CN105037538A - Optimized Fc fragment and optimizing method and application thereof - Google Patents

Abstract

The invention provides an optimized Fc fragment and an optimizing method and application thereof. The optimized Fc fragment is obtained by removing seven irregular curved amino acids at N ends of an Fc fragment. The optimizing method comprises the amplifying steps that RT-PCR is utilized for amplifying wild type Fc out of a blood sample, a primer is designed to cut off the seven amino acids at the N ends of the wild type Fc, and the optimized Fc fragment is amplified out, and further comprises expressing and purifying steps. The optimized Fc fragment and the optimizing method and application thereof are used for applying improvement with the Fc fragment. A provided Fcs framework is better in stability, and the producing, purifying and storing cost of a monoclonal antibody or Fc fusion protein can be lowered. The gathering resisting capacity is better, and clinical use risks caused by protein gathering can be lowered. The optimized Fc fragment framework can be applied to complete IgG or relevant Fc fusion protein and used for further studying the influence on the plasma half-life of relevant biological agents.

Description

The Fc fragment optimized and optimization method and application

Technical field

The present invention relates to bio-pharmaceutical engineer technology domain, particularly relate to the Fc fragment of optimization and optimization method thereof and application.

Background technology

Antibody, with its high specificity, highly sensitive, is widely used in every field, and especially life science and clinical treatment are as ELISA, Westernblot, immunofluorescence analysis, disease quick diagnosis and treat various disease as biotechnological formulation.Therefore, since entering 21 century, people also start research and development and the clinical application of more and more paying close attention to therapeutic antibodies medicine, this is because it is little to human body toxic side effect, have curative effect that is natural and high degree of specificity, and have createed huge Social benefit and economic benefit.

Simultaneously, fusion rotein based on Fc fragment is not only relatively little with its molecular weight and have good tissue permeability, and the Fc fragment of coupling can with the effect interactions of molecules such as Fc acceptor (FcRn), thus extend the plasma half-life of medicine and ADCC, CDC effect or phagolysis can be induced.

But these antibody drugs and Fc fusion rotein are as bioactive macromolecule, and it also has self limitation, as weak in poor stability, resistant to aggregation ability, in the processes such as its expression, purifying, storage, can tend to assemble or degraded.And the immunogenicity that protein aggregation causes, not only reduce drug effect, also bring very large risk to clinical application simultaneously.Therefore, raising antibody thermostability and resistant to aggregation ability are one of this field problem demanding prompt solutions.

Summary of the invention

For above-mentioned defect and the problem of prior art, the object of this invention is to provide the Fc fragment of optimization and optimization method thereof and application.Solve the thermostability of existing Fc fragment and the technical problem of resistant to aggregation ability.

In order to achieve the above object, the invention provides following technical scheme:

The Fc fragment optimized is that 7 the irregular curling amino acid amputation by being held by the N of Fc fragment obtain.

The Fc fragment related in the present invention can adopt the Fc fragment of the immunoglobulin (Ig) of different plant species, particularly, as Mammals etc., more specifically, as the mankind.

Optimization method, comprises and utilizes RT-PCR, from blood sample, amplify wild-type Fc, and 7 amino acid that the N of wild-type Fc holds by bamboo product primer block, and amplifies the amplification step optimizing Fc fragment.

Further, in described amplification step, for people source blood sample, the amplification method of wild-type Fc is: people source blood sample extracting cell total rna, and reverse transcription obtains cDNA; Be that template carries out PCR reaction with cDNA, obtain wild-type Fc gene fragment; The forward primer adopted in PCR reaction is 5-TCGCTACCGTGGT gGCC cAGGCGGCCgCACCTGAACTCCTGGGGGGAC-3, reverse primer is 5-GTGGTGGTGGTGGCCGGCCT gGCCTTTACCCGGaGACAGGGAGAGGCTC-3.

Further, in described amplification step, for people source blood sample, the amplification method of the Fc fragment of optimization is: with wild-type Fc gene fragment for template carries out PCR reaction, the Fc fragment be optimized; Wherein, the forward primer adopted in PCR reaction is 5-TCGCTACCGTGG tGGCCCAGGCGGCCcCGTCAGTCTTCCTCTTCC-3, reverse primer is 5-GTGGTGGTGGTGGCCGGCCT gGCCTTTACC cGGaGACAGGGAGAGGCTC-3.

Preferred technical scheme is, after amplification step completes, also comprise expression and purification step further; Described expression and purification step is specially: the optimization Fc fragment after being cut by SfiI enzyme carries out to prokaryotic expression carrier connecting, transform corresponding expressive host bacterium is optimized Fc bacterial strain; Carry out prokaryotic expression to optimization Fc bacterial strain again, purifying is optimized the target protein of Fc fragment.

Particularly, described prokaryotic expression carrier adopts expression vector pCom, and corresponding expressive host bacterium adopts intestinal bacteria HB2151.Or other prokaryotic expression carrier and corresponding expressive host bacterium.

Further, in expression and purification step, first adopt agarose gel electrophoresis to reclaim and optimize Fc fragment, then cut with SfiI enzyme and optimize Fc fragment, then glue recovery, obtain SfiI enzyme cut after optimization Fc fragment.

Further, in expression and purification step, prokaryotic expression concrete operations are as follows: will optimize Fc inoculation to 10mlSB substratum, in 37 DEG C, 250rpm shaking table cultivate 3 ~ 4 hours; Subsequently, be inoculated into 500mlSB substratum by 2% inoculum size, in 37 DEG C, 250rpm shaking table be cultured to OD600 reach 0.6 time, adding 250 μ l concentration is that 200 μ g/mlIPTG spend the night in 37 DEG C of abduction deliverings.

Particularly, consisting of of described SB substratum: containing 30g Tryptones, 20g yeast extract and 10gMOPS in 1L substratum, pH value 5MNaOH is adjusted to 7.0.

Further, in expression and purification step, purifying concrete operations are as follows: after prokaryotic expression, and collected by centrifugation thalline, resuspended with 50mlPBS for the first time, adds 250 μ l PXB room temperature treatment 30min, and recentrifuge collects supernatant; By collect supernatant filter after, with Ni-NTA filler purify optimize Fc albumen; The Fc albumen of the optimization after nickel post wash-out is diluted to 50ml, further with the Fc albumen that ProteinG filler difference purifying is optimized, subsequently with the Fc target protein that the ultra-filtration centrifuge tube ultrafiltration and concentration that molecular weight cut-off is 10kD is optimized; Wherein, centrifugal parameter is for the first time: 8000g, 4 DEG C, 15min; The parameter of recentrifuge is: 10000g, 4 DEG C, 15min.

The Fc of optimization of the present invention is used for the application of the transformation containing Fc fragment.

Further, the Fc of optimization of the present invention is used for the application containing the transformation of Fc fragment of the coupled product of the fusion rotein of the fusion rotein of full length monoclonal antibodies, antibody fragment and Fc, biological activity protein and Fc, bioactive compounds and Fc etc.

The present invention take Fc as skeleton, carries out directed modification transformation to it, finally obtains the new Fc that stability and resistance to aggregation be improved and clones.The Fc fragment skeleton of optimization provided by the invention can be applicable to complete IgG or relevant Fc fusion rotein and for studying further to the impact brought of the plasma half-life of associated biomolecule preparation.

The present invention is identified the secondary structure of Fc fragment optimized, existence form, stability, resistance to aggregation by methods such as CD, molecular sieve, fluorescence detector, ultraviolet spectrophotometer, ELISA, contrasts with wild-type Fc.ELISA experiment also proves that Fcs can combine with Fc acceptor (FcRn), therefore, modifies while improved Fcs improves self stability and does not destroy own biological effect.

Fcs skeleton provided by the invention, its advantage is: first, and relative to the Fc of wild-type, its stability is better, can reduce the production of monoclonal antibody or Fc fusion rotein, purifying, retain costs; Secondly, relative to the Fcs of wild-type, its resistant to aggregation ability is better, can reduce and cause Clinical practice risk because of the gathering of albumen.

In the present invention, prepare the transformation optimized the method for Fc and be not limited only to the Fc in SeqIDNo.3 sequence, also comprise the transformation of the Fc fragment to other all kinds of antibody molecule, such as, to dissimilar immunoglobulin (Ig), derive from the transformation of the immunoglobulin (Ig) of different plant species.

Accompanying drawing explanation

In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.

Fig. 1 is the structural representation of existing Fc fragment;

Fig. 2 is the protein immunoblot detected result figure of Fc, Fcs albumen in optimization method of the present invention;

Fig. 3 is the SDS-PAGE detected result figure of Fc, Fcs albumen in optimization method of the present invention after purifying;

Fig. 4 is the AKTA analytical results graphic representation of Fcs and Fc of the present invention;

Fig. 5 is the graphic representation of the circular dichroism CD spectrography detection protein molecular conformation of Fcs and Fc of the present invention;

Fig. 6 is the graphic representation of the circular dichroism CD spectrography detection thermostability of Fcs and Fc albumen of the present invention;

Fig. 7 is that the denaturant conditions stability inferior of Fcs and Fc albumen of the present invention analyzes matched curve figure;

Fig. 8 be the resistance to aggregation of Fc, Fcs albumen of the present invention OD320nm through time graphic representation;

Fig. 9 matched curve figure of OD405 under different concns that to be Fc, Fcs albumen of the present invention be combined at various ph values with FcRn.

Embodiment

Below in conjunction with embodiments of the invention, be clearly and completely described technical scheme of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.

Embodiment 1

The Fc fragment of optimization of the present invention is that modify it, after irregular curling 7 amino acid amputation of being held by the N of Fc fragment, the Fc finally obtaining the optimization that stability and resistance to aggregation are improved clones, called after Fcs with Fc gene fragment for skeleton.The Fc fragment of optimization of the present invention is represented below with " Fcs ".Wherein, Fc gene fragment can be Fc fragment in the immunoglobulin (Ig) of different plant species.

Below with the Fc fragment of the immunoglobulin (Ig) of the mankind for transformation object, the optimization method of the Fc fragment of optimization of the present invention is described, comprises the following steps:

Conveniently Fcs of the present invention (the Fc fragment of optimization) and the properties of Fc are compared, in the optimization method of the present embodiment 1, describe the amplification of Fc and the step of expression and purification simultaneously.

Step one, amplification Fc, Fcs gene fragment

Take people source blood sample, with TrizolReagent (Invitrogen company) extracting cell total rna, the cell total rna obtained is dissolved in the water of 50 μ L without RNA enzyme.Then use M-MLV Reverse Transcriptase kit (Promega company), adopt random primer, reverse transcription obtains cDNA.

According to the gene order (GenBank:KJ905798.1) of the Fc of IgG1, analyze its aminoacid sequence, the forward and reverse primer amplification object fragment of design Fc, Fcs gene: forward primer 1:5-TCGCTACCGTGGT gGCCCAGGCGGCCgCACCTGAACTCCTGGGGGGAC-3, forward primer 2:5-TCGCTACCGTGGT gGCCCAGGCGGCCcCGTCAGTCTTCCTCTTCC-3 (horizontal line is labeled as SfiI restriction enzyme site); Reverse primer: 5-GTGGTGGTGGTGGCCGGCCT gGCCTTTACCCGGaGACAGGGAGAGGCTC-3 (horizontal line is labeled as SfiI restriction enzyme site).

Be first that template carry out PCR reaction with reverse primer with cDNA with forward primer 1, obtain Fc gene fragment (nucleotide sequence is as shown in SEQIDNO:1); Again with forward primer 2 with reverse primer with Fc gene fragment for template carries out PCR reaction, thus to obtain containing Fcs gene fragment (nucleotide sequence is as shown in SEQIDNO:3).

Agarose gel electrophoresis reclaims Fc, Fcs gene fragment.

Step 2, expression also purifying Fc, Fcs

Cut Fc, Fcs gene and carrier pCom (Addgene) with SfiI enzyme, glue reclaims, and the Fc gene after being cut by SfiI enzyme, Fcs gene are connected with carrier pCom respectively, Transformed E .coliHB2151 competence, and picking mono-clonal send order-checking subsequently.According to sequencing result, the mono-clonal that picking sequence is correct is used for subsequent experimental.Wherein, prokaryotic expression carrier is not limited to above-mentioned carrier pCom, can also adopt other prokaryotic expression carrier and corresponding expressive host bacterium.

Fc, Fcs bacterial strain obtained above is inoculated into respectively 10mlSB substratum (in 1L substratum containing 30g Tryptones, 20g yeast extract and 10gMOPS, pH value 5MNaOH is adjusted to 7.0), in 37 DEG C, 250rpm shaking table cultivates 3 ~ 4 hours.Subsequently, be inoculated into 500mlSB substratum respectively by 2% inoculum size, in 37 DEG C, 250rpm shaking table be cultured to OD600 reach 0.6 time, adding 250 μ l concentration is that 200 μ g/mlIPTG spend the night in 37 DEG C of abduction deliverings.After expression, with mouse-anti Histag be first antibody, the sheep anti-mouse igg of HRP mark is two to resist for carrying out protein immunoblot detection, detected result is as shown in Figure 2.Swimming lane 1:Fc substratum; Swimming lane 2:Fc periplasmic space; Swimming lane 3:Fcs substratum; Swimming lane 4:Fcs periplasmic space; M: protein marker.The expression of obvious visible Fc, Fcs albumen from Fig. 2.

Subsequently, centrifugal (8000g, 4 DEG C, 15min) collects thalline, resuspended with 50mlPBS, adds 250 μ l PXB room temperature treatment 30min, and recentrifuge (10000g, 4 DEG C, 15min) collects supernatant.After the supernatant collected is filtered, with Ni-NTA filler (GE company) purifying Fc, Fcs albumen.Fc, Fcs albumen after nickel post wash-out is diluted to 50ml respectively, further with ProteinG filler purifying Fc, Fcs albumen respectively, subsequently with molecular weight cut-off be 10kD ultra-filtration centrifuge tube (MerckMillipore company) ultrafiltration and concentration Fc albumen (aminoacid sequence is as shown in SEQIDNO:2) and Fcs target protein (aminoacid sequence is as shown in SEQIDNO:4), its purity is verified, as shown in Figure 3 through polyacrylamide gel electrophoresis (SDS-PAGE).

The Fc albumen that the present invention obtains embodiment 1 and Fcs target protein have carried out multinomial performance test, carry out analysis contrast, prove the advantage of the FC gene fragment of optimization of the present invention.

(1) molecular conformation of Fc, Fcs and existence form

AKTA analyzes Fc, Fcs existence form: by Fc, Fcs protein concentration after purifying to 1mg/ml, PBS (pH7.4) is elution buffer, crosses ColumnSuperdex200Increase10/300GL, wherein, flow velocity is 1ml/min, and then detects its existence form.As shown in Figure 4, in figure, "●" represents Fcs albumen to the graphic representation that detection obtains, and " ■ " represents Fc albumen; Reference standard curve can find, these two molecular weight of albumen of Fc and Fcs are about 45kDa, and result shows that they exist with dimeric forms.

Circular dichroism CD spectrography detects protein molecular conformation: Fc, Fcs albumen after purifying is diluted to 50 μ g/ml, PBS (pH7.4) is contrast, its molar average ovality (i.e. circular dichroism) is detected under near ultraviolet end (wavelength X=190nm ~ 260nm) different wave length condition, and then analyzing proteins secondary structure.As shown in Figure 5, in figure, "●" represents Fcs albumen to detected result curve, and " ■ " represents Fc albumen; Analyze known, have an obvious trough at λ=218nm place, show that above-mentioned Protein secondary structure is beta sheet.

(2) stability of Fc, Fcs

A, thermostability: utilize circular dichroism spectrometer to compare Fc, Fcs albumen (50 μ g/ml) thermostability, PBS solution is blank, set temperature gradient condition is that every 2min raises 1 DEG C, termination reaction temperature is 94 DEG C, every 30s detects and record data at wavelength X=218nm place, for analysis.As shown in Figure 6, in figure, "●" represents Fcs albumen to the detection curve obtained, and " ■ " represents Fc albumen; When wavelength X=218nm, detect the difference of the molar absorptivity of its left and right circularly polarized light at different temperatures, and then compare its thermal stability difference, analyze known, the thermostability of modifying improved Fcs is higher than Fc 3.5 DEG C.

B, denaturant conditions stability inferior are analyzed: configuration concentration is the urea soln of 0M to 10M, and concentration gradient is 0.5M, totally 21 samples; Then Fc, Fcs albumen is added 4 DEG C of process of spending the night in the urea soln of different concns gradient, volume is 600 μ l, and Fc, Fcs final concentration of protein is 50 μ g/ml.Subsequently, excite at 280nm with fluorescence detection equipment, each sample fluorescence signal intensity is detected at 340nm place, compares the stability of Fc, Fcs albumen.Detected result matched curve as shown in Figure 7, in figure, the curve of " ■ " matching is the curve of Fcs, and the curve of "○" matching is the curve of Fc; Can find to compare Fc from figure, the fluorescent signal downtrending of Fcs is slower, shows that Fcs is more stable under denaturant conditions.

(3) resistance to aggregation of Fc, Fcs

Fc, Fcs final concentration of protein is adjusted to 1mg/ml, and 60 DEG C of water-bath thermal treatments, every 30min sampling, detect absorbancy and record data by ultraviolet spectrophotometer at λ=320nm place, the resistance to aggregation comparing Fc, Fcs albumen is strong and weak.Wherein, PBS solution is blank.Fc, Fcs albumen OD320nm obtained through time curve as shown in Figure 8, in figure, the curve of "●" matching is the curve of Fcs, and the curve of " ■ " matching is the curve of Fc; Can find that from figure Fcs signal ascendant trend is more slow, show that its resistant to aggregation ability is better.

(4) Fc, Fcs albumen and Fc acceptor (FcRn) avidity under condition of different pH is compared

Adopt ELISA to measure the combination of Fc, Fcs and FcRn, step is as follows:

Step 1 wraps quilt: with the FcRn albumen bag of 2 μ g/ml by costar96halfwells plank, stick preservative film, 4 DEG C of refrigerator overnight.

Step 2 is washed: sucking-off reaction solution, is dried by liquid in hole, fills it up with washings, soaks 3min and dries, three times repeatedly.

Step 3 is blockaded: in Sptting plate, every hole adds 100 μ l3% skim-milks (37 DEG C, 2h).

Step 4 is washed: sucking-off reaction solution, is dried by liquid in hole, fills it up with washings, soaks 3min and dries, three times repeatedly.

Step 5 sample: Fc, Fcs albumen is diluted to different pH (pH6.0, pH7.4) PBS damping fluid, final concentration is 50 μ g/ml, often organize 2 holes and add the concrete dilution albumen of 50 μ l (1:3 dilution), then plank is pasted preservative film and be statically placed in 37 DEG C of incubators 2 hours.

Step 6 is washed: with step 4, washes three times respectively by the PBS solution of different pH.

Step 7 adds HRP enzyme labelled antibody: add anti-flag monoclonal antibody, and every hole 50 μ l (1:1500 dilution), is then statically placed in 37 DEG C of incubators 2 hours.

Step 8 is washed: with step 6.

Step 9 develops the color: every hole adds 50 μ lABTS reaction substrates, detects after room temperature lucifuge process 15min.

Step 10 detects: detect OD405 with microplate reader device.

According to the OD405 value detected, under drawing different pH value, the matched curve of OD405 under different concns, as shown in Figure 9, in figure, the curve of the Fcs that the curve of " ▲ " matching is pH value when being 6, the curve of the Fcs that the curve of " ■ " matching is pH value when being 6, the curve of the Fcs that the curve of " △ " matching is pH value when being 7.4, the curve of the Fcs that the curve of " " matching is pH value when being 7.4.Analyze known, Fc and FcRn avidity under condition of different pH is different, when pH6.0, keeps high-affinity with FcRn; When pH7.4, reduce with FcRn avidity, and then the running balance of its plasma concentration can be maintained.In figure, data show, the Fcs through modifying transformation of the present invention still keeps the binding activities depending on different pH from FcRn.

The above; be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses; change can be expected easily or replace, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should described be as the criterion with the protection domain of claim.

Claims (10)

1. the Fc fragment optimized, is characterized in that: be that irregular curling 7 amino acid by hold by the N of Fc fragment amputate and obtain.

2. the optimization method of the Fc fragment optimized as claimed in claim 1, it is characterized in that: comprise and utilize RT-PCR, from blood sample, amplify wild-type Fc, 7 amino acid that the N of wild-type Fc holds by bamboo product primer block, and amplify the amplification step optimizing Fc fragment.

3. optimization method according to claim 2, is characterized in that: in described amplification step, and for people source blood sample, the amplification method of wild-type Fc is: people source blood sample extracting cell total rna, and reverse transcription obtains cDNA; Be that template carries out PCR reaction with cDNA, obtain wild-type Fc gene fragment; The forward primer adopted in PCR reaction is 5-TCGCTACCGTGGTGGCCCAGGCGGCCGCACCTGAACTCCTGGGGGGAC-3, and reverse primer is 5-GTGGTGGTGGTGGCCGGCCTGGCCTTTACCCGGAGACAGGGAGAGGCTC-3.

4. optimization method according to claim 3, is characterized in that: in described amplification step, and the amplification method of the Fc fragment of optimization is: with wild-type Fc gene fragment for template carries out PCR reaction, the Fc fragment be optimized; Wherein, the forward primer adopted in PCR reaction is 5-TCGCTACCGTGGTGGCCCAGGCGGCCCCGTCAGTCTTCCTCTTCC-3, and reverse primer is 5-GTGGTGGTGGTGGCCGGCCTGGCCTTTACCCGGAGACAGGGAGAGGCTC-3.

5. according to the optimization method one of claim 2 to 4 Suo Shu, it is characterized in that: after amplification step completes, also comprise expression and purification step; Described expression and purification step is specially: the optimization Fc fragment after being cut by SfiI enzyme carries out to prokaryotic expression carrier connecting, transform corresponding expressive host bacterium is optimized Fc bacterial strain; Carry out prokaryotic expression to optimization Fc bacterial strain again, purifying obtains the Fc fragment that target protein is optimized.

6. optimization method according to claim 5, it is characterized in that: in expression and purification step, first adopt agarose gel electrophoresis to reclaim and optimize Fc fragment, then cut with SfiI enzyme and optimize Fc fragment, again glue reclaim, obtain SfiI enzyme cut after optimization Fc fragment.

7. optimization method according to claim 5, is characterized in that: in expression and purification step, and prokaryotic expression concrete operations are as follows: will optimize Fc inoculation to 10mlSB substratum, in 37 DEG C, 250rpm shaking table cultivate 3 ~ 4 hours; Subsequently, be inoculated into 500mlSB substratum by 2% inoculum size, in 37 DEG C, 250rpm shaking table be cultured to OD600 reach 0.6 time, adding 250 μ l concentration is that 200 μ g/mlIPTG spend the night in 37 DEG C of abduction deliverings.

8. optimization method according to claim 5, is characterized in that: in expression and purification step, and purifying concrete operations are as follows: after prokaryotic expression, collected by centrifugation thalline for the first time, resuspended with 50mlPBS, add 250 μ l PXB room temperature treatment 30min, recentrifuge collects supernatant; By collect supernatant filter after, with Ni-NTA filler purify optimize Fc albumen; The Fc albumen of the optimization after nickel post wash-out is diluted to 50ml, further with the Fc albumen that ProteinG filler difference purifying is optimized, subsequently with the Fc target protein that the ultra-filtration centrifuge tube ultrafiltration and concentration that molecular weight cut-off is 10kD is optimized.

9. require the application of the Fc fragment of the optimization as described in 1 as profit, it is characterized in that: the Fc of optimization is used for the application containing the albumen of Fc fragment or the transformation of conjugate.

10. the application of the Fc fragment of optimization according to claim 9, is characterized in that: the Fc of optimization is used for the application of transformation of the fusion rotein of full length monoclonal antibodies, antibody fragment and Fc containing Fc fragment, biological activity protein and the fusion rotein of Fc or the conjugate of bioactive compounds and Fc.