

<400>361

<210>362

<211>51

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉the amino acid scope of Xaa=between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉the amino acid scope of Xaa=between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

＜223〉the amino acid scope of Xaa=between VH CDR2 and VH CDR3

<400>362

<210>363

<211>51

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉the amino acid scope of Xaa=between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉the amino acid scope of Xaa=between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

＜223〉the amino acid scope of Xaa=between VH CDR2 and VH CDR3

<400>363

<210>364

<211>51

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉the amino acid scope of Xaa=between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉the amino acid scope of Xaa=between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

＜223〉the amino acid scope of Xaa=between VH CDR2 and VH CDR3

<400>364

<210>365

<211>51

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

<223>VLCDR1-X-VLCDR3-X-VHCDR2-X-VHCDR3

<220>

<221>misc_feature

<222>(11)..(11)

＜223〉the amino acid scope of Xaa=between VL CDR1 and VL CDR3

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉the amino acid scope of Xaa=between VL CDR3 and VH CDR2

<220>

<221>misc_feature

<222>(39)..(39)

＜223〉the amino acid scope of Xaa=between VH CDR2 and VH CDR3

<400>365

<210>366

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉portion C H3 sequence

<400>366

<210>367

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉portion C H3 sequence

<400>367

<210>368

<211>1

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉portion C H3 sequence

<400>368

<210>369

<211>3

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉portion C H3 sequence

<400>369

<210>370

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉portion C H3 sequence

<400>370

<210>371

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉portion C H3 sequence

<400>371

<210>372

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉scorpion shape connexon

<400>372

<210>373

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉scorpion shape connexon

<400>373

<210>374

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉scorpion shape connexon

<400>374

<210>375

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉scorpion shape connexon

<400>375

<210>376

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉scorpion shape connexon

<400>376

<210>377

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉scorpion shape connexon

<400>377

<210>378

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti-CD 20 VL CDR1 (TRU-015)

<400>378

<210>379

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide

<220>

＜223〉anti-CD 20 VH CDR3 (TRU-015)

<400>379

Claims (98)

1. have the multivalence single strand binding protein of effector function, it comprises:

A. first binding domains, it is derived from immunoglobulin (Ig) or immunoglobulin-like molecule;

B., the constant subprovince of effector function is provided, and the constant subprovince of this immunoglobulin (Ig) is positioned at the C-end of described first binding domains;

C. scorpion shape connexon, it is positioned at C-end of described constant subprovince; And

D. second binding domains, it is derived from immunoglobulin (Ig) or immunoglobulin-like molecule, and is positioned at the C-end of described constant subprovince;

Therefore, described constant subprovince is positioned between described first binding domains and described second binding domains.

2. albumen as claimed in claim 1, wherein said first binding domains comprise the variable region of light chain that is derived from first immunoglobulin (Ig) and are derived from the variable region of heavy chain of second immunoglobulin (Ig).

3. albumen as claimed in claim 2, wherein said first immunoglobulin (Ig) is identical immunoglobulin (Ig) with described second immunoglobulin (Ig).

4. albumen as claimed in claim 1, wherein said second binding domains comprise the variable region of light chain that is derived from first immunoglobulin (Ig) and are derived from the variable region of heavy chain of second immunoglobulin (Ig).

5. albumen as claimed in claim 4, wherein said first immunoglobulin (Ig) is identical immunoglobulin (Ig) with described second immunoglobulin (Ig).

6. albumen as claimed in claim 1, wherein said first binding domains and the identical molecular target of described second binding domains identification.

7. albumen as claimed in claim 1, wherein said first binding domains and the identical epi-position of described second binding domains identification.

8. albumen as claimed in claim 1, the differing molecular target on wherein said first binding domains eukaryotic cell, prokaryotic cell prokaryocyte, virus, carrier or the object identical with the identification of described second binding domains.

9. albumen as claimed in claim 1, the relevant molecular target of eukaryotic cell, prokaryotic cell prokaryocyte, virus, carrier or object that wherein said first binding domains is different with entity with described second binding domains identification.

10. albumen as claimed in claim 1, at least one in wherein said first binding domains and described second binding domains discerned at least a molecular target, and described molecular target and eukaryotic cell, prokaryotic cell prokaryocyte, virus, carrier or object are uncorrelated.

11. albumen as claimed in claim 1, each in wherein said first binding domains, described second binding domains and the described constant subprovince is derived from human immunoglobulin.

12. albumen as claimed in claim 1, the target at least one identification cancer cells in wherein said first binding domains and described second binding domains.

13. albumen as claimed in claim 11, at least one identification in wherein said first binding domains and described second binding domains is selected from the target by the following group of forming: tumour antigen, B-cell target, TNF receptor superfamily member, Hedgehog family member, receptor tyrosine kinase, proteoglycan associated molecule, TGF-beta superfamily member, Wnt associated molecule, receptors ligand, T-cell target, dendritic cell target, NK cell target, monocyte/macrophage target and vasculogenesis target.

14. albumen as claimed in claim 13, wherein said tumour antigen are to be selected from the group of being made up of following: squamous cell carcinoma antigen 1, squamous cell carcinoma antigen 2, ovarian cancer antigen CA125, MUC-1, CTCL tumour antigen se1-1, CTCL tumour antigen se14-3, CTCL tumour antigen se20-4, CTCL tumour antigen se20-9, CTCL tumour antigen se33-1, CTCL tumour antigen se37-2, CTCL tumour antigen se57-1, CTCL tumour antigen se89-1, prostate specific membrane antigen, 5T4 cancer embryo trophoderm glycoprotein, the Orf73 Kaposi sarcoma simplexvirus of being correlated with, MAGE-C1, MAGE-B1 antigen, MAGE-B2 antigen, MAGE-2 antigen, MAGE-4a antigen, MAGE-4b antigen, colon cancer antigen NY-CO-45, LuCA NY-LU-12 varient A, cancer relevant surfaces antigen, gland cancer antigen A RT1, the relevant brain of secondary tumour-carcinoma of testis antigen, neural tumor veutro antigen 2, hepatocellular carcinoma antigen gene 520, tumor associated antigen CO-029, tumor associated antigen MAGE-X2, synovial sarcoma X breaking point 2, the squamous cell carcinoma antigen that the T cell is discerned, the colon cancer antigen 1 of serology definition, the breast cancer antigen NY-BR-15 of serology definition, the breast cancer antigen NY-BR-16 of serology definition, Chromogranin A; Parathyroid secretory protein 1, DUPAN-2, CA19-9, CA72-4, CA195 and L6.

15. albumen as claimed in claim 13, wherein said B cell target is to be selected from the group of being made up of following: CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138 and CDw150.

16. albumen as claimed in claim 13, wherein said TNF receptor superfamily member is selected from the group of being made up of following: 4-1BB/TNFRSF9, NGF R/TNFRSF16, BAFFR/TNFRSF13C, protect ossein/TNFRSF11B, BCMA/TNFR SF17, OX40/TNFRSF4, CD27/TNFRSF7, RANK/TNFRSF 11A, CD30/TNFRSF8, RELT/TNFRSF19L, CD40/TNFRSF 5, TACI/TNFRSF13B, DcR3/TNFRSF6B, TNF RI/TNFRSF 1A, DcTRAILR1/TNFRSF23, TNF RII/TNFRSF1B, Dc TRAIL R2/TNFRSF22, TRAILR1/TNFRSF10A, DR3/TNFRSF25, TRAIL R2/TNFRSF10B, DR6/TNFRSF21, TRAIL R3/TNFRSF10C, EDAR, TRAILR4/TNFRSF10D, Fas/TNFRSF6, TROY/TNFRSF19, GITR/TNFRSF18, TWEAK R/TNFRSF12, HVEM/TNFRSF14, XEDAR, lymphotoxin-beta R/TNFRSF3,4-1BB part/TNFSF9, lymphotoxin, APRIL/TNFSF13, lymphotoxin-beta/TNFSF3, BAFF/TNFSF13C, OX40 part/TNFSF4, CD27 part/TNFSF7, TL1A/TNFSF15, CD30 part/TNFSF8, TNF-α/TNFSF1A, CD40 part/TNFSF5, TNF-β/TNFSF1B, EDA-A2, TRAIL/TNFSF10, Fas part/TNFSF6, TRANCE/TNFSF11, GITR part/TNFSF18, TWEAK/TNFSF12 and LIGHT/TNFSF14.

17. albumen as claimed in claim 13, wherein said Hedgehog family member is selected from the group of being made up of Patched and Smoothened.

18. albumen as claimed in claim 13, wherein said receptor tyrosine kinase are to be selected from the group of being made up of following: Ax1, FGF R4, Clq R1/CD93, FGF R5, DDR1, Flt-3, DDR2, HGF R, Dtk, IGF-I R, EGF R, IGF-II R, Eph, INSRR, EphA1, Regular Insulin R/CD220, EphA2, M-CSF R, EphA3, Mer, EphA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R α, EphA7, PDGF R β, EphA8, Ret, EphB1, ROR1, EphB2, ROR2, EphB3, SCF R/c-kit, EphB4, Tie-1, EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF R1, VEGF R1/Flt-1, FGF R2, VEGF R2/Flk-1, FGF R3 and VEGF R3/Flt-4.

19. albumen as claimed in claim 13, wherein said proteoglycan associated molecule is to be selected from the group of being made up of proteoglycan and conditioning agent thereof.

20. albumen as claimed in claim 13, wherein said transforming growth factor (TGF)-beta superfamily member is to be selected from the group of being made up of following: activin RIA/ALK-2, GFR α-1, activin RIB/ALK-4, GFR α-2, activin RIIA, GFR α-3, activin RIIB, GFR α-4, ALK-1, MIS RII, ALK-7, Ret, BMPR-IA/ALK-3, TGF-β RI/ALK-5, BMPR-IB/ALK-6, TGF-β RII, BMPR-II, TGF-β RIIb, endothelial factor/CD105 and TGF-β RIII.

21. albumen as claimed in claim 13, wherein said Wnt associated molecule is to be selected from the group of being made up of following: Frizzled-1, Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1, Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6, sFRP-4, Frizzled-7, MFRP, LRP5, LRP6, Wnt-1, Wnt-8a, Wnt-3a, Wnt-10b, Wnt-4, Wnt-11, Wnt-5a, Wnt-9a and Wnt-7a.

22. albumen as claimed in claim 13, wherein said receptors ligand are to be selected from the group of being made up of following: 4-1BB part/TNFSF9, lymphotoxin, APRIL/TNFSF 13, lymphotoxin-beta/TNFSF3, BAFF/TNFSF13C, OX40 part/TNFSF4, CD27 part/TNFSF7, TL1A/TNFSF15, CD30 part/TNFSF8, TNF-α/TNFSF1A, CD40 part/TNFSF 5, TNF-β/TNFSF1B, EDA-A2, TRAIL/TNFSF10, Fas part/TNFSF6, TRANCE/TNFSF11, GITR part/TNFSF 18, TWEAK/TNFSF12, LIGHT/TNFSF14, amphiregulin, NRG1 isotype GGF2, the β cell growth factor, NRG1 isotype SMDF, EGF, NRG1-α/HRG1-α, Epigen, NRG1-β 1/HRG1-β 1, epiregulin, TGF-α, HB-EGF, TMEFF1/ soil is regulin-1 not, neuregulin-3, TMEFF2, IGF-I, IGF-II, Regular Insulin, activin A, activin B, activin AB, activin C, BMP-2, BMP-7, BMP-3, BMP-8, BMP-3b/GDF-10, BMP-9, BMP-4, BMP-15, BMP-5, Dpp albumen (Decapentaplegic), BMP-6, GDF-1, GDF-8, GDF-3, GDF-9, GDF-5, GDF-11, GDF-6, GDF-15, GDF-7, Aunar bright (Artemin), the special unit of knob (Neurturin), GDNF, Pu Saifen (Persephin), TGF-β, TGF-β 2, TGF-β 1, TGF-β 3, LAP (TGF-β 1), TGF-β 5, potentiality TGF-β 1, potentiality TGF-β bp1, TGF-β 1.2, Lefty, Nodal, MIS/AMH, acid FGF, FGF-12, basic FGF, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, neural pilin (Neuropilin)-1, PlGF, neural pilin-2, PlGF-2, PDGF, PDGF-A, VEGF, PDGF-B, VEGF-B, PDGF-C, VEGF-C, PDGF-D, VEGF-D and PDGF-AB.

23. albumen as claimed in claim 13, wherein said T-cell target is to be selected from the group of being made up of following: 2B4/SLAMF4, IL-2 R α, 4-1BB/TNFRSF9, IL-2 R β, ALCAM, B7-1/CD80, IL-4R, B7-H3, BLAME/SLAMF8, BTLA, IL-6 R, CCR3, IL-7 R α, CCR4, CXCR1/IL-8 RA, CCR5, CCR6, IL-10 R α, CCR7, IL-10 R β, CCR8, IL-12 R β 1, CCR9, IL-12 R β 2, CD2, IL-13 R α 1, IL-13, CD3, CD4, ILT2/CD85j, ILT3/CD85k, ILT4/CD85d, ILT5/CD85a, integrin alpha 4/CD49d, CD5, integrin alpha E/CD103, CD6, integrin alpha M/CD11b, CD8, integrin alpha X/CD11c, integrate plain β 2/CD18, KIR/CD158, CD27/TNFRSF7, KIR2DL1, CD28, KIR2DL3, CD30/TNFRSF8, KIR2DL4/CD158d, CD31/PECAM-1, KIR2DS4, CD40 part/TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CD83, leukotriene B4 R1, CD84/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, common γ chain/IL-2R γ, osteopontin, CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1, L-selects plain, CXCR3, SIRP β 1, CXCR4, SLAM, CXCR6, TCCR/WSX-1, DNAM-1, thymopoietin, EMMPRIN/CD147, TIM-1, EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas part/TNFSF6, TIM-4, Fc γ RIII/CD16, TIM-6, GITR/TNFRSF18, TNF RI/TNFRSF1A, particle cytolysin, TNF RII/TNFRSF 1B, HVEM/TNFRSF14, TRAIL R1/TNFRSF10A, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAIL R3/TNFRSF10C, IFN-γ R1, TRAIL R4/TNFRSF10D, IFN-γ R2, TSLP, IL-1RI and TSLP R.

24. albumen as claimed in claim 13, wherein said NK cell target is to be selected from the group of being made up of following: 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1, CD94, LMIR1/CD300A, CD69, LMIR2/CD 300c, CRACC/SLAMF7, LMIR3/CD300LF, DNAM-1, LMIR5/CD300LB, Fc ε RII, LMIR6/CD300LE, Fc γ RI/CD 64, MICA, Fc γ RIIB/CD32b, MICB, Fc γ RIIC/CD32c, MULT-1, Fc γ RIIA/CD32a, handle albumen-2/CD112, Fc γ RIII/CD16, NKG2A, FcRH1/IRTA5, NKG2C, FcRH2/IRTA4, NKG2D, FcRH4/IRTA1, NKp30, FcRH5/IRTA2, NKp44, class Fc acceptor 3/CD16-2, NKp46/NCR1, NKp80/KLRF1, NTB-A/SLAMF6, Rae-1, Rae-1 α, Rae-1 β, Rae-1 δ, H60, Rae-1 ε, ILT2/CD85j, Rae-1 γ, ILT3/CD85k, TREM-1, ILT4/CD85d, TREM-2, ILT5/CD85a, TREM-3, KIR/CD158, TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4/CD158d and ULBP-3.

25. albumen as claimed in claim 13, wherein said monocyte/macrophage target is to be selected from the group of being made up of following: B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, the common beta chain, integrin alpha 4/CD49d, BLAME/SLAMF8, integrin alpha X/CD11c, CCL6/C10, integrate plain β 2/CD18, CD155/PVR, integrate plain β 3/CD61, CD31/PECAM-1, latex element (Latexin), CD36/SR-B3, leukotriene B4 R1, CD40/TNFRSF5, LIMPII/SR-B2, CD43, LMIR1/CD300A, CD45, LMIR2/CD300c, CD68, LMIR3/CD300LF, CD84/SLAMF 5, LMIR5/CD300LB, CD97, LMIR6/CD300LE, CD163, LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN/CD147, MGL2, endothelial factor/CD105, bone element/GPNMB alive, Fc γ RI/CD64, osteopontin, Fc γ RIIB/CD32b, PD-L2, Fc γ RIIC/CD32c, Siglec-3/CD33, Fc γ RIIA/CD32a, SIGNR1/CD209, Fc γ RIII/CD16, SLAM, GM-CSF R α, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-γ R1, TLR4, IFN-γ R2, TREM-1, IL-1RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k, TREML1/TLT-1,2B4/SLAMF4, IL-10 R α, ALCAM, IL-10 R β, aminopeptidase N/ANPEP, ILT2/CD85j, the common beta chain, ILT3/CD85k, C1q R1/CD93, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, integrin alpha 4/CD49d, CCR5, integrin alpha M/CD11b, CCR8, integrin alpha X/CD11c, CD155/PVR, integrate plain β 2/CD 18, CD14, integrate plain β 3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4 R1, CD68, LIMPII/SR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97, LMIR2/CD300c, CD163, LMIR3/CD300LF, thromboplastin/tissue factor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR, endothelial factor/CD105, NCAM-L1, Fc γ RI/CD64, PSGL-1, Fc γ RIII/CD16, RP105, G-CSF R, L-selects plain, GM-CSF R α, Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-1, IL-6R, TREM-2, CXCR1/IL-8RA, TREM-3 and TREML1/TLT-1.

26. albumen as claimed in claim 13, wherein said dendritic cell target is to be selected from the group of being made up of following: CD36/SR-B3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-AI/MSR, CD5L, SREC-I, CL-P1/COLEC12, SREC-II, LIMPII/SR-B2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9,4-1BB part/TNFSF9, IL-12/IL-23p40,4-amino-1, the 8-naphthalimide, ILT2/CD85j, CCL21/6Ckine, ILT3/CD85k, 8-oxygen base (oxo)-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5, integrin alpha 4/CD49d, Aag, integrate plain β 2/CD18, AMICA, the langhans' cells differential protein, B7-2/CD86, leukotriene B4 R1, B7-H3, LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, C1q R1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB, CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD84/SLAMF5, MMR, CD97, NCAM-L1, CD2F-10/SLAMF9, bone element/GPNMB alive, Chem23, PD-L2, CLEC-1, RP105, CLEC-2, Siglec-2/CD22, CRACC/SLAMF7, Siglec-3/CD33, DC-SIGN, Siglec-5, DC-SIGNR/CD299, Siglec-6, DCAR, Siglec-7, DCIR/CLEC4A, Siglec-9, DEC-205, Siglec-10, lectin (Dectin)-1/CLEC7A, Siglec-F, lectin-2/CLEC6A, SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, Fc γ RI/CD64, TLR3, Fc γ RIIB/CD32b, TREM-1, Fc γ RIIC/CD32c, TREM-2, Fc γ RIIA/CD32a, TREM-3, Fc γ RIII/CD16, TREML1/TLT-1, ICAM-2/CD102 and capsaicine R1 (Vanilloid R1).

27. albumen as claimed in claim 13, wherein said vasculogenesis target is to be selected from the group of being made up of following: angiogenin (Angiopoietin)-1, class angiogenin 2, angiopoietin-2, class angiogenin 3, angiogenin-3, class angiogenin 7/CDT6, angiogenin-4, Tie-1, class angiogenin 1, Tie-2, angiogenine (Angiogenin), iNOS, thromboplastin/tissue factor, nNOS, CTGF/CCN2, NOV/CCN3, DANCE, OSM, EDG-1, Plfr, EG-VEGF/PK1, proliferating agent (Proliferin), endostatin (Endostatin), ROBO4, erythropoietin (Erythropoietin), thrombostondin-1, kassinin kinin statin (Kininostatin), thrombostondin-2, MFG-E8, thrombostondin-4, nitrogen protoxide (Nitric Oxide), VG5Q, eNOS, EphA1, EphA5, EphA2, EphA6, EphA3, EphA7, EphA4, EphA8, EphB1, EphB4, EphB2, EphB6, EphB3, Ephrin-A1, Ephrin-A4, Ephrin-A2, Ephrin-A5, Ephrin-A3, Ephrin-B1, Ephrin-B3, Ephrin-B2, acid FGF, FGF-12, basic FGF, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, FGF R1, FGF R4, FGF R2, FGF R5, FGF R3, neural pilin-1, neural pilin-2, arm plate albumen (Semaphorin) 3A, arm plate albumen 6B, arm plate albumen 3C, arm plate albumen 6C, arm plate albumen 3E, arm plate albumen 6D, arm plate albumen 6A, arm plate albumen 7A, MMP, MMP-11, MMP-1, MMP-12, MMP-2, MMP-13, MMP-3, MMP-14, MMP-7, MMP-15, MMP-8, MMP-16/MT3-MMP, MMP-9, MMP-24/MT5-MMP, MMP-10, MMP-25/MT6-MMP, TIMP-1, TIMP-3, TIMP-2, TIMP-4, ACE, IL-13 R α 1, IL-13, C1q R1/CD 93, integrin alpha 4/CD49d, VE-cadherins (Cadherin), integrate plain β 2/CD18, CD31/PECAM-1, KLF4, CD36/SR-B3, LYVE-1, CD151, MCAM, CL-P1/COLEC12, Fibronectin-2/CD112, thromboplastin/tissue factor, E-selects plain, D6, palatelet-selectin, DC-SIGNR/CD299, SLAM, EMMPRIN/CD147, Tie-2, endothelial factor/CD105, TNF RI/TNFRSF1A, EPCR, TNF RII/TNFRSF1B, erythropoietin R, TRAIL R1/TNFRSF10A, ESAM, TRAILR2/TNFRSF10B, FABP5, VCAM-1, ICAM-1/CD54, VEGF R2/Flk-1, ICAM-2/CD102, VEGF R3/Flt-4, IL-1RI and VG5Q.

28. at least one combines the target that is selected from by the following group of forming specifically: prostate specific membrane antigen (folic acid lytic enzyme 1) in the albumen as claimed in claim 1, wherein said first binding domains and described second binding domains, EGF-R ELISA, the acceptor of advanced glycosylation final product, IL-17A, IL-17F, P19, Dickkopf-1, NOTCH1, NG2, IgHE, IL-22R, IL-21, kind of starch β oligomer, kind of starch β forerunner albumen, NOGO acceptor (RTN4R), LDH receptor related protein 5, IL-4, muscle mass, very late antigen 4 and IGF-1R.

29. albumen as claimed in claim 12, wherein said cancer cells are the hematopoietic cells that transforms.

30. albumen as claimed in claim 29, at least one identification is selected from the target by the following group of forming in wherein said first binding domains and described second binding domains: B-cell target, T-cell target, dendritic cell target or NK-cell target.

31. albumen as claimed in claim 29, at least one identification is selected from the target by the following group of forming in wherein said first binding domains and described second binding domains: CD5, CD10, CD11c, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD30, CD38, CD45, CD70, CD80, CD86, CD103, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5, B-B2, B-B8 and B-cell antigen receptor.

32. albumen as claimed in claim 1, it comprises the sequence that is selected from by the following group of forming: SEQ ID NO:2,4,6,103,105,107,109,332,333,334 and 345.

33. albumen as claimed in claim 1, it comprises the sequence that is selected from by the following group of forming: SEQ ID NO:355,356,357,358,359,360,361,362,363,364 and 365.

34. albumen as claimed in claim 1, wherein said constant subprovince recognition effect cell FC acceptor.

35. albumen as claimed in claim 1, wherein said constant subprovince identification is selected from the effector cell's surface protein by the following group of forming: CD16, CD32a, CD32b, CD64, CD89, Fc ε R1, FcRn and pIgR.

36. albumen as claimed in claim 1, wherein said constant subprovince comprises C _H2Structural domain and C _H3Structural domain.

37. albumen as claimed in claim 36, wherein said CH ₃Structural domain is a truncation type, and comprises the C-terminal sequence that is selected from by the following group of forming: SEQ ID NO:366,367,368,369,370 and 371.

38. albumen as claimed in claim 1, wherein said scorpion shape connexon is derived from the immunoglobulin (Ig) hinge.

39. albumen as claimed in claim 38, wherein said scorpion shape connexon has reduced number but the halfcystine of non-zero with respect to the hinge of the described scorpion shape connexon of deriving, and one of them halfcystine is corresponding to the N-end hinge cysteine of described immunoglobulin (Ig) hinge.

40. albumen as claimed in claim 39, wherein said scorpion shape connexon is derived from the IgG1 hinge area, and wherein said connexon comprises 1 or 2 halfcystines, and wherein said in addition connexon keeps the halfcystine corresponding to the N-end hinge cysteine of described IgG1 hinge area.

41. albumen as claimed in claim 1, wherein said scorpion shape connexon is derived from the stem district of C-lectin.

42. albumen as claimed in claim 1, wherein said scorpion shape connexon comprises the sequence that is selected from by SEQ ID NO:373,374,375,376 and 377 groups of forming.

43. albumen as claimed in claim 1, wherein said scorpion shape connexon comprises the sequence that is selected from by SEQ ID NO:351,352,353 and 354 groups of forming.

44. albumen as claimed in claim 1, it further comprises and has at least about 5 amino acid whose connexons, described connexon is connected with described constant subprovince, and be connected with described first binding domains, thereby described connexon is positioned between described constant subprovince and described first binding domains.

45. albumen as claimed in claim 1, at least one binding affinity is less than 10 in wherein said first binding domains and described second binding domains ^-9M.

46. albumen as claimed in claim 1, at least one binding affinity is at least 10 in wherein said first binding domains and described second binding domains ^-6M.

47. albumen as claimed in claim 1, wherein said effector function are to be selected from the group of being made up of the cytotoxicity (ADCC) and the CDC (CDC) of antibody dependent cellular mediation.

48. albumen as claimed in claim 1, the wherein said albumen transformation period in the mankind's body is at least 28 hours.

49. composition pharmaceutically, it comprises albumen as claimed in claim 1 and pharmaceutically acceptable adjuvant, carrier or vehicle.

The proteic nucleic acid as claimed in claim 1 50. encode.

51. comprise the carrier of nucleic acid as claimed in claim 50.

52. comprise the host cell of carrier as claimed in claim 51.

53. prepare proteic method as claimed in claim 1, described method comprises:

A. the proteic nucleic acid as claimed in claim 1 of will encoding is introduced in the host cell; With

B. described host cell is hatched being suitable for expressing under the described proteic condition, thereby with the described albumen of horizontal expression of 1mg/ml at least.

54. method as claimed in claim 53, it further comprises and separates described albumen.

55. method as claimed in claim 53, wherein said host cell are to be selected from the group of being made up of following: the VERO cell, HeLa cell, Chinese hamster ovary celI, the COS cell, the W138 cell, bhk cell, the HepG2 cell, the 3T3 cell, the RIN cell, mdck cell, the A549 cell, the PC12 cell, the K562 cell, the HEK293 cell, the N cell, noctuid (Spodoptera frugiperda) cell is coveted on the meadow, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) cell, Pichia pastoris (Pichia pastoris) cell, fungal cell and bacterial cell.

56. prepare the method for the proteic nucleic acid as claimed in claim 1 of encoding, described method comprises:

A. coding source is covalently bound with 5 ' end of the polynucleotide of the constant subprovince of coding from 3 ' end of the polynucleotide of first binding domains of immune globulin variable region;

B. will encode scorpion shape connexon polynucleotide 5 ' end with the coding described constant subprovince polynucleotide 3 ' end covalently bound; And

C. coding source is covalently bound with 3 ' end of the polynucleotide of the described scorpion shape connexon of coding from 5 ' end of the polynucleotide of second binding domains of immune globulin variable region;

Thereby form the nucleic acid that coding has the multivalent binding proteins of effector function.

57. method as claimed in claim 56, the polynucleotide of wherein said coding first binding domains comprises the sequence that is selected from by the following group of forming: and SEQ ID NO:1 (anti-CD 20 variable region), SEQ ID NO:3 (anti--the CD28 variable region, V _L-V _HBe orientated) and SEQ ID NO:5 (anti--the CD28 variable region, V _H-V _LOrientation).

58. method as claimed in claim 56, it further comprises the connexon polynucleotide is inserted between the polynucleotide of the polynucleotide of described coding first binding domains and the constant subprovince of described coding, and described connexon polynucleotide encode has at least 5 amino acid whose peptide connexons.

59. method as claimed in claim 56, it further comprises the connexon polynucleotide is inserted between the polynucleotide of the polynucleotide of the constant subprovince of described coding and described coding second binding domains, and described connexon polynucleotide encode has at least 5 amino acid whose peptide connexons.

60. induce the method for target cell damage, described method comprises makes the target cell contact with the albumen as claimed in claim 1 of treatment significant quantity.

61. method as claimed in claim 60, wherein said target cell are the described albumen of organism that needs by having by contact in the body.

62. the method for treatment cell proliferation illness, described method comprise the albumen as claimed in claim 1 of the organism treatment significant quantity that needs are arranged.

63. method as claimed in claim 62, wherein said illness are to be selected from the group of being made up of cancer, autoimmune disorder, Rous sarcoma virus infection and inflammation.

64. method as claimed in claim 62, wherein said albumen is expression in vivo by the proteic nucleic acid as claimed in claim 1 of encoding by administration.

65. method as claimed in claim 62, wherein said proteic administration is by being selected from the approach by the following group of forming: intravenous injection, intra-arterial injection, intramuscular injection, subcutaneous injection, peritoneal injection and direct tissue injection.

66. improve the method for the symptom relevant with the cell proliferation illness, described method comprises the albumen as claimed in claim 1 of the organism treatment significant quantity that needs are arranged.

67. as the described method of claim 66, wherein said symptom is to be selected from the group of being made up of pain, heating, swelling and ankylosis.

68. treat the method for the infection relevant with infectious agent, described method comprises the albumen as claimed in claim 1 that the patient treatment of needs significant quantity is arranged, wherein said albumen comprises specifically the binding domains in conjunction with the target molecules of described infectious agent.

69. improve the method for the infection symptoms relevant with infectious agent, described method comprises the albumen as claimed in claim 1 that the patient treatment of needs significant quantity is arranged, and wherein said albumen comprises specifically the binding domains in conjunction with the target molecules of described infectious agent.

70. reduce method owing to the infection risk of infectious agent, described method comprises that the patient that infection risk is arranged prevents the albumen as claimed in claim 1 of significant quantity, and wherein said albumen comprises specifically the binding domains in conjunction with the target molecules of described infectious agent.

71. test kit, it comprises albumen as claimed in claim 1 and one and overlaps about giving the specification sheets of described albumen to treat the cell proliferation illness or to improve the symptom of described cell proliferation illness.

At least one combines a kind of antigen that is selected from by the following group of forming specifically: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and a kind of main II of histocompatibility complex class peptide 72. in the multivalence single strand binding protein as claimed in claim 1, wherein said first binding domains and described second binding domains.

73. as the described albumen of claim 72, one in wherein said first binding domains and described second binding domains combines CD20 specifically.

74. as the described albumen of claim 73, wherein said another binding domains is specifically in conjunction with a kind of antigen that is selected from by the following group of forming: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and a kind of main II of histocompatibility complex class peptide.

75. as the described albumen of claim 72, one in wherein said first binding domains and described second binding domains combines CD79b specifically.

76. as the described albumen of claim 75, wherein said another binding domains is specifically in conjunction with a kind of antigen that is selected from by the following group of forming: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and a kind of main II of histocompatibility complex class peptide.

77. as the described albumen of claim 72, one in wherein said first binding domains and described second binding domains combines a kind of main II of histocompatibility complex class peptide specifically.

78. as the described albumen of claim 77, wherein said another binding domains is specifically in conjunction with a kind of antigen that is selected from by the following group of forming: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and a kind of main II of histocompatibility complex class peptide.

79. as the described albumen of claim 72, one in wherein said first binding domains and described second binding domains combines CD22 specifically.

80. as the described albumen of claim 79, wherein said another binding domains is specifically in conjunction with a kind of antigen that is selected from by the following group of forming: CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86 and a kind of main II of histocompatibility complex class peptide.

81. as the described albumen of claim 72, wherein said albumen has a synergistic effect with respect to each binding domains effect summation for the target cell behavior, and wherein said albumen comprises a pair of binding domains and discerns a pair of antigen that is selected from by the following group of forming specifically: CD20-CD19, CD20-CD21, CD20-CD22, CD20-CD40, CD20-CD79a, CD20-CD79b and CD20-CD81.

82. as the described albumen of claim 72, wherein said albumen has an additive effect with respect to each binding domains effect summation for the target cell behavior, and wherein said albumen comprises a pair of binding domains and discerns a pair of antigen that is selected from by the following group of forming specifically: CD20-CD23, CD20-CD30, CD20-CD37, CD20-CD70, CD20-CD80, CD20-CD86, CD79b-CD37, CD79b-CD81, the main II of histocompatibility complex class peptide-CD30 and the main II of histocompatibility complex class peptide-CD72.

83. as the described albumen of claim 72, wherein said albumen has a retarding effect with respect to each binding domains effect summation for the target cell behavior, and wherein said albumen comprises a pair of binding domains and discerns a pair of antigen that is selected from by the following group of forming specifically: the CD20-II of main histocompatibility complex class peptide, CD79b-CD19, CD79b-CD20, CD79b-CD21, CD79b-CD22, CD79b-CD23, CD79b-CD30, CD79b-CD40, CD79b-CD70, CD79b-CD72, CD79b-CD79a, CD79b-CD80, CD79b-CD86, the CD79b-II of main histocompatibility complex class peptide, the main II of histocompatibility complex class peptide-CD19, the main II of histocompatibility complex class peptide-CD20, the main II of histocompatibility complex class peptide-CD21, the main II of histocompatibility complex class peptide-CD22, the main II of histocompatibility complex class peptide-CD23, the main II of histocompatibility complex class peptide-CD37, the main II of histocompatibility complex class peptide-CD40, the main II of histocompatibility complex class peptide-CD70, the main II of histocompatibility complex class peptide-CD79a, the main II of histocompatibility complex class peptide-CD79b, the main II of histocompatibility complex class peptide-CD80, the main II of histocompatibility complex class peptide-CD81, the main II of histocompatibility complex class peptide-CD86, CD22-CD19, CD22-CD40, CD22-CD79b, CD22-CD86 and the CD22-II of main histocompatibility complex class peptide.

84. differentiate at least one the method in the binding domains of multivalence binding molecule as claimed in claim 1, described method comprises:

(a) make anti-homotype antibody and discern the first antigenic antibody specifically and discern the second antigenic antibody specifically and contact;

(b) at least one the target that comprises in the described antigen is contacted with the described composition of step (a); And

(c) measure the activity of described target, wherein said activity is at least one of binding domains that is used for differentiating described multivalence binding molecule.

85. as the described method of claim 84, wherein said target is a sick cell.

86. method as claimed in claim 60, wherein compare with comprising described second binding domains but not comprising the infringement summation that the second antibody of described first binding domains brings out with the first antibody that comprises described first binding domains but do not comprise described second binding domains, described multivalence single strand binding protein brings out the infringement of collaborative amount to described target cell.

87. as the described method of claim 86, wherein said multivalence single strand binding protein comprises a pair of binding domains and discerns a pair of antigen that is selected from by the following group of forming specifically: CD19/CD20, CD20/CD21, CD20/CD22, CD20/CD40, CD20/CD79a, CD20/CD79b, CD20/CD81, CD21/CD79b, CD37/CD79b, CD79b/CD81, CD19/CL II, CD20/CL II, CD30/CL II, CD37/CL II, CD72/CL II and CD79b/CL II.

88. method as claimed in claim 60, wherein compare with comprising described second binding domains but not comprising the infringement summation that the second antibody of described first binding domains brings out with the first antibody that comprises described first binding domains but do not comprise described second binding domains, described multivalence single strand binding protein brings out the infringement of amount of suppression to described target cell.

89. as the described method of claim 88, wherein said multivalence single strand binding protein comprises a pair of binding domains and discerns a pair of antigen that is selected from by the following group of forming specifically: CD20/CL II, CD21/CD79b, CD22/CD79b, CD40/CD79b, CD70/CD79b, CD72/CD79b, CD79a/CD79b, CD79b/CD80, CD79b/CD86, CD21/CLII, CD22/CL II, CD23/CL II, CD40/CL II, CD70/CL II, CD80/CL II, CD86/CL II, CD19/CD22, CD20/CD22, CD21/CD22, CD22/CD23, CD22/CD30, CD22/CD37, CD22/CD40, CD22/CD70, CD22/CD72, CD22/79a, CD22/79b, CD22/CD80, CD22/CD86 and CD22/CL II.

90. method as claimed in claim 60, it further comprises multiple multivalence single strand binding protein.

91. as the described method of claim 90, wherein the binding domains of the binding domains of the first multivalence single strand binding protein and the second multivalence single strand binding protein brings out the infringement of collaborative amount to described target cell.

92. as the described method of claim 90, wherein the binding domains of the binding domains of the first multivalence single strand binding protein and the second multivalence single strand binding protein brings out the infringement of amount of suppression to described target cell.

93. composition, it comprises multiple multivalence single strand binding protein as claimed in claim 1.

94. as the described composition of claim 93, wherein the binding domains of the binding domains of the first multivalence single strand binding protein and the second multivalence single strand binding protein can bring out the infringement of collaborative amount to the target cell.

95. as the described composition of claim 93, wherein the binding domains of the binding domains of the first multivalence single strand binding protein and the second multivalence single strand binding protein can bring out the infringement of addition amount to the target cell.

96. as the described composition of claim 93, wherein the binding domains of the binding domains of the first multivalence single strand binding protein and the second multivalence single strand binding protein can bring out the infringement of amount of suppression to the target cell.

97. composition pharmaceutically, it comprises as the described composition of claim 93 and pharmaceutically acceptable carrier, thinner or vehicle.

98. test kit, it comprises as the described composition of claim 93 and a cover about the specification sheets of composition as described in giving with infringement target cell.

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