CN106065031A - Antibody based on hybridoma technology and high-flux sequence finds method - Google Patents
Antibody based on hybridoma technology and high-flux sequence finds method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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Abstract
The invention discloses a kind of method that generation combines the recombinant antibodies of at least one target antigen, and the recombinant antibodies being generated by.High flux antibody sequence measurement and the hybridoma antibody triage techniques of optimization are combined by the method producing antibody of the present invention, compared with traditional authentic monoclonal antibody technology, expand the range of choice of candidate antibodies gene, improve the efficiency that antibody structure optimizes, add the probability obtaining functional antibodies.
Description
Technical field
The present invention relates to biomedicine technical field, in particular it relates to one is based on hybridoma technology and high-flux sequence
The method producing the recombinant antibodies combining at least one target antigen, and the recombinant antibodies obtained by the method.
Background technology
Hybridoma technology is the most frequently used antibody digging technology, but its complex steps, and obtain monoclonal cell institute
The subcloning procedures needed time-consumingly long, cost height.
In recent years, high throughput sequencing technologies is increasingly being applied to the phenetic analysis of immune antibody variable region gene spectrum,
And the discovery of antigen-specific antibodies.The method is by the high-flux sequence to immune forward and backward immunoglobulin gene, it is thus achieved that
The antibody variable gene spectrum of immune animal.After immunity, the antibody gene of significant enrichment is deemed likely to and specific antigen phase
Close, choose the abundant heavy chain in antibody variable gene spectrum and light chain gene matches recombinant expressed antibody in vitro.Compare
Relatively antibody gene random pair is expressed, and uses the pairing of high abundance gene can improve the efficiency producing function antibody, reduces pairing
Blindness.Express and functional verification, expanded function antibody screen additionally, substantial amounts of candidate sequence also can be produced for antibody conjugates
The scale of scavenger.Relative to traditional hybridoma and display technique of bacteriophage, this technology evades the limitation of both technology,
The species limited including being only applicable to Mus and rabbit etc., the codon in prokaryotic expression system selects, and follow-up needs carries out humanization
Etc. technological difficulties.Meanwhile, antibody group sequence measurement can accelerate to obtain the process of hybridoma antibody gene.Final without obtaining
Monoclonal, can collect and check order at few cloning stages；Non-monoclonal sequence is corrected by secondary sequencing result.Compared to biography
Single/few clonal antibody information of generation order-checking detection of system, after can adding cell index sequence by PCR, secondary survey is used in mixing
Sequence carries out hybridoma antibody gene sequencing, improves flux, reduces cost.
But, the defect of this technology is: 1) cannot obtain natural antibody unpaired message；2) simple dependence gene abundance is joined
To causing, gene matching method is single, is paired into power low, and subsequent gene synthesis and pairing screening operation amount are big.
In recent years, analyzed the abundance of antibody variable region polypeptide by protein technique, obtain in conjunction with high-flux sequence anti-
The method of the sequence of body gene, has been applied to antibody producing.Use specific marker separation antibody to produce cell, pass through high pass
Amount monoclonal antibody sequencing technologies can obtain native heavy and light chain unpaired message.But monoclonal antibody order-checking and protein
Group analysis has the highest requirement for analysis of biological information method and experiment condition, and this adds the difficulty that antibody is excavated undoubtedly
Degree.Additionally, the acquisition of antibody variable gene spectrum there is also some technical difficulty.The coverage of antibody sequence specific primer and
Primer amplification efficiency differs, and causes antibody variable gene spectrum deleted antibodies gene information or information to there is skewed popularity, it is thus achieved that
High abundance sequence do not possess antigenic specificity；RACE method builds storehouse can overcome skewed popularity, but amplified fragments is longer to be difficult to
Survey logical, it is possible to lack part antibody sequence information.
Summary of the invention
The longest for existing hybridoma technology, antibody cell system screening scope is little, it is difficult to obtain high-affinity monoclonal
The defect of antibody, and antibody sequencing technologies exist build storehouse skewed popularity and the pairing defect such as blindness, the present invention will optimize
High flux antibody sequence measurement and hybridoma antibody triage techniques combine, with traditional authentic monoclonal antibody technology phase
Ratio, expands the range of choice of candidate antibodies gene, improves the efficiency that antibody structure optimizes, adds acquisition functional antibodies
Probability.
The present invention is achieved through the following technical solutions above-mentioned purpose:
First aspect, the invention provides the method producing the recombinant antibodies combining at least one target antigen, comprising:
(1) with described at least one target antigen immunity host subject；
(2) use the spleen cell through immune host subject, prepare hybridoma cell line, and obtain specific binding
The hybridoma antibody gene information of at least one target antigen described；
(3) use the lymphocyte of the host subject after immunity, carry out the high flux of immunoglobulin gene variable region
Order-checking, obtains the antibody variable gene at least one target antigen described and composes；
(4) choose hybridoma antibody heavy chain gene and match gene as the first candidate, and compose with from antibody variable gene
The light chain gene of middle selection matches gene as the second candidate and matches, to produce candidate's recombinant antibodies.
In a particular embodiment, in step (2), prepared hybridoma cell line can produce to be had with described target antigen
There is the monoclonal antibody of high-affinity, be preferably the binding constant with target antigen and be not higher than 10-9The monoclonal antibody of M.
In preferred embodiments, described hybridoma antibody gene information comprises heavy chain and the variable region of light chain base of antibody
Because of information.
In preferred embodiments, by following acquisition hybridoma antibody gene information:
With the total mRNA of hybridoma as template, Oligo dT is primer, through reverse transcription synthesis cDNA the first chain, then with
CDNA the first chain is template, with primer amplified antibody heavy chain variable region and the chain variable region gene of mixing, order-checking point
Analysis obtains hybridoma gene order, and hybridoma gene order carries out IgBlast analysis, gets rid of and derives from melting of composition hybridoma
Close antibody gene and the nonsense suppressor of companion's tumor self, obtain candidate's heavy chain and chain variable region gene.
In preferred embodiments, in step (3), described antibody variable gene spectrum comprises heavy chain and the light chain of antibody
Variable region gene information；
Preferably, described gene information is determined by high flux DNA sequencing；
It is further preferred that described high flux DNA sequencing carries out while being included in sequencing reaction synthesizing, connecting or miscellaneous
Hand over；And unique DNA order-checking, the order-checking of many polymerases group, nano-pore order-checking or a combination thereof；
It is further preferred that described high-flux sequence is by following enforcement:
With the total mRNA of cell as template, SMARTer RACE technology is used to synthesize the first chain cDNA；Design forward primer, and
According to antibody constant region sequences conserved regions design downstream primer, to expand antibody sequence variable region.
In preferred embodiments, in step (4), from antibody variable gene compose choose comprise high abundance CDR3 or
Person comprises light chain gene to described CDR3 the most similar for high abundance CDR3 and matches gene as the second candidate.Invention Crinis Carbonisatus
Existing, the light chain gene with high similarity CDR3 can produce function antibody with the pairing of same heavy chain gene.
In preferred embodiments, in composing from antibody variable gene, selected second candidate matches the CDR3 of gene
In the case of, it is prepared by the following the second candidate and matches the antibody variable region of gene:
From the sequencing result of the antibody gene segments comprising selected CDR3, choose successively the most abundant CDR2 and
CDR1 sheet segment information, then selects the most abundant variable region full length sequence comprising selected CDR2 and CDR1 sheet segment information, makees
It is that the second candidate matches the antibody variable sequences of gene.
In preferred embodiments, described method also includes reflecting gained antibody with at least one target antigen described
Fixed step, thus produces the recombinant antibodies combining at least one target antigen described.
In a particular embodiment, described authentication step include vivoexpression gained antibody scFv or Fab fragment or
IgG, is combined power test by gained scFv, Fab fragment or IgG with target antigen；
Preferably, described vivoexpression method including but not limited to: by prokaryotic cell, phage display method or ferment
Mother system expresses scFv or Fab fragment, or by mammalian cell exogenous gene expression Fab or IgG；
Preferably, described method of testing includes but not limited to ELISA and/or SPR.
In the context of the present invention, term " high-affinity " refers to that affinity of antibody constant is not higher than 10-9M。
In the context of the present invention, term " high abundance " refers to that the abundance in antibody variable gene is composed is higher than 1%.
In the context of the present invention, term " the most similar " refers to have with object to be compared on amino acid levels
Sequence iden higher than 80%.
Second aspect, the invention provides a kind of recombinant antibodies prepared according to the method described in above-mentioned first aspect.
In one embodiment of the invention, inventor obtains the functional restructuring of selectively targeted HA and resists
Body, the variable region of heavy chain of described recombinant antibodies includes such as SEQ ID NO:40, the CDR3 shown in 42 and 50, and described restructuring resists
The variable region of light chain of body includes such as SEQ ID NO:20, the CDR3 shown in 53,68 and 103.
In another specific embodiments of the present invention, inventor obtains the functional heavy of selectively targeted PD-L1
Group antibody, the variable region of heavy chain of described recombinant antibodies includes the CDR3 as shown in SEQ ID NO:84, and described recombinant antibodies
Variable region of light chain include such as SEQ ID NO:23, the CDR3 shown in 89 and 105.
Compared with prior art, the method have the advantages that
The present invention combines hybridoma technology and antibody sequencing technologies, by specific selection mode, will obtain from hybridoma
The light chain gene pairing obtained in the antibody heavy chain gene taken and antibody variable gene spectrum is expressed, and obtains functional antibodies.Institute
The antibody library preparation flow of the optimization used, can intactly obtain the antibody variable gene spectrum of immunized subject, experiment
Result has the highest repeatability；The bioinformatic analysis method of the optimization used can obtain the abundance of CDR3 rapidly
Information and accurately antibody gene variable region total length information.The hybridoma technology that compares and simple dependence antibody gene abundance select
The method of pairing candidate sequence, the antibody of the present invention finds that method can improve candidate and match the range of choice of gene, overcomes and join
Problem to blindness, improves successfully pairing and obtains the efficiency of functional antibodies.
Accompanying drawing explanation
Fig. 1 shows the flow chart of the method producing recombinant antibodies of the present invention.
Fig. 2 shows variable region of heavy chain and the agarose gel electrophoresis figure of variable region of light chain of amplification in embodiment 2；Wherein,
VH represents that variable region of heavy chain, VL represent that variable region of light chain, M represent D2000marker (Takara).
Fig. 3 shows the Bioanalyzer qualification result of heavy chain and variable region of light chain sequencing library prepared by embodiment 2.
Fig. 4 shows the dependency of CDRH3 abundance between bone marrow and the spleen tissue of sample GW02-5.
Fig. 5 shows the dependency of CDRL3 abundance between bone marrow and the spleen tissue of sample GW02-5.
Fig. 6 show each spleen tissue of sample GW02-3 repeat between the dependency of CDRH3 abundance.
Fig. 7 show each spleen tissue of sample GW02-3 repeat between the dependency of CDRL3 abundance.
Fig. 8 show each spleen tissue of sample GW02-5 repeat between the dependency of CDRH3 abundance.
Fig. 9 show each spleen tissue of sample GW02-5 repeat between the dependency of CDRL3 abundance.
Figure 10 shows the affinity analysis between GW01 antigen polypeptide and pairing antibody；Numeric representation OD490nm ripple in figure
Long absorption value；Figure inner conical base portion solid line represents the natural pairing of hybridoma antibody.
Figure 11 shows the affinity analysis between GW02 antigen polypeptide and pairing antibody；Numeric representation OD490nm ripple in figure
Long absorption value；Figure inner conical base portion solid line represents the natural pairing of hybridoma antibody.
Figure 12 is the result schematic diagram of the antibody conjugates efficiency of display GW01 antigen and GW02 antigen.
Detailed description of the invention
For ease of understanding the present invention, further illustrate below by way of the mode enumerating specific embodiments and specific embodiment
Technical scheme.Those skilled in the art are it will be clearly understood that cited specific embodiments and embodiment are only side
Assistant solves the present invention, is not construed as the concrete restriction to the present invention.
Such as, in a specific embodiment, the present invention uses following program:
1) using standard immunization protocol at the 0th day and the 21st day twice immune mouse, before taking immunity, mice serum is as the moon
Property comparison；Within after initial immunity the 35th day, take blood examination and survey serum titer, reach serum titer to merge the mice booster immunization required,
Within after initial immunity the 38th day, carry out cell fusion and prepare hybridoma.After fusion about 10 14 days, take hybridoma supematant and carry out gram
Grand detection, separates sub-clone, the sub-clone that screening affinity is the highest.
2) with the mRNA of hybridoma as template, Oligo dT is primer, reverse transcription synthesis cDNA the first chain, then with
CDNA the first chain is template, uses heavy chain and light chain degenerate primer PCR amplification VH and VL gene (Tiller et al., 2009), institute
Table 1 below is seen with primer sequence.Employing standard PCR program amplified hybridization tumor antibody sequence variable region, PCR primer cloning and sequencing,
Antibody gene is carried out IgBlast analysis, rejects pseudogene and the gene stopped in advance.
Table 1
3) use Trizol cracking spleen and medullary cell and extract RNA, using SMARTer RACE (Clontech) skill
Art synthesizes the first chain cDNA；Design forward primer and according to antibody constant region sequences conserved regions design downstream primer, expand antibody
Sequence variable region.One section of sequential design forward universal primer is had with cDNA chain 5 ' end, and at forward universal primer 5 ' end
Connect Illunina MiSeq forward sequence measuring joints.According to murine constant region conserved sequence reverse primer, and add at primer 3 end
Illumina MiSeq backward sequencing joint.Primer sequence information is shown in Table 2.
Table 2
Amplification heavy chain and sequence of light chain variable region, use Illumina Miseq 2 × 250 order-checking, carry out after disposal data
IgBlas analyzes, and obtains the aminoacid sequence of complementary determining region 3 (complementary determining region 3, CDR3)
Row, according to the abundance ranking of sequence, select and comprise high abundance CDR3 or comprise the most similar to described high abundance CDR3
The antibody light chain gene of CDR3 matches gene (that is, specifically described herein to " the second candidate match gene ") as light chain candidate；Choose
Hybridoma heavy chain gene matches gene (that is, specifically described herein to " the first candidate match gene ") as heavy chain candidate；By selected
Heavy chain candidate match gene and light chain candidate and match gene in mammalian cell, match expressions, test pairing antibody and spy
The affinity of hapten.
The separation of embodiment 1 hybridoma antibody sequence and qualification
Use following program:
(1) amplification culture myeloma cell so that it is be in exponential phase when merging.Merge the same day, collect cell, meter
Number, standby.
(2) take serum titer and reach agglutinin of blood HA antigen polypeptide (hereafter representing with code name GW01) and the immunity inspection of standard
Make an inventory of each 2 immune mouses of PD-L1 antigen polypeptide (hereafter representing with code name GW-02) and prepare hybridoma, be respectively designated as GW01-
1, GW01-3 and GW02-3, GW02-5.Excision eyeball of mouse is taken a blood sample, and separates serum as positive control blood during antibody test
Clearly.Draw neck to put to death mice, the aseptic mouse spleen that takes, separating Morr. cell splitting erythrocyte, obtain pure bone-marrow-derived lymphocyte.Carefully
Myeloma cell is mixed in the proper ratio by born of the same parents' fusion with bone-marrow-derived lymphocyte, makes two kinds of cells merge under fusion agent effect.Melt
After cell HAT culture medium after conjunction is resuspended, subpackage contains 96 porocyte culture plates of macrophage.Put 37 DEG C, 5%CO2Training
Cultivate in supporting case.HT culture medium of changing cell HAT culture medium culturing 2 Zhou Houyong after fusion continues to cultivate 2 weeks, then changes
R1640 complete medium.
(3) after merging about 10-14 days, take hybridoma supematant and carry out for the first time cloning detection, hole is changed fresh culture.
Continue to cultivate 3 days, take hybridoma supematant and carry out cloning detection for the second time, hole is changed fresh culture.Positive colony is expanded training
Support to 48 orifice plates.Positive colony is carried out cloning simultaneously, will be diluted by positive colony, by the cell inoculation one of 1, every hole
Block 96 orifice plate, 200ul/ hole.Continuing to cultivate 3 days, take hybridoma supematant and carry out cloning detection for the third time, amplification culture is positive simultaneously
Clone.Frozen positive colony cell strain, collects supernatant simultaneously and carries out hypotype qualification.
(4) by 23 GW01 with high-affinity identified and 18 GW02 monoclonal hybridoma system recoveries
After, through amplification culture to 1 × 107Individual cell, extracts total serum IgE with Trizol (Invitrogen company), enters by shop instruction
OK.Use Reverse Transcription box SuperScriptIII (Life Technologies) that 2 μ g total serum IgE reverse transcriptions are become cDNA the 1st
Chain, is carried out by shop instruction.Using universal primer clonal antibody weight, chain variable region gene, universal primer sees above
Table 1；As a example by chain variable region gene is cloned, mixed in equal amounts light chain primer, take 2 μ l mix primer, 2 μ l cDNA are template,
Pfu enzyme (GENEWIZ) prepares reaction system, after response procedures is 98 DEG C of C 30s of denaturation, carries out 98 DEG C of C 8 seconds, 58 DEG C of C 15
Second, the amplification program of 72 DEG C of C 30 seconds totally 30 circulations, then 72 DEG C of C are incubated 2 minutes.Use BciVI removal non-functional gently
Chain gene, enzyme action system is as follows: BciVI, 1 μ l, PCR primer 1 μ g, and 10 times of buffer 5 μ l then moisturizings are to 50 μ l, 37 DEG C of C enzymes
Cutting through night, electrophoresis reclaims and the fragment of purification about 400bp, and is cloned into carrier T, and sequence of light chain is identified in order-checking.Use
Similar steps, uses heavy chain primer PCR to expand heavy chain variable region gene.Agarose gel electrophoresis separation PCR primer, reclaims purification
400bp-500bpPCR product, and it is cloned into sequencing vector to check order, the sequence inputting IMGT data base that will record
Be analyzed, get rid of and derive from the antibody gene of fusion partner tumor self and the nonsense suppressor constituting hybridoma, obtain heavy chain and
The variable region of light chain gene.
The preparation of embodiment 2 antibody sequencing library and the analysis of antibody sequence
It is respectively adopted embodiment 1 and prepares the remaining spleen tissue of hybridoma and thin from the bone marrow of same immune mouse
Born of the same parents, carry out the preparation of antibody sequencing library.
Specifically, being ground by described spleen tissue in liquid nitrogen, extract total serum IgE, concrete extraction procedure is by extracting reagent
Shop instruction is carried out；Through the mice of immunity, gather tibia from same and femur removes muscle and fatty tissue, with injection
Device washes out bone marrow and is collected in the middle of centrifuge tube in Trizol injects bone, extract total serum IgE, and concrete extraction procedure is by extracting examination
The shop instruction of agent is carried out.
Use antibody variable region forward primer and heavy chain and variable region of light chain downstream primer, with sample 5 ' RACE product as mould
Plate, amplification heavy chain and variable region of light chain, prepare the sequencing library of heavy chain and variable region of light chain respectively.
PCR amplification system and amplification program are shown in table 3 below and table 4 respectively.
Table 3
Table 4
Barcode is added by the method for PCR, for distinguishing the sequencing result of different sample at amplified production end.PCR
Amplification system and amplification program are shown in table 5 below and table 6 respectively.
Table 5
Table 6
The purification of amplified production: add the AMPure Beads of 0.8 times of volume in PCR primer, mixing room temperature stands
5min；Brief centrifugation is placed on magnetic frame upper 5 minute；Abandon supernatant, add after 70% dehydrated alcohol washes twice and carefully discard remnants
Ethanol, room temperature is placed and within 10 minutes, is treated ethanol volatilization completely.Adding 30 μ l 10mM Tris (pH8.0) and mix beads, room temperature is quiet
Putting 5 minutes, eluted dna is also transferred in new pipe.
By the agarose gel electrophoresis result of above-mentioned PCR amplification derived heavy chain variable region and variable region of light chain as shown in Figure 2.
Additionally, use Agilent 2100Bioanalyzer to analyze system detection library inserts size and content, institute
Obtain the Bioanalyzer qualification result in heavy chain and library, variable region of light chain as shown in Figure 3.
Illumina MiSeq 2 × 250 (GENEWIZ) is used to carry out high-flux sequence.To heavy chain and variable region of light chain literary composition
Each of storehouse sets appropriate order-checking number, after data produce, gets rid of incomplete sequence, by the complete R2 sequence comprising CDR3
Row carry out IgBlast comparison, produce CDR3 region amino acid sequence information bank.Owing to CDR3 sequence is that different antibodies gene is close to only
The indications of one, can assess its abundance in antibody variable gene is composed according to the quantity of the CDR3 sequence obtained.With sample
As a example by product GW02-5, comparing antibody variable gene spectrum (see Fig. 4-5, table 7-8) coming from spleen and bone marrow, result shows two
Plant the dependency of CDRH3 or CDRL3 abundance in tissue close, R2Value is respectively 0.69 and 0.71；Wherein ranking be positioned at antibody can
The dependency of the high abundance CDR3 becoming district's gene profile top ten is relatively low, it means that under the conditions of reinforced immunological, high-flux sequence
Result can reflect bone marrow and antibody cell kind and the difference of quantity in spleen tissue.
Table 7
Table 8
Additionally, the dependency of CDRH3 and the CDRL3 aminoacid sequence abundance that also compares between each repetition is (see Fig. 6-figure
9), result shows that CDRH3 and the CDRL3 sequence abundances between each repetition has the highest dependency, illustrates that experimental system is steady
Fixed.
Embodiment 3 hybridoma and the comparison of antibody variable gene spectral sequence and the selection of candidate's matched sequence
Total analyzes 18 GW02 and 23 GW01 monoclonal hybridoma systems, submits to hybridoma antibody gene variable
District carries out IgBlast, determines CDR3H and CDR3L sheet segment information.CDR3H and CDR3L is composed with corresponding antibody variable gene
Relatively, major part hybridoma CDR3H and CDR3L is included in the middle of antibody variable gene spectrum, and part CDR3 is corresponding
Order-checking bar number exceedes the 1% of order-checking total number, belongs to high abundance CDR3.The number of high abundance CDR3 and hybridoma CDR3 are anti-
Ratio in body variable region gene spectrum is shown in table 9 below.
Table 9
In table 9, CDR3 > 1%: exceed the quantity of the hybridoma CDR3 of antibody order-checking bar number 1%；CDR3 quantity: choose
Hybridoma CDR3 quantity；% always checks order bar number: hybridoma CDR3 ratio in antibody order-checking bar number.
Further analyze and show, hybridoma natural pairing CDR3H and CDR3L abundance in antibody library heterogeneity.
The abundance of part CDR3H and CDR3L is close, and the such as abundance of sample GW01-3-11/GW01-3-3CDR3H TRGDY is
9.58%, the abundance of pairing CDR3L WQGTHFPWT is 5.57%；The abundance difference of portion paired is relatively big, with sample GW02-5
As a example by, CDR3H ARAYGDYGFIY and ASLLDF of GW02-5-191 and GW02-5-231, their abundance is respectively 6.78%
With 3.02%；But the abundance of pairing CDR3L FQGSLAPST and LQYDEIPYT is respectively 0.22% and 0.23%；Additionally,
The abundance of CDR3L WQGTHFPWT and QQYNSYPLT of GW02-5-5/5-24/5-43 and GW02-5-10 8.64% He respectively
7.21%, the abundance of pairing CDR3H is 0.58% and is close to 0.And WQGTHFPWT and QQYNSYPLT is at two antigen GW-
All exist with high abundance in 01 and GW-02.Thus it is presumed that antibody gene is likely to be of multiple matching form, comprise high abundance
The antibody gene of CDR3, or comprise all possible pairing formation functional antibodies between high abundance and low abundance CDR3 antibody gene.
Table 10 below-table 13 is each hybridoma sequence enrichment analysis result in antibody library.
Table 10
Table 11
Table 12
Table 13
In above table, VH: variable region of heavy chain；VL: variable region of light chain.
Choose 6 hybridoma antibody heavy chain genes for two antigens, in composing with antibody variable gene, comprise high abundance
The light chain gene of CDR3 and the hybridoma antibody light chain gene of natural pairing match expression in vitro.The hybridoma heavy chain selected
Gene C DR3 is all concurrently present in the middle of antibody variable gene spectrum, and wherein three CDR3, TRGDY,
ARSGDAYYFAWFAY and ARGGGA ranking in the middle of antibody variable gene is composed is respectively the 1st, 2 and 10；Its excess-three bar
CDR3 belongs to low abundance CDR3.In the antibody natural pairing light chain of its correspondence 2 comprise high abundance CDR3 remaining 4 and comprise low
Abundance.The light chain candidate comprising high abundance CDR3 chosen matches gene at least one immunity receptor antibody variable gene spectrum
Middle ranking is positioned at top ten.
GW01 and GW02 candidate CDR3H and CDR3L pairing gene information is shown in table 14 below and table 15 respectively.Table 14
Table 15
Candidate matches full length gene information source in hybridoma gene sequencing, or is chosen by antibody variable region full length sequence
Choosing method obtains, and first such as light chain gene variable region K10 and K11 determine candidate CDR3 in the sequencing result of gene variable region
Sequence, continuing, you select high abundance CDR2 and CDR1 sequence successively, determine the sequence information of CDR1, CDR2 and CDR3 with this, so
After obtain from sequencing result and comprise the high abundance fragment determining CDR1, CDR2 and CDR3, obtain antibody variable region total length ammonia
Base acid sequence and nucleotide sequence information.De novo synthesis antibody gene expression of matching in vitro.
Compare candidate and match the CDR3 region sequence of light chain gene, at portion gene, there is higher similarity, such as K5,
Similarity between K8, K9, K10 reaches 78%-89%, and they are likely to be in antibody forming process the hyper mutation formed
Body, the enrichment in antibody variable gene is composed might mean that they are relevant to specific antigen.
Embodiment 4GW01 and the vivoexpression of GW02 pairing antibody
By the variable region of candidate's heavy chain and the light chain method construction expression frame by bridging PCR, expression cassette includes promoter,
Variable region and people's antibody constant region.The plasmid comprising heavy chain and light chain expression frame is matched and transfects 293FT cell, carry out external table
Reach and antibody function analysis.Collect 293FT cell and adjust cell density and inoculate 48 orifice plates to 1.2 × 105/ hole；37 DEG C, 5%
CO2Incubator overnight incubation, transfects when cell density length to 60-80%.By 0.25 μ g heavy chain and 0.75 μ g light chain plasmids
Incubated at room 5 minutes, after then mixing with transfection reagent, form transfection composite in incubated at room 20min.Transfection is combined
Thing adds cell hole, mixes gently.37 DEG C, 5%CO2Incubator is cultivated 72 hours.Collect supernatant ELISA detection activity.First
Anti-human igg (Fc) is coated liquid with pH 9.6 carbonic acid respectively with detection antigen and is diluted to 10 μ g/ml, 96 hole ELISA Plate every hole bags
By 100 μ l, 4 DEG C, overnight；Or 37 DEG C, 2 hours；Then closing with 4% defatted milk powder-PBS, 300 μ l/ holes, 37 DEG C process 1
～2 hours.Abandon liquid in enzyme mark hole, add 48 hours culture supernatant 100 μ l/ holes of transfection after washing three times with PBST, at 37 DEG C
Manage 1 hour, set culture medium and PBS control.Abandon liquid in enzyme mark hole, wash three times with PBST, be separately added into HRP goat-anti people
IgG (Fc), 1:2000；With HRP goat anti-human igg, 1:5000；100 μ l/ holes, 37 DEG C process 1hr.Abandon liquid in enzyme mark hole, use
PBST washes five times, adds OPD nitrite ion, 100 μ l/ holes, and lucifuge develops the color；Microplate reader reads OD490 wavelength absorption value.
The pairing affinity of antibody analysis result of antigen GW01 and GW02 the most as shown in FIG. 10 and 11, antigen GW01 and
The antibody conjugates efficiency analysis result of GW02 is the most as shown in figure 12.
Result shows, comes from the middle of 44 heavy chains/light chain pairing of two immune host subject, it is thus achieved that 7 have parent
With the antibody of power, wherein having 3 is the natural pairing of hybridoma antibody gene；Remaining 4 is hybridoma heavy chain gene and comprise height
The restructuring pairing of abundance CDR3 light chain, heavy chain gene GW02-5-H2 and 3 light chains K5, K9 and K11 match all formed have affine
The antibody of power, wherein QQYNSYPLT (K5) broadly falls in the middle of the variable region gene of two kinds of target antigen immunity host subject is composed
High abundance CDR3L, it is 89% with the similarity of QQYNSYPFT (K9) CDR3, and the similarity of gene variable region is 84%, meaning
And there is the light chain gene of high similarity CDR3 may match with same heavy chain, form function antibody.Also imply that and can adopt
Forming a small libraries by the sequence that pairing antibody CDR3 district similarity is high, directly the affinity maturation for antibody provides and optimizes
Selection.
3 antibody creating affinity as expected in 6 natural pairings of hybridoma, illustrate hybridoma antibody sequence
Qualification be accurately, external pairing express and Elisa experimental technique be also effective.
The above result illustrates, in conjunction with hybridoma antibody technology and high flux antibody sequencing technologies, chooses hybridoma weight
Chain gene is expressed with high abundance CDR3 light chain gene pairing from antibody variable gene spectrum, can be with Extensive pairing candidate gene
The range of choice, improves the efficiency that antibody structure optimizes, and adds the probability obtaining functional antibodies.Hybridoma heavy chain GW02-
5H2 can produce affinity antibodies with the pairing of multiple high abundance light chains, illustrates that some heavy chain gene also has and preferably matches spy
Property, functional antibodies may be produced with multiple light chain gene pairings with high similarity CDR3.
Applicant states, the present invention illustrates the product of the present invention, detailed preparation technology and use thereof by above-described embodiment
On the way, but the invention is not limited in above-mentioned detailed preparation technology and purposes, i.e. do not mean that the present invention have to rely on above-mentioned in detail
Preparation technology and purposes could be implemented.Person of ordinary skill in the field is it will be clearly understood that any improvement in the present invention, to this
The equivalence of each raw material of invention product is replaced and the interpolation of auxiliary element, concrete way choice etc., all falls within the protection of the present invention
Within the scope of scope and disclosure.
Claims (10)
1. generation combines the method for recombinant antibodies at least one target antigen, comprising:
(1) with described at least one target antigen immunity host subject；
(2) use the spleen cell through immune host subject, prepare hybridoma cell line, and obtain specific binding described
The hybridoma antibody gene information of at least one target antigen；
(3) use the lymphocyte of the host subject after immunity, carry out the high-flux sequence of immunoglobulin gene variable region,
Obtain the antibody variable gene at least one target antigen described to compose；
(4) choose hybridoma antibody heavy chain gene and match gene as the first candidate, and select in composing from antibody variable gene
The light chain gene selected matches gene as the second candidate and matches, to produce candidate's recombinant antibodies.
Method the most according to claim 1, wherein, in step (2), prepared hybridoma cell line produces and described target
Antigen has the monoclonal antibody of high-affinity；
Preferably, described hybridoma antibody gene information comprises heavy chain and the chain variable region gene information of antibody；
Preferably, by following acquisition hybridoma antibody gene information:
With the total mRNA of hybridoma as template, Oligo dT is primer, synthesizes cDNA the first chain through reverse transcription, then with cDNA the
One chain is template, and with primer amplified antibody heavy chain variable region and the chain variable region gene of mixing, sequencing analysis obtains
Hybridoma gene order, carries out IgBlast analysis by hybridoma gene order, gets rid of and derives from the fusion partner constituting hybridoma
The antibody gene of tumor self and nonsense suppressor, obtain candidate's heavy chain and chain variable region gene.
Method the most according to claim 1 and 2, wherein, in step (3), described antibody variable gene spectrum comprises antibody
Heavy chain and chain variable region gene information；
Preferably, described gene information is determined by high flux DNA sequencing；
It is further preferred that described high flux DNA sequencing carries out synthesizing, connect or hybridizing while being included in sequencing reaction；
And unique DNA order-checking, the order-checking of many polymerases group, nano-pore order-checking or a combination thereof；
Preferably, described high-flux sequence is by following enforcement:
With the total mRNA of cell as template, SMARTer RACE technology is used to synthesize the first chain cDNA；Design forward primer, and according to
Antibody constant region sequences conserved regions design downstream primer, to expand antibody sequence variable region.
4., according to the method described in any one of claim 1-3, wherein, in step (4), choose from antibody variable gene is composed
Comprise high abundance CDR3 or comprise the light chain gene to described CDR3 the most similar for high abundance CDR3 and join as the second candidate
To gene.
5. according to the method described in any one of claim 1-4, wherein, selected second candidate in composing from antibody variable gene
In the case of the CDR3 of pairing gene, it is prepared by the following the second candidate and matches the antibody variable region of gene:
From the sequencing result of the antibody gene segments comprising selected CDR3, choose the most abundant CDR2 and CDR1 sheet successively
Segment information, then selects the most abundant variable region full length sequence comprising selected CDR2 and CDR1 sheet segment information, as second
Candidate matches the antibody variable sequences of gene.
6. according to the method described in any one of claim 1-5, wherein, also include with at least one target antigen described, gained being resisted
Body carries out the step identified.
Method the most according to claim 6, described authentication step includes scFv or the Fab fragment of vivoexpression gained antibody
Or IgG, gained scFv, Fab fragment or IgG are combined power test with target antigen；
Preferably, described vivoexpression method including but not limited to: by prokaryotic cell, phage display method or yeast system
System expresses scFv or Fab fragment, or by mammalian cell exogenous gene expression Fab or IgG；
Preferably, described method of testing includes but not limited to ELISA and/or SPR.
8. the recombinant antibodies prepared according to the method described in any one of claim 1-7.
Recombinant antibodies the most according to claim 8, it is the recombinant antibodies of selectively targeted HA, the weight of described recombinant antibodies
Chain variable region includes such as SEQ ID NO:40, the CDR3 shown in 42 and 50, and the variable region of light chain of described recombinant antibodies includes
Such as SEQ ID NO:20, the CDR3 shown in 53,68 and 103.
Recombinant antibodies the most according to claim 8, it is the recombinant antibodies of selectively targeted PD-L1, described recombinant antibodies
Variable region of heavy chain include the CDR3 as shown in SEQ ID NO:84, and the variable region of light chain of described recombinant antibodies include as
SEQ ID NO:23, the CDR3 shown in 89 and 105.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779500A (en) * | 2017-10-25 | 2018-03-09 | 上海药明生物技术有限公司 | A kind of sequence measurement and primer sequence of quick obtaining Rat hybridoma cell monoclonal antibodies sequence |
| CN108456682A (en) * | 2017-02-17 | 2018-08-28 | 苏州金唯智生物科技有限公司 | A kind of screening technique of monoclonal antibody and its application |
| WO2018153320A1 (en) * | 2017-02-21 | 2018-08-30 | 上海君实生物医药科技股份有限公司 | Anti-pd-l1 antibody and application thereof |
| CN114854686A (en) * | 2022-05-12 | 2022-08-05 | 中国农业大学 | T cell subset for specifically identifying salmonella typhimurium and application thereof |
| CN117004700A (en) * | 2023-10-07 | 2023-11-07 | 北京爱博生生物技术有限公司 | Method for high-throughput sequencing of monoclonal antibody variable region genes, composition and kit used by method |
-
2016
- 2016-01-14 CN CN201610023897.9A patent/CN106065031A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| CZERWINSKI M.等: "A molecular approach for isolating high-affinity Fab fragments that are useful in blood group serology", 《TRANSFUSION》 * |
| 余馨 等: "高通量DNA测序技术在抗体新药研发中的应用", 《药学学报》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108456682A (en) * | 2017-02-17 | 2018-08-28 | 苏州金唯智生物科技有限公司 | A kind of screening technique of monoclonal antibody and its application |
| WO2018153320A1 (en) * | 2017-02-21 | 2018-08-30 | 上海君实生物医药科技股份有限公司 | Anti-pd-l1 antibody and application thereof |
| US11267890B2 (en) | 2017-02-21 | 2022-03-08 | Shanghai Junshi Biosciences Inc. | Anti-PD-L1 antibody and application thereof |
| CN107779500A (en) * | 2017-10-25 | 2018-03-09 | 上海药明生物技术有限公司 | A kind of sequence measurement and primer sequence of quick obtaining Rat hybridoma cell monoclonal antibodies sequence |
| CN114854686A (en) * | 2022-05-12 | 2022-08-05 | 中国农业大学 | T cell subset for specifically identifying salmonella typhimurium and application thereof |
| CN117004700A (en) * | 2023-10-07 | 2023-11-07 | 北京爱博生生物技术有限公司 | Method for high-throughput sequencing of monoclonal antibody variable region genes, composition and kit used by method |
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