CN106188305A - There is the bivalent antibody of the single domain Fab being fused to conventional Fab fragment - Google Patents

Abstract

The present invention provides a kind of bivalent antibody, comprise (a) and contain the light chain of antibody and the Fab (Fab) of Partial heavy, and (b) is fused to the single domain Fab (VHH) of C end of described light chain or described Partial heavy, wherein said Partial heavy does not comprise CH2 or CH3 territory.The bivalent antibody of the present invention can killing tumor cell in vivo, suppress tumor growth, and can be by producing with escherichia coli expression for soluble protein form in vitro.

Description

There is the bivalent antibody of the single domain Fab being fused to conventional Fab fragment

Technical field

The present invention relates to a kind of bivalent antibody, particularly there is the single domain Fab being fused to conventional Fab fragment Bivalent antibody.

Background technology

The man-made protein that bispecific antibody (BsMAb, BsAb) is made up of the fragment of two kinds of different monoclonal antibodies, thus Can be coupled to two kinds of dissimilar antigens.Such as, in cancer immunotherapy, it is simultaneously bonded to carefully through the BsMAbs of through engineering approaches Cellular toxicity cell and target (such as tumor cell) to be killed.

At least bispecific antibody of three types has been suggested or has tested, including trifunctional antibody, the Fab that is connected chemically With double specific T cell convergence bodies.In order to overcome the difficult point in manufacture, have been developed for being referred to as the first generation of trifunctional antibody BsMAb.It is made up of two heavy chains and two light chains, each from different antibody.The two Fab district is for two kinds of antigens. Fc district is made up of two heavy chains and forms the 3rd binding site, thus gains the name.

Having designed other kinds of bispecific antibody to solve some problem, such as, the half-life is short, immunogenicity and cell The factor discharges the side effect caused.They include: Jin You Fab district composition the Fab being connected chemically, and all kinds bivalence and The single-chain variable (scFv) (fusion protein in two kinds of antibody variable territories of simulation) of trivalent.The form of recent development is double special Different T cell convergence body (BiTE) and the antibody of four senses.

Although it is progressive to achieve these, but the main challenge of bispecific antibody yet suffers from, such as improve manufacture efficiency, Retain immunogenicity and maintain the half-life.

Summary of the invention

In one embodiment, the invention provides bivalence or trivalent antibodies, this antibody includes the antigen knot of routine Close fragment (Fab) and one or two single domain antigen combines (VHH) fragment.Each Fab and VHH has the spy to certain antigen The opposite sex, they may be identical or different.

In antibody producing and purification, such a kind of bivalence relative to conventional antibody decrease in molecular weight or trivalent resist Body, presents a significant advantage.Although its size is little, but unexpectedly, such antibody still can be effectively incorporated into Two different antigens, it is achieved biological function expected from it.Although bispecific antibody disclosed in these is heterodimer, Know that they are unfavorable for expressing and producing, but the most unexpectedly, they can be expressed easily at bacterial cell (such as E.coli), Cause generating soluble protein.On the contrary, other known bispecific antibodies produced by E.coli are the most insoluble.In addition, Because the heavy chain of Fab and light chain will not combine (that is, heavy chain combine heavy chain or light chain combines light chain) with themselves, so Production avoid the formation of unexpected heterodimer or at least minimizes it, causing intended heterodimer yield high.

Therefore, an embodiment of the invention provides a kind of bivalent antibody, and described bivalent antibody comprises (a) and comprises anti- The light chain of body and the Fab (Fab) of Partial heavy and (b) are fused to described light chain or Partial heavy C-end Single domain antigen combine (VHH) fragment, wherein said Partial heavy does not include CH2 or CH3 domain.

In some aspects, VHH segment composition to described Partial heavy.In some aspects, this antibody comprises further It is fused to the 2nd VHH fragment of described light chain.

In some aspects, Fab fragment and VHH fragment are each with the specificity to tumor cell or immunocyte.? Some aspect, described Fab fragment has the specificity to tumor antigen, and described VHH fragment has the spy to immunocyte The opposite sex.In some aspects, described tumor antigen selected from CEA, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, mucoprotein, TAG-72, CIX, PSMA, FBP, GD2, GD3, GM2, VEGF, VEGFR, Integrin, α V β 3, α 5 β 1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and tendon are raw Albumen.In some aspects, described tumor antigen is CEA or Her2.

In some aspects, described VHH fragment has mammalian T cell or the specificity of mammal NK cell.At certain A little aspects, VHH fragment has specificity to the antigen selected from CD3, CD16, CD19, CD28 and CD64.In some aspects, described anti- Former is CD16 or CD3.

In some aspects, described VHH fragment do not comprise in Kabat position 37,44,45 and 47 respectively Val, Gly, Leu and Trp residue.

In one embodiment, present invention also offers many nucleoside of the antibody comprising the invention of one or more code book The host cell of acid.In some aspects, host cell is bacterial cell or yeast cells.In some aspects, host cell is E.coli。

In another embodiment, the invention provides one and prepare soluble antibody method, described method comprises to be made The antibody that the host of the present invention and results are expressed in cell.

Further, in one embodiment, the present invention provides a kind of method of tumor treated in patient, the party Method includes the antibody using the present invention to described patient.

Accompanying drawing explanation

Fig. 1 a is a kind of bivalent antibody, and described antibody includes Fab (Fab) and single domain Fab (VHH).This Fab fragment includes having variable domain (VH) and the Partial heavy of constant domain (CH1), and have variable domain (VL) and The light chain of constant domain (CL).Heavy chain and light chain are connected by disulfide bond.VHH segment composition is to the C end of heavy chain.

Fig. 1 b is a kind of trivalent antibodies, and this antibody includes Fab (Fab) and two single domain antigen binding fragments Section (VHH and VHH ').Described Fab fragment includes having variable domain (VH) and the Partial heavy of constant domain (CH1), and have can Variable domain (VL) and the light chain of constant domain (CL).Heavy chain and light chain are connected by disulfide bond.VHH and VHH ' fragment is each fused to heavy chain C end with light chain.

Fig. 2 a-f illustrates bispecific antibody S-Fab design and preparation.The bacterial expression structure of (a) strand.This structure bag Contain: PelB signal sequence, Humanized CD 3-resisting (UTCH1 clone) VH or VL, CH1 or CL and anti-CEA VHH.Flag label or His8 label is added to C end, to help Protein Detection and purification.(b) comparison Fab (HCBF4/5) and S-Fab by coexpression (HCB5/6) scheme.C () and (b), SDS-PAGE Coomassie blue stain and immune-blotting method are affine through immobilization Ni-NTA Purifying protein after chromatography.Upper figure, Ni-NTA affinity chromatography (M, molecular weight ladder；SF, sucrose component；PF, pericentral siphon component；P, Micropore ball；FT, penetrates liquid；W, washing liquid；E, eluent；E1,100nM imidazoles, E2,200nM imidazoles, E3,300nM imidazoles, E4, 400nM imidazoles).Arrow shows the HCBF4/5 (left) and HCBF5/6 (right) of purification；Middle figure, immunoblotting is resisted by anti-His HCBF5 is surveyed in health check-up；Figure below, immunoblotting is by anti-Flag antibody test HCBF4 (left) and HCBF6 (right).E () is through second time Purifying protein after immobilization Ni-NTA affinity chromatograph.(f) gel permeation chromatography illustrate electrophoresis to 60kd HCBF4/5 and HCB5/6.Upper figure, the protein labeling of gel filtration.

Fig. 3 a-c illustrates the S-Fab of purification and is capable of identify that T cell and CEA antigen.A () is thin at the Jurkat that CD3 is positive On born of the same parents, HCBF5/6 (50ug/ml) (black line) and the flow cytometry of comparison OKT3 antibody (red line), gray area is There is no the cell of dyeing, and blue line is the cell of the most anti-Flag-FITC dyeing.(b) and (c), HCBF4/5 and HCB5/6 The SPR being bound to CEA analyzes.

Fig. 4 a-e illustrate S-Fab by T cell rely on and tumor antigen dependence in the way of killing tumor cell.Exist In the case of S-Fab and Fab of shown concentration, human T-cell mixes with the ratio of 10:1 (a) and 5:1 (b) with tumor cell.Carefully After born of the same parents hatch 48 hours, such as method and measurement cytotoxicity as described in material.C () CEA immunoblotting in different cell lines divides Analysis.D () S-Fab HCBF5/6 is at HT29 (open squares), LS174T (hollow triangle), DLD-1 (open diamonds) cell In EC50.(e). combining AntiCD3 McAb affects S-Fab HCB5/6 activity.There is the HCBF5/6 of shown concentration and compareing Fab's In the case of, use T cell and LS174T (10:1) to carry out cell toxicity test (* P < 0.05, T test, 100nM HCBF4/5 couple Without HCBF4/5).All of error line represents the standard deviation of three same experiments.Data represent at least three independent experiments it One.

Fig. 5 illustrates S-Fab and suppresses tumor growth in vivo.NOD/SCID mice (n=5/ often group) subcutaneous transplantation LS174T and the PBMC of people.Subsequently, mice PBS (solid diamond, PBS treats, and transplants) is given without PBMC；Only PBMC (hollow three Dihedral, PBS treats, and PBMC transplants)；HCBF4/5 (hollow circle, HCBF4/5 treats, and PBMC transplants)；These data represent 5 The average gross tumor volume of mice.Error line represents standard deviation. and (* P < 0.05, T test, HCBF5/6 are than other three groups).

Fig. 6. built by the bacterial expression of E.coli purification Her2-S-Fab. (a) Her2-S-Fab and comparison Her2-Fab Body, each construct contains the signal sequence pelB for periplasmic expression；Anti-Her2-VL-CL and anti-Her2-VH-CH1 construct Coexpression produce Her2-Fab；The coexpression of anti-Her2-VL-CL and anti-Her2-VH-CH1-anti-CD16VHH construct produces Her2-S-Fab；For ease of Protein Detection and purification, the C-terminal in each construct connects Flag label or His8 label. B the coomassie brilliant blue staining result of () two-step method Her2-S-Fab after purification, M represents molecular weight marker, and unit is kD；C () is coagulated The size of glue filtering chromatogram display Her2-S-Fab is about 65kD.

Fig. 7 .Her2-S-Fab identifies Her2 positive cell.Her2-PE antibody (a), Herceptin (b), comparison Fab (c) and Her2-S-Fab (d) flow cytometric analysis results in different cell lines.

The cytotoxicity of Fig. 8 .Her2-S-Fab induced NK cell mediation.A () is with Her2-Fab, Her2-of 10 μ g/ml S-Fab or Herceptin carry out cytotoxicity experiment to CHO and SKOV3 cell line.Data are the meansigma methodss of three experiments, by mistake Difference bar represents standard deviation.Black indicates without CHO during NK cell；Lycoperdon polymorphum Vitt represents the CHO adding NK；Cord indicates thin without NK The SKOV3 of born of the same parents；It is that striped represents the SKOV3 that with the addition of NK.(b-f) different cell lines have been carried out the cell toxicant of dose dependent The analysis of effect: CHO (b)；MDAMB435(c)；MCF7(d)；SKBR3 (e) and SKOV3 (f).Her2-Fab、Her2-S-Fab Or the concentration of Herceptin is 0.01ng/ml to 10 μ g/ml.Data are the meansigma methodss of three experiments, and error bars represents standard Difference.

Fig. 9 .Her2-S-Fab suppresses tumor growth in vivo.NOD/SCID mice (n=5/ group, male) is by subcutaneous vaccination SKOV3 cell (2x10⁶) and the human PBMC (2x10 of fresh separated⁷) mixture.After two hours, lumbar injection PBS, comparison Her2-Fab (1mg/kg) or Her2-S-Fab (1mg/kg) is as first administration (D₀).At subsequently 6 days (D1, D2, D3, D4, D5 And D6), to mice subcutaneous (i.p.) injection PBS (dotted line)；Her2-Fab (1mg/kg, dotted line) or Her2-S-Fab (1mg/ Kg, solid line).Data represent the mean tumour volume of 5 mices.Error bars represents standard deviation (* P < 0.05, t test, Her2- S-Fab is relative to other two groups).

Detailed description of the invention

Definition

Should be noted that term " a kind of " entity refers to this entity one or more, such as, " a kind of bivalent antibody " quilt It is interpreted as representing one or more bivalent antibody.Similarly, these terms " a kind of ", " one or more " and " at least one " exist This is used interchangeably.

" homology " or " homogeneity " or " similarity " refer between two peptide sequences or two nucleic acid molecules Sequence similarity.Measure homology by comparing in the position of each sequence, each sequence perhaps to omparison purpose and It is compared.When the position of this comparative sequences is occupied by identical base or aminoacid, then these molecules in that position are Homology.Degree of homology between sequence becomes along with the total coupling of these sequences or the quantity of homologous position." uncorrelated " or " nonhomologous " sequence refer to that one of sequence with the present invention has the homogeneity less than 40%, preferably shorter than 25% Homogeneity.

(such as, polynucleotide or polynucleotide region (or polypeptide or peptide zone) have certain percentage ratio with another sequence 60%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) " sequence iden " refers to work as During comparison, when relatively the two sequence, the base (or aminoacid) of this percentage ratio is identical.Use known in the art soft Part program can determine that the percentage ratio of this comparison and homology or sequence iden.

Term " polynucleotide of equivalent " refers to have a kind of certain journey with a kind of with reference to nucleotide sequence or its complementary series Degree homology or the nucleotide sequence of sequence iden.In one aspect, the autoploid of nucleic acid can hybridize so far nucleic acid or it is mutual Complementary series.Similarly, " polypeptide of equivalent " refers to have to a certain degree homology or sequence with the aminoacid sequence compareing polypeptide The polypeptide of homogeneity.In some aspects, the homogeneity of sequence be at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.In some respects, this equivalent sequence keeps the activity (such as epi-position combination) of control sequence or structure is (such as Salt bridge).

Each polypeptide disclosed herein or polynucleotide, its equivalent is also intended.In one aspect, Yi Zhongduo The equivalent of peptide includes the change (that is, lack, increase or replace) of amino acid residue.On the one hand, a peptide species equivalent bag Include the change of not more than two kinds of amino acid residues.On the one hand, the equivalent of a peptide species includes not more than 3,4 or 5 kind of amino The change of acid residue.In some respects, this amino acid change is positioned at the polypeptide active to reference is not on crucial residue.Right The residue of polypeptide active key can be by mutation site-specific analysis, even sequence alignment (because this sequence be high Degree is guarded) and tested easily.

" antibody " used herein or " antigen-binding polypeptides " refer to that specifically identifying and be attached to one or more resists Former polypeptide or polypeptide complex.Antibody is whole antibody and its any Fab or strand.Therefore, term " antibody " Including at least containing any albumen of molecule or the peptide of an immunoglobulin part, a part for this immunoglobulin molecules has It is bound to the biological activity of antigen.Such example includes, but not limited to the complementarity of weight/light chain or its ligand binding moiety Determine district (CDR), heavy chain or variable region of light chain, heavy chain or constant region of light chain, framework (FR) district or its any part or combine egg White is at least some of.Term antibody also comprises and once activates polypeptide or the polypeptide complex i.e. possessing antigen binding capacity.

Term used herein " antibody fragment " or " Fab " are parts for antibody, such as F (ab')₂、F (ab)₂, Fab', Fab, Fv, scFv etc..Regardless of structure, the same antigen knot that antibody fragment is all identified with complete antibody Close.This term " antibody fragment " includes aptamers, enantiomorph (spiegelmers), binary.Term " antibody fragment " also includes appointing What synthesis or genetically engineered albumen, these albumen by be bound to specific antigen with form a species complex and as antibody Equally work.

The antibody of the present invention, antigen-binding polypeptides, its variant or derivant include but not limited to polyclonal, monoclonal , polyspecific, the mankind, people source, spirit lengthization (primatized) or be fitted together to antibody, single-chain antibody, epi-position tie Close fragment (such as, Fab, Fab' and F (ab')₂, Fd, Fvs, scFv s (scFv)), single-chain antibody, disulfide bond connect Fvs (sdFv) fragment in VK or VH territory, Fab the expression library fragment generated and antiidiotype (anti-Id) antibody, are comprised (including that anti-Id antibody the most disclosed herein is to LIGHT antibody).The immunoglobulin of the present invention or antibody molecule can be Any type (such as, IgG, IgE, IgM, IgD, IgA and IgY), kind (such as, IgGl, IgG2, IgG3, IgG4, IgAl and Or the immunoglobulin molecules of subclass IgA2).

Light chain is divided into kappa or lambda (Κ, λ).Each heavy chain classes can be combined with kappa or lambda light chain. On the whole, light chain and heavy chain covalent bond each other, exempt from when being produced by hybridoma, B cell or engineered host cell During epidemic disease globulin, " tail " portion of two heavy chains is bonded to each other by disulfide bond or the non-covalent bond of covalency.In heavy chain, amino Acid sequence runs to the C end in the bottom of each chain from the N end at Y conformation fork end.

Light chain and heavy chain are divided into the district of the homology of 26S Proteasome Structure and Function.Term " constant " and " variable " are by function Property ground use.In this respect it is to be understood that light-chain variable domain (VK) and heavy chain variable domain (VH) all determine that antigen is known Not and specificity.On the contrary, the constant domain of light chain (CK) and heavy chain (CH1, CH2 or CH3) gives important biological nature, such as Secrete, through Placenta Hominis mobility, the combination of Fc receptor, complement combination etc..By convention, the numbering of constant region domain is along with they changes Must increase away from when antigen binding site or antibody amino groups end.N end portion is variable domain, and C end portion is constant domain；CH3 With the c-terminus that CK domain contains light chain and heavy chain the most respectively.

As it has been described above, variable region allows antibody optionally to identify and the specifically epi-position on conjugated antigen.Namely Say, the VK domain of antibody and VH domain, or the subclass of complementary determining region (CDR), determine the anti-of three-dimensional in conjunction with to be formed The variable domain of former binding site.This quaternary antibody structure defines the antigen binding site of each the arm end being presented on Y. More specifically, antigen binding site by three CDR on every VH and VK chain determine (that is, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3).In some instances, such as, Camelidae or work based on Camelidae immunoglobulin it are derived from Some immunoglobulin molecules of journey, entire immunoglobulin molecule may be only made up of heavy chain, and does not have light chain, sees Such as Hamers-Casterman et al., Nature 363:446-448 (1993).

In natural antibody, be present in each antigen binding domain six " complementary determine territories " or " CDR " be short, Discrete aminoacid sequence, they are positioned to form antigen when antibody presents 3-d modelling in aqueous environments specifically Binding domain.The aminoacid (claiming " framework region ") of the remainder in antigen binding domain shows relatively low intermolecular transmutability.Structure Frame district mainly uses beta sheet conformation, and CDR forms ring, and these rings connect beta sheet structure, constitute beta sheet in some cases A part for structure).Therefore, framework region is used for forming support, its by the non-covalent interaction of interchain by CDR with just True location, direction.The antigen binding domain formed by the CDR arranged defines the epi-position complementation on immunoreactivity antigen Surface.This complementary surface promotes antibody to be noncovalently bound to its homologous epitopes.For any given heavy chain or light chain Variable domain, those of ordinary skill in the art can differentiate these aminoacid comprising CDR and framework region the most respectively because They (are seen " Sequences of Proteins of Immunological Interest, " by accurate definition Kabat,E.,et al.,U.S.Department of Health and Human Services,(1983)；And Chothia and Lesk, J.MoI.Biol., 196:901-917 (1987), in being incorporated by reference herein in their entirety).

If using in the art for a term and/or accept two or more definition, then in this article, Unless otherwise stated, the definition of this term is intended to include all such connotations.One specific example is, term is " mutually Mend determine district " (" CDR ") be used for be described in the variable region of heavy chain polypeptide and light chain polypeptide in all find discontinuous antigen knot Close site.This specific region is described the most in the following documents: Kabat et al., U.S.Dept.of Health and Human Services, " Sequences of Proteins of Immunological Interest " (1983) and They are incorporated into herein by Chothia et al., J.MoI.Biol.196:901-917 (1987) by quoting.According to Kabat and Chothia for the definition of CDR, it overlap including amino acid residue when contrasting each other or sub-group.But, Any definition about antibody or the CDR of its variable region is used to be considered as at present invention definition and the term that uses In the range of.Comprise as defined in any of the above quotes document the Suitable amino acid residues of CDR by listed below as a comparison In form.The accurate residue quantity comprising specific CDR changes according to sequence and the size of CDR.Variable region ammonia for antibody For base acid sequence, those skilled in the art can determine the residue comprising specific CDR by conventional method.

Kabat etc. also define the numbering system of the variable domain sequence being applicable to any antibody.Those skilled in the art Can will should " Kabat numbering " system apply to any variable domain sequence, without depending on beyond sequence itself clearly Any experimental data.As it is used herein, " Kabat numbering " refers to the numbering system illustrated in the following documents: Kabat et al.,U.S.Dept.of Health and Human Services,“Sequence of Proteins of Immunological Interest”(1983)。

" specific binding " or " right ... to have specificity " typically refers to antibody and is bound to epi-position by its antigen binding domain, And this combination needs the certain complementarity between antigen binding domain and epi-position.Define according to this, when antibody resists via it Former binding domain can be bound to defined epitope more quickly compared to for being bound to random incoherent epi-position, then claim this to resist Body " specific binding " is to this epi-position.Term " specificity " is used to quantitative specific antibodies and combines the most affine of defined epitope Property.Such as, for given epi-position, antibody " A " is considered have higher specificity than antibody " B ", or antibody " A " combines Specificity to epi-position " C " is higher than its specificity being bound to the epi-position " D " being correlated with.

Terms used herein " treats " therapeutic or the preventative means of referring to, wherein object is prevented or slows down (alleviation) The progress of less desirable Pathologic changes or disorder, such as cancer.Useful or desired clinical effectiveness includes, but not limited to The alleviating of symptom, the reduction of disease degree, stable (not the deteriorating) of morbid state, the delay of progression of disease or slow down, disease The improvement of state or mitigation and alleviate (either local or entirety), no matter whether these results are detectable or not Detectable." treat " and also refer to showed prolonged survival compared with not accepting to treat intended survival period.The object needing treatment includes Have suffered from disease or the object of disease and tend to suffer from this disease or the object of disease or need to prevent this disease or disease Object.

" object " or " individual " or " animal " or " patient " or " mammal " refer to any desired diagnosis, prognosis or treatment Object, particularly mammalian object.Mammalian object includes people, performing animal, farming animals and zoo animal, sports Animal or house pet, such as Canis familiaris L., cat, Cavia porcellus, rabbit, rat, mice, horse, cattle, milch cow etc..

Term " have treatment need patient " or " having the object that treatment needs " include can from use the present invention antibody or The object be benefited in compositions, such as mammalian object, to realize such as detection, diagnotor and/or the purpose for the treatment of.

Bivalence and trivalent antibodies

In one embodiment, the present invention provides bivalent antibody, and it has one of them chain being fused to Fab fragment Single domain Fab (VHH).When another chain of Fab fragment is also fused to single domain Fab (VHH '), then This antibody is trivalent.

The bivalence of the present invention or trivalent antibodies can be monospecific, bispecific or tri-specific.As Fab and VHH has the specificity being bound to same antigen, then this antibody is monospecific.If it is special that they have different combinations The opposite sex, then this antibody is bispecific.If VHH, VHH ' and Fab is each has different binding specificities, then should Antibody is tri-specific.

As demonstrated in test example, the exemplary types of bi-specific antibody is the antibody targeting two not synantigens, One of them antigen is present in tumor cell or microorganism, and another is present on immunocyte.When being applied to individuality, this Plant bi-specific antibody and be specifically bound to tumor cell or microorganism, be specifically bound to immunocyte (as carefully simultaneously Born of the same parents' poison cell).This dual combination may result in combined tumor or microorganism and killed by the immune system of host.

A. the unforeseeable character of the bi-specific antibody of the present invention

Because VHH fragment is little and much shorter than conventional antibody, previously it is suspected that when VHH is fused to Fab fragment, big Albumen can suppress the binding ability of VHH due to steric effect.And, it is suspected that this chimeric protein is the most not Can stablize.

But, beyong contemplation, the function of the bivalent antibody of the present invention is had limited by the steric effect suspected Impact.And, the overall affinity of this antibody (need not Fc fragment) is suitable with conventional antibody.Another surprising part is VHH can be effectively bonded to its target, regardless of whether it in the same direction (i.e. the N end of VHH be connected to Fab fragment heavy chain or The N end of light chain) or (i.e. the C end of VHH is connected to the heavy chain of Fab fragment or the N end of light chain) is fused to Fab in a reverse direction Fragment.

Advantages of the present invention is also embodied in cell expression efficiency and protein stability.Although it is generally acknowledged little antibody ratio more Big antibody is easier in cell produce, but heterodimer antibody produces for antibody and proposes particularly challenge.This At least can interact at intraor extracellular because of the protein chain that two (or three or four) are different, cause interference with, thus drop The low efficiency of host cell expression system, and add the unstability of albumen.Therefore, there is the different of whole heavy chain and light chain Source homodimeric antibody is often expressed as in two independent cells, or use common light chain to reduce the mispairing of light chain and heavy chain, This causes generating non-functional antibody.

Even also there are these problems less than those bi-specific antibodys of bi-specific antibody disclosed by the invention.Example As, bispecific T cell convergence body (BITE) is the fusion protein being made up of two typical single chain variable fragments.Although being less than (usually 55KDa) bi-specific antibody disclosed herein (about 65KDa), but BITE cannot be with soluble form in bacterial cell Express.At present, BITE expresses in mammalian cell, and it is much more expensive.

Unforeseeable, as demonstrated in test example, when being expressed in E.coli cell, up to about 20% this Bright bi-specific antibody is solvable (based on Western hybridization analysis).As it has been described above, these antibody are more much bigger than BITE. But, although the BITE of bacterial expression is fairly insoluble, but the antibody of the present invention can be produced by antibacterial easily.Therefore, this result is Surprising and be beyong contemplation.

Therefore, the antibody of the present invention has relatively small size compared with conventional antibody, and can effectively (especially It is in the way of large-scale production) the most such as yeast and bacterial host produce.This stability of these antibody, solubility and Half life, is significantly better than those bi-specific antibodys developed this area.

Knowable to description before, the antibody exhibits of the present invention goes out high antibacterial productivity ratio, stability and joint affinity.This The example planting bivalence and trivalent antibodies is illustrated in Fig. 1 a and Fig. 1 b respectively.

Fig. 1 a shows have Fab part and the bivalent antibody of VHH part.Fab part includes having variable domain (VL) and perseverance The light chain of localization (CL).Fab part also includes having variable domain (VH) and the heavy chain of constant domain (CH1).It is different from common antibody Heavy chain, this heavy chain does not include any one in CH2 territory and CH3 territory or two.Fab fragment is functional element, can be special Property is bound to antigen.Structurally, VH and VL comprises and is suitable to this species specific CDR, CH1 and CL to have suitable aminoacid residual Base makes them connect to each other to form required Fab structure.

Fig. 1 a shows that VHH segment composition is to the C end in the CH1 territory of the heavy chain of Fab fragment.It is readily appreciated that, one In a little embodiments, VHH fragment also can be fused to light chain.When being fused to Fab fragment, VHH and Fab can have identical N end To C extreme direction so that whole chain can be expressed with single polynucleotide.In another embodiment, the C end of VHH can connect It is connected to the heavy chain of Fab fragment or the C end of light chain.

Similarly, Fig. 1 b shows the trivalent antibodies of the present invention, and it includes Fab part and two VHH (VHH and VHH '), Each VHH is fused to a chain of Fab fragment.

The Fab part of antibody can easily identify and prepare.Such as, light chain and the sequence of heavy chain can from include two identical Fab fragment any conventional antibody in identify.Conventional antibody can derive from animal or humanization, and can be further by this Known to field, method is modified.

" single domain Fab " or " single domain antibody fragment " or " VHH " are that one can be bound to antigen without joining The Fab of standby light chain.VHH initially with the isolated in form of single Fab from single domain antibody (sdAb).First Individual known single domain antibody is isolatable from camel (Hamers-Casterman et al., Nature 363:446-8 (1993)), it After be isolatable from selachian.Camel is produced without the functional antibodies of light chain, their single N terminal domains (VHH) conjugated antigen And match (see Harmsen and Haard, App Microbiol Biotechnol., 77:13-22 (2007) without domain Summary).Single domain antibody does not include CH1 territory, and in conventional antibody, CH1 territory interacts with light chain.

VHH comprises and constitutes four framework regions (FR1-FR4) of core texture of immunoglobulin domains and relate to resisting Three complementary determining regions (CDR1-CDR3) of former combination.Compared to people's VH domain, VHH framework region demonstrates and people's VH structure The high sequence homology (> 80% in territory).See Harmsen and Haard, 2007, it further describes title: " VHH's is the most special The feature of levying property is (the 37th, 44,45 and 47, four FR2 positions；Kabat numbers) aminoacid replacement at place, they Conventional VH domain is conservative, and relates to and the hydrophobic interaction of VL domain ".VHH be generally of these with And other the conservative position of conventional VH camber different aminoacids (as Leu11Ser, Val37Phe or Tyr, Gly44Glu, Leu45Arg or Cys, Trp47Gly).

Harmsen and Haard, 2007 also describe: the CDR of VHH has characteristic feature known to some.Such as, The N end portion of CDR1 is more variable than conventional antibody.And, some VHH has the CDR3 of prolongation, its generally by with CDR1 or Cysteine in FR2 forms extra disulfide bond and stablizes, and causes CDR3 ring to fold on the whole interface of VL before.Beautiful The specific subfamily (VHH3) of continent camel VHH is possibly together with a CDR3 extended, and it is by the cysteine shape of the 50th with FR2 Extra disulfide bond is become to stablize.

Known in the art have many sdAb, and easily can prepare from the animals such as such as camel.Based on these sdAb, Their VHH can be identified easily and prepare.Table 1 lists the non-limitative example of many VHH and sdAb.Therefore, at some In embodiment, the present invention provides and comprises each such open sequence or the polypeptide of its equivalent and encode each polypeptide Polynucleotide.In one aspect, this polypeptide comprises the aminoacid sequence of SEQ ID NO:13, or has one, two or three Aminoacid insertion/disappearance/substituted aminoacid sequence.

Single domain Fab (VHH) that table 1. is exemplary and single domain antibody (sdAb)

From upper table it will be evident that some region of these sequences is high conservative (such as FR1-3), and CDR is more Add variable.

B. monospecific, bispecific and three-specific antibody

When Fab fragment and VHH have identical binding specificity, bivalent antibody can be monospecific, or works as When they have different binding specificities, bivalent antibody can be bispecific.Similarly, trivalent antibodies can be Dan Te The opposite sex, bispecific or tri-specific.

Bi-specific antibody can be configured to target different antigen.Such as, between Fab fragment and VHH fragment, one Having the specificity to the first immunocyte, another can target the second immunocyte；One has the first tumor cell Specificity, and another has the specificity to the second tumor cell；One has the specificity to immunocyte, another tool Have microorganism, infection cell, tumor cell, inflammatory cell, apoptotic cell or the specificity of foreign cell (indefinite).

Similarly, trivalent antibodies can configure on demand.In one aspect, Fab, VHH have with VHH ' fragment identical anti- Former or the specificity of epi-position.In yet another aspect, Fab fragment has a specificity to a kind of antigen or epi-position, and VHH and VHH ' Fragment has another kind of antigen or the specificity of epi-position.In yet another aspect, Fab fragment has but and VHH ' identical with VHH Different binding specificities.In yet another aspect, each of which has different binding specificities.

The nonrestrictive example of the integrated structure of bivalence and trivalent antibodies is listed in the following table.

The example arrangement of table 2. bivalent antibody

The example arrangement of table 3. trivalent antibodies

Fragment	Binding specificity	Fragment	Binding specificity	Fragment	Binding specificity
						Fab	Immunocyte	VHH	The second immunocyte	VHH’	The second immunocyte
Fab	Immunocyte	VHH	The second immunocyte	VHH’	The third immunocyte
						Fab	Tumor cell	VHH	The second tumor cell	VHH’	The second tumor cell
Fab	Tumor cell	VHH	The second tumor cell	VHH’	The third tumor cell
						Fab	Immunocyte	VHH	Tumor cell	VHH’	Tumor cell
Fab	Immunocyte	VHH	Tumor cell	VHH’	The second tumor cell
						Fab	Tumor cell	VHH	Immunocyte	VHH’	Immunocyte
Fab	Tumor cell	VHH	Immunocyte	VHH’	The second immunocyte
						Fab	Tumor cell	VHH	Immunocyte	VHH’	The second tumor cell
Fab	Immunocyte	VHH	Tumor cell	VHH’	The second immunocyte

C. target is combined

In some embodiments, Fab or VHH (or VHH ') fragment of bivalent antibody has the immunity spy to tumor antigen The opposite sex.

" tumor antigen " is the antigenic substance produced in tumor cell, i.e. it causes the immune response of host.Tumor resists Former can be used for tumor cell and be the potential candidate substances for the treatment of of cancer.Internal normal albumen is not antigenic. But, some albumen produces or overexpression in upsilonmorigenic process, thus seeming for health is " external source ".This can Including hidden well immune normal albumen, generally with the albumen of indivisible generation, generally only on specific growth rank The albumen of section generation or structure be adorned albumen due to sudden change.

Known in the art have many tumor antigens, by screening it will be readily seen that many new tumor antigens.Tumor antigen Non-limitative example include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, glutinous Albumen, TAG-72, CIX, PSMA, folic acid-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, α V β 3, α 5 β 1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin.

In some respects, Fab or VHH fragment has compared to the overexpression on tumor cell of corresponding non-tumor cell The specificity of albumen." corresponding non-tumor cell " refers to have the non-swollen of same cell type with the cell in tumor cell source Oncocyte.It should be noted that such albumen is not necessarily different from tumor antigen.Nonrestrictive example includes carcinoembryonic antigen (CEA), its overexpression is in most of colon and rectum carcinomas, breast carcinoma, pulmonary carcinoma, cancer of pancreas and gastrointestinal cancer；Adjust protein receptor (HER-2, neu or c-erbB-2), it is generally excessive in breast carcinoma, ovarian cancer, rectal cancer, pulmonary carcinoma, carcinoma of prostate and cervical cancer Express；EGF-R ELISA, it (includes breast carcinoma, head and neck cancer, lung cancer in non-cellule type and front at a series of entity tumors Row adenocarcinoma) interior overexpression；Asialoglycoprotein-receptors；TfR；The serine stretch protein expressed on hepatocyte Enzyme inhibitor combined enzyme agent receptor；The fibroblast growth factor acceptor of overexpression on pancreatic ductal adenocarcinoma cell (FGFR)；Vascular endothelial growth factor receptor (VEGFR) for anti-angiogenic rebirth gene therapy；It is selectively over being expressed in The folacin receptor of the ovarian cancer of the non-mucus type of 90%；The polysaccharide-protein complex (glycocalyx) of cell surface；Carbon aquation Compound receptor；And polymeric immunoglobulin receptor, it is of value to gene delivery to airway epithelial cell, and to pneumonopathy (such as cystic fibrosis) treats attractive.

In one aspect, VHH fragment has specificity to CEA or Her2.The representational sequence of this VHH is SEQ ID NO:1 or 6 (table 1).In some respects, VHH include the aminoacid sequence of SEQ ID NO:1 or 6 and have one or two or three Individual insertion, lack or replace.In one aspect, VHH includes SEQ ID NO:7 aminoacid sequence (anti-EGFR 1), and optionally Ground has one or two or three insertions, lacks or replace.

In some respects, Fab or VHH fragment has specificity to microorganism (such as virus or antibacterial).Microorganism non- Limitative examples includes antimicrobial surface receptor and endotoxin.Endotoxic example include, but not limited to lipopolysaccharide (LPS) and Fat oligosaccharide (LOS).

In some respects, VHH fragment includes the aminoacid sequence selected from SEQ ID NO:8-11 (table 1), or optionally There is one or two or three insertions, lack or replace.

In some respects, another fragment of bivalent antibody has specificity to immunocyte.In one aspect, this immunity is thin Born of the same parents select free T cell, B cell, mononuclear cell, macrophage, neutrophilic granulocyte, dendritic cell, phagocyte, NKT Cell, eosinophilic granulocyte, basophil and the group of mastocyte composition.

In one aspect, this another fragments specific ground identifies antigen, described antigen select free CD3, CD16, CD19, The group of CD28 and CD64 composition.In one aspect, the 2nd VHH identifies CD3 or CD16 specifically.

In one aspect, this another fragment has specificity to CD16 or CD3.The representative series of VHH fragment is SEQ ID NO:2,3,4,5,12 and 13 (table 1), or optionally there is one or two or three insertions, lack or replace.

In some respects, Fab part includes: one or two is selected from the aminoacid sequence of SEQ ID NO:14-25 (table 4) Row, or optionally there is one or two or three insertions, lack or replace.

Table 4.Fab sequence example

Table 5 below illustrates the aminoacid sequence of an embodiment of the bispecific antibody of the present invention.

Table 5. targeting CD3 (at Fab) and the bispecific antibody of CEA (at VHH)

Any antibody described above or polypeptide can include extra polypeptide, such as, coded by guidance further The signal peptide of the secretion of polypeptide, antibody constant region as described herein or other heterologous polypeptides as described herein.

It will be appreciated by the skilled addressee that antibody disclosed herein can be modified so that they are in aminoacid sequence Be different from row they its derivative natural Binding peptide.Such as, polypeptide or the aminoacid sequence of a kind of specific protein it are derived from Can be similar, such as, have the concordance of certain percent to homing sequence, such as it may have with homing sequence 60%, the concordance of 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.

Additionally, nucleotide or the replacement of amino acid whose conservative can be carried out in " nonessential " aminoacid district, lacks or inserts.Example As, be derived from specific protein polypeptide or aminoacid can be the same with homing sequence, except one or more single aminoacid replacement, Insert or disappearance (such as, one, two, three, four, five, six, seven, eight, nine, ten, 15, two Ten or more single aminoacid replacement, insert or lack).In some embodiments, relative to homing sequence, it is derived from The polypeptide of specific protein or aminoacid sequence have one to five, one to ten or one to two ten single aminoacid replacement, insertion Or disappearance.

In some embodiments, antigen-binding polypeptides comprise the aminoacid sequence or not generally being connected with antibody or Some.Exemplary modification is described in detail below.Such as, the fragment of the present invention can comprise flexible linker sequence, or can It is modified to the addition of funtion part (such as PEG, medicine, toxin or labelling).

The antibody of the present invention, its variant or derivant include adorned derivant, and the most any types of molecules is covalently bound To antibody, as long as this covalently bound does not stop antibodies to epi-position.Such as, but being not intended to limit, antibody can be modified, Such as glycosylation, acetylation, Pegylation, phosphorylation, amidatioon, by known protection group or blocking group derivatization, egg White enzyme action, it is connected to cell ligand or other albumen etc..Any repaiied by what known technology can be implemented in a large amount of chemical modification Decorations, include but not limited to metabolism synthesis of special chemical cracking, acetylation, formylated, tunicamycin etc..Additionally, antibody can To comprise one or more atypical amino acids.

In other embodiments, the antigen-binding polypeptides of the present invention can comprise conservative aminoacid replacement.

In " conservative aminoacid replacement ", an amino acid residue is replaced by the amino acid residue with similar side chain. The amino acid residue families with similar side chain is defined in this area, including basic side chain (such as lysine, essence ammonia Acid, histidine), acid side-chain (such as aspartic acid, glutamic acid), uncharged polar side chain (such as glycine, Radix Asparagi acyl Amine, glutamine, serine, threonine, tyrosine, cysteine), non-polar sidechain (such as alanine, valine, bright ammonia Acid, isoleucine, proline, phenylalanine, methionine, tryptophan), β branched building block (such as threonine, valine, different bright ammonia Acid) and beta-branched side (such as tyrosine, phenylalanine, tryptophan, histidine).Therefore, non-at immunoglobulin polypeptides Necessary amino acid residue is preferably to be replaced by other amino acid residues from same side chain family.At another embodiment In, amino acid chain can by structure is similar but on the order of side chain family member or composition, different chains replaces.

Following table provides the substituted unrestriced example of conserved amino acid.In table, similarity scores 0 or higher shows Two kinds of amino acid whose conservative replacements.

	C	G	P	S	A	T	D	E	N	Q	H	K	R	V	M	I	L	F	Y	W
																					W	-8	-7	-6	-2	-6	-5	-7	-7	-4	-5	-3	-3	2	-6	-4	-5	-2	0	0	17
Y	0	-5	-5	-3	-3	-3	-4	-4	-2	-4	0	-4	-5	-2	-2	-1	-1	7	10
																					F	-4	-5	-5	-3	-4	-3	-6	-5	-4	-5	-2	-5	-4	-1	0	1	2	9
L	-6	-4	-3	-3	-2	-2	-4	-3	-3	-2	-2	-3	-3	2	4	2	6
																					I	-2	-3	-2	-1	-1	0	-2	-2	-2	-2	-2	-2	-2	4	2	5
M	-5	-3	-2	-2	-1	-1	-3	-2	0	-1	-2	0	0	2	6
																					V	-2	-1	-1	-1	0	0	-2	-2	-2	-2	-2	-2	-2	4
R	-4	-3	0	0	-2	-1	-1	-1	0	1	2	3	6
																					K	-5	-2	-1	0	-1	0	0	0	1	1	0	5
H	-3	-2	0	-1	-1	-1	1	1	2	3	6
																					Q	-5	-1	0	-1	0	-1	2	2	1	4
N	-4	0	-1	1	0	0	2	1	2
																					E	-5	0	-1	0	0	0	3	4
D	-5	1	-1	0	0	0	4
																					T	-2	0	0	1	1	3
A	-2	1	1	1	2
																					S	0	1	1	1
P	-3	-1	6
																					G	-3	5
C	12

In some embodiments, antibody can be incorporated in curative preparation, prodrug, peptide, albumen, enzyme, virus, fat Class, biological effect instrumentality, pharmaceutical preparation or PEG.

These antibody can be combined or be fused to curative preparation, and this therapeutic preparation can include detectable label (such as radioactive label), immunomodulator, hormone, enzyme, oligonucleotide, photosensitive therapeutic agent or diagnostic agent, cytotoxic agent (medicine or toxin), ultrasound enhancing agent, nonradioactive labeling, combinations thereof and other preparations known in the art.

Carry out traget antibody by coupleding to chemiluminescent compound, then this antibody can be detected.Then, by detection The existence of fluorescence (occurring in chemical reaction process), determines the existence of the antigen-binding polypeptides of chemiluminescent labeling.Extremely The example of useful chemiluminescent labeling compound is luminol, different luminol, theromatic acridinium ester, imidazoles, acridinium salt And oxalate.

The polynucleotide of encoding antibody and the method preparing antibody

Coding bispecific antibody, its variant or the polynucleotide of derivant or the nucleic acid that present invention provides separation divide Son.

The polynucleotide of the present invention can encode on identical polynucleotide molecule or on independent polynucleotide molecule Whole VHH, its variant or derivants.Additionally, the polynucleotide of the present invention can be on identical polynucleotide molecule or solely On vertical polynucleotide molecule encoding antibody or VHH, its variant or a part for derivant.

In some embodiments, the antibody of preparation will not be in causing harmful immunity on animal subject (such as human body) Reaction.In one embodiment, the technology using field accreditation modifies the antigen-binding polypeptides of the present invention, its variant or derivative Thing is to reduce their immunogenicity.Such as, antibody can be lengthization humanized, clever, go immunity, or can prepare Chimeric antibody.

The binding specificity of the bispecific antibody of the present invention can pass through analyzed in vitro (such as, immuno-precipitation, radioimmunity Measure (RIA) or immunoenzyme connection absorption method (ELISA)) determine.

Production system and method

Present invention also offers the method and system of bi-specific antibody for producing the present invention.Well known in the art suitable Human cell's (such as Chinese hamster ovary celI), mammalian cell and bacterial cell is included in the cell producing antibody.Come with bacterial cell Produce bispecific antibody and there is significant challenge.But, as being shown in an embodiment, when expressing antibody in bacterial cell Time, even two peptide chains are all expressed in same cell, the antibody of generation is the most also solvable.

Therefore, in one embodiment, the invention provides comprise one or more code book invention double special resist The host cell of the polynucleotide of two chains of body.In one aspect, mononucleotide construct (such as plasmid) includes two volumes Code sequence.In yet another aspect, the present invention provides two kinds of different polynucleotide constructs, and each encodes one of them many nucleoside Acid chain.In one embodiment, present invention also offers the host of two polypeptide chains of the bispecific antibody comprising the present invention Cell.

In some respects, described host cell is human cell.In some respects, described host cell is that mammal is thin Born of the same parents.In some respects, described host cell is yeast cells.In some respects, described host cell is bacterial cell, including G+ With G-bacterial cell.Representational bacterial cell includes, but not limited to escherichia coli and Salmonella typhimurium.

In some aspects, the method that the present invention also provides for preparing the bi-specific antibody of the present invention.In one aspect, the party Method needs express two peptide chains of this antibody in host cell and extract antibody from cell pyrolysis liquid.Additionally, the present invention Also provide for the bi-specific antibody obtained by these methods.

Treatment and diagnostic method

As it has been described above, the bi-specific antibody of the present invention, its variant or derivant can be used for and cancer or infectious disease In some relevant treatment and diagnostic method.

The invention further relates to therapy based on antibody, it relates to being applied to the bi-specific antibody of the present invention suffer from Person, such as animal, mammal and human body, to treat one or more diseases as herein described or symptom.The treatment of the present invention Property compositions include, but not limited to the antibody (including its variant as herein described and derivant) of the present invention and code book invention The nucleic acid of antibody (including its variant as herein described and derivant) or polynucleotide.

The antibody of the present invention can be used to treatment, suppress or prevent following disease, imbalance or disease, including with such as with increasing Add the relevant disease of cell survival or inhibited apoptosis or relevant malignant disease, imbalance or the disease of imbalance (such as cancer), institute State cancer to include but not limited to follicular lymphoma, (include with the cancer of p53 sudden change and the tumor of hormone dependant but do not limit In colon cancer, cardiac tumor, cancer of pancreas, melanoma, one-tenth retina tumor, glioblastoma, pulmonary carcinoma, colorectal cancer, carcinoma of testis, stomach Cancer, one-tenth neuroblastoma, myxoma, muscular tumor, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, cartilage Sarcoma, adenocarcinoma, breast carcinoma, carcinoma of prostate, Kaposi sarcoma)；Autoimmune disorder (combine by such as multiple sclerosis, Sjogren Simulator sickness, Grave disease, Hashimoto thyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet is sick, Crohn sick, Polymyositis, systemic lupus erythematosus (sle), Ia glomerulonephritis, autoimmune gastritis, autoimmune platelet subtract Few property purpura and rheumatoid arthritis)；With viral infection (such as herpesvirus, poxvirus and adenovirus), inflammation, transplanting The anti-host disease of thing (acute and/or chronic), acute transplant rejection and chronic transplant rejection.The antigen of the present invention combines many Peptide, its variant or derivant are used for suppressing the development of cancer, evolution and/or transfer, particularly at paragraph above or subsequently In the cancer listed.

Antibody or its variant or the derivatives for treatment of the available present invention, prevent, diagnose and/or prognosis and cell survival increase Other relevant disease or disease, include but not limited to, the progress of following disease and/or transfer: malignant tumor and relevant disease Disease, such as leukemia (acute leukemia (and as acute lymphoblastic leukemia, acute myelocytic leukemia (include myeloblast, Promyelocytic leukemia, Myelomonocyte, mononuclear cell and leukaemia)) and chronic leukemia (such as chronic myelocytic (granulocyte) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (as sick in Hodgkin and non- Hodgkin is sick), multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease and entity tumor (include but do not limit In: sarcoma and cancer (as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, ridge lock tumor, angiosarcoma, Endotheliosarcoma, lymphosarcoma, lymphatic endothelia sarcoma, synovioma, mesothelioma, Ewing tumor, leiomyoma, rhabdomyosarcoma, colon Cancer, cancer of pancreas, thymic carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, spiroma, papillary carcinoma, Papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cancer of biliary duct, choriocarcinoma, spermatogonium cancer, embryo Tire cancer, Wilm tumor, cervical cancer, testicular tumor, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, shape cell Tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, Meningioma, melanoma, neuroblastoma with become retinal cells film tumor)).

The antibody of the present invention also can affect the elimination of microorganism and for treating by targeting microorganism and immunocyte The infectious disease caused by microorganism or killing microorganisms.In one aspect, this microorganism is that virus (includes that RNA and DNA is sick Poison), gram positive bacteria, gram negative bacteria, protozoacide or fungus.

The dosage special for any given patient and therapeutic scheme will depend upon which many factors, and (include being used is special Antigen-binding polypeptides, its variant or derivant, the age of patient, body weight, general health, sex, diet, time of application, excretion Rate, the associating of medicine and the order of severity of treated specified disease).Medical personnel belong to this to the judgement of such factor In the those of ordinary skill determination range of field.This dosage also can be based on treated individual patient, route of administration, the class of formula Type, the feature of the compositions used, the order of severity of disease and the effect expected.The dosage used can pass through this area institute The principle of known pharmacology and pharmacokinetics determines.

Bispecific antibody, the application process of variant include but not limited to: Intradermal, intramuscular, abdominal cavity, vein, skin Under, intranasal, peridural and oral cavity route.Antigen-binding polypeptides or compositions can facilitate approach to use by any, Such as, by infusion or bolus in ection, by the absorption of epithelium or mucosa protection layer, (such as oral mucosa, rectum and intestinal are glutinous Film etc.)；It can be used together with other biological active ingredient.Therefore, medicinal group of antigen-binding polypeptides of the present invention is comprised Compound can with direct oral cavity, rectum, parenteral, intravaginal, abdominal cavity, locally (as by powder, ointment, drop or skin plaster), contain Clothes are used or are used by the spraying of oral cavity or nose.

Term used herein " parenteral " refers to the mode used, and this mode includes intravenous, intramuscular, abdomen Chamber, intrasternal, subcutaneous and IA injection and infusion.

Using can be system or local.Further, it may be contemplated that the antibody of the present invention is drawn by any suitable pathways Entering to central nervous system, these approach include: the injection in Intraventricular and sheath；(storage such as it is connected to by intraventricular catheter Storage (such as Ommaya bin)) ICV injection can be promoted.May be used without pulmonary administration, such as, sucked by use Device or aerosol apparatus and the formula with aerosol are administered.

Bispecific antibody or the compositions local application of the present invention are probably desirable to the region needing treatment； This can be realized by such as (but not limited to) in the following manner: in operation, local infusion, local application (are used in combination the most after surgery Wound dressing), inject, by conduit, by suppository or (implant is porous, non-porous, impermeable by implant Or gelatinous material, including thin film (such as pellosil) or fiber)).Preferably, when the albumen (bag including the present invention Include antibody) time, should be noted the use non-adsorbable material of this albumen.

The antibody of the present invention inflammation, immunity or malignant disease, imbalance or disease treatment, suppress and prevent in effectively Amount, can be determined by the clinical technology of standard.Furthermore, it is possible to optionally use external test to help to identify optimal agent Weight range.The dosage accurately used in formula also will depend upon which the approach of administration and the serious of disease, imbalance or disease Property, and should determine according to the situation of the judgement of practitioner and each patient.Effective dose can be external from being derived from Or animal model test system dose-response curve reason out.

As general suggestion, the dosage of the antigen-binding polypeptides giving the present invention of patient is usually 0.1mg/kg extremely 100mg/kg weight in patients, 0.1mg/kg to 20mg/kg weight in patients, or 1mg/kg to 10mg/kg weight in patients.Overall next Saying, due to the immunoreation of the polypeptide to external source, in human body, the antibody of people has longer than the antibody from other kinds Half-life.Therefore, relatively low dosage and the lower administration frequency of people's antibody is typically possible.And, the antibody of the present invention Administration frequency and dosage can strengthen the absorption of these antibody by modifying (the most esterified) and tissue penetration (such as enters Enter brain) reduce.

For treat infection or malignant disease, disease or imbalance method (comprise give the antibody of the present invention, its variant or Derivant), before human body, the most tested, in acceptable animal model, then carry out internal test, to obtain The treatment that must expect or prophylactic activity.Suitably animal model (including transgenic animal) is ripe to those of ordinary skill in the art Know.Such as, show that the experiment in vitro of the treatment effectiveness of antigen-binding polypeptides as herein described includes that antigen-binding polypeptides is carefully Effect in born of the same parents system or tissue of patient sample.Utilize technology known to those skilled in the art (such as public in elsewhere herein The test opened) can determine that antigen-binding polypeptides effect in cell line and/or tissue sample.According to the present invention, can be used to Cell culture invitro tests (wherein patient's group to determine the need for using the in vitro tests of specific antigen-binding polypeptides to include Tissue samples grows in the medium and is exposed to or otherwise gives antibody) and observe this antibody at this tissue sample Effect on product.

In further embodiment, the compositions of the present invention and antitumor agent, antiviral agent, antibacterial or antibiotic Preparation or antifungal preparation are co-administered.These preparations being known in the art any can in the present compositions by Give.

In another embodiment, the compositions of the present invention is combined with chemotheraping preparation and is given.Can be with the combination of the present invention The chemotheraping preparation that thing gives together includes but not limited to: antibiotic derivatives is (such as amycin, bleomycin, daunomycin, anti- Line rhzomorph), estrogen antagonist (such as tamoxifen), antimetabolite (such as fluorouracil, 5-FU, ammonia methylpurine, floxuridine, dry Disturb element α-2b, glutamic acid, plicamycin, mercaptopurine and 6-thioguanine), cytotoxic agent (such as carmustine, BCNU, Lip river Mo Siting, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, methylbenzyl hydrazine, mitomycin, busulfan, cisplatin and length Spring new alkali sulfate), hormone (such as medroxyprogesterone, estramustine phosphate sodium, ethinylestradiol, estradiol, tumer ground pregnant Ketone, first hydroxytestosterone, stilphostrol, chlorotrianisene and testolactone), nitrogen mustard derivatives (melphalan, benzenebutanoic acid nitrogen Mustard, dichloromethyldiethylamine (chlormethine), thio-tepa), steroid and goods (betamethasone sodium phosphate) and other (such as Daccas Bar piperazine, asparaginase, mitotane, vincristine sulfide, vincaleucoblastine sulfide and etoposide).

In other embodiment, the compositions of the present invention is combined with cytokine and is given.Can be with the combination of the present invention The cytokine that thing is given together includes but not limited to: IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD 40, CD40L and TNF-α.

In other embodiment, the compositions of the present invention and other treatment or preventive therapy (such as radiotherapy) connection Conjunction gives.

Compositions

Present invention also offers pharmaceutical composition.Such compositions comprises antibody and the acceptable carrier of effective dose. In a detailed description of the invention, term " pharmaceutically acceptable " means by federal or state government regulator's license Or listed by American Pharmacopeia or other pharmacopeia being conventionally recognized by for animal, more particularly, for people's.Further Ground, " pharmaceutically acceptable carrier " will be generally nontoxic solid-state, semi-solid or the filler of liquid, diluent, encapsulating Material or any kind of adjuvant.

Term " carrier " refer to drug use by diluent, adjuvant, excipient or carrier.Such pharmaceutical carrier Can be aseptic liquid, such as water and oil, including oil, animal, plant or the oil in synthesis source, such as Oleum Arachidis hypogaeae semen, Semen sojae atricolor Oil, mineral oil, Oleum sesami etc..When pharmaceutical composition by intravenous injection be administered time, water is first-selected carrier.Saline solution and aqueous Glucose can also be used as the carrier of liquid phase with glycerite, particularly for injectable solution.Suitably medicine Thing excipient includes: starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, Chalk, silica gel, sodium stearate, list are hard Glycerol, Talcum, sodium chloride, defatted milk powder, glycerol, propylene, ethylene glycol, water, ethanol etc..If it is required, compositions A small amount of moistening or emulsifying agent or pH buffer agent, such as acetate, citrate or phosphate can also be comprised.It is also contemplated by adding Enter antibiotic preparation (such as benzylalcohol or essence of Niobe)；Antioxidant (such as ascorbic acid or sodium sulfite)；Chelating agen (second Ethylenediamine tetraacetic acid (EDTA)) and it is used for the preparation (such as sodium chloride or dextrose) that isotonia regulates.These compositionss can take solution, The forms such as suspension, tablet, pill, capsule, powder, controlled-release formulation.Said composition can use usual binding agent and carrier (example Such as triglyceride) and as suppository formulations.Formula of oral can include the carrier of standard, such as, the mannitol of pharmaceutical grade, lactose, Starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate etc..At Remington's Pharmaceutical Sciences In the example (being incorporated into this by quoting) of suitable pharmaceutical carrier is described by E.W.Martin.Such compositions The antigen-binding polypeptides (the preferably form of purification) of the upper effective dose for the treatment of and appropriate number of carrier will be comprised, in order to for patient Suitable administering mode is provided.This formula should be suitable for the mode being administered.This parental generation (parental) preparation can be loaded on ampoule In the multi-dose vials that bottle, disposable syringe or glass or plastics are made.

In one embodiment, it is formulated as being suitable to be administered intravenously in the medicine of the mankind according to routine Compositions.The compositions being commonly used for intravenously administrable is the solution in the aqueous buffer solution of sterile isotonic.When necessary, group Compound may also comprise solubilizing agent and local anesthetic (such as lignocaine, to alleviate injection site pain).Component is typically single Solely or mix and provide in a unit, such as, in hermetic container (the such as peace of the quantity of lined out activity preparation Small jar or sachette) in freeze dried powder or without aqueous concentrate.When compositions is given by infusion, it be dispersed among containing with or without In the pharmaceutical grade water of bacterium or the infusion bottle of saline.When compositions is given by injection, it is possible to provide being used for an of ampoule is injected Sterilized water or saline, in order to this composition can be mixed before administration.

The compositions of the present invention can be formulated into neutrality or the form of salt.Pharmaceutically acceptable salt include by the moon from The salt that son is formed, such as those come from the salt of hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid etc., and the salt formed by cation, example As those come from sodium, potassium, ammonium, calcium, hydrated ferric oxide., 2-aminopropane., triethylamine, ethyl oxyethylamine, histidine, procaine etc. Salt.

Experimental example

The cytotoxicity that embodiment 1. bispecific antibody induction T-is cell-mediated

This embodiment offers a kind of new bispecific antibody form, by the list by targeting tumor cell positive for CEA Territory anti-CEA VHH is connected to combine the anti-CD3Fab of T cell and forms.Such antibody (S-Fab) can raise T cell to CEA- Positive tumor cell.By making T cell participate in, it is thin that this S-Fab antibody kills tumor positive for CEA the most effectively Born of the same parents.In heteroplastic model, this S-Fab antibody presents strong tumor inhibitory effect, show such strategy for In many immunotherapies.Further, this S-Fab antibody can be expressed and purification from E.coli effectively and stably.

This embodiment make use of the combination of single domain antibody (VHH) and Fab fragment to produce heterodimer antibody.This In form, anti-CEA VHH is connected to the C end of the Partial heavy of anti-CD 3 antibodies (VH-CH1), then produced VH-CH1-VHH chain forms S-Fab bispecific antibody with anti-CD3 light chain (VL-CL) pairing.CEA is chosen as target, be because of For it generally in cancer (colon cancer, cancer of pancreas, gastric cancer, esophageal carcinoma, pulmonary carcinoma, breast carcinoma, uterus carcinoma, ovarian cancer, carcinoma of endometrium) Middle overexpression.

This S-Fab antibody is expressed and purification from E.coli.Experiment in vitro confirms the tumor cytotoxicity merit of S-Fab Can be specific to the tumor cell that CEA-is positive and be to rely on T cell.In vivo, this S-Fab antibody suppression swells Tumour development.Therefore, such antibody formation presents the new model of the bispecific antibody as immunotherapeutic agent.

Method and material

Fab design and protein purification

Build S-Fab albumen HCBF5/6 and comparison Fab HCBF4/5 such as Fig. 2 a-b.Anti-based on disclosed sequent synthesis CD3 (humanized UCHT1) gene (being shown in Table 5).Camel anti-CEA VHH sequence is cloned into the C end in CH1 territory.Signal sequence quilt Interpolation is used for periplasmic expression to the N end of each chain.

The plasmid of recombinant antibodies is entered BL21 (DE3) competent cell by cotransformation, and is selecting with suitable antibiotic Coexpression in the LB culture medium selected.For protein expression, 1L culture grows 20h to OD600 0.8-1.0 (0.1mM in 16 DEG C IPTG induces).Bacterial cell is at 4 DEG C, and 4400rpm is centrifuged 30min and forms cell mass.In freezing sucrose solution (20mMTris-HCl pH 8.0；25% (w/v) sucrose；1mM EDTA；1mM PMSF) with 1:4 (m/v) re-suspended cell agglomerate Carry periplasmic extract.This suspension is 8, and 500g is centrifuged 20min, and supernatant is sucrose component.Cell mass is in freezing In periplasmic solution (5mM MgCl2) resuspended, suspension is 8, and 500g is centrifuged 20min, and supernatant is pericentral siphon component.

By two steps of the immobilized metal affinity chromatography by C end His8-labelling, from sucrose component and pericentral siphon component Blending ingredients in this albumen of purification.

As described in manufacturer, GE Superdex 200Increase 10/300GL is used to carry out gel filtration.Gel The protein labeling filtered is purchased from Sigma (MWGF1000).

Cell line and animal

All cells system (includes positive for CEA people rectum cancer cell system HT29, DLD-1 and LS174T, the people that CEA is negative Rectum cancer cell system SKOV3, and JurkatT cell line) purchased from Chinese Academy of Sciences's Type Tissue Collection (China, on Sea).At 5%CO₂, in 37 DEG C of moist incubators, HT29 and SKOV3 containing 10% hyclone (Gibco, Life Technologies, China) and DMEM culture medium (Gibco, the Life of 1% penicillin/streptomycin (Hyclone) Technologies, China) middle cultivation, LS174T and Jurkat contains 10% hyclone (Gibco, Life Technologies, USA) and RPMI-1640 culture medium (Gibco, the Life of 1% penicillin/streptomycin (Hyclone) Technologies, China) middle cultivation.Nod/SCID mice is purchased from Zhongshan University's Experimental Animal Center.Human blood gathers, moves Thing is taken care of and zoopery is ratified by Zhongshan University.

T cell is separated from PBMC

Ficoll density centrifugation is used to prepare human PBMC from healthy donor.Use EasySep^TMPeople's CD3 positive selects examination Agent box (STEMCELL Technologies, Inc., Vancouver, Canada) purified T cell.The T cell separated is incubated at In the culture medium of RPMI1640 completely containing 10%FBS and 1% penicillin/streptomycin, it is placed in 37 DEG C, 5%CO₂Moist cultivation Case.

Surface plasma body resonant vibration (SPR) test that CEA and S-Fab interacts

CEA full-length proteins (ab742, Abcam) be used for immobilization chip (GLC chip, #33D211O1, Bio-Rad).After immobilization, at 25 DEG C, with PBST (the 10mM Na of 5 μ l/min flow velocitys₃PO₄, 150mM NaCl, 0.01% Tween, pH=7.4) as electrophoretic buffer, carry out the transactional analysis to different samples.Use ProteOn^TM XPR 36 (Bio-rad) collect data, use Protein On evaluation software (Bio-rad) to analyze these data with applicable conjunction Suitable combination model.Kinetic constant is measured according to the matching combined with phase of dissociating.KD is derived from the ratio of these constants.Positive right According to (CEA/CD66e (CB30) Mouse mAb (#2383, Cell Signaling) is used for optimizing this process.

Flow cytometry

In order to check the combination of S-Fab Yu CD3 cell surface marker, 1 × 10⁶Jurkat cell/sample is centrifuged collection (1000rpm, 5min), then cleans with PBS+0.1%BSA ice-cold for 1ml.Cell mass is at PBS+0.1% ice-cold for 100ul In BSA resuspended.Anti-CD 3 antibodies (R&D systems, MAB100), HCBF4/5 and HCBF5/6 (S-Fab) are added to final concentration 50ug/ml.Cell and one resists hatches one hour on ice.Cell is cleaned twice with ice-cold PBS+0.1%BSA.Then, Sheep-anti-Mus Alexa 488 or anti-Flag-FITC is added to final concentration 10ug/ml.Cell is hatched on ice one hour again.Clearly Wash after twice of cell, carry out flow cytometry.

Cell toxicity test

SKOV3, HT29 and LS174T are used as target cell.The T cell of the non-pre-stimulation of human PBMC or separation is used as effect Answer cell.The target cell (5000 cells) of 100ul is transferred to each hole (in quadruplicate) of 96 orifice plates.At 6 hours After hatching, isopyknic CD3⁺T cell is added to each hole with the ratio of E:T (10:1 or 5:1).Be subsequently adding from The antibody of the shown concentration of 0.01ng/ml to 10ug/ml.After hatching at 48 hours, cell PBS washes twice.10ul CCK8Kit is subsequently fed to each hole.By " Magellan " software use TECAN Infinite F50 survey Amount.Target cell survival rate (%) calculates according to this formula: [(survival target cell (sample)-culture medium)/(survival target cell is (right According to)-culture medium)] × 100.

In competitive trials, LS174T is used as target cell.Effector lymphocyte is 10:1 to the ratio of target cell.HCBF4/ 5 and HCBF5/6 add with the ratio of 1:1,1:10 and 1:100.Then, cell toxicity test proceeded as above.

In vivo test

Non-obese diabetes/severe combined immunodeficiency (NOD-SCID) Mouse feeder is in Zhongshan University's laboratory animal The heart, under the conditions of aseptic and standardized environment, (20-26 DEG C of room temperature, 40%-70% relative humidity, and 12 hours circadian rhythms) is little Mus accepts autoclaved food, bedding and padding and aseptic drinking water.

From cell culture fluid results LS174T human colon cancer cell, wash once with PBS, resuspended in PBS, then with The PBMC mixing just separated from healthy donor.Cell suspension by subcutaneous injection in right side, cumulative volume 0.4mL/ mice, be mixed with 1x10⁶LS174T cell and 5x10⁶Human PBMC.In LS174T/T cell transplantation one hour after, lumbar injection (i.p.) give antibody Or vehicle Control (PBS).Then, these animals were treated ensuing 7 day every day.Tumor is substantially formed at after transplanting about 8 By 10 days.Measure gross tumor volume with slide gauge in two perpendicular directions, use this formula (width²X length)/2 calculating Volume.All results represent with the arithmetic mean of instantaneous value of each group.

Result

S-Fab bispecific antibody builds and purification

Designed by anti-CEA single domain antibody and the connection of the anti-CD3Fab of routine bispecific antibody S-Fab (Fig. 2 a, 2b).The anti-CD3Fab of this unit price is expected to identify T cell and raise T cell.At the C end of VH-CH chain, anti-CEA VHH with Fab melts Close.The expression of S-Fab is suitable with the expression compareing AntiCD3 McAb Fab.In order to promote expression and the purification of S-Fab, S- Two chains of Fab are cloned into two different plasmids；Periplasmic expression is used for this complex of purification (Fig. 2 c, 2d).

Similar to Fab, this S-Fab is purified with the form of heterodimer.Immunoblotting (Fig. 2 c, 2d) presents this partly Antibody is folded into a complete Fab really.After second time Ni-NTA affinity purification, the purity of this albumen is more than 90% (figure 2e).Then, gel filtration is used for analyzing this purifying protein.Light chain becomes complete Fab antibody (Fig. 2 e), molecule with heavy chain combinations Amount (～65KD with～50KD) molecular weight as expected is the same.

S-Fab identifies antigens c D3 and CEA

In order to detect whether this antibody purification can identify these antigens, carry out flow cytometry to characterize CD3 antigen Combination with S-Fab HCBF5/6.For control antibodies OKT3, it was observed that the positive table in the Jurkat cell that D3 is positive Reach (Fig. 3 a).S-Fab HCBF5/6 also has the positive expression (Fig. 3 a) in Jurkat cell, shows that HCBF5/6 can be in conjunction with To Jurkat cell.This low-intensity is likely due to what the unit price of Fab caused.

In order to confirm the combination of S-Fab Yu CEA, this embodiment uses SPR to measure the combination of S-Fab Yu CEA.For right According to FabHCBF4/5, it is not detected by the combination (Fig. 3 a) with CEA.S-Fab HCBF5/6 be can be observed when kd～12.5nM Strong bonded (Fig. 3 b) with CEA.

HCBF5/6S-Fab kills the tumor cell of specific expressed CEA

In order to assess S-Fab whether can target tumor cell with realize T cell mediation cell killing, carried out cell toxicant Property measure.At E:T 10:1 (Fig. 4 a) or the ratio of 5:1 (Fig. 4 b), for S-Fab (HCBF5/6) or comparison CD3Fab (HCBF4/5) cytotoxicity of the cell line (SKOV3) negative to CEA is not the most observed.For the cell line that CEA is positive (LS174), comparison AntiCD3 McAb Fab (HCBF4/5) does not has killing functions of immunocytes；But S-Fab (HCB5/6) can kill with inducing cell (Fig. 4 b).For CEA positive cell line (HT29), comparison CD3Fab (HCBF4/5) itself gets final product killing tumor cell, but, S-Fab (HCBF5/6) can induce higher cell killing activity (Fig. 4 a and 4b) than HCBF4/5.

Immunoblotting assay shows that LS174T cell has higher CEA than HT29 cell and expresses (Fig. 4 c).At dose response In experiment, compared on HT29 cell, S-Fab HCBF5/6 has lower IC50 (Fig. 4 d) on LS174T cell, enters One step ground confirms that the cell killing for S-Fab HCBF5/6 mediation of expressing of CEA is important.

T cell combines the cell killing activity of mediation S-Fab

Essential in order to confirm the cell killing that S-Fab HCBF5/6 is induced by T cell further, competed Property measure.CD3⁺T cell and LS174T use with the ratio of E:T=10:1.When 1nM HCBF5/6 is used for tumor cell and T During cell, cause effective cell killing (Fig. 5).But, when applying the Fab HCBF4/5 of increase, cell killing is imitated Should reduce.Owing to single FabHCBF4/5 does not have impact to cell killing, these data show that S-Fab HCBF5/6 needs to combine Cell killing is guided to T cell.When the combination of T cell is by the competition isolation of control antibodies HCB4/5, then induction of relatively low Killing functions of immunocytes.

S-Fab suppresses tumor growth in vivo

This embodiment tests whether S-Fab HCBF5/6 can suppress tumor growth in vivo.LS174T cell and people PBMC mixes, and then transplants under NOD-SCID mouse epithelial.Then, the S-Fab HCBF5/6 of these mice 20ug, or make PBS or comparison Fab HCBF4/5 (n=5) for matched group process.For only by the tumor of PBMC treatment or containing comparison Fab The tumor of the PBMC treatment of HCBF4/5 does not observe Tumor growth inhibition.Mice for treating with PBMC and HCBF5/6 is seen Observe obvious Tumor growth inhibition (Fig. 5).In the mice treated with PBMC and HCBF5/6, the mice of only 2/5ths Define tumor.Tumor growth is not observed in other three groups of mices.These data validations S-Fab HCB5/6 can be special Suppress the tumor growth in transplanted tumor mice different in naturely.

This embodiment offers the new form (S-Fab) of bispecific antibody.Bispecific antibody is as strong immunity Therapeutic Method has caused great interest.Various forms for their anti-tumor activity have been suggested and have ground Study carefully.Among them, because the anti-tumor activity that BITE is strong by being directly targeted induced t cell, so BITE (two scFvs The antibody merged) present wide prospect.Blinatumomab (targeting the BITE antibody of CD19 and CD3) has been allowed to It is used clinically for B cell leukemia.But, scFv fusion protein (being used by technology similar with other for BITE) is had The trend assembled and the difficult problem expressed in bacterial cell.

A kind of new bispecific antibody (S-Fab) is based on conventional Fab (Fig. 2 b), and wherein single domain antibody is connected to VH- The C end of CH1.Being different from BITE or other scFvs, the anti-CD3 part of S-Fab is the native form of Fab in our current research, because of This more they tends to stable.Test some different anti-cd 3 antibodies, show owing to being derived from the AntiCD3 McAb Fab of UTCH1 clone E.coli. more preferably express and solubility, thus it is used in our current research.Owing to this AntiCD3 McAb Fab can be in E.coli Expressing and generate, it greatly reduces the complexity that ScFv expresses, and ScFv typically requires the mammalian cell expression system of costliness.

S-Fab provides the new model of bispecific antibody, and it has multiple advantage.Use natural human anti-in the present invention CD3Fab, T cell can participate in killing tumor cell.

The cytotoxicity that embodiment 2. bispecific antibody induction NK-is cell-mediated

This embodiment offers a kind of new bispecific antibody form Her2-S-Fab, by by anti-for single domain CD16VHH even Be connected to Herceptin (Trastuzumab,) C-terminal of Fab is built-up.Her2-S-Fab can be through antibacterial Cell is expressed and purification.In vitro in cell experiment, Her2-S-Fab can kill Her2 by recruitment NK cell-specific and cross table The cancerous cell reached.Compared with Herceptin, it was observed that the tumor cytotoxicity effect strengthened.In vivo study shows, Her- 2-S-Fab can suppress tumour progression.

Method and material

Fab design and protein purification

The construct of Her2-S-Fab and comparison Her2-Fab is shown in Fig. 6 a, by standard DNA clone technology, first Anti-Her2VL-CL and VH-CH1 of chemosynthesis Herceptin (being shown in Table 5), and it is cloned into pET21a carrier and pET26b carrier In.Subsequently, VHH-CD16 is cloned into the C-end of the anti-Her2VH-CH1 of Herceptin.Signal sequence is added at N-end PelB expresses to realize periplasmic.The Heterodimerization of VL-CL/VH-CH1-VHH (CD16) defines Her2-S-Fab, and The Heterodimerization of VL-CL/VH-CH1 defines Her2-Fab.

Periplasmic protein purification in E.coli is summarized as follows.Encode two plasmids of not homopolypeptide by cotransformation extremely Have in BL21 (DE3) cell of suitable antibiotic labelling.Inducible protein is expressed and extracts periplasmic.By Ni-NTA fine jade Lipolysaccharide affinity chromatography and anti-igg CH1 affinity purification are purified into anti-from sucrose with the mixture of periplasmic composition Her2Fab or Her2-S-Fab (Fig. 6 b).Use GE Hiload 16/600Superdex 200pg by manufacturer (GE) explanation Carry out gel filtration.The protein label of gel filtration is purchased from Sigma (MWGF1000).

Cell line and animal

Her2 positive cancer cell system SKBR3 (human breast cancer cell), BT474 (human breast cancer cell), SKOV3 (people's ovary Cancerous cell), MCF7 (human breast cancer cell) and Her-2 negative carcinoma cell lines MDAMB435, MDAMB468, Chinese hamster ovary Cell (CHO) is purchased from Chinese Academy of Sciences's Type Culture Collection (Shanghai).SKBR3, SKOV3 and MDAMB435 cell is incubated at interpolation 10%HI hyclone (Gibco, Life Technologies, USA) and 1% penicillin/streptomycin (HyClone) In DMEM culture medium (Gibco, Life Technologies, China)；BT474 and Chinese hamster ovary celI are incubated at and with the addition of 10%HI The RPMI-1640 training of hyclone (Gibco, Life Technologies, USA) and 1% penicillin/streptomycin (HyClone) Support in base (Gibco, Life Technologies, China)；MDAMB468 cell is incubated at L15 (Gibco, Life Technologies, China) in, it is placed in the CO2 incubator of temperature 37 DEG C, humidity 5%.

Nod/SCID mice is purchased from Zhongshan University's Experimental Animal Center.Human blood gathers, Animal Care and zoopery by Zhongshan University ratifies.

Hemocyte separates

Ficoll density centrifugation is used to prepare human peripheral blood mononuclear cell (PBMC) from healthy donor.Use EasySep^TM NK cells of human beings enrichment kit (STEMCELL Technologies, Inc., Vancouver, Canada) purified NK cells.Point From NK cell be incubated in the complete RPMI1640 culture medium containing 10%FBS and 1% penicillin/streptomycin, be placed in 37 DEG C, 5%CO₂Humidified incubator.

Flow cytometry

When tissue culture cells degrees of fusion reaches 80-90%, add 0.25% trypsinization.1500rpm from The heart presses 1 × 10 in 5 minutes⁵Cell/sample collection cell, then cleans twice with PBS+0.1%BSA ice-cold for 3ml.Cell mass In the PBS+0.1%BSA that 500ul is ice-cold resuspended.Each test tube is separately added into Herceptin, Her-2Fab or Her2- S-Fab resists as one；Sheep-Anti-Human IgG (H+L) Alexa Fluor 488 (Invitrogen, cat#A11013) resists as two. HER2/neu-PE (BD, cat#340552) is as comparison.Clean after twice of cell, carry out flow cytometry.

Cell toxicity test

SKBR3, BT474, MCF7, SKOV3, MDAMB435, MDAMB468 and Chinese hamster ovary celI are by as target (T) cell.People The NK cell (without stimulating) of PBMC cell or separation is used as effect (E) cell.Target cell (5000 cells) quilt of 100 μ l It is transferred to each hole (in triplicate) of 96 orifice plates.After hatching at 12 hours, isopyknic NK cell is with the ratio of E:T (10:1) Example is added to each hole.It is subsequently adding the antibody of shown concentration from 0.01ng/ml to 10 μ g/ml.After hatching at 72 hours, Use CCK8 reagent (Dojindo, CK04) that cell survival rate is carried out quantitative analysis.Target cell survival rate (%) is according to this formula Calculate: [(survival target cell (sample)-culture medium)/(survival target cell (comparison)-culture medium)] × 100.

Tumor growth in vivo inhibition test

From cell culture fluid, gather in the crops SKOV3 cell, wash once with PBS, resuspended in PBS, then donate with from health The PBMC mixing that person has just separated.Cell suspension, is mixed in NOD/SCID right side of mice, cumulative volume 0.2mL/ mice by subcutaneous injection There is 2x10⁶SKOV3 cell and 1x10⁷Human PBMC.After SKOV3/PBMC cell transplantation 2 hours, lumbar injection (i.p.) resisted Body or vehicle Control (PBS).Then, treated these animals ensuing 6 day every day and observe mice state.Use slide gauge Measure gross tumor volume in two perpendicular directions, use formula (width²X length)/2 calculating volumes.All results are with each The arithmetic mean of instantaneous value of group represents.

Result

The purification of Her2-S-Fab and Her2-Fab

The two-step method that is purified by of Her2-Fab and Her2-S-Fab realizes, and first by Ni-NTA-agarose, re-uses Anti-CH1 affinity purification (Fig. 6 b).Because VHH is relatively small and be solubility, adds anti-CD16VHH and do not interfere with anti- The expression of Her2Fab and dissolubility.The dissolubility of Her2-S-Fab and expression are with to compare Her2-Fab suitable (0.6mg/L).In order to determine that Her2-S-Fab is the most correctly folded into heterodimeric thing, gel filtration is used to analyze The albumen of purification.The major part of albumen defines single peak.Light chain and heavy chain form complete Fab antibody, and molecular weight is about 65kD (Fig. 6 c) and about 50kD (data are not shown), similar to the expection molecular weight of Her2-S-Fab and Her2-Fab respectively, table Bright major part Her2-S-Fab the most correctly folds.

Her2-S-Fab identifies Her2 positive cell

Whether can be bound to Her2 positive expression cell for checking Her2-S-Fab, utilize HER2 positive and HER2 is negative Cell carries out flow cytometry.Result shows, use compare anti-Her2 antibody, Her2 negative cells CHO, MDAMB435 and MDAMB 468 has low-down, even without dyeing；MCF7 has low-down Her2 to express；And BT474, SKBR3 and SKOV3 cell has high Her2 and expresses (Fig. 7 a).

Herceptin can dye HER2 express cell (Fig. 7 b).Her2-Fab and Her2-S-Fab can be bound to HER2 positive cell (Fig. 7 c and 7d), and demonstrate similar fluorescence intensity change, show to compare Fab and Her2-S-Fab tool There is similar binding ability.Her2-Fab with Her2-S-Fab also shows that in the staining pattern that Herceptin is identical, intensity Relatively low, this is consistent with the unit price character of Her2-Fab and Her2-S-Fab.

The cytotoxicity of Her2-S-Fab induced NK cell mediation

In order to evaluate the cytotoxicity of Her2-S-Fab, Her2-Fab, Her2-S-Fab and Herceptin by with The NK cell of cancerous cell and fresh separated is cultivated together.Under the concentration of 10 μ g/ml, even if there is NK cell, HER2-is negative Cell line CHO is not the most observed cytotoxicity (Fig. 8 a).For cell line SKOV3 of HER2 process LAN, when there is not NK During cell, only Herceptin reduces cell proliferation, and survival rate is 72% (Fig. 8 a), Her2-S-Fab or Her2-Fab is to carefully Born of the same parents' survival rate is without impact.But, when there is NK cell, the cytotoxicity that Her2-S-Fab induction is strong, and compare toltrazuril The cytotoxicity of monoclonal antibody is higher (survival rate 10.70%vs33.12%) (Fig. 8 a).The cytotoxicity of Her2-S-Fab depends on Anti-CD16, even if because individually Her2-Fab does not has cytotoxicity (Fig. 8 a) in the case of there is NK cell yet.

For evaluating Her2-S-Fab effect on tumor cell further, we determine different antibodies on cancerous cell Dose response.Her2-Fab has no or only little impact to cell survival rate, regardless of whether how Her2 expression status (schemes 8b-f).Herceptin only has slight tumor inhibition effect (figure when without NK cell to Her2 high expressing cell SKOV3 8b-f).In the case of there is NK cell, SKBR3 and SKOV3 cell is caused strong by Herceptin and Her2-S-Fab Cytotoxicity, and in dose dependent, but (Fig. 8 b-is not acted on for HER2 negative cells system MDAMB435 and CHO f).MCF7 (Her2 low expression cancerous cell) is needed to the antibody ability inducing cell toxic action of high concentration, show Her2-S- The activity of Fab depends on the expression of Her2.In MCF7, SKBR3 and SKOV3 cell, Her2-S-Fab demonstrates more appropriate than song The cytotoxicity that pearl monoclonal antibody is higher, shows that Her2-S-Fab is more effective with Herceptin.

Her2-S-Fab suppresses tumor growth in vivo

Adoptive transfer model is used to test whether Her2-S-Fab can suppress tumor growth in vivo.First will SKOV3 cell and human PBMC's mixing with cells, subcutaneous transplantation is in NOD/SCID mice subsequently.Mice is subsequently with PBS, Her2-Fab Or Her2-S-Fab process.For the mice processed with Her2-Fab, do not observe the suppression of tumor growth.But, for Her2-S-Fab, it was observed that powerful Tumor growth inhibition phenomenon (Fig. 9).

Therefore, Her2-S-Fab can be used to make NK cell guide Her2 overexpressing cell.Her2-S-Fab can be at body Interior and external killing Her2 positive cancer cell effectively.

The all patents quoted in description and other lists of references are all one skilled in the art of the present invention They are incorporated into herein with integral form by quoting by the expression of level, including any form therein and accompanying drawing, as With each list of references separately through quoting and being incorporated into herein with its integral form as.

Those skilled in the art's meeting it is readily appreciated that the present invention can easily transform and obtain those described herein purpose and Advantage and lie in those purposes herein and advantage.Retouch with the form of the representative of current preferred mode in this article Method, variant and the compositions stated are exemplary, are not intended to limit the scope of the present invention.For those skilled in the art For, they can be made a change or use it for other purposes, but this is included in such as this of claims definition In bright scope.

Additionally, when describing some features or the aspect of the present invention with Ma Kushi group or other substitute group forms, One skilled in the art will recognize that, the present invention is also with this Ma Kushi group or any single member of other groups or member The form of subgroup is described.

And, unless there are phase antirepresentation, otherwise when some embodiments, provides various quantitative value, any by taking Two different values, as the end points of a value range, describe other embodiments.These value ranges are also at the model of the present invention Within enclosing.

At the appropriate time, the content the most illustratively described can lack not specifically disclosed herein any one Or implement in the case of multiple element, one or more restrictive condition.It is therefoie, for example, in the case of every kind herein, term " include ", " substantially by ... composition " and " Consists of " in either of which can be substituted by other two terms.Therefore, Should be understood that, although the present invention is specifically disclosed by preferred implementation and optional feature, but those skilled in the art Idea disclosed herein can be made modifications and variations, and these modifications and variations still fall within the basis that claims define Within the scope of invention.

Claims (16)

1. a bivalent antibody, comprises (a) and contains the light chain of antibody and the Fab (Fab) of Partial heavy, and B () is fused to the single domain Fab (VHH) of the C end of described light chain or described Partial heavy, wherein said Partial heavy Do not comprise CH2 or CH3 territory.

Bivalent antibody the most according to claim 1, wherein said VHH fragment is fused to described Partial heavy.

Bivalent antibody the most according to claim 2, wherein said bivalent antibody comprises further and is fused to the second of light chain Plant VHH fragment.

4., according to the bivalent antibody described in any one in claims 1 to 3, wherein said Fab fragment and described VHH fragment are every One all has the specificity to tumor cell or immunocyte.

Bivalent antibody the most according to claim 4, wherein said Fab fragment has specificity, and institute to tumor antigen State VHH fragment and immunocyte is had specificity.

Bivalent antibody the most according to claim 5, wherein said tumor antigen selected from CEA, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, mucoprotein, TAG-72, CIX, PSMA, folic acid combine egg In vain, GD2, GD3, GM2, VEGF, VEGFR, integrin, α V β 3, α 5 β 1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin.

Bivalent antibody the most according to claim 5, wherein said tumor antigen is CEA or Her2.

Bivalent antibody the most according to claim 5, wherein said VHH fragment is to mammalian T cell or mammal NK Cell has specificity.

Bivalent antibody the most according to claim 5, wherein said VHH fragment to selected from CD3, CD16, CD19, CD28 and The antigen of CD64 has specificity.

Bivalent antibody the most according to claim 9, wherein said antigen is CD16 or CD3.

11. exist according to the bivalent antibody according to any one of Claim 1-3 or claim 5 to 9, wherein said VHH fragment Val, Gly, Leu and Trp residue is not the most comprised at Kabat site 37,44,45,47.

12. 1 kinds of host cells, its bivalent antibody comprised described in one or more coding any one of the claims is many Nucleotide.

13. host cells according to claim 12, wherein said host cell is bacterial cell or yeast cells.

14. host cells according to claim 12, wherein host cell isE. coli。

15. 1 kinds of methods preparing soluble antibody, described method comprises the place cultivated according to any one of claim 12-14 Chief cell, and the antibody that results are expressed in described cell.

The application in the medicine of preparation treatment cancer of the bivalent antibody described in any one of 16. claim 1-11.