RU2577226C2 - Methods for making bispecific antibodies against cd3*cd19 in flexybody format in mammalian cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. Method for producing a recombinant monoclonal antibody against CD3*CD19 in flexybody format. Constructed recombinant expression plasmid DNA encoding a bispecific antibody against the CD3*CD19. Resulting DNA is transfected with CHO cell strain M, and the resulting cell line is cultured. Cultivation is carried out in a perfusion mode in the presence of feeder additives CB1 to CB5 and 1: 1 ratio, with a volume of additive of 25 % by volume of the nutrient medium. Submitted monoclonal antibody against CD3*CD19 flexybody format having two polypeptide chains non-covalently associated with the amino acid sequence of SEQ ID №1 or non-covalently associated two chain polypeptide having the amino acid sequence of SEQ ID №2, obtained in this way. Also provided a recombinant expression plasmid DNA pOG776, encoding a polypeptide having the sequence bispecific antibody in flexybody format against CD19*CD3, having a size of 10,618 bp, and a recombinant expression plasmid DNA pOG854, encoding a polypeptide with the sequence of the bispecific anti-CD3 * CD19, having a size of 10,618 bp with the physical map shown in fig. 6.

EFFECT: invention provides a high yield of protein of pharmaceutical quality, suitable for the production of drugs.

6 cl, 8 dwg, 3 tbl, 6 ex

Description

The invention relates to biotechnology, in particular to an industrial method for the production of bispecific antibodies against CD3 * CD19 format "flexibody" in mammalian cells.

Bispecific antibodies (BAPs) against CD3 * CD19 are currently being developed primarily for the treatment of malignant diseases of B cells or depletion of B cells, including non-Hodgkin lymphoma (WO 2010037835, RU 2228202), B-cell leukemia (RU 2008129080), acute lymphoblastic leukemia (WO 20100052013), etc.

In Russia alone, more than 20,000 new cases of non-Hodgkin's lymphoma (NHL) are reported annually. Less than 5% of patients survive for 5 years, despite the fact that the incidence of non-Hodgkin's lymphomas in Russia is 4: 100,000 people per year. The standard of treatment for NHL in Russia is the combination of rituximab and chemotherapeutic drugs. However, more than 20% of the initially diagnosed patients and about 50% of patients with recurrent lymphoma stop responding to rituximab therapy, and in the initially responding patients, the effectiveness of rituximab decreases with subsequent courses of therapy due to the development of resistance (Br. J. Haematol. 2011, v.155 , 426-437).

Current studies (e.g., J Clin Oncol, 2011, 29: 2493-8; Cancer, 2010, 116: 5568-74; Science, 2008, 321: 974-7) give every reason to believe that drugs based on bispecific antibodies against CD3 * CD19 promise to become more effective in the treatment of the above diseases.

The increased efficiency is explained by the fact that BAPs act as adapters that physically connect T cells with tumor cells and trigger a powerful signaling cascade of the T-cell receptor complex by binding to the invariant component of the CD3 T-cell receptor. In this case, the second (target) antigen-specific fragment recognizes CD19. Those. BAP can temporarily connect a T cell and a cancer cell by simultaneously binding to CD3 and the target antigen. In this case, the activation and proliferation of T cells, the formation of a cytolytic synapse with the release of cytotoxic granules and the secretion of cytokines occur. Targeted lysis of cancer cells includes perforation of the target cell membrane involving perforin and subsequent granzyme-induced apoptosis. It should be noted that T cells activated with BAP recognize antigens on the surface of a cancer cell in the same way as ordinary monospecific antibodies. Thus, it is possible to redirect the cytotoxic function of T cells against tumor cells, and BAP activity is already observed at relatively low (picomolar) concentrations. Binding of BAP only to a T cell in the absence of a target cell does not cause T cell activation.

Currently, the most successful BAT clinic is the anti-CD19 / anti-CD3 (CD19 * CD3) blinatumomab preparation (blinatumomab, MT103; a product from Micromet), whose action is based on the mobilization of unstimulated primary T cells against CD19 ⁺ lymphoma cells. Blinatumomab consists of two domains: the anti-CD19 scFv domain from the HD37 hybridoma and the anti-CD3 scFv domain from the TR66 hybridoma. To obtain antibodies, the VLCD19-VHCD19-VHCD3-VLCD3 DNA fragment is expressed in DHFR-negative CHO cells (patent RU 2228202).

But blinatumomab is relatively small and quickly eliminated from the bloodstream. Therefore, to achieve stable concentrations of the drug in the blood, continuous intravenous administration of the drug is used for 4-8 weeks in one cycle, which is quite difficult for the patient. To overcome these shortcomings, a new antibody format was proposed called flexibody, the technology of which is described in detail in patent EP 1293514. Flexibody consists of two pairs of variable domains VH and VL, which are located on the same polypeptide chain and connected by linkers, the length of which determines the possibility the formation of multimeric structures: dimers, trimers and tetramers. The most promising option is when the resulting polypeptide is two non-covalently bound molecules of a bispecific antibody. In this case, flexibody has a molecular weight of about 120 kDa and is capable of divalently binding to both the target cell and the effector T cell, which allows increasing the therapeutic potential of the molecule (Kratz F, Senter P, Kiprijanov S. Bispecific Antibodies and Immune Therapy Targeting in Drug Delivery in Oncology: From Basic Research to Cancer Therapy. Wiley, 2011) and increase elimination time from the body.

Such a flexibody molecule has a modular structure in which the diabody module serves to dimerize a single chain molecule. By pairing complementary VH and VL domains located on different peptide chains, functional Fv modules of the same specificity are formed, and a stable non-covalent protein dimer is obtained.

Examples of bispecific antibodies known from the prior art (Blood, (ASH Annual Meeting Abstracts), 2009, 114: 840) indicate that the effective therapeutic concentration of drugs derived from the use of multivalent antibodies is significantly lower compared to classic anti-cancer antibodies drugs. This contributes to good tolerance, the possibility of implementing long-term courses of treatment and reducing the risk of side effects. In addition, the volume and cost of producing bispecific antibodies is lower. Therefore, the development of a new method for the production of such antibodies will significantly reduce the scale and cost of producing drugs based on them and, most importantly, make these drugs available to all patients in need of such therapy.

A known method of producing bespecifically flexibody against CD3 * CD19 in E. Coli cells, strain RV308 (patent EP 1293514), comprising the following stages: transformation of cells with plasmids containing the target protein sequence; cultivation in 2xYT medium containing 50 μg / ml ampicillin and 100 mM glucose at 28 ° C until an optical density of 0.8 is reached (at a wavelength of 600); after which the cells are centrifuged, resuspended in fresh medium and grown in the presence of IPTG at 20C for 18-20 hours. To obtain a fraction of soluble periplastic proteins, the bacterial sediment is separated, resuspended in a buffer containing EDTA, HCl and sucrose (pH 8.0). The suspension is subjected to successive stages of centrifugation and dialysis, and finally the recombinant product is concentrated by precipitation with ammonium sulfate, after which several chromatographic purification steps are carried out.

However, this method is not convenient enough for industrial production, since it is designed to produce small amounts of antibodies, it contains a large number of stages. In addition, the isolation of the protein developed in E. Coli requires the introduction of additional purification steps due to the presence of large quantities of bacterial endotoxins. The description of the patent indicates the theoretical possibility of producing antibodies of flexibody format in mammalian cells, however, there is no example of such a possibility.

Closest to the claimed method is a method for producing antibodies against CD3 * CD19 in CHO cells, described in patent RU 2228202 for producing antibody blinatomumab. In this method, an anti-CD19 * CD3 antibody was obtained from Chinese hamster ovary (CHO) cells, which were transfected with an expression vector (pEF-DHFR) encoding the antibody, and further containing a hexahistidine tag and a FLAG tag. Transfection was performed using electroporation. Then the cells were grown in serum-free medium in a hollow fiber fermenter. Purification of the supernatant was carried out using sequential steps: affinity chromatography on a Ni-NTA column, cation exchange chromatography, cobalt chelate affinity chromatography, and gel filtration at the final stage. Received about 4 mg of antibody per 1 l of supernatant. Such low yields (4 mg of antibody per 1 liter) negatively affect the cost and complexity of the production of GLF. The disadvantages of the prototype can also include the presence of hexahistidine and FLAG tags, which are additional antigenic determinants, and therefore require confirmation of the immune safety of the target molecule.

The present invention solves the problem of constructing plasmid DNA containing the sequence of bispecific antibodies against CD3 * CD19 format flexibody and the development of a method for the industrial production of bispecific antibodies against CD3 * CD19 format flexibody in mammalian cells. The developed method allows to obtain about 8-13 mg of antibody per 1 liter of supernatant, which is approximately 2-3 times higher than the prototype yield.

The problem is solved by constructing recombinant plasmid DNA pOG766 with a size of 10618 bp containing Bis1 antibody and recombinant plasmid DNA pOGS854 with a size of 10 618 bp containing Bis2 antibody, as well as selecting the optimal conditions for the production process, allowing to obtain antibodies in the format flexibody in mammalian cells with a stably high yield sufficient for industrial production. The proposed method for producing antibodies of flexibody format against CD3 * CD19 in CHO cells also allows you to expand the arsenal of methods for producing such antibodies.

DETAILED DESCRIPTION OF THE INVENTION

For expression in CHO cells, two sequences of antibodies Bis1 and Bis2 were constructed (Figure 1 and Figure 2). Based on these amino acid sequences, the codon composition was optimized for expression in CHO cells. Similar work was done to optimize the sequence of blinatumomab or MT-103 (Figure 3), which was used as a control.

Transfection vectors were constructed using commercially available plasmids pSUREtech191 (Selexis, Switzerland). Design schemes are shown in Fig.4 and Fig.5.

Plasmid pOG766 (Figure 6) encoding a Bis1 polypeptide is characterized by the following features:

- consists of 10618 bp,

- has a molecular weight of 6.4 MDa,

- encodes a polypeptide of a bispecific antibody of flexibody format against CD3 * CD19, BiS1

- provides resistance of mammalian cells transfected with the indicated plasmid to puromycin;

- contains the following elements:

- CMVe / EFa pro - enhancer of the CMV virus and promoter of transcription of the gene for elongation factor alpha,

- BGH pA - signal polyadenylation of bovine growth hormone,

- sv40 enh - enhancer of the SV40 virus,

- SGE 1 - element of attachment to the nuclear matrix,

- Amp - β-lactamase gene,

- puro - puromycin resistance gene.

- contains unique recognition sites for the following restriction endonucleases: KpnI (7 bp), AgeI (460 bp). SacI (1206 bp), HindIII (1590 bp), XbaI (3135 bp), SpeI (3932 bp), PvuII (4835 bp), AccI (5800 bp) bp), BsrGI (6612 bp), AhdI (8138 bp), SfiI (9774 bp), BsiWI (9912 bp), NotI (10456 bp).

Plasmid pVS854 (Fig.7) encoding a Bis2 polypeptide is characterized by the following features:

- consists of 10618 bp,

- has a molecular weight of 6.4 MDa,

- encodes a polypeptide bispecific antibody format flexibody against CD3 * CD19, BiS2,

- provides resistance of mammalian cells transfected with the indicated plasmid to puromycin,

- contains the following elements:

- CMVe / EFa pro - enhancer of the CMV virus and promoter of transcription of the gene for elongation factor alpha,

- BGH pA - signal polyadenylation of bovine growth hormone,

- sv40 enh - enhancer of the SV40 virus,

- SGE 1 - element of attachment to the nuclear matrix,

- Amp - β-lactamase gene,

- puro - puromycin resistance gene.

- contains unique recognition sites for the following restriction endonucleases: KpnI (7 bp), AgeI (460 bp). SacI (1206 no), HindIII (1590 no), XbaI (3135 bp), SpeI (3932 bp), PvuII (4835 bp), AccI (5800 bp), BsrGI ( 6612 bp), AhdI (8138 bp), SfiI (9774 bp), BsiWI (9912 bp), NotI (10456 bp).

Transfection was performed using a commercially available strain of CHO-M (Selexis). The choice of cell line is due to the presence of an optimal glycosylation profile for the synthesis of human proteins, stability, safety and suspension conditions for the cultivation of this cell line, which is the most important parameter for the production of therapeutic antibodies.

Applicants have found that in the case of cultivation of antibodies, bioreactors can achieve higher densities of cell cultures than during the process in shaker flasks, probably due to better pH-statization and oxygenation. In addition, it turned out that in this case, using the perfusion cultivation process to obtain the producers of the target proteins Bis1 and Bis2, allows to achieve significantly better performance than the Batch process. It was also possible to influence the increase in protein yield in the cultures of Bis1 and Bis2 producers by enriching the nutrient medium with an optimally balanced set of feeder additives. As a result, in addition to increasing productivity, we were able to achieve a high technological process, including the possibility of linear scaling, which is a huge advantage for industrial production.

When scaling up the processes of isolation and purification of the target product, a scheme was developed that included the affinity stage (CaptoL) with a yield of more than 90%, cation exchange chromatography on SP-Sepharose (yield more than 80%), gel filtration on Sephacryl S300 (yield ~ 55%) and MonoQ (yield ~ 60%). The development and optimization of this scheme made it possible to isolate bispecific antibodies in quantities sufficient to study their pharmacokinetic and pharmacological properties.

Description of drawings

Figure 1. Amino Acid Sequence of Bispecific Design

Bisi with orientation V _L ^αCD19 -L1-V _H ^αCD19 -L2-V _H ^αCD3- L3-V _L ^αCD3

Figure 2. Amino acid sequence of Bis2 bispecific construct with orientation V _H ^αCD19 -L1-V _L ^αCD19 -L2-V _L ^αCD3- L3-V _H ^αCD3

Figure 3. Amino acid sequence of blinatumomab (MT103).

Figure 4. Scheme for obtaining the plasmid pOG766 encoding the antibody Bis12.

Figure 5. Scheme for obtaining plasmid pVS854 encoding an antibody Bis2.

6. Plasmid pOG766 containing the coding sequence of Bis1.

7. Plasmid pVS854 containing the coding sequence of Bis2.

Fig. 8. Results of a comparative ADCC test of bispecific antibody samples.

Description of sequences

SEQ ID NO: 1 — amino acid sequence of Bis1 anti-CD3 * CD19 antibody in flexibody format with orientation V _L ^αCD19 -L1-V _H ^αCD19 -L2-V _H ^αCD3 -L3-V _L ^αCD3

SEQ ID NO: 2 — amino acid sequence of Bis2 anti-CD3 * CD19 antibody flexibody format with orientation V _H ^αCD19 -L1-V _L ^αCD19 -L2-V _L ^αCD3 -L3-V _H ^αCD3

SEQ ID NO: 3 - optimized nucleotide sequence of Bis1 anti-CD3 * CD19 antibody flexibody format with orientation V _L ^αCD19 -L1-V _H ^αCD19 -L2-V _L ^αCD3 -L3-V _H ^αCD3

SEQ ID NO: 4 - optimized nucleotide sequence of Bis2 anti-CD3 * CD19 antibody in flexibody format with orientation V _H ^αCD19 -L1-V _L ^αCD19 -L2-V _L ^αCD3 -L3-V _H ^αCD3

The invention is illustrated by the following examples.

Example 1. Construction of recombinant plasmid DNA.

For cloning, commercially available pSUREtech vectors containing the SGE genetic element were selected to enhance expression as a result of the close proximity of the target transgene with a highly active nuclear transcription complex.

For convenience of cloning the target gene into the pSUREtech vectors, the HindIII and XbaI restriction sites were added at the 5 ′ and 3 ′ ends of the sequences, respectively. At the 5′-end of the sequence, Kozak consensus was added; at the 3′-end, 2 stop signals. The signal peptide MGWSLILLFLVAVATRVLS was added at the N-terminus of the sequence. Plasmid pOG766 was obtained by cloning, in which pSUREtechl91 processed by HindIII, XbaI was used as a vector; the insert was a fragment of pDAD22-1 - HindIII, XbaI containing the coding sequence of Bis1. Plasmid pVS854 was obtained by cloning, in which pSUREtechl91 processed by HindIII, XbaI was used as a vector; the insert was a fragment of pDAD22-2 - HindIII, XbaI containing the coding sequence of Bis2. Schemes of the obtained expression vectors are shown in Fig.6 and 7.

The resulting plasmids were verified by restriction mapping. After that, sequential double transfection was carried out, followed by selection for puromycin resistance using standard methods.

Example 2. The selection of the optimal method of cultivation.

To select the optimal method of cultivation, we compared the productivity and growth characteristics of cell cultures - producers of Bis1, Bis2 and MT 103 in the Batch process and perfusion cultivation process.

Cultivations were carried out in shaker bottles with a working volume of 50 ml, on a basic nutrient medium SFM4 CHO; temperature - 37 ° С, 5% СО ₂ in the gas phase of the incubator.

Model perfusion was carried out by daily centrifugation of cell cultures (at 100 g), weaning of the supernatant and resuspension of cells in the same volume of fresh nutrient medium.

Implemented 6-day Batch cultivation and 12-day perfusion cultivation. The effectiveness of the processes was evaluated, given:

- The number of cells;

- The percentage of viable cells;

- The content of target proteins in the culture fluid by ELISA.

The results obtained during the experiments are presented in Table 1, which shows that the use of the perfusion process allowed to increase productivity in the case of producers MT103 and Bis1 by about 5 times, and in the case of Bis2 - by almost 20 times.

Table 1 Comparison of growth characteristics and productivity of cultures of producer cells Bis1, Bis2 and MT 103 in Batch and perfusion cultivation processes Producer Process The duration of the process, day Peak cell concentration, mln / ml The proportion of viable cells at the end of the cultivation process,% of the total number The concentration of the target protein in QOL, mg / l Volumetric production for 6 days, mg Bis1 Batch 6 10,2 79 23.1 23.1 ** Perfusion* 12 17.0 86 18.9 per day on average per process, 39.7 per day at peak productivity 18.9 × 6 = 113.4 Bis2 Batch 6 8.0 74 5.9 5.9 ** Perfusion* 12 18.9 81 21.4 per day on average per process, 37.4 per day at peak productivity 21.4 × 6 = 128.4 MT103 Batch 6 7.1 69 15.1 15.1 ** Perfusion* 12 20,0 68 12.9 per day on average per process, 22.6 per day at peak productivity 12.9 × 6 = 77.4 * 100% volume of medium per day; ** In terms of 1 liter.

Example 3. The study of the effect of feeder additives in the basic nutrient medium on the productivity of cell cultures in the perfusion cultivation process.

The aim of the experiment was to study the effect of Cell Boost feeder additives on the productivity of cell cultures (producers of Bis1, Bis2 and MT 103) in the perfusion cultivation process in CHM SFM4 medium.

As a result of the performance optimization for target producers, the best result was obtained using a combination of Cell Boost I (CB1) and Cell Boost 5 (CB5) in a 1: 1 ratio, with an additive volume = 25% of the volume of the nutrient medium.

Table 2 presents a summary of the selected combination of CB1 and CB5 in a 1: 1 ratio, with an additive volume of 25% of the volume of the nutrient medium.

The cultivation was carried out in shaker bottles with a working volume of 50 ml, temperature 37 ° C, 5% CO ₂ in the gas phase of the incubator. Model perfusion was carried out:

- Daily centrifugation of cell cultures (at 100 g);

- Weaning supernatant;

- Resuspension of cells in the same volume of fresh nutrient medium.

table 2 The influence of feeder additives in the basic nutrient medium on productivity in the perfusion process of cultivation of the producers of target proteins Producer The composition of the nutrient medium Peak cell concentration, mln / ml The concentration of the target protein mg / l / day Bis1 SFM4 CHO 17.7 18.8 average per process, 38 at peak productivity SFM4 CHO + CB1 + CB5 57.7 41.3 on average per process, 62.2 at peak productivity Bis2 SFM4 CHO 19,2 21.3 on average per process, 31.0 at peak productivity SFM4 CHO + CB1 + CB5 43.3 29.0 average per process, 37.0 at peak productivity MT103 SFM4 CHO 18.1 13.1 on average per process, 35.5 at peak productivity SFM4 CHO + CB1 + CB5 21.9 13.7 on average per process, 39.0 at peak productivity

Table 2 shows that the enrichment of the nutrient medium with feeder additives led to a significant (2–3 times) increase in protein yield in the cultures of Bis1 and Bis2 producers. However, the same approach in the case of producer MT103 did not give any effect.

Based on the experiments, the final methodology of the cultivation process, described in Example 4, was developed.

Example 4. The cultivation of bespecifically antibodies against CD3 * CD19 format flexibody in CHO cells.

Stage 1 - Depreservation of cell culture

After thawing the ampoule with the CHO-M cell culture by the producer of the target protein (Bis1 or Bis2), the concentration of cells is calculated and their viability is determined. In order to grow, cells are seeded in a culture vial in growth medium SFM4CHO (HyClone).

Stage 2 - Obtaining a seed inoculum

After de-preservation, the culture-producer of the target protein (Bis1 or Bis2) is subcultured to 100 ml of SFM4CHO medium volume with a culture density of 5.1 × 10 ⁶ cells / ml and 98% viability.

Stage 3 - Cell Culture in the Sartorius CultiBag 10L Bioreactor

The culture obtained in Step 2 is transplanted into a disposable Sartorius cultibag 10L perfusion bioreactor with a working volume of 1 l, of which:

- 0.9 l of growth medium SFM4CHO (Hyclone);

- 0.1 l of cell suspension;

- seeded cell concentration in the reactor 0.5 × 10 ⁶ cells / ml, viability of 99%;

- cultivation temperature 37 ° C;

- rocking speed 16-25 kach. / min;

- tilt angle of 6-8 °;

- the concentration of dissolved oxygen was maintained at a level of not less than 30% of saturation by enriching the supplied air mixture with oxygen and changing the swing parameters.

On the 3rd day of cultivation in the bioreactor, the cell culture volume is adjusted to 5 l with SFM4CHO growth medium, the concentration of cells is calculated and their viability is determined, which should be higher than 95%.

On the 4th day of cultivation in the bioreactor, with a culture viability above 95%, perfusion is initiated by the selected feeder combination CB1-CB5 with a perfusion ratio of 0.5 working volume of the bioreactor per day (2.5 l of growth medium).

On the 7th day of cultivation in the bioreactor, with a culture viability above 95%, the perfusion ratio increases to 1 working volume of the bioreactor per day (5 l of medium), the temperature of cultivation decreases to 32 ° C;

Until the end of cultivation (15th day), the perfusion ratio and the composition of the nutrient medium remain unchanged.

Total Received:

47.5 l of perfusate with a total content of the target protein Bis1 = 2117 mg.

36 l of perfusate with a total content of 1134 mg of Bis2.

Example 5. Isolation and purification of bespecifically antibodies against CD3 * CD19 format flexibody.

Capto L Affinity Chromatography (CV - 35 ml)

Buffer A: PBS, pH 7.2

Buffer B: PBS, 5% iso-Propanol, pH 7.2

Buffer C: 0.1 M Na-Citrate, pH 2.4

Column: HiScale 26/20

Before applying the culture fluid, the sorbent was balanced with 3-5 column volumes (CV) of buffer A. All washes were performed until the optical density, conductivity, and pH reached the baseline.

Before applying to Capto L, 5 M NaCl was added to the initial QOL to a concentration of 0.15 M and 0.5 M sodium phosphate, pH 7.4-7.6, to a concentration of 0.02 M. The conditioned QOL was applied to the column at a speed of 170 cm / h After application, the sorbent was washed from unbound proteins with buffer A (3-5 CV), buffer B (at least 10 CV), then with buffer A (at least 5 CV).

The fraction containing the target protein was eluted with buffer C at a rate of 55 cm / h. The eluate was measured pH (should be less than 3.0) and left for 1 hour at room temperature for viral inactivation.

The column was regenerated by successive washes with 0.01 M NaOH (contact time 15-30 minutes), buffer A (until the pH turned neutral) and preserved with 20% ethanol (3-4 CV).

Chromatography on SP-Sepharose (CV - 70 ml)

Buffer D: 0.04 M MES, 0.004 M EDTA, pH 5.5

Buffer E: 0.02 M MES, 0.02 M NaCl, pH 5.8

Buffer F: 0.02 M MES, 0.02 M NaCl, 5% iso-Propanol, pH 5.8

Buffer G: 0.02 M MES, 0.5 M NaCl, pH 5.8

Column: HC 50/20

Before applying the protein solution, the columns were equilibrated with buffer E. The eluate from the previous step, after incubation at room temperature, was diluted 3 times with buffer D and titrated with a 1 M Tris solution to pH 5.8. The resulting solution was applied to the column at a speed of 60 cm / h. After application, the SP-Sepharose column was washed sequentially with E (5 CV), F (10 CV) and E (5 CV) buffers.

The fraction containing the target protein was eluted with buffer G at a rate of 15 cm / h. The regeneration of SP-Sepharose is carried out by successive washing with 2 M NaCl, water, 0.5 M HCl, water, 0.5 M NaOH, water.

Gel filtration on Sephacryl S300 (CV - 320 ml)

Buffer H: PBS, pH 7.2

Column: HiPrep 26/60 (pre-wired)

The column was pre-equilibrated with N. buffer. The eluate from the previous step was applied in parts (application volume not more than 15 ml) at a speed of 22 cm / h. The collection of the main protein fraction was started 160-163 ml after the start of application of the sample (depending on the height of the previous peak of the oligomeric forms) and until the optical density dropped to 30 mAu. The column was regenerated by successive washes with 0.5 M NaOH and water.

Mono Q Chromatography (CV - 1 ml)

Buffer I: 0.02 M Na-Phosphate, 0.02 M His, 1.8% Mannitol, 0.01% Tween 80, pH 7.3

Buffer J: 0.02 M Na-Phosphate, 0.02 M His, 1.8% Mannitol, 0.1% Triton X100, pH 7.3

Buffer K: 0.02 M Na-Phosphate, 0.02 M His, 1.8% Mannitol, 5% iso-Propanol, pH 7.3

Buffer L: 0.02 M Na-Phosphate, 0.02 M His, 1.8% Mannitol, 0.01% Tween 80, 0.5 M NaCl, pH 7.3

Column: HiTrap 5/50

Before applying the protein solution, the column was equilibrated with buffer I. The eluate from the previous step was diluted 3 times with buffer I. No more than 15 mg of total protein was applied at a speed of 300 cm / h. After application, the Mono Q column was washed with buffer I (10 CV), then washed successively with buffers J, I, K according to the scheme (30 CV J, 10 CV I, 30 CV K, 10 CV I).

The fraction containing the main dimeric form of the target protein was eluted with a gradient with buffer step L at a speed of 300 cm / h. A gradient of 0-100% buffer L was set for 60 CV, then when the conductivity reached 16.75 ± 0.05 mS / cm ² for BIS1 (14.55 ± 0.05 mS / cm ² for BIS2), the corresponding percentage of buffer L was fixed and the peak of the target protein was collected with 35 mAu at the beginning and up to 20% of the maximum optical density of the peak.

The regeneration of Mono Q is carried out by successive washing with 2 M NaCl, water, 0.5 M NaOH, water, 0.1 M HCl, water.

As a result of these steps, 582.7 mg of Bis1 and 295.2 Bis2 were obtained from 47.5 L and 36 L of the supernatant, respectively.

Example 6. The study of the biological activity of bespecifically molecules in the ADCC test

To determine the biological activity of the obtained bispecific antibodies, an antibody-dependent cell-mediated cytotoxicity test (ADCC) was tested and tested, which allows in vitro simulating the immune response process that occurs in a living organism. As a result of this analysis, such a parameter is determined for antibodies as the concentration of the active substance at which a half biological effect is observed, which is a qualitative characteristic of antibody-like structures specific for cell receptors

In order to study the biological activity of the bispecific molecules Bis1, Bis2 and MT103, a mononuclear fraction of human blood was used.

Preparation of effector PBMCs of human cells.

To isolate the mononuclear fraction from 50 ml of donated blood, platelets and erythrocytes were purified by gradient centrifugation in ficol 1.077. The cell mass was diluted in 50 ml of phosphate buffer and put into 10 ml tubes, layering on 5 ml of pre-applied ficoll. After a 20-minute centrifugation at 2500 rpm, a cell fraction was taken from the monocyte ring. Cells were washed twice with RPMI1640 medium with 10% FBS, followed by precipitation as a result of 5-minute centrifugation at 1600 rpm at + 4C. After resuspension in 10 ml of medium, the pellets of each tube were pooled, then the amount of PBMC was counted.

Preparation of target cells RAji (CD19 +).

Cells were grown in DMEM / F12 medium with 10% ETS and glutamine. Before the experiment, the concentration of the target cells was counted. During the analysis, an appropriate number of tablets with targeted Raji cells is prepared, at the rate of 15 thousand cells per well. For this, the cells were washed twice in PBS, followed by resuspension of the pellet in RPMI1640 medium. Then, the calcein AM dye was added to the cells, followed by a 30-minute incubation in a CO ₂ shake incubator. Cells were washed from the dye, followed by a change of medium to medium with probenicide, which prevents the metabolized dye from leaving the cells through transport systems.

10-fold dilutions of antibody bispecific samples were prepared on the basis that the final concentration should be from 10 μg / ml to 0.000001 μg. Human effector cells (PBMCs) and diluted bispecific samples were added to target cell plates (Raji). After 3 hours of incubation under 5% CO ₂ condition, 20 μl of a 9% aqueous solution of Triton X100 was added to control wells (maximum cell killing value) and data analysis was performed using plate fluoroscan (fluorescence 488 nm exc., 518 nm yen.).

The result, calculated using the GraphPad Prizm package, is also presented in Fig. 8 and in Table 3.

Table 3 Bis1 Bis2 MT103 EC50 (mg / ml) 21.6 * 10 ^-8 46.4 * 10 ^-8 82.7 * 10 ^-8 R square 0.95 0.97 0.97 % max 60.1 55.6 48.36

Based on the data obtained, it can be seen that the Bis1 and Bis2 samples reach the highest cell killing maximum (% max) and are more active (lower IC50) in comparison with the control MT103 molecule.

Claims (6)

1. A method of producing a recombinant monoclonal antibody against CD3 * CD19 in flexibody format, comprising constructing a recombinant expression plasmid DNA encoding a bispecific anti-CD3 * CD19 antibody, transfection of the obtained DNA strain of CHO M cells and culturing the obtained cell line, characterized in that the cultivation is carried out in perfusion mode in the presence of feeder additives CB1 and CB5 in a ratio of 1: 1, with the volume of the additive constituting 25% of the volume of the nutrient medium. 2. The method according to claim 1, characterized in that the expression plasmid pOG776 encoding a polypeptide with a sequence of bispecific anti-CD3 * CD19 flexibody format antibody having a size of 10 618 bp with the physical map shown in FIG. 6, and consisting of the following elements:
- an optimized sequence encoding a polypeptide of a bispecific antibody of flexibody format against CD3 * CD19, SEQ ID No. 3,
- enhancer of the CMV virus and transcription promoter of the gene for elongation factor alpha, CMVe / EFa pro,
- bovine growth hormone polyadenylation signal, BGH pA,
- enhancer of the SV40 virus, sv40 enh,
- an attachment element to the nuclear matrix, SGE 1,
- cassettes for the expression in bacteria cells of the β-lactamase gene, which provides resistance to ampicillin,
- cassettes providing resistance to puromycin,
- Unique recognition sites of the following restriction endonucleases:
KpnI (7 bp), AgeI (460 bp), SacI (1206 bp), HindIII (1590 bp), XbaI (3135 bp), SpeI (3932 bp) p.), PvuII (4835 bp), AccI (5800 bp), BsrGI (6612 bp), AhdI (8138 bp), SfiI (9774 bp), BsiWI (9912 bp), NotI (10456 bp). 3. The method according to claim 1, characterized in that the expression plasmid pOG854 encoding a polypeptide with a bispecific anti-CD3 * CD19 antibody sequence having a size of 10,618 bp with the physical map shown in FIG. 6, and consisting of the following elements:
- an optimized sequence encoding a polypeptide of a bispecific flexibody anti-CD3 * CD19 format antibody, SEQ ID No. 4,
- enhancer of the CMV virus and transcription promoter of the gene for elongation factor alpha, CMVe / EFa pro,
- bovine growth hormone polyadenylation signal, BGH pA,
- enhancer of the SV40 virus, sv40 enh,
- an attachment element to the nuclear matrix, SGE 1,
- cassettes for the expression in bacteria cells of the β-lactamase gene, which provides resistance to ampicillin,
- cassettes providing resistance to puromycin,
- Unique recognition sites of the following restriction endonucleases:
KpnI (7 bp), AgeI (460 bp), SacI (1206 bp), HindIII (1590 bp), XbaI (3135 bp), SpeI (3932 bp) p.), PvuII (4835 bp), AccI (5800 bp), BsrGI (6612 bp), AhdI (8138 bp), SfiI (9774 bp), BsiWI (9912 bp), NotI (10456 bp). 4. Monoclonal anti-CD3 * CD19 antibody of flexibody format, containing two non-covalently linked polypeptide chains with the amino acid sequence of SEQ ID No. 1 or two non-covalently linked polypeptide chains with the amino acid sequence of SEQ ID No. 2, obtained by the method according to any one of claims. 1-3. 5. Recombinant expression plasmid DNA pOG776 encoding a polypeptide with the sequence of the bispecific anti-CD3 * CD19 flexibody format antibody having a size of 10 618 bp with the physical map shown in FIG. 6, and consisting of the following elements:
- an optimized sequence encoding a polypeptide of a bispecific antibody of flexibody format against CD3 * CD19, SEQ ID No. 3,
- enhancer of the CMV virus and transcription promoter of the gene for elongation factor alpha, CMVe / EFa pro,
- bovine growth hormone polyadenylation signal, BGH pA,
- enhancer of the SV40 virus, sv40 enh,
- an attachment element to the nuclear matrix, SGE 1,
- cassettes for the expression in bacteria cells of the β-lactamase gene, which provides resistance to ampicillin,
- cassettes providing resistance to puromycin,
- Unique recognition sites of the following restriction endonucleases:
KpnI (7 bp), AgeI (460 bp), SacI (1206 bp), HindIII (1590 bp), XbaI (3135 bp), SpeI (3932 bp) p.), PvuII (4835 bp), AccI (5800 bp), BsrGI (6612 bp), AhdI (8138 bp), SfiI (9774 bp), BsiWI (9912 bp), NotI (10456 bp). 6. Recombinant expression plasmid DNA pOG854 encoding a polypeptide with a bispecific anti-CD3 * CD19 antibody sequence having a size of 10,618 bp with the physical map shown in FIG. 6, and consisting of the following elements:
- an optimized sequence encoding a polypeptide of a bispecific flexibody anti-CD3 * CD19 format antibody, SEQ ID No. 4,
- enhancer of the CMV virus and transcription promoter of the gene for elongation factor alpha, CMVe / EFa pro,
- bovine growth hormone polyadenylation signal, BGH pA,
- enhancer of the SV40 virus, sv40 enh,
- an attachment element to the nuclear matrix, SGE 1,
- cassettes for the expression in bacteria cells of the β-lactamase gene, which provides resistance to ampicillin,
- cassettes providing resistance to puromycin,
- Unique recognition sites of the following restriction endonucleases:
KpnI (7 bp), AgeI (460 bp), SacI (1206 bp), HindIII (1590 bp), XbaI (3135 bp), SpeI (3932 bp) p.), PvuII (4835 bp), AccI (5800 bp), BsrGI (6612 bp), AhdI (8138 bp), SfiI (9774 bp), BsiWI (9912 bp), NotI (10456 bp).

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