RU2683549C1 - SYSTEM FOR EXPRESSION OF FAB-FRAGMENTS OF ANTIBODIES IN METHYLOTROPHIC YEAST PICHIAPASTORIS, ON THE BASIS OF RECOMBINANT PLASMID DNA Ab-HCh-HIS/pPICZ_α_A AND Ab-LCh-LAMBDA/pPICZα_A, INTENDED TO CLONE VARIABLE DOMAINS OF ANTIBODY HEAVY AND LIGHT CHAINS, RESPECTIVELY - Google Patents

Abstract

FIELD: biotechnologies.SUBSTANCE: invention relates to the biotechnology. Proposed system for the expression of Fab fragments of antibodies, consisting of recombinant plasmid DNA Ab-HCh-HIS/pPICZ_α_A and Ab-LCh-LAMBDA/pPICZα_A. Transformation of P. Pastoris yeast with these plasmid DNAs allows obtaining purified Fab fragments of antibodies with a yield of up to 30 % with a purity of more than 95 % and stability of at least 60 days when stored at +4 °C.EFFECT: transformation of the yeast with the indicated plasmid DNAs allows the preparation of purified Fab fragments of antibodies.3 cl, 2 dwg, 2 ex, 1 tbl

Description

The invention relates to the field of genetic engineering and biotechnology, namely to: recombinant plasmid DNA Ab-HCh-HIS / pPICZ_α_A and Ab-LCh-LAMBDA / pPICZ_α_A, allowing to clone any variable domains of heavy and light chains, respectively, the expression system of Fab fragments of antibodies with a lambda isotype of the light chain in the cotransformation of both genetic constructs with pre-built variable domains of the heavy and light chains of interest into the methylotrophic yeast cells Pichia pastoris, to obtain Fab fragments of antibodies with the lambda isotype ohm light chain from a culture medium without proteolytic degradation of the final drug. The invention can be used in scientific research and biotechnology to obtain recombinant Fab fragments of antibodies that require a high degree of purity, homogeneity and reproducibility of experimental batches of samples.

The object of the invention is a system of two modified recombinant plasmid DNA Ab-HCh-HIS / pPICZ_α_A and Ab-LCh-LAMBDA / pPICZα_A, designed for cloning of the variable domains of the heavy and light chains of antibodies, respectively, followed by cotransformation of a mixture of both genetic structures in an equimolar ratio cells of methylotrophic yeast Pichia pastoris and obtaining stable recombinant Fab fragments of antibodies with lambda isotype of the light chain.

A known method for producing single-chain antibodies in P. pastoris cells as part of a fusion protein with a constant domain of mouse immunoglobulin (Wang DD, Su MM, Sun Y, Huang SL, Wang J, Yan WQ. Protein Expr Purif. 2012 Nov; 86 (1) : 75-81). In this work, we used a single-cystronic vector system. The authors were able to obtain a product yield of 60 mg / l using an 80 liter fermenter. The disadvantage of this system is the production of antibodies in the form of a single chain fragment, since there is evidence of differences in the levels of specific activity between Fab fragments and single chain antibodies in favor of the former (Zakharov A.V., Smirnov I.V., Serebryakova M.V. and other // Molecular Biology. 2011.V. 45, No. 1, P. 86-95).

A known system for producing a Fab fragment of an antibody as part of a monocistronic vector containing a KEX2 protease hydrolysis site (Burtet RT1, Santos-Silva MA, Buss GA, Moraes LM, Maranhao AQ, Brigido MM. J Biochem. 2007 Dec; 142 (6): 665- 9). As a result, the authors were able to obtain a functionally active Fab fragment, but the expression level did not exceed 10 mg / l of culture medium.

A system for producing a Fab fragment of an antibody is described, consisting of two monocistronic vectors encoding the light and heavy chains of antibodies, respectively (Zakharov A.V., Smirnov I.V., Serebryakova M.V. et al. // Molecular Biology. 2011 T. 45, No. 1, S. 86-95). The system made it possible to obtain up to 15 mg / L of the active Fab fragment, but subsequently a low stability of the obtained protein preparations was discovered, presumably due to the co-isolation and cleavage of Fab fragments by the host cell proteinases. Light chains may belong to the kappa or lambda isotype and correspond to one constant domain. It was shown that the light chain isotype can have a significant effect on the structure of the recombinant Fab fragment of the antibody, while the variant with the lambda light chain isotype has greater conformational rigidity and thermal stability (Ponomarenko N, Chatziefthimiou SD, Kurkova I, Mokrushina Y, et al. Role of κ → λ light-chain constant-domain switch in the structure and functionality of A17 reactibody. Acta Crystallogr D Biol Crystallogr. 2014; 70: 708-19).

The present invention is to provide a system for the expression of Fab fragments of antibodies with high stability of the final protein preparations. The problem is solved by creating two modified recombinant plasmid DNA Ab-HCh-HIS / pPICZ_α_A and Ab-LCh-LAMBDA / pPICZ_α_A, which, unlike the described closest analogues (Zakharov A.V., Smirnov I.V., Serebryakova M. .V. Et al. // Molecular Biology. 2011.V. 45, No. 1, P. 86-95 and Ponomarenko NA, Chatziefthimiou SD, Kurkova IN et al. // Acta Crystallogr. Section D. 2014. Vol. 70, N3. P. 708-719) the marker sequences of the 3xFLAG epitope in the case of the heavy chain and the c-myc epitope in the case of the light chain were deleted in order to minimize the proteolytic degradation of the final recombinant product, the Fab fragment of the antibody and - during its operating time in a yeast system and chromatographic purification. The degradation sites of Fab fragments correspond to two types of endogenous proteases of P. pastoris yeast - protease A (EC 3.4.23.8) and carboxypeptidase Y (EC 3.4.16.5). Degradation sites such as His-X, Asn-X, Asp-X, Gln-X, Glu-X are abundantly represented in the nearest analogues in the 3xFLAG and c-myc epitope sequences located at the C-ends of the heavy and light chains, respectively. Also, changes were made in the polylinker regions of the recombinant plasmid DNA Ab-HCh-HIS / pPICZ_α_A and Ab-LCh-LAMBDA / pPICZ_α_A, namely, the recognition site for the BsmBI restriction endonuclease was saved, but shifted in both cases by 12 nucleotides Sequence cleavage directly in the region encoding the leader peptide in the Ab-LCh-LAMBDA / pPICZα_A recombinant plasmid DNA was added with unique restriction sites between sequences encoding the leader peptide and the light chain constant domain. Thus, the recombinant plasmid DNA Ab-HCh-HIS / pPICZ_α_A and Ab-LCh-LAMBDA / pPICZ_α_A (Fig. 1) contain unique restriction sites BsmBI and XhoI, Ace65I for cloning any variable domains of the heavy chain and BsmBI and KpnInd, Av II, KpnInd for cloning any variable domains of the light chain of antibodies between sequences encoding the leader peptide and the constant domain of the heavy or light chain and are intended for subsequent cotransformation into methylotrophic yeast Pichia pastoris and obtain stable recombinant Fab fragments of antibodies with lambda isotype of the light chain . In the presence of the indicated variants of restriction sites in the sequence of the variable domain of the heavy or light chain for amplification by PCR using primers, sites for type II restriction endonucleases (BspMI, BsaI, etc.) can be introduced, which then form compatible sticky vectors when cloned into recombinant vectors ends.

The designation "pPICZ_α_A" in the recombinant plasmid DNA Ab-HCh-HIS / pPICZ_α_A and Ab-LCh-LAMBDA / pPICZ_α_A (Fig. 1) reflects the content of structural elements of the genetic construct pPICZαA (Invitrogen, USA) on the basis of which the closest analogues were obtained ( Zakharov A.V., Smirnov I.V., Serebryakova M.V. et al. // Molecular Biology. 2011.V. 45, No. 1, P. 86-95 and Ponomarenko NA, Chatziefthimiou SD, Kurkova IN et al. // Acta Crystallogr. Section D. 2014. Vol. 70, N3. P. 708-719), namely, the inducible promoter of the wild-type yeast P. pastoris alcohol oxidase gene AOX1, the native terminator and the AOX1 gene polyadenylation signal, follower awn gene Sh ble, the product of which provides resistance to the antibiotic zeocin transformant, the nucleotide sequence of the signal peptide of the alpha-factor from S. cerevisiae, required for secretion of the expressed product into the culture medium. The sequence encoding the signal peptide of the alpha factor has been changed, namely, 12 nucleotides encoding two EA repeats have been deleted. The removal of the EAEA sequence during processing of the recombinant product of the STE13 yeast cell dipeptidyl aminopeptidase is often ineffective, therefore, in the present invention, the C-terminus of the alpha-factor peptide terminates in the Kex2 protease recognition site, and in order to clone the variable domains of the heavy and light chains of antibodies in conjunction with the Kex2 site there is a recognition site for restriction endonuclease II type BsmBI, which ensures the preservation of the native sequence of the N-end of the heavy and light chains of Fab fragments antibodies.

The recombinant Ab-HCh-HIS / pPICZ_α_A plasmid (Fig. 1A) contains the nucleotide sequence of the first constant domain of the human immunoglobulin γ1 chain (CH1), part of the hinge region, a six-histidine cluster sequence for protein purification using metal affinity chromatography. These structural features are reflected in the designation "Ab-HCh-HIS".

The recombinant plasmid Ab-LCh-LAMBDA / pPICZ_α_A (Fig. 1B) contains the nucleotide sequence, 12 fragments, as well as the constant lambda-2 domain of the light chain ending in a stop codon. These structural features are reflected in the designation "Ab-LCh-LAMBDA".

In the recombinant plasmid DNA Ab HCh-His / pPicZαA (Fig. 1A) for expression of antibody heavy chain fragments, the C-terminus of the constant domain gene is fused to the 6xHis sequence, which is used to purify the protein in the first stage using metal chelate chromatography. In the recombinant plasmid DNA Ab-LCh_lambda / pPICZαA (Fig. 1B), a stop codon was introduced before the 6xHis sequence to express antibody light chain fragments at the C-terminus of the constant domain gene. The synthesis of a full-sized light chain ends before the 6xHis cluster and, thus, it is expressed in its native form and is purified only in combination with the heavy chain in the form of a Fab fragment.

The technical result of the cotransformation of Ab-HCh-HIS / pPICZ_α_A and Ab-LCh-LAMBDA / pPICZ_α_A plasmid DNAs with previously built variable domains of the heavy and light chains of the antibody into P. pastoris yeast cells is the production of Fab fragments of antibodies with subsequent chromatographic purification with access to 30%, purity more than 95% and stability for at least 60 days when stored at + 4 ° C.

The invention is illustrated by the following graphic materials: FIG. 1. Schematic representation of modified genetic constructs for expression of Fab fragments of antibodies in the yeast system of P. pastoris. (A) - recombinant plasmid DNA Ab HCh-His / pPicZ_α_A. (B) - recombinant plasmid DNA Ab-LCh_lambda / pPICZ_α_A.

FIG. 2. Electrophoretic analysis of the purity (A) of preparations of the Fab fragment of the A5 antibody lambda at different stages of chromatographic isolation (B) of purified preparations of the Fab fragment of the antibody A17, with observed degradation at the C-terminal epitopes. In Figure A: 1 - eluate after metal-chelate chromatographic purification, 2 - main fraction after separation of the sample by cation exchange chromatography, 3 - fraction after gel filtration, 4 - slip, not binding to a hydrophobic sorbent, 5 - Fab fragment eluate with a hydrophobic sorbent, M is a molecular weight marker. In Figure B: 1 - preparation of a Fab fragment of an antibody containing epitopes at the C-ends of c-myc and 3xFLAG chains immediately after the cleaning procedure, 2 - after storing the sample at + 4 ° C for a week, 3 - after storing the sample at + 4 ° C for two weeks. Table. Characterization of the technique for isolating preparations of recombinant Fab fragments of antibodies.

The invention is illustrated by the following examples, which should not be construed as limiting the present invention.

EXAMPLE 1

Creating a system of genetic constructs Ab-LCh_kappa / pPICZ_α_A and Ab-LCh_lambda / pPICZ_α_A.

The genetic constructs are optimized based on the previously obtained vectors pPICZαA / Hch and pPICZαA / Lch (Zakharov A.V., Smirnov I.V., Serebryakova M.V. et al. // Molecular Biology. 2011. V. 45, No. 1, S. 86-95) and pPICZαA / Jλ2 (Ponomarenko NA, Chatziefthimiou SD, Kurkova IN et al. // Acta Crystallogr. Section D. 2014. Vol. 70, N3. P. 708-719). To obtain the optimized Ab HCh-His / pPicZαA vector for expression of antibody heavy chain fragments, the first constant domain of the human immunoglobulin γ1 chain (CH1) and part of the hinge region are amplified by polymerase chain reaction (PCR) using the pPICZαA / Hch construct using specific primers. The resulting PCR product is cloned into the original vector pPICZαA / Hch at the restriction sites Acc65I and SalI with the elimination of the 3xFLAG epitope. Similarly, to obtain the constructs Ab-LCh_kappa / pPICZαA and Ab-LCh_lambda / pPICZαA, J fragments and constant domains of light chains are amplified using specific primers by PCR and then ligated into the pPICZαA / Lch vector at XhoI-SalI and 65 restriction sites, respectively . At the same time, the C-myc epitope is eliminated at the C-terminus and the stop code is introduced using primers. Based on the created vectors, constructs for expression of the A5 antibody, A5_lambda / pPICZαA, A5_kappa / pPICZαA and A5-HCh-His / pPicZαA, are obtained. Amplification of the variable domains is carried out using specific primers from the previously obtained construct encoding a single chain antibody, A5ScFvFLAG / pHEN2 (Reshetnyak AV, Armentano MF, Ponomarenko NA et al. // J. Am. Chem. Soc. 2007. Vol. 129, N51. P. 16175-16182) (Fig. 1).

When constructing the Ab HCh-His / pPicZαA vector (Fig. 1A) for expression of antibody heavy chain fragments, the 3xFLAG epitope sequence is deleted and the C-terminus of the constant domain gene is combined with the 6xHis sequence, which will be used to purify the protein in the first stage using metal chelate chromatography. When cloning, universal restriction sites BsmBI and XhoI, as well as Acc65I, are retained for embedding any variable domains of the heavy chain. In the Ab-LCh_lambda / pPICZαA vector (Fig. 1B), in addition to the removal of the c-myc epitope, a stop codon is introduced before the 6xHis sequence. The synthesis of a full-sized light chain will end before the 6xHis marker and, thus, it will be expressed in its native form and purified only in combination with the heavy chain in the form of a Fab fragment. To clone the variable domains of the light chain into the Jl2 region, universal restriction sites KpnI, AvrII, and HindIII are introduced into the Jl2 region and the recognition site for BsmBI is maintained.

EXAMPLE 2

Obtaining a producer strain of a recombinant Fab fragment in P. pastoris yeast.

The cells of P. pastoris strain GS115 are co-transformed by electroporation using the preliminarily linearized Pmel restriction site constructs A5-HCh-His / pPicZα paired with A5_lambda / pPICZαA or A5_kappa / pPICZαA. The selection of transformants is carried out on the antibiotic zeocin, a clone producer with a maximum level of production is selected by analyzing the total proteins of the culture medium in 12% SDS-PAGE.

Preparative production of the Fab fragment of antibody A5 is carried out in 3-9 L of BMMY nutrient medium using 750 ml rocking flasks according to the manufacturer's recommendations (Invitrogen) with some modifications: the starting culture density for methanol induction is A600 = 5.0, methanol is added to concentrations of 0.5% every 24 hours, at the same time sorbitol up to 0.15% is also added, expression is carried out for three days.

The culture medium obtained after expression of the Fab fragment of antibody A5 is centrifuged to remove cells. Next, ultrafiltration is carried out using 0.45 mm and 0.22 mm filtration modules and the medium is concentrated using a 10 kD Pelicon2 ultrafiltration module. The concentrated solution of the Fab fragment is subjected to metal chelate chromatography using a Ni-NTA Superflow sorbent (QIAGEN) according to the protocol of the manufacturer. The resulting eluate was dialyzed against 20 mM CH3COONa pH 6.0. The second purification step is carried out using cation exchange chromatography using a MonoS 5/50 GL column (GE Healthcare) according to the manufacturer's instructions. For application, a buffer containing 20 mM CH3COONa pH 6.0 is used; elution is carried out with a NaCl salt gradient from 0 to 0.35 M in 20 mM CH3COONa pH 6.0 in 20 column volumes at a flow rate of 1 ml / min. Next, the fractions containing the Fab fragment were purified by gel filtration on a HiLoad 26/600 Superdex75 pg column (GE Healthcare) in a buffer containing 20 mM NaxH3-xPO4, pH 7.4, 300 mM NaCl. For further purification, hydrophobic chromatography was performed using HiTrepPhenyl HP 5ml (GE Healthcare) in a buffer of 1 M (NH4) 2S04, 20 mM NaxH3-xPO4, pH 7.4. Isocratic elution is carried out with a solution of 20 mM NaxH3-xPO4, pH7.4. After each stage of purification, SDS page electrophoresis is carried out under non-reducing conditions according to the standard Lemmli method, followed by assessment of the purity of the drug using densitometric analysis. The cleaning results are presented in the Table.

In FIG. 2A shows an electrophoretic analysis of chromatographic fractionation of a Fab fragment of an A51ambda antibody. The resulting final sample (Fig. 2A, lane 5) does not change the properties of electrophoretic mobility and remains stable for at least 60 days when stored at + 4 ° C. In FIG. 2B presents a comparative electrophoregram of a purified sample of a Fab fragment containing 3xFLAG and c-myc marker epitopes at the C-termini of the chains and undergoing gradual degradation under the same storage conditions. Thus, the system we developed allows us to obtain highly purified, homogeneous, and stable preparations of recombinant Fab fragments of antibodies of therapeutic value.

Claims (3)

1. A system for expressing Fab fragments of antibodies in P. pastoris methylotrophic yeast, consisting of the Ab-HCh-HIS / pPICZ_α_A and Ab-LCh-LAMBDA / pPICZ_α_A recombinant plasmid DNAs shown in FIG. 1 and containing the plasmid pPICZαA with the following elements in the order of their location: the wild-type yeast P. pastoris inducible promoter of the alcohol oxidase gene AOX1, the nucleotide sequence of the signal peptide of the alpha factor from S. cerevisiae ending in the recognition site for Kex2 protease and necessary for secretion expressed products into the culture medium, unique restriction sites BsmBI and XhoI, Acc65I for cloning the variable domains of the antibody heavy chain in plasmid DNA Ab-HCh-HIS / pPICZ_α_A or BsmBI and KpnI, AvrII, HindIII for cloniro of the variable domains of the light chain of antibodies in the Ab-LCh-LAMBDA / pPICZ_α_A plasmid DNA, the nucleotide sequence encoding part of the hinge region and the first constant domain of the human immunoglobulin heavy chain (CH1) γ1, as well as the six-histidine cluster sequence (6xHis) directly ending -codon and necessary for purification of the final protein preparation using metal affinity chromatography in Ab-HCh-HIS / pPICZ_α_A plasmid DNA or a nucleotide sequence encoding part of the J2 region region and the constant domain of the lambda-2 light chain of the isotype (32X2) of a human immunoglobulin ending with a stop codon in the Ab-LCh-LAMBDA / pPICZ_α_A plasmid DNA, a native terminator and AOX1 alcohol oxidase gene polyadenylation signal, the Sh ble gene sequence, the product of which ensures the stability of transformants to the antibiotic zeocin, the origin of pUC replication. 2. A system for expressing Fab fragments of antibodies in P. pastoris methylotrophic yeast according to claim 1, wherein any cloned antibody variable domains form an open reading frame — a sequence of the alpha-factor signal peptide from S. cerevisiae ending in a recognition site for Kex2 protease, the sequence antibody heavy chain variable domain, sequence of the first constant domain of the heavy immunoglobulin γ1 heavy chain (CH1) of a human immunoglobulin, six-histidine cluster sequence (6xHis) or signal peptide sequence an alpha-factor tyda from S. cerevisiae ending with a recognition site for Kex2 protease, an antibody light chain variable domain sequence, a human immunoglobulin isotype light chain constant domain sequence of lambda-2. 3. A system for expressing Fab fragments of antibodies in P. pastoris methylotrophic yeast according to claim 2, wherein the recombinant Ab-HCh-HIS / pPICZ_α_A and Ab-LCh-LAMBDA / pPICZ_α_A plasmid DNAs with pre-integrated antibody heavy and light chain variable regions of interest are transformed into cells of methylotrophic yeast Pichia pastoris.

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