CN102827277B - Anti-human serum albumin single-chain antibody and method for connecting polypeptide drugs to carbon end thereof - Google Patents

Anti-human serum albumin single-chain antibody and method for connecting polypeptide drugs to carbon end thereof Download PDF

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CN102827277B
CN102827277B CN201210125323.4A CN201210125323A CN102827277B CN 102827277 B CN102827277 B CN 102827277B CN 201210125323 A CN201210125323 A CN 201210125323A CN 102827277 B CN102827277 B CN 102827277B
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chain antibody
chain
sequence
antibody
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CN102827277A (en
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杨艳坤
龙泉
张芃芃
杨冬
刘冰
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BAIMING (SUZHOU) BIOLOGICAL TECHNOLOGY Co Ltd
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Abstract

The invention discloses an anti-human serum albumin single-chain antibody and a method for connecting polypeptide drugs to a carbon end of the anti-human serum albumin single-chain antibody. According to the anti-human serum albumin single-chain antibody a, a VH (Variable fragments of heavy chain) chain area of the single-chain antibody has a CDR (Complementarity-Determining Region) amino acid sequence of a complementary recognition domain, shown as C-SEQ (Sequence)-05, C-SEQ-06 and C-SEQ-07; a VL (Variable fragments of light chain) chain area of the single-chain antibody has a CDR amino acid sequence of a complementary recognition domain, shown as C-SEQ-08, C-SEQ-09 and C-SEQ-010; and the amino sequence of the single-chain antibody and the amino sequences of polypeptide drugs are connected from a nitrogen end of the single-chain antibody by a protein linking hinge shown as C-SEQ-01 or C-SEQ-03. The single-chain antibody disclosed by the invention has small molecular weight, high antibody specificity, weak immunogenicity and good water solubility, and is easily massively produced through a fermenting way. The invention also discloses a composition manufactured by using the single-chain antibody disclosed by the invention; and the composition can effectively prolong a half-life period of the polypeptide drugs.

Description

AHS's albumin single-chain antibody and carbon teminal thereof connect the method for polypeptide drugs
Technical field
The present invention relates to a kind of single-chain antibody, particularly relate to a kind of method that AHS's albumin single-chain antibody and carbon teminal thereof connect polypeptide drugs.
background technology
At present, worldwide present the situation of rapid popularization taking polypeptide as main bio-pharmaceutical.
Polypeptide class bio-pharmaceutical drug effect is remarkable, and can treat the hereditary class disease that chemical compound lot similar drug cannot take effect.But one of polypeptide class bio-pharmaceutical greatest problem is in use that the fading period of this type of medicine in human body is generally shorter, causes medicine frequency relatively high.Bring larger treatment cost and medication misery to patient.Therefore, the transformation period of prolongation polypeptide class bio-pharmaceutical just becomes biopharmaceutics field problem urgently to be resolved hurrily.
At present mainly contain and comprise and build that mutant, PEG are modified and with high molecular weight protein fusion etc. for extending the method for polypeptide class bio-pharmaceutical transformation period.These methods can extend the transformation period of polypeptide class bio-pharmaceutical effectively, but also have some problems.Structure by mutant can reduce the susceptibility of polypeptide to lytic enzyme conventionally, effectively extend the transformation period of polypeptide class bio-pharmaceutical, but the mutant grown of a lot of transformation period can change the activity of medicine, obtain one and can meet transformation period requirement, the mutant that does not change again pharmaceutical activity is simultaneously very difficult.Therefore, this technology is not suitable in protein drug field wide popularization and application, PEGization is modified and can in the stability that improves protein drug, be reduced its immunogenicity, but there is the bioactive potential possibility that affects polypeptide class bio-pharmaceutical, simultaneously, the polypeptide class bio-pharmaceutical of processing through PEGization is carried out to comparatively difficulty of purifying, increased difficulty and the production cost in production process.By with have compared with long half-lift human body protein directly to merge be the effective ways of a kind of practicable prolong drug transformation period, but this amalgamation mode has increased the molecular weight of polypeptide class bio-pharmaceutical greatly, bring a lot of problems to production and purifying, the increase of molecular weight also can change some pharmacokinetic properties of medicine, may make it be difficult to reach target spot position, and then affect drug effect.
summary of the invention
For solving the problems of the technologies described above, the invention provides a kind of single-chain antibody of effectively prolong half-life and the pharmaceutical composition that uses this single-chain antibody to be combined to form.
One of object of the present invention is to provide a kind of specific anti-human serum albumin (HSA) single-chain antibody.
Two of object of the present invention is to provide a kind of method that contains described specific anti-human serum albumin (HSA) single-chain antibody and polypeptide drug formation pharmaceutical composition, and the profit method of prolong drug transformation period in this way.
The VH chain region that a kind of single-chain antibody is provided in first object of the present invention, its complementary determining region CDR has the aminoacid sequence that is selected from lower group of CDR:
CDR1: C-SEQ-05
CDR2: C-SEQ-06
CDR3: C-SEQ-07
Described single-chain antibody VH chain region is taking the aminoacid sequence shown in C-SEQ-02 as a sequence preferably.
In first object of the present invention, the present invention also provides a kind of VL chain region of single-chain antibody, and its complementary determining region CDR has the aminoacid sequence that is selected from lower group of CDR:
CDR1: C-SEQ-08
CDR2: C-SEQ-09
CDR3: C-SEQ-010
Described single-chain antibody VL chain region is taking the aminoacid sequence shown in C-SEQ-04 as a sequence preferably.
In first object of the present invention, the present invention also provides a kind of single-chain antibody.Its VL chain region has complementary cog region as specified in claim 1; Its VL chain region has complementary cog region as specified in claim 3; Its VH chain is taking the aminoacid sequence shown in C-SEQ-02 as a sequence preferably.Its VH chain is taking the aminoacid sequence shown in C-SEQ-04 as a sequence preferably.Its VH chain region is used the albumen as shown in C-SEQ-01 or shown in C-SEQ-03 to be connected chain connection with VL chain region.
In second object of the present invention, provide a kind of single-chain antibody and polypeptide drugs to be connected to form the method for attachment of composition.It uses the albumen connection hinge as shown in C-SEQ-01 or as shown in C-SEQ-03 to be connected with polypeptide drug from the carbon teminal of single-chain antibody.
In second object of the present invention, the present invention also provides a kind of method that effectively extends the bio-pharmaceutical transformation period by above-mentioned single-chain antibody.It adopts the carbon teminal of single-chain antibody of the present invention and medicine to form composition.It contains above-mentioned and the aminoacid sequence of single-chain antibody and the sequence of any one polypeptide drug.
Compared with prior art beneficial effect of the present invention is: the single-chain antibody in the present invention it to have molecular weight little, antibodies specific is strong, a little less than immunogenicity, good water solubility, be easy to by fermentation mode scale operation, the features such as product homogeneity is strong, single-chain antibody is applicable to merging with polypeptide class bio-pharmaceutical very much, produce in enormous quantities by ferment overall expression process of mushroom, human serum albumin molecular weight is 150Kda, transformation period in human body reaches 19 days, the composition forming by itself and Humanized anti-human serum albumin single chain antibody fragments, can effectively extend the transformation period of polypeptide class bio-pharmaceutical, simultaneously, because single-chain antibody molecular weight is very little, the molecular weight of the antibody-polypeptide class bio-pharmaceutical molectron after amalgamation and expression itself still can remain on and be less than 20Kda lower level.Avoid potential immunogenicity, also reduced difficulty and the production cost in production process.
brief description of the drawings
Fig. 1 is electrophoresis detection result schematic diagram;
Fig. 2 carries out cell in vitro dosing test result schematic diagram;
Fig. 3 is that Humanized anti-human serum albumin single-chain antibody and drug conjugates are in Mice Body build-in test result schematic diagram.
Embodiment
Below the specific embodiment of the present invention is described in further detail.Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
Embodiment mono-, obtains the single-chain antibody in the present invention:
1, build human antibody library
From Humanized single chain antibody storehouse, screening has the antibody variable region sequence of specific recognition capability to human serum albumin, human antibody library is by gathering heavy chain immunoglobulin synthetic in periphery lymphocyte and many gene fragments of light chain, with the method amplification of polymerase chain reaction, and montage is to phage vector, be exemplified below with primer, one for finding the primer example of light chain CDR: the reverse primer of forward primer [5'-GATATCCAACTTACCCAATCC-3'] [5'-CCTCTTTATTTCTACCTTGG-3'], find the primer example of heavy chain CDR for one: forward primer [5'-GAAGTCCAGCTGCTCG-3'], reverse primer [5'-CGAAGAGACTGTGACTAGCGT-3'].
Antibody molecule fragment is by phage expression and be showed in phage surface, utilizes human serum albumin (HSA) for antigen, the antibody that is revealed in phage surface to be screened, and obtains the antibody of specificity the best.
2, antibody screening
Add in the LB nutrient solution of 19.5mL in-86 DEG C of cell strain suspensions 500 μ L frozen, 37 DEG C of temperature, 250rpm frequency, incubation 16 hours under 0.5L flask condition, with whizzer with the rotating speed of 12000rpm by centrifugal the cell suspension after incubation, discard the supernatant liquor after centrifugal, centrifugal sediment is resuspended in LB nutrient solution, reach 10 11titre more than/mL, as suspension A, is coated on the human serum albumin of purifying (HSA) in 50mL polyoxyethylene Tissue Culture Flask, and suspension A is added in above-mentioned Tissue Culture Flask, forms 10 9/ mL phage particle concentration, at 37 DEG C of temperature, incubation 1 hour, discards the nutrient solution in Tissue Culture Flask, uses the PBS washed cell culturing bottle wall 5 times that is dissolved with 0.5% concentration Tween-20, adds 10 of 2.0mL in culturing bottle 3the EColi cell of/mL, 37 DEG C of temperature, 250rmp frequency, incubation 16 hours under condition, is cycled to repeat whole process 5 times.
The cell that above-mentioned process screening is obtained is with 10 5/ mL concentration is uniformly coated on the agar plate that contains 0.1% kantlex and cultivates, single cell strain 1056 strains of making even on plate are transferred on 11 96 orifice plates and continue to cultivate, 1 strain of every hole, 37 DEG C of temperature, 250rmp frequency, incubation 16 hours under condition, after incubation finishes, in board-like whizzer with the speed of 5000rmp centrifugal 30 minutes, get supernatant preservation.
Get above-mentioned supernatant 5 μ L, add 96 orifice plates coated with human serum albumin solution's incubation of 20 μ g/mL concentration, incubation 1 hour at 37 DEG C of temperature, with the PBS washing microwell plate 5 times that is dissolved with 0.5% concentration Tween-20, add the anti-human antibody of rabbit of horseradish enzyme labelling, incubation 1 hour at 37 DEG C of temperature, then with the PBS washing microwell plate 5 times that is dissolved with 0.5% concentration Tween-20.Add the DAB developer of 200 μ L and the hydrogen peroxide of 1 μ L, at 37 DEG C of temperature, incubation reads 560nm wavelength absorption photometric after 20 minutes, chooses the corresponding antibody variable region clone of several sample aperture that absorption photometric is the strongest and makes sample of the ScFv single-chain antibody of recombinating for next step.
3, avidity test
In antibody screening process, obtain the clone that 294 antigen-antibody reactions are positive, utilize the single-chain antibody of recombined human single-chain antibody purification system ProteinL affinity chromatography separation and purification restructuring, single-chain antibody after purifying is carried out to avidity test, avidity test adopts conventional Scatchard avidity analytical method, obtains 3 clones that avidity is the strongest: 9.12 × 10 -7m; 3.09 × 10 -6m and 9.70 × 10 -6m, select in the LB nutrient solution that is seeded in 100mL that these 3 avidity are the strongest, incubation 10 hours under the condition of 37 DEG C of temperature, 250rmp frequency, recycling isopropylthiogalactoside (IPTG) inducing culture 10 hours, choose the wherein the highest strain of expression amount, called after 10D7 carries out the analysis of encoding sequence and setting up of expression vector.
Embodiment bis-, the analysis of the encoding sequence of single-chain antibody, restructuring and the setting up of expression vector:
The 10D7 strain producing in embodiment 1 is bred in LB nutrient solution, then utilize DNA extraction test kit by the plasmid purification in cell strain, shear and 2% agargel electrophoresis purifies and separates by restriction enzyme, obtain variable region of heavy chain encoding sequence, obtain variable region of light chain encoding sequence with method, antibody variable region, employment source universal primer carries out PCR amplification, then the amplified production of acquisition being delivered to Eurofin company checks order, the DNA sequence dna that order-checking obtains and the result of protein sequence are presented in table 2.1 and table 2.2, wherein the listed sequence of table 2.1 is the light chain region (C-SEQ-04) of humanization AHS albumin single-chain antibody, and variable region sequences (C-SEQ-08, C-SEQ-09 and C-SEQ-010), the listed sequence of table 2.2 is the heavy chain region (C-SEQ-03) of humanization AHS albumin single-chain antibody, and variable region sequences (C-SEQ-05, C-SEQ-06 and C-SEQ-07).
Table 2.1: preferred embodiments, i.e. a C-SEQ-04 of sequence of light chain (VL).(comprising C-SEQ-08, C-SEQ-09 and C-SEQ-010)
GAT ATC CAA CTT ACC CAA TCC CCA AGT AGC
D I Q L T Q S P S S
[FR1 5 10
CTT TCG GCC TCG GTT GGA GAC CGC GTC ACG
L S A S V G D R V T
15 20
ATT ACA TGT CGT GCC TCA CAG TAT ATC GGG
I T C R A S Q Y I G
FR1] [No.8] 25 30
CGA TAC CTA AGG TGG TAC CAA CAG AAG CCG
R Y L R W Y Q Q K P
[No.8] [FR2 40
GGC AAA GCT CCT AGG CTC CTA ATC TAC GAT
G K A P R L L I Y D
45 FR2] [No.9]
TCG TCA GTG CTG CAA AGT GGT ATA CCC AGT
S S V L Q S G I P S
55 [No.9] [FR3 60
AGG TTC AGT GGC TCG GGT TCG GGT ACT GAC
R F S G S G S G T D
65 70
TTC ACC CTA ACC ATT TCA TCC CTC CAA CCC
F T L T I S S L Q P
75 80
GAA GAC TTC GCA ACG TAC TAC TGC CAG CAA
E D F A T Y Y C Q Q
85 FR3] [No10] 90
AAG TAT CTA CCC CCG TAC ACC TTT GGC CAG
K Y L P P Y T F G Q
95 [No10] 100
GGT ACC AAG GTA GAA ATA AAG AGG
G T K V E I K R
105 108
Table 2.2: preferred embodiments, i.e. a C-SEQ-02 of sequence of heavy chain table (VH).(comprising C-SEQ-05, C-SEQ-06 and C-SEQ-07)
GAA GTC CAG CTG CTC GAA TCA GGT GGC GGC
E V Q L L E S G G G
[FR1 5 10
CTG GTG CAA CCT GGA GGG TCG CTC AGA CTT
L V Q P G G S L R L
15 20
TCG TGT GCC GCC TCA GGT TTC ACC TTT TGG
S C A A S G F T F W
25 FR1]
GCC TAT CCC ATG TCC TGG GTA AGG CAA GCC
A Y P M S W V R Q A
[No.5] [No.5] [FR2 40
CCT GGT TGG GGG CTA GAA TGG GTC TCC ACC
P G K G L E W V S T
45 FR2] [No.6]
ATC TCCCCA TTC GGC TCC ACA ACA TAC TAT GCC
I SP F G S T T Y Y A
55 60
GAC TCG GTG AAG GGA CGC TTC ACC ATA TCA
D S V K G R F T I S
[No.6] [FR3 70
CGA GAC AAC TCC AAA AAT ACA CTA TAC CTC
R D N S K N T L Y L
75 80
CAA ATGAAC TCCCTT CGAGCC GAA GAT ACT GCT GTG TAT
Q MN SL RA E D T A V Y
85 90
TAT TGT GCC AAA GTC AGA TCAATG CGCCCT TATAAG TTT
Y C A K V R SM RP YK F
FR3] [No.7] 100
GAT TAC TGG GGT CAA GGC ACG CTA GTC ACA
D Y W G Q G T L V T
[No.7] 105 110
GTC TCT TCG
V S S
113
Embodiment tri-, single-chain antibody is light, the link of sequence of heavy chain and be connected and make drug regimen with the polypeptide class bio-pharmaceutical exendin-4 for the treatment of type-II diabetes:
Light chain of antibody region and the heavy chain regional DNA of having determined sequence is by directly synthesizing or the method realization of recombinant PCR links, its hinge fraction is that the VH chain region of single-chain antibody has the corresponding DNA sequence dna of SEQ ID No.2, the working method of recombinant PCR used is the general ordinary method in this area, to entrusting the synthetic sequence of Invitrogen company to check order, the antibody sequencing result that links sequence light, heavy chain completing by the method for recombinant PCR with us is identical, and sequencing result is as shown in table 3.1:
3.1: the DNA sequence dna of a better sequence of the Humanized anti-human serum albumin single-chain antibody that order-checking obtains
GAT ATC CAA CTT ACC CAA TCC CCA AGT AGC
[V L] 5 10
CTT TCG GCC TCG GTT GGA GAC CGC GTC ACG
15 20
ATT ACA TGT CGT GCC TCA CAG TAT ATC GGG
[No.8] 25 30
CGA TAC CTA AGG TGG TAC CAA CAG AAG CCG
[No.8] 40
GGC AAA GCT CCT AGG CTC CTA ATC TAC GAT
45 [No.9]
TCG TCA GTG CTG CAA AGT GGT ATA CCC AGT
55 [No.9] 60
AGG TTC AGT GGC TCG GGT TCG GGT ACT GAC
65 70
TTC ACC CTA ACC ATT TCA TCC CTC CAA CCC
75 80
GAA GAC TTC GCA ACG TAC TAC TGC CAG CAA
85 [No10] 90
AAG TAT CTA CCC CCG TAC ACC TTT GGC CAG
95 [No10] 100
GGT ACC AAG GTA GAA ATA AAG AGG GGT GGA
105 [V L] [No.2] 110
GGT GGA TCG GGT GGA GGT GGA TCG GGT GGA
115 120
GGT GGA TCG GAA GTC CAG CTG CTC GAA TCA
[No.2] [V H] 125 130
GGT GGC GGC CTG GTG CAA CCT GGA GGG TCG
135 140
CTC AGA CTT TCG TGT GCC GCC TCA GGT TTC
145 150
ACC TTT TGG GCC TAT CCC ATG TCC TGG GAT
[No.5] 155 [No.5] 160
AGG CAA GCC CCT GGT TGG GGG CTA GAA TGG
165 170
GTC TCC ACC ATC TCCCCA TTC GGC TCC ACA ACA
[No.6] 175 180
TAC TAT GCC GAC TCG GTG AAG GGA CGC TTC
185 [No.6] 190
ACC ATA TCA CGA GAC AAC TCC AAA AAT ACA
195 200
CTA TAC CTC CAA ATGAAC TCCCTT CGAGCC GAA GAT ACT
205 210
GCT GTG TAT TAT TGT GCC AAA GTC AGA TCAATG
215 [No.7] 220
CGCCCT TATAAG TTT GAT TAC TGG GGT CAA GGC ACG
225 230
CTA GTC ACA GTC TCT TCG
[No.7] [V H]
By repeating the method for recombinant PCR, can obtain Humanized anti-human serum albumin single-chain antibody and be connected with the polypeptide class bio-pharmaceutical exendin-4 for the treatment of type-II diabetes the sequence of making pharmaceutical composition, this sequence also can obtain by directly synthetic method, the Humanized anti-human serum albumin single-chain antibody that restructuring PCR method is obtained is connected the order-checking of making pharmaceutical composition with the polypeptide class bio-pharmaceutical exendin-4 for the treatment of type-II diabetes, obtain in corresponding DNA sequence dna preferably one, sequencing result is as shown in table 3.2:
Table 3.2: the Humanized anti-human serum albumin single-chain antibody that order-checking obtains is made the sequence of pharmaceutical composition at the polypeptide class bio-pharmaceutical exendin-4 of carbon teminal link treatment type-II diabetes
GAT ATC CAA CTT ACC CAA TCC CCA AGT AGC
[V LRise] 5 10
CTT TCG GCC TCG GTT GGA GAC CGC GTC ACG
15 20
ATT ACA TGT CGT GCC TCA CAG TAT ATC GGG
[No.8 rises] 25 30
CGA TAC CTA AGG TGG TAC CAA CAG AAG CCG
[No.8 only] 40
GGC AAA GCT CCT AGG CTC CTA ATC TAC GAT
45 [No.9 rises]
TCG TCA GTG CTG CAA AGT GGT ATA CCC AGT
55 [No.9 only] 60
AGG TTC AGT GGC TCG GGT TCG GGT ACT GAC
65 70
TTC ACC CTA ACC ATT TCA TCC CTC CAA CCC
75 80
GAA GAC TTC GCA ACG TAC TAC TGC CAG CAA
85 [No10 rises] 90
AAG TAT CTA CCC CCG TAC ACC TTT GGC CAG
95 [No10 only] 100
GGT ACC AAG GTA GAA ATA AAG AGG GGT GGA
105 [V LOnly] [No.2 rises] 110
GGT GGA TCG GGT GGA GGT GGA TCG GGT GGA
115 120
GGT GGA TCG GAA GTC CAG CTG CTC GAA TCA
[No.2 only] [V HRise] 125 130
GGT GGC GGC CTG GTG CAA CCT GGA GGG TCG
135 140
CTC AGA CTT TCG TGT GCC GCC TCA GGT TTC
145 150
ACC TTT TGG GCC TAT CCC ATG TCC TGG GAT
[No.5 rises] 155 [No.5 only] 160
AGG CAA GCC CCT GGT TGG GGG CTA GAA TGG
165 170
GTC TCC ACC ATC TCCCCA TTC GGC TCC ACA ACA
[No.6 rises] 175 180
TAC TAT GCC GAC TCG GTG AAG GGA CGC TTC
185 [No.6 only] 190
ACC ATA TCA CGA GAC AAC TCC AAA AAT ACA
195 200
CTA TAC CTC CAA ATGAAC TCCCTT CGAGCC GAA GAT ACT
205 210
GCT GTG TAT TAT TGT GCC AAA GTC AGA TCAATG
215 [No.7 rises] 220
CGCCCT TATAAG TTT GAT TAC TGG GGT CAA GGC ACG
225 230
CTA GTC ACA GTC TCT TCG CGA GGT CGA GGT
[No.7 only] [V HOnly] [No.1 rises] 240
CGA GGT CGA GGT CGA TCC CGA GGT GGA GGT
245 250
TCC CAT GGT GAG GGA ACT TTC ACT AGC GAC
[No.1 only] [exendin4 rises] 255 260
CTC TCA AAG CAG ATG GAG GAG GAA GCT GTC
265 270
AGG CTT TTC ATC GAA TGG TTG AAG AAC GGC
275 280
GGA CCT TCG TCA GGA GCC CCA CCA CCG TCG
285 [exendin4 only]
Embodiment tetra-, expression and the order-checking of being connected of single-chain antibody and polypeptide class biological medicament and expression vector, clone and albumen:
Utilize restriction enzyme HindIII and BamHI that the DNA sequence encoding of setting up in example 3 is inserted in expression vector pMG18.Be configured to Humanized anti-human serum albumin single-chain antibody is connected the pharmaceutical composition sequence of making expression vector with the polypeptide class bio-pharmaceutical exendin-4 for the treatment of type-II diabetes.
The expression vector with antibody and medication combined gene that utilizes aforesaid method to build is proceeded to intestinal bacteria, complete the conversion to e. coli host cell, will in the LB substratum that is inoculated in 500mL after transforming, ferment.
Product after purifying is checked order, obtain in sequence preferably one, sequencing result is as shown in table 4.1:
Table 4.1: Humanized anti-human serum albumin single-chain antibody is connected with the polypeptide class bio-pharmaceutical exendin-4 for the treatment of type-II diabetes the protein sequence of making pharmaceutical composition
【DIQLTQSPSSLSASVGDRVTITC RASQYIGRYLR WYQQKPGKAPRLLIY DSSVLQS GIPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQKYLPPYT FGQGTKVEIKR GGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTFS AYPMS WVRQAPGKGLEWVS TISPFGSTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK VRSMRPYKFDY WGQGTLVTVSS RGRGRGRGRSRGGGS HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS】。
Embodiment five, utilizes the host cell manufacture order chain antibody that transformed and the production process of medicinal composition:
1. bacterial classification preparation
On the freshly prepared Lu Bo LB flat board that contains tsiklomitsin, be evenly coated with Escherichia coli bacteria liquid, cultivate 16 hours for 37 DEG C, the finely disseminated coli strain of picking, in the substratum that contains tsiklomitsin at LB, 37 DEG C of shaking tables are cultivated, 16 hours.The 1:100 of above-mentioned incubated overnight is inoculated in the LB substratum of 200ml, 37 DEG C of shaking tables are cultivated and are used as fermented bacterium for about 8 hours.
2. fermented liquid preparation and sterilizing
Detect fermentor tank tank body operation conditions, use pH value reference liquid to demarcate fermentor tank pH probe, demarcate feed supplement flow rate pump.According to formulated fermention medium 20L in table 1, the online sterilizing 20min of 1210C, simultaneously in high-pressure sterilizing pot to pipeline, defoamer, phosphoric acid carries out autoclaving (ammoniacal liquor can not autoclaving), treat that fermented liquid is cooled to 37 DEG C, adjust fermentor tank air flow and stirrer rotating speed to maximum value operation five minutes, while demarcating this, dissolved oxygen amount is 100%, Ensure Liquid liquid formula preparation stream Ensure Liquid liquid 2.5L, be divided in four feed supplement bottles, and use pressure kettle sterilizing, the complete connection line of sterilizing.
3. fermentation flow process, stream add strategy and processing condition
Prepare according to the method described above fermentor tank and substratum, drop to after design temperature until fermented liquid temperature, after 300C, the intestinal bacteria bacterial classification (1:100) in 20L fermentor tank of inoculation 200ml extinction concentration A600=1, add appropriate tsiklomitsin simultaneously, inoculation time is recorded as to the beginning of fermentation time, fermentation parameter is set as: temperature T=25-370C, acidity-basicity ph=6.0-7.5 rotating speed S=600-1500rpm, air flow is V=1-5vvm, ferment in 16-18 hour fermented liquid when significantly fluctuation appears in dissolved oxygen (DOT), start stream and add nutritive medium, flow velocity is 50ml/h/L-200mL/h/L, ferment and after 16-20 hour, add the appropriate IPTG of 1mol/L, be 0.1mM-10mM to final concentration, adjust leavening temperature T=18-25 DEG C simultaneously, flow acceleration is 25ml/h-50mL/h/L, fermentation is measured fermented liquid A600 place light absorption value for judging thalline amplification situation during carrying out, after proceeding to 60-80 hour, fermentation collects fermented liquid, clean tank body, finish fermentation.
4. fermentation liquor treatment
Fermented liquid adopts osmotic pressure ballistic method to process release target protein.Fermented liquid is collected supernatant liquor through 5000rpm centrifugal treating after 60 minutes, use sucrose, the resuspended precipitation thalline of ethylenediamine tetraacetic acid (EDTA) damping fluid, leave standstill after 16 hours according to recentrifuge, collect centrifuged supernatant (OSI), and use 1mM magnesium ion solution outstanding precipitation again, 4 DEG C of concussions are spent the night, recentrifuge, discards thalline, collects supernatant liquor (OSII), abandon and process rear thalline, merge collection supernatant liquor and OSI, OSII and process supernatant liquor.
5. antibody purification
Protein purification adopts cationic exchange prepacked column to carry out purifying, preparation of samples: fermented liquid supernatant and OSI and OSII are after 1:50 dilution, high speed centrifugation 30min, get the PB post dialysed overnight of supernatant to 0.02mM, subsequently protein solution is carried out to 12000rp 40C centrifugal 30 minutes, again through the membrane filtration of 0.22 μ m, each CMM prepackage ion exchange column uses 0.01mM PB damping fluid balance, adopt the PB balance pillar wash-out foreign protein of 10 times of volume 0.02mM parallel with baseline to A280 detection line, use arginic acid salt damping fluid stepwise elution, detect eluted protein amount (A280) collects each several part albumen and detects by the detection of SDS-PAGE electrophoresis detection and ELSIA after concentrated simultaneously, detection display scFv antibody activity is good.
6. result
Through the fermentation reaction of 80 hours, according to the strict zymotechnique flow process of above-mentioned parameter control, in fermenting process by 16 hours, 48 hours, 64 hours, the bacterium fluid samples concentration of 72 hours points detects, and obtains A600 place absorbancy and is respectively: 70.0,82.8,130,99.8, fermented liquid, through osmotic pressure shock treatment, is divided into three part OS1, OS2 and fermented liquid supernatant, utilize recombinant protein content in 12% each component of SDS-PAGE electrophoresis detection, electrophoresis result shows that antibody all has high efficient expression in OSI and fermented liquid supernatant, as shown in Figure 1.
Detect the humanization antiserum(antisera) albumin single-chain antibody of fermentation reaction generation and the protein expression situation of medicinal composition by SDS-PAGE method.M: albumen Standard chi; 1: purifying sample 1 μ L; 2, purifying sample 0.1 μ L.
Embodiment six, the transformation period in cell tests has obtained prolongation to data validation medicine in vitro:
The several antibody clonings that obtain in embodiment mono-are linked according to the described method of embodiment 3 with polypeptide class biological medicament Exendin-4, carry out Expression product according to the method for embodiment 6, on people's cell that the pharmaceutical composition that the Humanized anti-human serum albumin single-chain antibody of producing acquisition is connected with the polypeptide class bio-pharmaceutical Exendin-4 for the treatment of type-II diabetes is cultivated in vitro, carry out dosing test, observe the transformation period (T) of drug metabolism, data presentation, compare with the simple Exendin-4 medicine that is not connected Humanized anti-human serum albumin single-chain antibody of the present invention, the transformation period of the pharmaceutical composition that Humanized anti-human serum albumin single-chain antibody is connected with polypeptide class bio-pharmaceutical Exendin-4 obtains significantly and extends, utilize statistics software R Stats to carry out statistical test to obtain cloning the conclusion (P<0.001) of 10D7-Exendin-4 mixture transformation period and the difference highly significant of simple Exendin-4 transformation period, as shown in Figure 2.
The transformation period in cell tests has obtained prolongation in vitro for data validation Humanized anti-human serum albumin single-chain antibody and drug conjugates.
Embodiment seven. data validation Humanized anti-human serum albumin single-chain antibody and the transformation period of drug conjugates in Mice Body build-in test have obtained prolongation.
According to the experiment of mouse pharmacokinetics, adopt subcutaneous administration sc method, measure the mixture transformation period, metering is 0.1mg/kg, and subcutaneous administration mean half-life is 95 h by analysis.Significantly be longer than the 2-9 h of simple exendin-4, as shown in Figure 3.
Data validation Humanized anti-human serum albumin single-chain antibody and drug conjugates have obtained prolongation in the transformation period in Mice Body build-in test
Comprehensively above-mentioned, single-chain antibody in the embodiment of the present invention can utilize PCR from the DNA sequence fragment corresponding with above-mentioned each aminoacid sequence, the method of recombination method or synthetic obtains the complete DNA sequence fragment corresponding with single-chain antibody, then or vivoexpression interior by organism obtains, also can directly directly obtain by amino acid recombination method or synthetic method, in the method that uses the DNA sequence expression corresponding with aminoacid sequence to obtain, once obtain relevant sequence, just above-mentioned DNA sequence can be cloned as in carrier, proceed to again cell, then by promoting the increment of host cell to obtain more relevant sequence.The invention still further relates to the carrier that comprises the DNA sequence that above-mentioned each aminoacid sequence is corresponding.These carriers can, for transformed host cell, can be expressed aminoacid sequence of the present invention.Host cell can be prokaryotic cell prokaryocyte, as Bacillus coli cells; Or eukaryotic cell, as yeast cell and mammalian cell.
Only the preferred embodiment of the present invention from the above; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvement and modification, these improve and modification also should be considered as protection scope of the present invention.
Annex:
Sequence TXT file and claims are the sequence synopsis in specification sheets.
[VL][FR1] 【DIQLTQSPSSLSASVGDRVTITC】
[VL][FR2] 【WYQQKPGKAPRLLIY】
[VL][FR3] 【GIPSRFSGSGSGTDFTLTISSLQPEDFATYYC SYMGDRFDY】
[VL][FR4] 【FGQGTKVEIKR】
[VL][CDR1] 【RASQYIGRYLR】 [C-SEQ-08]
[VL][CDR2] 【DSSVLQS】 [C-SEQ-09]
[VL][CDR3] 【QQKYLPPYT】 [C-SEQ-010]
[Linker 1] 【GGGGSGGGGSGGGGS】[C-SEQ-01]
[VH][FR1] 【EVQLLESGGGLVQPGGSLRLSCAASGFTFS】
[VH][FR2] 【WVRQAPGKGLEWVS】
[VH][FR3] 【RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK】
[VH][FR4] 【WGQGTLVTVSS】
[VH][CDR1] 【AYPMS】 [C-SEQ-05]
[VH][CDR2] 【TISPFGSTTYYADSVKG】 [C-SEQ-06]
[VH][CDR3] 【VRSMRPYKFDY】 [C-SEQ-07]
[Linker2] 【RGRGRGRGRSRGGGS】 [C-SEQ-03]
[Exendin-4] 【HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS】
[C-SEQ-02] =
[VH][FR1]+[VH][CDR1]+[VH][FR2]+[VH][CDR2]+[VH][FR3]+[VH][CDR3]+[VH][FR4]
[C-SEQ-04] =
[VL][FR1]+[VL][CDR1]+[VL][FR2]+[VL][CDR2]+[VL][FR3]+[VL][CDR3]+[VL][FR4]
A kind of manifestation of [ScFv]=
[C-SEQ-04]+[C-SEQ-01]+[C-SEQ-02] or
[C-SEQ-04]+[C-SEQ-03]+[C-SEQ-02]
A kind of manifestation of antibody pharmaceutical compositions:
[C-SEQ-04]+[C-SEQ-01]+[C-SEQ-02]+[C-SEQ-03]+[Exendin-4]
Only the preferred embodiment of the present invention from the above; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvement and modification, these improve and modification also should be considered as protection scope of the present invention.
SEQUENCE LISTING
<110> Baiming (Suzhou) Biotechnology Co., Ltd.
<120> AHS albumin single-chain antibody and carbon teminal thereof connect the method for polypeptide drugs
<130> 201210125323.4
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 15
<212> PRT
<213> composition sequence
<400> 1
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 2
<211> 120
<212> PRT
<213> composition sequence
<400> 2
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr
20 25 30
Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Ser Pro Phe Gly Ser Thr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Val Arg Ser Met Arg Pro Tyr Lys Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 3
<211> 15
<212> PRT
<213> composition sequence
<400> 3
Arg Gly Arg Gly Arg Gly Arg Gly Arg Ser Arg Gly Gly Gly Ser
1 5 10 15
<210> 4
<211> 117
<212> PRT
<213> composition sequence
<400> 4
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Tyr Ile Gly Arg Tyr
20 25 30
Leu Arg Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ser Ser Val Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Ser Tyr Met Gly Asp Arg Phe Asp
85 90 95
Tyr Gln Gln Lys Tyr Leu Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys
100 105 110
Val Glu Ile Lys Arg
115
<210> 5
<211> 5
<212> PRT
<213> composition sequence
<400> 5
Ala Tyr Pro Met Ser
1 5
<210> 6
<211> 17
<212> PRT
<213> composition sequence
<400> 6
Thr Ile Ser Pro Phe Gly Ser Thr Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 7
<211> 11
<212> PRT
<213> composition sequence
<400> 7
Val Arg Ser Met Arg Pro Tyr Lys Phe Asp Tyr
1 5 10
<210> 8
<211> 11
<212> PRT
<213> composition sequence
<400> 8
Arg Ala Ser Gln Tyr Ile Gly Arg Tyr Leu Arg
1 5 10
<210> 9
<211> 7
<212> PRT
<213> composition sequence
<400> 9
Asp Ser Ser Val Leu Gln Ser
1 5
<210> 10
<211> 9
<212> PRT
<213> composition sequence
<400> 10
Gln Gln Lys Tyr Leu Pro Pro Tyr Thr
1 5
<210> 11
<211> 39
<212> PRT
<213> composition sequence
<400> 11
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210> 12
<211> 30
<212> PRT
<213> composition sequence
<400> 12
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
20 25 30
<210> 13
<211> 14
<212> PRT
<213> composition sequence
<400> 13
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
1 5 10
<210> 14
<211> 32
<212> PRT
<213> composition sequence
<400> 14
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
1 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys
20 25 30
<210> 15
<211> 11
<212> PRT
<213> composition sequence
<400> 15
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10

Claims (2)

1. a single-chain antibody, is characterized in that, its VL chain region has complementary cog region, and described complementary cog region has the aminoacid sequence that is selected from lower group of CDR;
CDR1: C-SEQ-08
CDR2: C-SEQ-09
CDR3: C-SEQ-010 ；
The complementary cog region of its VH chain region tool, described complementary cog region has the aminoacid sequence that is selected from lower group of CDR;
CDR1: C-SEQ-05
CDR2: C-SEQ-06
CDR3: C-SEQ-07 ；
Its VH region is with the aminoacid sequence shown in C-SEQ-02;
Its VL region is with the aminoacid sequence shown in C-SEQ-04;
Its VH chain region and VL chain region are used the albumen chain connection as shown in C-SEQ-01 or as shown in C-SEQ-03.
2. a single-chain antibody and polypeptide drug are connected to form the method for pharmaceutical composition, it is characterized in that, the sequence that it contains single-chain antibody and any polypeptide drug, its VH chain region of described single-chain antibody is taking the aminoacid sequence shown in C-SEQ-02 as a kind of manifestation; Its VL chain region is taking the aminoacid sequence shown in C-SEQ-04 as a kind of manifestation, and described single-chain antibody uses the albumen as shown in C-SEQ-01 or shown in C-SEQ-03 to be connected the nitrogen end connection of hinge from single-chain antibody with the aminoacid sequence of polypeptide drug.
CN201210125323.4A 2012-04-26 2012-04-26 Anti-human serum albumin single-chain antibody and method for connecting polypeptide drugs to carbon end thereof Active CN102827277B (en)

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