CN106075433A - Many targeted nanos antibody polymerization chain junctor that a kind of probiotic bacteria that can be used for treatment of cancer is expressed - Google Patents
Many targeted nanos antibody polymerization chain junctor that a kind of probiotic bacteria that can be used for treatment of cancer is expressed Download PDFInfo
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Abstract
The present invention relates to many targeted nanos antibody Nanobody (Nb that a kind of probiotic bacteria is expressed, or VHH) polymeric chain junctor, it is formed by connecting by polypeptide link Linker for the VHH monomer nano antibody of target spot HER2, EGFR and VEGF by specificity, and by the DNA transfer load of described many targeted nanos antibody polymerization chain junctor in probiotic oral.Its probiotic oral bacterial strain can survive in human body gastrointestinal system certain time, expresses and discharge many targeted nanos antibody of polymerization link, for treatment gastric cancer.The nano antibody polymeric chain junctor of the present invention has played that nano antibody volume is little, stability is high, acid and alkali-resistance environment, the advantage that can penetrate tissue, overcome nano antibody in the short defect of people's Half-life in vivo, start the new paragon of oral antibody drug treatment gastric cancer.The nano antibody polymeric chain junctor of the present invention has played the effect of many targeted inhibition growth of cancer cells, and antitumaous effect mixes enhancing decades of times or even hundreds of times at random than simple monomeric igg.
Description
Technical field
The present invention relates to the polymeric chain junctor of a kind of nano antibody, utilize food stage probiotic bacteria to be expressed by genetic modification and receive
Rice antibody polymerization chain junctor target stomach cancer tumor targets is to treat gastric cancer.
Background technology
Gastric cancer is one of common malignant tumor of digestive system, Most patients when diagnosis already at the terminal stage of a disease.
In world wide, annual mortality of gastric carcinoma number accounts for the 2nd of tumor mortality number.The annual death rate of every 100,000 populations of China's gastric cancer
It is 25.21%, in various malignant tumor, accounts for first place.Chemotherapy is still the primary treatments of late gastric cancer, but the curative effect of chemotherapy
Difficult fully up to expectations, local recurrence and metastasis are the main causes of postoperative 5 years survival rates of impact.Existing chemotherapeutic levels arrives
One bottleneck, optimum response rate is less than 50%, and middle position Overall survival rarely exceeds 12 months, it is desirable to have breakthrough, and Biotherapeutics becomes
For necessary selection.
Molecular targeted therapy is the new treatment means emerged in treatment blood and entity tumor in recent years.Along with to depositing
It is that gastric cancer occurs, develops and the research of transfer process Middle molecule biological mechanism, the most this treatment means is applied to
The clinical practice of curing gastric cancer.Current this therapeutic strategy mainly includes EGFR inhibitor, VEGF inhibitor, HER2 inhibitor
Deng, how species specific molecular biosciences targeted therapy means achieve greater advance in Comprehensive Therapy on Gastric Carcinoma.
EGF-R ELISA (EGFR) is the transmembrane receptor of a kind of multi-functional glycoprotein, be tyrosine kinase growth because of
One member of sub-receptor family, with ligands specific such as epidermal growth factor (EGF), transforming growth factor α (TGF-α), two-way
Regulation albumen (AR), beta cell element (BTC) etc. combine, by the self phosphorylation activated receptor of corresponding tyrosine kinase,
Thus a plurality of signal transduction pathway such as MAPK, PI3K, c-Src in activated cell etc. promote tumour cell division, migrate and
Promote tumor angiogenesis.EGFR has activation in various degree to express in quite a few tumor, such as gastric cancer, knot directly
Intestinal cancer, head and neck scale carcinoma, cancer of pancreas, pulmonary carcinoma, breast carcinoma, renal carcinoma and Gliblastoma etc..Therefore, its specific part is selected to make
For target spot, conducted by interference EGFR signal, reach to suppress tumor proliferation, infiltration and the purpose of metastasis, divide for gastric cancer
Sub-targeted therapy provides new thinking.
Representing medicine is Herceptin (trastuzumab, herceptin), handkerchief trastuzumab (pertuzumab,
Omnitarg).EGFR and the HER2 belonging to erbB family member can form heterodimer, can by HER2 kinase activator phosphorylation
Enabling signal transduction strengthens malignant transformation of cells and promotes tumour progression.Herceptin is U.S. FDA approval listing in 1998
The derivative humanization mAb of the first recombinant DNA, affect the transmission of growth signals by being combined with HER2 receptor-specific, it
VEGF and other angiogenesis factors activity can also be lowered.Within 2002, enter China market, trade name
" Trastuzumab ".Handkerchief trastuzumab is second filial generation recombinant humanized monoclonal Anti-HER 2, II binding domain outer with HER2 receptor born of the same parents
In conjunction with the dimerization stoped between other HER2 receptors.Scholar is had to use chromogenic in situ hybridization (CISH) to detect 131 examples
Stomach organization specimen and 100 example cardiac carcinoma tissue specimen, as a result 16 examples (12.2%) gastric cancer specimen and 24 examples (24%) carcinoma of gastric cardia mark
This HER2/neu overexpression.Herceptin treatment can make HER2 breast cancer patients with positive OS extend, but HER2 is in breast carcinoma
In expression rate be also only 20～30%, HER2 is expressed as 6～35% in gastric cancer, and Herceptin application in gastric cancer is
Gratifying results are obtained through clinically.
Tumor growth has clear and definite blood vessel dependency, and tumor obtains nutritional labeling by new vessels from host, again warp
Cross blood vessel and carry tumor cell to host, strengthen the metastasis ability of tumor stove.Therefore angiogenesis inhibitor is also gastric cancer molecule
The study hotspot of targeted therapy.One of great advantage of this kind of medicine is will not to produce tumor drug resistance, in combined chemotherapy very
Favorably.There is multiple angiogenesis inhibitor at present just in clinic trial.VEGF VEGF is current known effect
The strongest promotees the cytokine that blood vessel endothelium generates.Multiple studies have shown that, VEGF expression in progressing gastric carcinoma is obvious
Increase, closely related with the infiltration metastasis of tumor and prognosis.
Cancer drives its growth and diffusion often by many A signal pathways, thus, compound targeted therapy will carry
High therapeutic effect, reduces the possibility developed immunity to drugs.
In recent years, molecular targeted therapy achieves some achievements in the treatment of gastric cancer, but owing to preparation is the most multiple
The reason such as miscellaneous, expensive, range of application receives certain restriction.And the appearance of nano antibody, bring common molecular target
Not having the advantage that to medicine, its molecular weight is little, and structure function character is unique, provides newly to the targeted therapy of gastric cancer
Treatment means and Research Thinking.
There is the heavy chain antibody of disappearance light chain in nature in camel body, cloning the single domain antibody that its variable region obtains is
Little functional antigen binding fragment, relative molecular mass (Mr) be only 15KD, referred to as nano antibody, have molecular mass little,
The feature such as stability is strong, solubility good, easy expression, immunogenicity are low.The genetic engineering antibody of this miniaturization basic research,
Have broad application prospects in developing new drug and the diagnosis of disease and treatment.Nano antibody is as a kind of small-sized genetic engineering
Antibody, its special structure makes it have the peculiar property not available for common antibody: water solublity height, good stability, targeting
By force, humanization is simple and easily obtains and expresses.And three big effect (surface, size and the macroscopic quantum tunnelings that nano antibody is unique
Effect) so that it is all it is substantially better than conventional antibodies at a lot of aspects.
Human body intestinal canal exists 100,000,000,000,000 antibacterials, about about 1Kg.Almost all of intestinal bacteria all can be found at colon,
Collectively constitute intestinal microbial population.Each antibacterial is unicellular, about different more than 100 kind.Only more than 40, kind is main
Antibacterial composition intestinal microbial population, is probiotic bacteria mostly, and remaining is to may result in disease flora.Probiotic bacteria is for ensureing that body health is to closing
Important, immunity of organisms can be improved, reduce serum cholesterol, facilitating digestion system health.
Probiotic bacteria (probiotics) is to be colonizated in human body gastrointestinal system, can produce definite health efficacy thus improve place
Main microecological balance, the active beneficial microbe useful to host of performance beneficial effect.The antibacterial useful to human body or fungus
Mainly have: clostridium butyricum, lactic acid bacteria, bacillus bifidus, bacillus acidophilus, actinomycetes, yeast etc..
Nineteen eighty-three U.S. Tufts, university two distinguished professor Sherwood Gorbach and Barry Goldin was from healthy human body
Isolate LGG (lactobacillus rhamnosus).LGG is one of widest probiotic bacteria of human research, and it is subordinated to Lactobacillus, is
One of Body normal flora, it is possible to tolerance animal alimentary canal environment, in humans and animals intestinal, field planting is up to two weeks, activity by force,
Intestinal Stickiness is high, colonization ability is strong, energy efficiently cholesterol reducing, promotes cell division, plays regulating intestinal canal flora, prevent and control
Treat diarrhoea, get rid of toxin, pre-anti-caries and improve the effects such as immunity of organisms.
Probiotic bacteria can coexist with host, and can produce the material promoting host intestine microecological balance effect, more pathogenic
Bacterium and subtract toxic bacteria there is bigger application advantage.Digestive is the main place that probiotic bacteria is lived away from home, at probiotic bacteria and entity
There is hypoxia metabolism district in tumor, therefore can utilize the anaerobe carrier as therapy of tumor of hypoxia.Anaerobe conduct
The carrier of gene therapy, has that tumor-targeting is strong, safety high, is a kind of novel gene therapy vector.When detesting
When oxygen bacterium carries tool medicative genes of interest, can effectively suppress the growth of solid tumor cell.
Prebiotics is the prebiotic strain of scalable or the material of activity.General prebiotics provides " food " to probiotic bacteria, it is possible to
Decomposed by beneficial bacteria in intestinal and absorb, promote beneficial bacteria growth and breeding.Prebiotics includes oligofructose, partially protein class
Material and protein hydrolysate, dextrinosan, natural plant extracts class and polysaccharide bifidus factor etc..
The material that oligofructose is naturally-occurring, has good promotion bacillus bifidus proliferation activity, in inducing intestinal
Probiotics amount reproduction with bacillus bifidus as representative, improves enteric microorganism environment, improves body immunity, and can be in conjunction with intestinal
Interior noxious substance, and promote to excrete, additionally, also have reduction serum cholesterol levels, reduce blood glucose and decompose carcinogenic
The functions such as thing.Research finds, it will not cause dental caries, the most also use as water soluble dietary fiber, and in addition, it is also
Can use in some need the food of low-temperature heat.
Root according to the survey, has numerous protein hydrolysate and partially protein class material inside bifidus factor, these
Substance advantageous is in the growth of promotion bacillus bifidus, and these protein substances or the common component of protein hydrolysate, that is just
Being the peptide of sulfur-bearing, this sugar polypeptide may advantageously facilitate bacillus bifidus and grows in intestinal, if the disulfide bond of the peptide of these sulfur-bearings
It is reduced or alkylation, then it can be made to lose activity, do not have the effect promoting bifidobacterium growth, therefore, it can
Know the substantial connection of the disulfide bond in these protein substances and protein hydrolysate and bacillus bifidus healthy growth.
Dextrinosan is tetrose, pentasaccharides, dextrinose combine the glucose of a part and formed, the most on the market
Applying more, great majority are used in various types of beverages and food.It can use with substituting saccharose, this is because its sweet taste is sweet and refreshing,
Can reduce again food sugariness reaches to benefit the purpose of health simultaneously, therefore adds glycosuria to as natural additive
Patient's food is more satisfactory, and it will not cause dental caries.Finding according to research, it is also equipped with heat resisting
Property, the performance of this heat resisting makes it be convenient to be added in acidic beverages the process accepting food high temperature sterilizing.
Research report shows, there is the material that can promote bifidobacterium growth in a large number, such as in natural plant extracts
Tea polyphenols in Folium Camelliae sinensis, Rhizoma amorphophalli hydrolyzed solution, Thallus Laminariae (Thallus Eckloniae), Jerusalem artichoke powder etc. possess this function.Bifidus factor there are polysaccharide thing
Matter, these materials include the nitrogenous polysaccharide in Brown algae Sulfated saccharides, Radix Dauci Sativae, krestin etc..
Summary of the invention
It is an object of the invention to provide many targeted nanos antibody polymerization chain junctor that a kind of probiotic bacteria is expressed, to be applied to stomach
The targeted therapy of cancer.
Foregoing invention purpose of the present invention be by using probiotics strain as many targeted nanos antibody Nanobody (Nb, or
VHH) delivery vehicles of polymeric chain junctor medicine, by the DNA transfer load of many targeted nanos antibody polymerization chain junctor in oral benefit
Express nano antibody polymeric chain junctor in raw bacterium, make probiotic bacteria survive in human body gastrointestinal system certain time by genetic modification,
Under the support of prebiotics regulates, express and release nano antibody polymeric chain junctor biological medicament target stomach cancer at human body gastrointestinal system
The Mutiple Targets of tumor, in suppression tumor, EGFR, HER2, VEGF signal path realizes.
Specifically, many targeted nanos antibody polymerization chain junctor that probiotic bacteria of the present invention is expressed is to be received by specific monomer
Meter Kang Ti VHH by polypeptide link Linker connect and compose nano antibody polymerization connector, and using probiotic oral bacterial strain as
The delivery vehicles alive of described nano antibody polymerization connector medicine, wherein, described monomer nano antibody VHH is with HER2
(human epidermal growth factor receptor 2 or HER2/neu)、EGFR(epidermal growth
Factor receptor) and/or antibody Nb-that VEGF (vascular endothelial growth factor) is target spot
EGFR, Nb-HER2 and/or Nb-VEGF.
Further, the monomer nano antibody VHH in described many targeted nanos antibody polymerization chain junctor can complete people source
Change transformation, to reduce its immunogenicity.
In the present invention, described many targeted nanos antibody polymerization chain junctor is by monomer nano antibody Nb-HER2, Nb-EGFR
And/or couplet, trimerization chain junctor or four that Nb-VEGF is coupled together by polypeptide link Linker in any combination gather
Chain junctor.Including: Nb-EGFR polypeptide link Linker Nb-HER2；Nb-EGFR polypeptide link Linker Nb-
VEGF；Nb-HER2 polypeptide link Linker Nb-VEGF；Nb-EGFR polypeptide link Linker Nb-HER2 polypeptide
Link Linker Nb-VEGF；Nb-VEGF polypeptide link Linker Nb-EGFR polypeptide link Linker Nb-
HER2 polypeptide link Linker-Nb-VEGF etc., and other any combination of coupling including three target spot nano antibodies
Body, trimerization chain junctor or four poly chain junctors.It is little that the nano antibody polymeric chain junctor of the above-mentioned composition of the present invention has played nano antibody
Volume, high stability and acid and alkali-resistance, the advantage such as tissue, many targeting anticancer compound can be penetrated, overcome selectively targeted receiving
Meter Kang Ti, in the short defect of people's volume lifetime, can greatly improve the therapeutic effect of gastric cancer.
In many targeted nanos antibody polymerization chain junctor of the present invention, described monomer nano antibody links Linker by polypeptide
End to end, tail tail or head connect.Its connected mode has multiple, and Fig. 1 lists 26 kinds of on-link mode (OLM)s that most probable occurs.
Further, many targeted nanos antibody polymerization chain junctor of the present invention is by different polypeptide link Linker
Connect the different nano antibody polymeric chain junctor that monomer nano antibody is formed.Wherein for connecting monomer nano antibody VHH's
Polypeptide link Linker includes short Linker, middle Linker or long Linker, and specifically, described short Linker is by less than 15 ammonia
Base acid forms, and middle Linker is made up of 15-25 aminoacid, and long Linker is made up of 26-60 aminoacid.Hereinafter list
The sequence of a part of polypeptide link Linker, it should be clear that, the sequence of the polypeptide link Linker of the present invention includes
But it is not limited to listed polypeptide link Linker sequence.
The probiotic oral bacterial strain of present invention application food stage, expresses above-mentioned compound people including lactobacillus or bacillus bifidus
The nano antibody group in source, and it is made into oral formulations, with production and the delivery of beneficially probiotic bacteria humanization nano antibody.
Wherein, described lactobacillus includes bacillus acidophilus (Lactobacillus acidophilus), lactobacillus casei
(Lactobacillus casei), Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus bulgaricus
(Lactobacillus bulgaricus), Deshi Lactobacillus (Lactobacillus delbrueckii), Lactobacillus fermenti
(Lactobacillus fermentium), Lactobacillus gasseri (Lactobacillus gasseri), lactobacillus helveticus
(Lactobacillus helveticus), Lactobacillus johnsonii (Lactobacillus johnsonii), Lactobacillus paracasei
(Lactobacillus paracasei), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus reuteri
(Lactobacillus reuteri), lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus salivarius
(Lactobacillus salivarius), and lactococcus lactis Lactococcus, Lactococcus lactis subsp.lactis
(Lactococcus lactis subspecies lactis) etc..
And then, the present invention also includes selectivity prebiotic substance, described selectivity prebiotics in described oral formulations
Material includes bifidus factor, to help growth and the activity of specific bacterial strain.
Further, described selectivity prebiotic substance also includes oligofructose, protein substance and protein water
One or more in hydrolysis products, dextrinosan, natural plant extracts, polysaccharide bifidus factor.
Preferably, described oral formulations is made the medicine including the forms such as capsule, tablet or oral liquid formulations by the present invention
Agent type.And then, it is also possible to described oral formulations is made food form and takes, such as Yoghourt, beverage, cookies etc..
Many targeted nanos antibody polymerization chain junctor that the probiotic oral present invention provided is expressed is as anti-gastric cancer medicament
Application, its probiotic bacteria can be survived certain time in human body gastrointestinal system by genetic modification, expressed and discharge many targeted nanos
Antibody polymerization chain junctor, using as anti-gastric cancer medicine many targeted therapies gastric cancer.
The present invention characteristic according to cancer target and the essential condition mechanism of tumor growth, initiate and design Mutiple Targets nanometer
Antibody polymerization chain junctor, they many integrations (two one, the Trinity or quaternity), various different anti-by associating
Cancer mechanism, has played the effect of many targeted inhibition growth of cancer cells simultaneously.In many targeted nanos antibody polymerization chain junctor one
Point nano antibody (both nano antibodies of HER2 and EGFR target spot) can be done directly on cancer cell surfaces and the inside HER2 and
EGFR, stops growth of cancer cells signal path；Another part (both nanometers of VEGF target spot of nano antibody polymeric chain junctor simultaneously
Antibody) at the signal path of cancerous cell periphery prevention tumor neovasculature generation, and tumor growth can be suppressed to support system to be formed, make
Become cancer cell-apoptosis, as shown in Figure 2.The antitumaous effect of many targeted nanos antibody polymerization chain junctor is combined than simple monomeric igg
The antitumaous effect decades of times to be strengthened of (the most multiple but non-polymeric chain connects the random of nano antibody and mixes) or even hundreds of times.
Compared with prior art, the invention have the advantages that.
1) present invention probiotic bacteria express many targeted nanos antibody polymerization chain junctor played nano antibody volume little,
The advantages such as molecular weight is little, simple in construction, heterologous are low, high stability and acid and alkali-resistance, by monomer nano antibody with last a long time
Micel link merges, and improves the nano antibody existence time in blood, overcomes selectively targeted nanometer and resists
Body, in the short defect of people's volume lifetime, can penetrate tissue by continuous print, form the growth of association mechanism anticancer, significantly carry
The therapeutic effect of high gastric cancer.
2) production and the use cost of many targeted nanos antibody polymerization chain junctor that probiotic bacteria of the present invention is expressed are the lowest
Honest and clean.
3) many targeted nanos antibody polymerization chain junctor that the probiotic bacteria of the present invention is expressed has the couplet of uniqueness with multi-joint
Shape, it is provided that the multi-functional gastric cancer tumor of the many mechanism of many targeting is treated.
4) present invention initiates the VHH nano antibody of particular design and is combined chain junctor group, can play and be formed comprehensive many mechanism
Eliminating and hinder cancerous cell growth effect, both composite Nano antibody population can play simultaneously and carry out Mutiple Targets anticancer growth
And promote the effect of cancer cell-apoptosis, significantly improve nano antibody curing gastric cancer effect.
5) many targeted nanos antibody polymerization chain junctor that probiotic bacteria of the present invention is expressed utilizes food stage probiotic bacteria to pass through gene
Nano antibody polymeric chain junctor target stomach cancer tumor targets is expressed in transformation, and this oral food level genetic modification probiotic bacteria can survive in
Regular hour in human body gastrointestinal system, under the regulation of prebiotics assists, express at human body gastrointestinal system and release many targeting
Nano antibody polymeric chain junctor, to reach to treat the effect of gastric cancer.
Accompanying drawing explanation
Fig. 1 lists coupling or the cascade schematic diagram of the chain junctor of targeted nano antibody polymerization more than 26 kinds.
Fig. 2 gives many targeting of two one, the Trinity or the coupling of quaternity various ways or cascade
The interaction of nano antibody polymeric chain junctor and cancer cell antigen and spatial distribution schematic diagram.
Composite Nano antibody includes that the nano antibody couplet of targeting HER2, VEGF, EGFR or concatemer composite Nano resist,
Wherein connect the polypeptide link Linker of monomer VHH be divided into short, in, long three classes, short Linker is formed by less than 15 aminoacid,
Middle Linker is made up of 15-25 aminoacid, and long Linker is made up of 26-60 aminoacid.
In Fig. 1, n ' represents the head of humanization monomer nano antibody, and c ' represents the afterbody of humanization monomer nano antibody.
Each humanization monomer nano antibody by polypeptide link Linker end to end, tail tail or head connect.
Detailed description of the invention
Following embodiment is only the preferred technical solution of the present invention, is not used to the present invention is carried out any restriction.For
For those skilled in the art, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, made
Any modification, equivalent substitution and improvement etc., should be included within the scope of the present invention.
One of main technologies of the present invention is the probiotics strain by genetic modification, can be at human body gastrointestinal system table
Reach many targeted nanos antibody polymerization chain junctor, both target stomach cancer tumor targets HER2 (human epidermal growth
Factor receptor 2, orHER2/neu), EGFR (epidermal growth factor receptor) and VEGF
(vascular endothelial growth factor), prepares special probiotic oral gastric cancer medicament preparation.
One of main technologies of the present invention is to be carried by many targeted nanos antibody polymerization chain junctor of the present invention
For target stomach cancer oncotherapy.These nano antibody polymeric chain junctors are couplet or the concatemers of nano antibody VHH.The present invention
Described nano antibody couplet or concatemer include the nano antibody couplet or many of targeting HER2, VEGF, EGFR respectively
Conjuncted.
Wherein, nano antibody VHH sequence of the present invention is:
Containing 128 amino acid whose VHH-α HER2 sequences 1:DVQLVESGGG, SVQGAAGGSL, RLSCAASDIT,
YSTDCMGWFR, QAPGKEREGV, ATINNGRAIT, YYADSVKGRF, TISQDNAKNT, VYLQMNSLRP, KDTAIYYCAA,
RLRAGYCYPA, DYSMDYWGKG, TQVTVSSG.
Containing 126 amino acid whose VHH-α HER2 sequences 2:DVQLEESGGG, SVQTGGSLRL, SCAASGYTYS,
SACMGWFRQG, PGKEREAVAD, VNTGGRRTYY, ADSVKGRFTI, SQDNTKDMRY, LQMNNLKPED, TATYYCATGP,
RRRDYGLGPC, DYNYWGQGTQ, VTVSSG.
Containing 132 amino acid whose VHH-α VEGF sequences 1:MAQVQLQESG, GGSVQDGGSL, RLSCAASGYA,
YDTYYMGWFR, QAPGKEREWV, AGITSLVSGV, AYYKYYTDSV, KGRFTIFRDD, DKNTVDLQMN, SLKPEDTAIY,
YCAASRSGLR, ARLLRPELYE, YWGQGTQVTV, SS.
Containing 129 amino acid whose VHH-α VEGF sequences 2:MAQVQLQESG, GGSVQAGGSL, RLSCVASGDT,
YSSACMGWFR, QAPGKEREGV, ATICTSTSMR, TRYYADAVKA, RFTISQDNAK, NTVYLQMNSL, KPEDIAMYYC,
ATGHTVGSSW, RDPGAWRYWG, QGTQVTVSS.
Containing 138 amino acid whose VHH-α EGFR sequences 1:QVQLQESGGG, LVQPGGSLRL, SCAASGRTFS,
SYAMGWFRQA, PGKQREFVAA, IRWSGGYTYY, TDSVKGRFTI, SRDNAKTTVY, LQMNSLKPED, TAVYYCAATY,
LSSDYSRYAL, PQRPLDYDYW, GQGTQVTVSS, LEHHHHHH.
The most non-humanized nano antibody VHH sequence, can be by the aminoacid sequence of its naturally occurring VHH sequence domains
More than one amino acid residue in row replaces with the amino acid residue that conventional people's VH sequence domains corresponding position exists.
Many targeted nanos antibody polymerization chain junctor that the probiotic bacteria for the treatment of gastric cancer of the present invention is expressed is by above-mentioned specificity
Monomer nano antibody VHH is formed by connecting by polypeptide link Linker different in size.Humanized nano antibody target stomach cancer
The target spot of tumor includes HER2 (human epidermal growth factor receptor 2, orHER2/neu), EGFR
(epidermal growth factor receptor) and VEGF (vascular endothelial growth factor).
Current most widely used aminoacid link Linker is four glycine and the combination of a serine
, and repeated the combination (GGGGSGGGGSGGGGS) of 3 times (GGGGS).In addition, of the present invention for
The aminoacid link Linker of link nano antibody VHH monomer can also include the amino acid chain shown in following Linker-Seq
Connect, but be not limited to this:
Linker1:GPcPcPc GlySer-polyPro (Glyc)-polyPro (Glyc)-polyPro (Glyc)
Linker2:GPPcP GlySer-polyPro-polyPro (Glyc)-polyPro
Linker3:GPGcP GlySer-polyPro-GlySer (Glyc)-polyPro
Linker4:GPPP GlySer-polyPro-polyPro-polyPro
Linker5:GPbP GlySer-polyPro-b2m-polyPro
Linker6:GPbG GlySer-polyPro-b2m-GlySer
Linker7:PbGbG polyPro-b2m-GlySer-b2m-GlySer
Linker8:GPbGbP GlySer-polyPro-b2m-GlySer-b2m-polyPro
Linker9:GPUG GlySer-polyPro-Ub-GlySer
Linker10:GPZP GlySer-polyPro-ZAG-polyPro
Linker11:GGZGZP GlySer-GlySer-ZAG-GlySer-ZAG-polyPro
Linker12:GcGcP GlySer (Glyc)-GlySer (Glyc)-polyPro
Linker13:cTPR3 (G4S) 3-cTPR3-(G4S) 3
Linker14:cTPR6 (G4S) 3-cTPR6-(G4S) 3
Linker15:cTPR9 (G4S) 3-cTPR9-(G4S) 3
Linker16:cTPR12 (G4S) 3-cTPR12-(G4S) 3
Linker17:GSn (G4S) n (n=1,2,3,4 ... 9).
Compound humanization nano antibody polymeric chain junctor or compound humanization nanometer that probiotic bacteria in the present invention is expressed resist
Each humanization monomer nano antibody in body group is connected end to end by amino acid chain, tail tail or head, forms many integration.Its
Three kinds of different nano antibodies end to end, the mode most probable that is connected of tail tail or head occur have 26 kinds more than, as Fig. 1 retouches
State.The mode that compound humanization nano antibody group is connected by different amino acid chains will form different shapes, then produce
The different effects hindering cancerous cell to grow up.
The present invention initiates the VHH humanization nano antibody composite joint group of particular design, both nano antibody couplets or many
The anti-group of conjuncted composite Nano, contains humanization HER2 nano antibody, humanization VEGF nano antibody and humanization EGFR nanometer
Antibody.In humanization nano antibody composite joint group, humanization HER2 nano antibody and humanization EGFR nano antibody are directly made
It is used on cancerous cell, plays Mutiple Targets anticancer growth function.And humanization VEGF nano antibody will act at cancerous cell
With its peripheral extent, stop the raw formation of blood vessel of cancerous cell periphery, produce blood vessel formation against function, thus promote cancerous cell to wither
Die.Humanization nano antibody composite joint group or nano antibody polymeric chain junctor form many integration, and its anticancer grows
To have than the anticancer growth of the humanization nano antibody of each random dispersion with the effect promoting cancer cell-apoptosis
More than effect decades of times or even hundred times.
The present invention utilizes food stage to take and can survive in the probiotic strain of human body gastrointestinal system, through genetic engineering
Transformation produces and release active nano antibody, delivers gastrointestinal environment in human body, enters blood circulation, arrives inside tumor.
Probiotic bacteria of the present invention includes lactobacillus or bacillus bifidus.Its probiotic oral preparation also includes selectivity
Prebiotic substance, the prebiotics such as including bifidus factor, to help growth and the activity of specific bacterial strain.
The prebiotics being simultaneously administered orally with probiotic bacteria can also include oligofructose, partially protein class material and protein water
Hydrolysis products, dextrinosan, natural plant extracts class, polysaccharide bifidus factor etc..
The present invention is initiated the Mutiple Targets nano antibody polymer of particular design and is turned by technique for gene engineering ways and means
Enter probiotic bacteria to express and release.Being currently available that DNA proceeds to biological probiotic bacteria method and has several, the most domestic patent is (open
Number CN 103656633A) described in proceed to method by what the restructuring of Cre/lox locus specificity realized.In order to make external source
Protein or polypeptide can need the nucleotide sequence to its encoding gene according to lactic acid at the smooth and high efficient expression of lactic acid bacteria
The use Preference of codon is optimized by bacterium, to remove rare codon.
Probiotic bacteria live vector has been widely used for various immunity exploitation.European Union patent " Oralrecombinant
Lactobacilli vaccines (EP1084709A1) " disclose a kind of method that not antigen is opened up on lactic acid bacteria surface.As can
With the lactic acid bacteria vaccine of commercialization, antigen shows simply one of multiple sport technique segments at lactobacillus cell wall.European Union's patent
“Lactobacilli harboring aggregation and mucin binding genes as
Vaccinedelivery vehicles (EP1066375B1) " disclose a kind of Lactobacillus reuteri cell wall and show anti-
Former method.United States Patent (USP) " Live bacterial vaccine (US2009/0324638A1) " discloses a kind of for breast
Acid bacterium secretion or cell wall display protein matter or the method for polypeptide.
Nano antibody VHH monomer of the present invention and the above-mentioned molecule lasted a long time link Linker merge one
Rise, the nano antibody VHH existence time in blood can be improved, make up nano antibody VHH monomer half-life short defect.
Claims (12)
1. many targeted nanos antibody polymerization chain junctor that probiotic bacteria is expressed, is to be passed through by specific monomer nano antibody VHH
Polypeptide link Linker connects and composes nano antibody polymerization connector, and gathers using probiotic oral bacterial strain as described nano antibody
Closing the delivery vehicles alive of chain junctor medicine, wherein, described monomer nano antibody VHH is with HER2, EGFR and/or VEGF as target
Antibody Nb-EGFR, Nb-HER2 and/or Nb-VEGF of point.
2., according to the many targeted nanos antibody polymerization chain junctor described in claim 1, described monomer nano antibody VHH is completed people
Sourceization is transformed, to reduce its immunogenicity.
3., according to the many targeted nanos antibody polymerization chain junctor described in claim 1, its nano antibody polymeric chain junctor is by list
Body nano antibody Nb-HER2, Nb-EGFR and/or Nb-VEGF are coupled together by polypeptide link Linker in any combination
Couplet, trimerization chain junctor or four poly chain junctors.
4., according to the many targeted nanos antibody polymerization chain junctor described in claim 3, described monomer nano antibody passes through polypeptide chain
Connect Linker end to end, tail tail or head connect.
5. according to the many targeted nanos antibody polymerization chain junctor described in claim 1, described for connecting monomer nano antibody VHH
Polypeptide link Linker be short Linker, middle Linker or long Linker, wherein, described short Linker is by less than 15 amino
Acid composition, middle Linker is made up of 15-25 aminoacid, and long Linker is made up of 26-60 aminoacid.
6., according to the many targeted nanos antibody polymerization chain junctor described in claim 1, described probiotic oral bacterial strain includes breast bar
Bacterium or bacillus bifidus.
7., according to the many targeted nanos antibody polymerization chain junctor described in claim 6, wherein said lactobacillus includes addicted to yogurt
Bacillus, lactobacillus casei, Lactobacillus crispatus, Lactobacillus bulgaricus, Deshi Lactobacillus, Lactobacillus fermenti, Lactobacillus gasseri, auspicious
Scholar's lactobacillus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, lactobacillus rhamnosus, saliva breast
Bacillus, and lactococcus lactis, Lactococcus lactis subsp.lactis.
8. an oral formulations for many targeted nanos antibody polymerization chain junctor that probiotic bacteria is expressed, containing benefit described in claim 1
Many targeted nanos antibody polymerization connector that raw bacterium is expressed, also includes selectivity prebiotic substance, institute in described oral formulations
State selectivity prebiotic substance and include helping the growth of specific bacterial strain and the bifidus factor of activity.
9., according to the oral formulations described in claim 7, described selectivity prebiotic substance also includes oligofructose, protein-based
One or several in material and protein hydrolysate, dextrinosan, natural plant extracts, polysaccharide bifidus factor
Kind.
10., according to the oral formulations described in claim 7, it is characterized in that described oral formulations is made capsule, tablet or mouth
Take liquid formulation.
11. according to the oral formulations described in claim 7, it is characterized in that described oral formulations is made Yoghourt, beverage or cookies.
Many targeted nanos antibody polymerization chain junctor that probiotic oral described in 12. claim 1 is expressed is as anti-gastric cancer medicament
Application.
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