Recombinant single-strand trispecific antibody anti-cea/cd3/cd28, produced through genetic engineering

FIELD: chemistry.

SUBSTANCE: proposed is a recombinant single-strand trispecific antibody for treating tumours which express CEA. The said antibody consists of a series of three antibody fragments: anti-CEA-scFv, anti-CD3-scFv and VH CD28-antibody, linked by two intermediate linkers (intermediate linker Fc and intermediate linker HSA). If necessary, a c-myc-mark or (His)6-mark can be added at the C-end. Described is DNA, which codes the antibody, expression vector based on it and E.coli cell, containing the vector.

EFFECT: use of the invention is more beneficial in clinical use compared to bispecific antibodies and known trispecific antibodies, makes easier clearing and expression of an antibody, which can further be used in treating CEA-mediated tumours.

10 cl, 21 dwg, 11 ex

 

Introduction

The technical field to which the invention relates.

The present invention relates to the field of production of recombinant genetically engineered antibodies, more specifically, to obtain a linear single-stranded recombinant thespecification anti-CEA/D3/CD28 antibodies (scTsAb), the methods used for construction, expression and purification scTsAb, vectors and cells of the host E. coli that contain the specified scTsAb.

The level of technology

For the activation of T lymphocytes requires two signals in vivo: 1) the interaction between the MHC/antigenic peptide, located on antigen presenting cells (APC), and a complex of TCR/CD3 on T lymphocytes generates a first signal; and 2) the interaction between co-stimulating a receptor on the APC and co stimulatory molecule on T lymphocytes creates a second co-stimulating signal. In General, it is believed that the existence of only the first signal cannot stimulate T-lymphocytes (Baxter and Hodgkin, 2002; Bernard et al., 2002).

There are two types of T lymphocytes, including cytotoxic T lymphocytes (CTL) and T-helper cells (TH). CTL is the main effector cells for the cellular immune response, whereas T is involved in the cellular immunological response indirectly through the secretion of cytokines (such as interleukin-2 (IL-2)). So it is to antitumor immunity is mainly cellular immunity, the design of anticancer drugs for specific CTL activation is critical for antitumor immunotherapy (Foss, 2002).

In practice designed some of recombinant bespecifically (BsAb) anti-TAA/D3 antibodies to provide the first signal to activate CTL and targeting of activated CTL environment of tumor cells to induce their specific mortality. Of these antibodies, some bespecifically antibodies are already enrolled in the clinic (Daniel, Kroidl et al., 1998; Holliger, Manzke et al., 1999; Loffler, Kufer et al., 2000; Manzke, Tesch et al., 2001; Manzke, Tesch et al., 2001; Dreier, Lorenczewski et al., 2002; M. Fang, 2002; Dreier, Baeuerle et al., 2003; Loffler, Gruen et al., 2003; Fan, Zhao et al., 2004). It has been proven that they activate specific T-lymphocytes and induce appreciable specific cytolysis of tumor cells. However, most of these antibodies are unable to fully activate T-lymphocytes and can sometimes lead to activation induced cell death (AICD) of T-lymphocytes (Daniel, Kroidl et al., 1998).

To overcome the above disadvantages are designed BsAb another anti-TAA/CD28-BsAb. In the presence of anti-TAA/CD3 BsAb cells CTL get a double trigger signals, which induce more effective specific cytolysis of tumor cells. However, there are several disadvantages with the combined use of two BsAb. The drawbacks include the two times the number of stages of expression and purification of antibodies and, as a consequence, increase the cost of production, and the problem of "partnership" of the two BsAb in clinical application. Thespecification antibody (TsAb) with three specificnosti binding (TAA, CD3 and CD28) can replace the two above BsAb in providing dual trigger signals (Jung, Brandl et al., 2001; Kodama, Suzuki et al., 2002) and may have an advantage over them in the process of expression, purification and clinical application.

To date there are three types of TsAb. Such TsAb include 1) chemically conjugated TsAb (Jung, Freimann et al. 1991; Tutt, Stevenson et al., 1991; French, 1998; Wong, Vakis et al., 2000); 2) recombinant polymer TsAb (Atwell, Breheney et al., 1999; Dolezal, Pearce et al., 2000; Schoonjans, Willems et al., 2000; Schoonjans, Willems et al., 2000; Kortt, Dolezal et al., 2001; Schoonjans, Willems et al., 2001; Willems, Leoen et al., 2003) and 3) single-stranded recombinant TsAb (scTsAb) (Li-ping Ju-long et al., 2003). It is believed that the third type TsAb has advantages compared with other in respect to simplicity of construction, expression and purification. As cancer embryonic antigen (the carcinoembryonic antigen (CEA) is TAA broad-spectrum (Ganjei, Nadji et al., 1988; Horie, Miura et al., 1996; Kuo, Tsai et al., 1996; Feil, Wechsel et al., 1999; Kammerer, Thanner et al., 2003; Shang-zhi, Chang-cheng et al., 2004), scTsAb containing anti-CEA antibodies, can be used for the prevention or treatment of various tumors in the clinic.

The invention

In the present invention the introduction of anti-BEHOLD the antibodies in the anti-CEA/CD3/CD28-scTsAb provides the ability to distinguish tumor cells from normal cells in vivo and to avoid or reduce non-specific cell death, caused by activated T-lymphocytes.

Because CEA is widely expressed in many tumor cells, another aspect of the present invention relates to the widespread use of these antibodies for the treatment or prevention of various tumors in the future.

Another aspect of the present invention relates to a method of constructing scTsAb.

Amino acid sequence (SEQ ID NO: 1) murine single-chain anti-CEA-fragment variable region, located in the CEA-scTsAb are listed below:

Another aspect of the present invention relates to a vector for the expression of CEA-scTsAb: CEA-scTsAb/pTRI.

Additional aspects of the present invention relate to a method of promoting cytoplasmic expression of the above soluble scTsAb at lower temperatures and relate to the purification method above scTsAb using DEAE-anion exchange chromatography.

However, in the context of the present invention other aspects and advantages of the invention will be obvious to experts in the field, especially on the basis of the description in the "Examples"section.

Brief description of drawings

Figure 1. Chart of the method of constructing the frame DNA for multiple cloning using the-W PCR with overlapping. Numbers 2 through 11 represent different synthetic polymer fragments of nucleic acids. The symbols "a, b, C, D, E, I, II, III, IV, UP, DOWN" the meaning of the various intermediate products design. The designation "WHOLE" means the final product.

Figure 2. Detection of PCR products with overlapping by agarose gel electrophoresis. Lane 1: PCR product with overlapping; lane 2: marker DL2000 DNA (Dalian Takara Biothech.).

Figure 3. The sequence of frames DNA for multiple cloning.

Figure 4. Method of constructing CEA-scTsAb.

Figure 5. Schematic maps vectors to construct and expression of CEA-scTsAb.

6. Identification of the method of design by agarose gel electrophoresis. Lane 1: PCR product, amplificatory with empty vector pTRI; lane 2: PCR product, amplificatory with vector CD28 VH/pTRI; lane 3: PCR product, amplificatory with vector CD3scFv/CD28 VH/pTRI; lane 4: PCR product, amplificatory with vector CEA-scTsAb/pTRI; lane 5: marker DL2000 DNA (Dalian Takara Biotech.).

7. Shows a diagram of the process of designing a murine anti-CEA-scFv by PCR with overlapping. Numbers 1-22 mean different synthetic polymer fragments of nucleic acids; the Characters "a, b, C, D, E, F, G, H, I, J, K, a, b, C, d, e, f, g, I, II, III, IV, UP, DOWN" means intermediate products design. The symbol "WHOLE" means the final product.

p> Fig. Detection of PCR product with overlap using agarose gel electrophoresis. Tracks 1 and 9: DL2000 DNA markers (Dalian Takara Biotech.); track 2-5: intermediates I, II, III, IV; lanes 6 and 7: intermediate products UP and DOWN; lane 8: the final product is WHOLE.

Fig.9. SDS-page downregulation of soluble CEA-scTsAb. Track 1: the residue after ultrasonic treatment containing downregulation of CEA-scTsAb/pTRI; track 2: adosados after ultrasonic treatment, containing the products of expression of CEA-scTsAb/pTRI; lane 3: standard molecular weight proteins (Shanghai Biochemistry Institute); lane 4: sediment after ultrasonic treatment, containing the products of expression of empty vector pTRI; track 5: adosados after ultrasonic treatment, containing the products of expression of empty vector pTRI. Strip CEA-scTsAb indicated by arrows in their respective tracks.

Figure 10. Western blotting of soluble expressed CEA-scTsAb. Lane 1: standard molecular weight proteins (NEB); lane 2: sediment after ultrasonic treatment, containing the products of expression of CEA-scTsAb/pTRI; lane 3: sediment after ultrasonic treatment, containing the products of expression of empty vector pTRI; lane 4: adosados after ultrasonic treatment, containing the products of expression of CEA-scTsAb/pTRI; track 5: adosados after ultrasonic treatment containing products is xpressia empty vector pTRI.

11. SDS-PAG CEA-scTsAb, purified by anion-exchange chromatography on DEAE. Track 1: adosados after ultrasonic treatment, containing the products of expression of empty vector pTRI; track 2: adosados after ultrasonic treatment, containing the products of expression of CEA-scTsAb/pTRI; lane 3: flow through the column with anion-exchange chromatography on DEAE; lane 4: elution of NaCl in anion-exchange chromatography on DEAE; lane 5: elution NaOH in anion-exchange chromatography on DEAE; lane 6: standard molecular weight proteins (Shanghai Biochemistry Institute). Strip CEA-scTsAb indicated by arrows in their respective tracks.

Fig. The results of ELISA (enzyme-linked immunosorbent assay) CEA-scTsAb. In the figure from top to bottom the first curve: 10 µg/ml membrane antigen Jurkat; the second curve: 1 µg/ml purified CEA (R&D); the third curve: 1 μg/ml CD28 Chimera-Fc (R&D); the fourth curve: without antigens.

Fig. FCM binding of CEA-scTsAb with a variety of tumor cells. The shaded peak is a negative control without addition of CEA-scTsAb.

Fig. FCM binding of CEA-scTsAb with Jurkat cells and RVMS mononuclear cells of peripheral blood (RVMS). The shaded peak is a negative control without addition of CEA-scTsAb.

Fig. The influence of the ratio E/T (effector cells/target cells) on a specific cytolysis of tumor cells, inducir the bath CEA-scTsAb, which was determined in the MTT assay. In the figure from top to bottom the first curve: E/T = 10; the second curve: E/T = 5, the third curve: E/T = 1. Effector cells: RVMS; target cells: tumor cells SW1116.

Fig. The effect of the concentration of CEA-scTsAb on specific cytolysis of tumor cells in the MTT assay. There are four step phase-specific cytolysis of tumor cells. In the first phase, from 6 μg/ml to 12 μg/ml, there is a negative correlation between the effectiveness of specific cytolysis of tumor cells and the concentration of CEA-scTsAb, and the peak is reached at 6 µg/ml In the second phase, from 750 ng/ml to 6 μg/ml, there is a direct correlation, and the minimum efficiency is observed at 750 ng/ml In the third phase, from 24 ng/ml, 750 ng/ml, there is a return to negative correlation. In the fourth phase, from 24 ng/ml to zero, again manifested a direct correlation.

Fig. The effect of the concentration of CEA-scTsAb on the proliferation of effector cells determined in the MTT assay. There are 3 speed phase change of the stimulation index (SI). In the first phase, from 750 ng/ml to 12 μg/ml, there is a direct correlation between SI and the concentration of CEA-scTsAb, and the smallest value is achieved at 750 ng/ml In the second phase, from 50 ng/ml, 750 ng/ml, there is a negative correlation, and the peak is reached at 50 ng/ml In the third phase, 50 ng/ml to zero, there is a return to the direct correlation.

Fig. Morphological change of various cells in the process of specific cytolysis of tumor cells induced by CEA-scTsAb. (A) Tumor cells SW1116 after 20 hours of cultivation; (B)-(I) a Mixture of SW1116 (target cells) and RVS (effector cells) adding CEA-scTsAb. E/T=5. (C) Attached to the target cells begin to detach; (C) Effector cells aggregate at the surface of target cells; (D) TsAb appeared on the surface of target cells; (E) Membrane of target cells are partially destroyed; (F) Membrane of target cells completely destroyed; (G)to(I) target Cells disintegrate into fragments.

Fig. Scheme of mechanism-specific cytolysis of tumor cells induced by CEA-scTsAb. Upper diagram: structure of CEA-scTsAb; lower diagram: the diagram of a mechanism-specific cytolysis of tumor cells induced by CEA-scTsAb; CEA-scTsAb simultaneously associated with both cell-target and effector cell, thus leading to activation of effector cells, which provides double the signals that cause specific death of target cells.

Fig. Micrograph of killed target cells (SW1116). Tracks a, b and C represent three different States of tumor cell death, respectively: necrosis, apoptosis of late-stage apoptosis at an early stage.

Fig. FCM (PI/annexin V-FITZ (isothiocyanate fluorescein)) specific titoli is and tumor cells. Four sectors represent different States of tumor cells: the living cells in the lower left quadrant (LL); cells at an early stage of apoptosis in the lower right hand quadrant (LR); cells at the late steel apoptosis in the upper right hand quadrant (UR); necrotic cells in the upper left quadrant (UL). The sample without the addition of CEA-scTsAb: LL (90,17%), LR (1.66 per cent), UL (5,94%), UR (2,33%). The sample with the addition of 50 ng/ml of CEA-scTsAb: LL (52,83%), LR (16,12%), UL (9,80%), UR (each holding 21.25%).

Detailed description of the invention

In the description of the present invention, all terms easily understood by professionals in this area, except for those, which is explained in detail. Some of the terms used in the description are listed below:

Linear single-stranded recombinant thespecification antibody is a single linear molecule, constructed with three different specificnosti binding of antigens by way of genetic engineering. In particular, linear single-stranded recombinant thespecification anti-CEA/D3/D28-antibody is a single linear molecule constructed by merging fragments of three different antibodies (anti-CEA antibodies, anti-CD3 antibody, anti-CD-28-antibodies), which are separated by two linkers (Fc-linker and HSA linker) (Min Fang, 2003). Can be added with-ICC-tag and a (His) 6-tag to the C-end of the molecule to detect activity or additional purification (Hengen, 195; Fan, Villegas et al., 1988). Fragments of the antibodies listed in this description may be a single-chain fragments of variable regions (scFv) antibodies, Fab fragments of antibodies or single domain antibody fragments (VHor VL). More specifically, CEA-scTsAb design sequential merge anti-CEA-scFv, Fc-linker, anti-D3-sFv, HSA linker and anti-CD28-VNwith c-myc-tagged and His-6-tag at the C-end. This block has two advantages:

1. Antibody-based model of activated T-cells, two signals and has the ability to fully activate T cells.

2. CEA is TAA broad-spectrum, therefore, the CEA has broad prospects for use in the prevention and treatment of many tumors.

Method of induction of cytoplasmic expression of soluble CEA-scTsAb in cold areas proposed in the present invention, requires the induction of bacterial hosts 0.4 mm IPTG at 30°C. In the case of this method can be considerably reduced education Taurus inclusions, and about 50% downregulation of CEA-scTsAb is soluble. Soluble downregulation of CEA-scTsAb can be used in the next stage of purification directly without denaturation or renaturation, which can reduce production costs and increase output.

The single-stage method of purification, at which collect passing the sweat is to anion exchange chromatography on DEAE, requires application of soluble products of expression on a column of anion exchange resin DEAE at pH 8.0. Then almost all proteins not of interest, can be absorbed, while most of CEA-scTsAb out in passing the stream with a purity of about 75%.

The method according to the invention is described below:

First design of the original vector pTRI with the introduction of new specific sites for multiple clone (MCS). Then the DNA fragment encoding the anti-CD28 VH, amplified in PCR with vector CD28 VH/pTMF, both ends of which add specific pairs of restriction sites NdeI/kpni restriction sites. The same method of preparing a DNA fragment encoding the anti-D3-scFv, with a site restriction ScaI/SalI. The DNA fragment encoding the anti-CEA-scFv, with the restriction sites XhoI/EcoRI cut of CEA-scFv/pTMF. Finally, all three fragments consistently give pTRI getting end vector of CEA-scTsAb/pTRI. DNA fragments encoding the anti-CEA-scFv, anti-CD3 scFv and anti-CD28-University, have sequentially from N-Terminus to the C-end.

After transformation of E. coli BL21(DE3) and induction using IPTG at a low temperature (30°C) soluble CEA-scTsAb is expressed in the cytoplasm. The first antibody is purified using a single phase anion-exchange chromatography on DEAE. Specificity of binding of three antigens (CEA, CD3, CD28) detected in ELISA. Specificity of binding with puhelimella identify using FCM after conjugation CEA-scTsAb with FITC. The cytolysis of tumor cells and proliferation of T-lymphocytes induced by CEA-scTsAb, examined by MTT-assay. Morphological changes of tumor cells recorded on the microphotographs in the inverted microscope. Necrosis and apoptosis of tumor cells induced by CEA-scTsAb, visualize using FCM with double staining, PI/annexin-V-FI, and fluorescence microscopy.

Examples

Example 1. Obtaining a DNA fragment containing multiple cloning sites using PCR with overlapping.

Diagram of the method shown in figure 1. All the synthesized fragments used according to the invention are set forth below:

Processing phase:

Stage 1. As shown in figure 1, pairs of mixed fragments (2-11) and carry out the elongation reaction, as described below.

Reaction mixture: synthetic fragments, 1 μl (each); 10 x buffer for PCR, 2 μl; dNTP (2 mmol/ml each) (Dalian Takara Biotechnology Co. Ltd.) 2 ál; Taq (1 unit) (Dalian Takara Biotechnology Co. Ltd.) 0,5 µl; distilled water, 14 ál.

Reaction conditions: pre-denaturation at 94°C for 1 min; denaturation at 94°C for 30 seconds; annealing at 45°C for 30 seconds, elongation at 72°C for 30 seconds; 10 cycles.

All products collected without treatment and directly used for the next the stage.

Stage 2. As shown in figure 1, pairs mixed products (a, b, C, D, E) with stage 1 and carry out the elongation reaction, as described below.

Reaction mixture: products with stage 1, 10 µl (each).

Reaction conditions: pre-denaturation at 94°C for 1 min; denaturation at 94°C for 30 seconds; annealing at 45°C for 30 seconds, elongation at 72°C for 30 seconds; 10 cycles.

All products were applied to agarose gel electrophoresis (1%) and was purified using the kit for purification of DNA from gel (Watson Biotech. Inc.).

Stage 3. As shown in figure 1, pairs mixed products (I, II, III, IV) from step 2 and perform the elongation reaction, as described below.

The reaction mixture: the products from stage 2, and 1 µl (each); 10 x buffer for PCR, 2 μl; dNTP (2 mmol/ml each) (Dalian Takara Biotechnology Co. Ltd.), 2 ál; Taq (1 unit) (Dalian Takagi Biotechnology Co. Ltd.), 0,5 µl; distilled water, 13 ml.

Reaction conditions: pre-denaturation at 94°C for 1 min; denaturation at 94°C for 30 seconds; annealing at 45°C for 30 seconds, elongation at 72°C for 30 seconds; 10 cycles.

All products were applied to agarose gel electrophoresis (1%) and was purified using the kit for purification of DNA from gel (Watson Biotech. Inc.).

Stage 4. As shown in figure 1, pairs mixed products (UP, DOWN) with stage 3 and carry out reaction amplification, as indicated by the who.

Reaction mixture: products with stage 3, and 1 µl (each), primers (synthetic fragments 1 and 12), 1 μl; 10 x buffer for PCR, 2 μl; dNTP (2 mmol/ml each) (Dalian Takara Biotechnology Co. Ltd.), 2 ál; Taq (1 unit) (Dalian Takara Biotechnology Co. Ltd.), 0,5 µl; distilled water, 12 μl.

Reaction conditions: pre-denaturation at 94°C for 1 min; denaturation at 94°C for 30 seconds; annealing at 45°C for 30 seconds, elongation at 72°C for 50 seconds; 25 cycles.

The final products (493 BP) were separated by electrophoresis in agarose gel (1%) (figure 2) and was purified using the kit for purification of DNA from gel (Watson Biotech. Inc.). Sequence, restriction sites and components shown in figure 3.

Example 2. The design of CEA-scTsAb

The method of construction shown in figure 4, schematic maps of all vectors used in the method shown in figure 5.

(1) Construction of vector pTRI:

The DNA fragment containing multiple cloning sites and an empty vector pTMF (Zhang et al., 2002) cut NcoI/BamHI and ligated together. The products of ligation were used to transform E. coli strain TOP10 (Invitrogen). Plasmids isolated from transformed bacterial cells, called pTRI.

Cleavage by restriction enzymes, ligation, acquisition and transformation of TOP10 competent cells was carried out as follows:

The cleavage reaction enzymes R is stricly: in the volume of 20 µl of 1 µg pTMF or DNA fragment, containing multiple cloning sites, was digested according to the instructions Promega. The products were separated by electrophoresis in agarose gel (1%) and was purified using the kit for purification of DNA from gel (Watson Biotech. Inc.).

The ligation reaction: 50-100 ng of the split vector and 3-10 larger amount (molar ratio) cleaved DNA fragments were mixed in 20 μl of solution containing 2 μl of 10 x buffer for DNA ligase T4; 1 unit of DNA ligase T4 (Dalian Takara Biotechnology Co. Ltd.) and the required amount of distilled water. The ligation reaction was carried out at 16°C during the night.

Preparation of competent cells TOP10: TOP10 bacteria (Invitrogen Co.) was inoculable in 2 ml of LB medium (10 g/l of tryptone (GIBCO Co.), 5 g/l yeast extract (GIBCO Co.), 5 g/l NaCl, pH 7.5) and incubated overnight at 37°C on a rocking chair. Transferred in 20-40 ml of LB medium in a ratio of 1:100, incubated at 37°C with shaking until reaching And6000.3 to 0.4 (approximately 2.5 hours). Cooled on ice for 15 minutes and centrifuged at 4°C with a speed of 4000 rpm./min for 10 minutes. Sediment resuspendable in 10 ml pre-cooled solution of 0.1 mol/ml CaCl2(Sigma Co.) and cooled on ice for 20 minutes. After re-centrifugation at 4°C with a speed of 4000 rpm./min for 10 minutes to precipitate carefully resuspendable in 1-2 ml of pre-cooled solution, content is the future of 0.1 mol/l CaCl 2and 12% glycerol. Divided into aliquots of 200 μl in THE tube, and kept at -80°C.

Transform: Mixture for ligation was added to 200 μl of competent cells. After careful mixing and cooling on ice for 30 minutes, the cells were placed in a water bath at a temperature of 42°C for 100 seconds, then cooled on ice for 2 minutes. Once added to a mixture of 0.8 ml of LB medium, the mixture was shaken at 37°C (<150 rpm./min) for 45 minutes for the cells to recover. The cells were centrifuged at 10000 rpm./min for 1 minute, resuspendable in 50-100 ml of LB medium, were sown for a Cup of LB-K (10 g/l of tryptone, 5 g/l yeast extract, 5 g/l NaCl, 15 g/l agar (SIGMA Co.), 50 µg/ml kanamycin (SIGMA Co.), pH 7.5) and incubated at 37°C during the night.

Selection of positive clones: Collected individual clones on the Cup LB-K and then individually transferred into 2 ml of LB medium-K (10 g/l of tryptone, 5 g/l yeast extract, 5 g/l NaCl, 15 g/l agar (SIGMA Co.), 50 µg/ml kanamycin (SIGMA Co.), pH 7.5). After shaking at 37°C over night plasmids were isolated using the kit for plasmid isolation (Watson Biotechnologies, Inc) according to standard instructions. Positive clones were identified in PCR, using the selected plasmids as matrices. The reaction mixture for PCR: 0.1 to 1 ál of plasmid DNA (approximately 20-200 ng), 10 pmol above primer (T7-up: 5'-TAATACGACTCACTATAGGGGA-3') (SEQ ID NO: 17), 10 p the ol following set of primers (T7-down: 5'-GCTAGTTATTGCTCAGCGG-3')(SEQ ID NO: 18), 2 µl of 10 x Taq buffer; 2 μl of a solution containing 2 mmol/ml dNTP; 1 unit of Taq enzyme, 12 μl of distilled water. The PCR reaction conditions: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 40 seconds, annealing at 53°C for 40 seconds; and elongation at 72°C for 40 seconds; 25 cycles. At the end of 5 µl of PCR product were separated by electrophoresis in agarose gel (1%). As shown in Fig.6, the PCR product had a size of approximately 500 BP

(2) Construction of CD28 VH/pTRI:

The DNA fragment encoding the anti-CD28-VN, amplified from plasmid CD28 VH/pTMF (Cheng, Wang et al., 2002), using P1 (P1: 5'-TCACATATGCAGGTACAGCTACAG-3') (SEQ ID NO: 19) as above primer and P2 (P2: 5'-TTCGCTAGCGGAAGATACGGTACCA-3') (SEQ ID NO: 20) as located below the primer. The restriction sites NdeI/NheI was inserted in the 5'- and 3'-end, respectively, by PCR.

The reaction mixture for PCR: 1 μl of each primer, and 2 ál of dNTP (each 2 mmol/ml); 2 μl of 10 x buffer pfu; 100 ng plasmid CD28 VH/pTRI; 0,3 µl of enzyme pfu (Promega Co.); distilled water was added to a final volume of 20 µl. The PCR reaction conditions: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds, elongation at 72°C for 50 seconds; 25 cycles. PCR products were purified by agarose gel electrophoresis (1%), using the kit for purification of DNA in the gel (Watson Biotech. Inc.).

The above PCR product and plasmid pTRI simultaneously uncoupled NdeI/NheI (Promega Co.). Split the PCR product (approximately 350 BP) and pTRI (about 5300 BP) and ligated together and transformed strain E. coli TOP10. Plasmids isolated from positive clones, named CD28 VH/pTRI and identified in a PCR product of approximately 750 BP (as shown in Fig.6). Every operation required in this case, as described in (1).

(3) Constructing CD3 scFv/CD28 VH/pTRI:

The DNA fragment encoding the anti-CD3 scFv amplified from plasmid CD3-scFv/pTMF (Liu X.F.1996) using P1 (P1: 5'-AAGAGTACTGAGGTGAAGCTGGTGG-3) (SEQ ID NO: 21) as above primer and P2 (P2: 5'-GAAGTCGACAGCGCGCT TCAGTTCCAG-3) (SEQ ID NO: 22) as located below the primer. Restriction enzymes cut sites. ScaI and SalI, was inserted in the 5'-end and 3'end respectively during PCR.

The reaction mixture for PCR: 1 μl of each primer, and 2 ál of dNTP (each 2 mmol/ml); 2 μl of 10 x buffer pfu; 100 ng plasmid CD28 VH/pTRI; 0,3 µl of enzyme pfu (Promega Co.); distilled water was added to a final volume of 20 µl. The PCR reaction conditions: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds, elongation at 72°C for 50 seconds; 25 cycles. PCR products were purified by agarose gel electrophoresis (1%), using the kit for purification of DNA in the gel (Watson Biotech. Inc.).

The above PCR product and plasmid CD28 VH/pTRI simultaneously uncoupled ScaI/ScaII (Promega Co.). Split the PCR product (approximately 750 BP) and CD28 VH/pTRI (approximately 5700 BP) and ligated together and transformed strain E. coli TOP10. Plasmids isolated from positive clones, called

CD3 scFv/CD28-VH/pTRI and identified in a PCR product of approximately 1400 BP (as shown in Fig.6). Every operation required in this case, as described in (1).

(4) Construction of CEA-scTsAb/pTRI:

Design of anti-CEA-scFv in PCR with overlapping:

Anti-CEA-scFv was designed by the binding of VH(variable region of the heavy chain) and VL(variable region of the light chain) monoclonal anti-CEA antibodies (Koga, Kanda et al., 1990) with the polypeptide GGGGSGGGGSGGGGS (SEQ ID NO: 23). The complete amino acid sequence of anti-CEA-scFv back translated into a DNA sequence according to the table preferred the use of E. coli codons (Nakamura, Gojobori et al., 2000), which was divided into 22 complementary oligonucleotide. Synthesized 22 of the oligonucleotide, as described below, and collected in a DNA fragment encoding the anti-CEA scFv using PCR with overlapping.

Processing phase:

Stage 1. As shown in Fig.7, in pairs mixed fragments (1-2) and was carried out by reaction of elongation, as indicated below.

Reaction mixture: synthetic fragments, 1 μl (each);

10 x buffer for PCR, 2 μl; dNTP (each 2 mmol/ml) (Dalian Takara Biotechnology Co. Ltd.) 2 ál; Taq (1 unit) (Dalian Takara Biotechnology Co. Ltd.) 0,5 µl; distilled water, 14 ál.

Reaction conditions: pre-denaturation at 94°C for 1 min; denaturation at 94°C for 30 seconds; annealing at 45°C for 30 seconds, elongation at 72°C for 30 seconds; 10 cycles.

All products collected without treatment and directly used for the next stage.

Stage 2. As shown in Fig.7, in pairs mixed products (a, b, D, E, G, H, J, K) with stage 1 and was carried out by reaction of elongation, as described below.

Reaction mixture: products with stage 1, 10 µl (each).

Reaction conditions: pre-denaturation at 94°C for 1 min; denaturation at 94°C for 30 seconds; annealing at 45°C for 30 seconds, elongation at 72°C for 30 seconds; 10 cycles.

All products were applied to agarose gel electrophoresis (1%) and was purified using the kit for purification of DNA from gel (Watson Biotech. Inc.). Fragments "a" and "g" have a size of approximately 120 BP fragments "C" and "e" had a size of approximately 170 BP fragments "d" and "f" had a size of about 100 BP

Stage 3. As shown in Fig.7, in pairs mixed products (a, b, C, d, e, f, g) from stage 2 and was carried out by reaction amplification, as indicated the ANO below. The primers S1 and S6 matched pair "a" and "b"; primers S7 and S12 matched pair "C" and "d"; primers S13 and S16 correspond "e", the primers S17 and 22 matched pair of "f" and "g".

The reaction mixture: the products from stage 2, and 1 µl (each); primers, 1 μg (each); 10 x buffer for PCR, 2 μl; dNTP (2 mmol/ml each) (Dalian Takara Biotechnology Co. Ltd.), 2 ál; Taq (1 unit) (Dalian Takara Biotechnology Co. Ltd.), 0,5 µl; distilled water, 12 μl.

Reaction conditions: pre-denaturation at 94°C for 1 min; denaturation at 94°C for 30 seconds; annealing at 45°C for 30 seconds, elongation at 72°C for 30 seconds; 25 cycles.

All products were applied to agarose gel electrophoresis (1%) and was purified using the kit for purification of DNA from gel (Watson Biotech. Inc.). The fragment I had a size of approximately 200 BP; fragment II had a size of approximately 250 BP; III fragment had a size of about 140 BP fragment IV had a size of approximately 230 BP

Stage 4. As shown in Fig.7, in pairs mixed products (I, II, III, IV) with stage 3 and was carried out by reaction amplification, as described below. The primers S1 and S12 correspond to a pair of I and II; primers S13 and 22 matched pair III and IV.

Reaction mixture: products with stage 3, and 1 µl (each); primers, 1 µl (each); 10 x buffer for PCR, 2 μl; dNTP (2 mmol/ml each) (Dalian Takara Biotechnology Co. Ltd.), 2 ál; Taq (1 unit) (Dalian Takara Biotechnology Co. Ltd.), 0,5 µl; distilled the ode, 12 μl.

Reaction conditions: pre-denaturation at 94°C for 1 min; denaturation at 94°C for 30 seconds; annealing at 45°C for 30 seconds, elongation at 72°C for 30 seconds; 25 cycles.

All products were applied to agarose gel electrophoresis (1%) and was purified using the kit for purification of DNA from gel (Watson Biotech. Inc.). Slice UP had a size of approximately 430 BP; fragment of a DOWN had a size of approximately 340 BP

Stage 5. As shown in Fig.7, in pairs mixed products (UP and DOWN) with stage 4 and was carried out by reaction amplification, as described below. The primers S1 and 22 matched pair UP and DOWN.

The reaction mixture: the product from step 4, and 1 µl (each); primers, 1 µl (each); 10 x buffer for PCR, 2 μl; dNTP (2 mmol/ml each) (Dalian Takara Biotechnology Co. Ltd.), 2 ál; Taq (1 unit) (Dalian Takara Biotechnology Co. Ltd.), 0,5 µl; distilled water, 12 μl.

Reaction conditions: pre-denaturation at 94°C for 1 min; denaturation at 94°C for 30 seconds; annealing at 45°C for 30 seconds, elongation at 72°C for 30 seconds; 25 cycles.

All products were applied to agarose gel electrophoresis (1%) and was purified using the kit for purification of DNA from gel (Watson Biotech. Inc.). The WHOLE fragment had a size of approximately 750 BP

The scheme of the above stages of the method are shown in figure 7, the results of PCR identification shown on Fig.

The above products the PCR and plasmid pTMF simultaneously uncoupled XhoI/EcoRI (Promega Co.). Split the PCR product (approximately 750 BP) and split the product pTMF (approximately 5200 BP) and ligated together and transformed strain E. coli TOP10. Plasmids isolated from positive clones, named CEA-scFv/pTMF and identified in a PCR product of approximately 750 BP every operation required in this case, as described in (1).

Plasmid CEA-scFv/pTMF and plasmid CD3-scFv/CD28-VH/pTRI simultaneously uncoupled XhoI/EcoRI (Promega Co.). Product splitting the first plasmid (approximately 750 BP) and the product of the splitting of the second plasmid (approximately 6000 BP) and ligated together and transformed strain E. coli TOP10. Plasmids isolated from positive clones were named CEA-scTsAb/pTRI and identified in a PCR product of approximately 2100 BP (as shown in Fig.6). Every operation required in this case, as described in (1).

Example 3. Cytoplasmic expression of soluble CEA-scTsAb induced at low temperature.

(1) Transformation of E. coli strain lines BL21 (DE3) (Novagen) plasmid CEA-scTsAb/pTRI

Competent cells of E. coli BL21 (DE3) was prepared according to the method described in example 2. Plasmid CEA-scTsAb/pTRI was isolated using the kit for plasmid isolation (Watson Biotech. Inc.). Subsequent transformation procedures and identification of positive clones was carried out in accordance with example 2.

(2) Induced expression at low t is mperature

A single clone of BL21 (DE3)containing plasmid CEA-scTsAb/pTRI, collected from a Cup LB-K and inoculable 5 ml of LB medium. After culturing at 37°C overnight on a shaker culture was transferred into 250 ml of LB medium-K in the ratio of 1/100, were shaken at 37°C until A600or =0.6. Added IPTG (Takara Biotech. (Dalian)) to a final concentration of about 0.4 mmol/l for the induction of expression of soluble product at 30°C for 4 hours. Bacterial cells were collected by centrifugation at 12000 rpm./min for 10 minutes and then resuspendable in phosphate-buffered saline (PBS: 8 g NaCl, 0.2 g KCl, 1.44 g of Na2HPO4, 0.24 g KH2PO4, a pH of 7.4, 1 liter) (1/5 volume of culture medium). Thus the cytoplasmic soluble CEA-scTsAb was released in adosados obtained by centrifugation, lysed by ultrasound cells. In addition, the expression of CEA-scTsAb in the form of a soluble product and the expression of CEA-scTsAb in Taurus inclusions were identified in pampering SDS-page and Western-blots according to "Molecular cloning: a laboratory manual" (Sambrook and Russel, 2001). The results were photographed using the system for registration and analysis of Alpha-Image 2200 (Amerikan Alpha Innotech Company). As shown in figure 10, the expression of soluble CEA-scTsAb approximately 70% of the total expression. As obtained after ultrasonic treatment adosados can be directly used for the next stages of purification and assays of activity in vitro without the need for denaturation or renaturation, the cost and time of receipt can be significantly reduced.

Example 4. Purification of CEA-scTsAb by one-step anion exchange chromatography on DEAE

250 ml of culture medium containing bacterial cells expressing CEA-scTsAb, was centrifuged at 12000 rpm./min at 4°C for 10 minutes. Sediment resuspendable in 50 ml of the buffer used for equilibration of the column with DEAE when anion-exchange chromatography (20 mmol/l NaCl, 20 mmol/l Tris-HCl, pH 8.0) for further ultrasonic treatment. After the second centrifugation at 12000 rpm./min at 4°C for 10 minutes adosados containing downregulation of soluble CEA-scTsAb directly used at the stage of purification.

20 ml of anion exchange resin DEAE (Amersham Bioscience) suspended in 100 ml of buffer for equilibration and filled column 16×20 cm (Shanghai Hua-mei). The column was balanced with 5 volumes of buffer to balance at a rate of 1 ml/min will Throw the above adosados inflicted with a speed of 0.25 ml/min of Purified CEA-scTsAb was in a passing stream. The column was washed or suirable 2 volumes of eluting buffer (500 mmol/l NaCl, 20 mmol/l Tris-HCl, pH 8.0) at a rate of 0.25 ml/min and purified 2 volumes of 500 mmol/l NaOH at a rate of 0.5 ml/min During the stage of regeneration of the column was balanced by 2 volumes of buffer to balance at a rate of 1 ml/min

The result is clean, you who implemented during electrophoresis passing through the column fractions in pampering SDS-page, shown figure 11. In result, most of the bacterial protein in nadeshiko can be removed as the result of one stage anion-exchange chromatography on DEAE. CEA-scTsAb was 70% passing through a column fractions.

Then the purified sample were dialyzed against PBS at 4°C over night. The protein concentration was determined by the method of Bradford (Ausubel 1999). After addition of sodium azide (0.05% of wt./about.) and fucose (0.15 mol/l) cialisovernight the solution was divided into aliquots of 1 ml and stored at -80°C.

Example 5. Determination of binding specificity with respect to the three antigens (CEA, CD3, CD28) in ELISA

Preparation of antigen Jurkat cell membrane: 5×106the Jurkat cells (American type collection of cultures, ATSS, TIB-152) were collected by centrifugation at 1000 g for 10 minutes. Sediment cells suspended in 0.5 ml PBS and literally sonification. To nadeshiko obtained by centrifugation, treated with ultrasound cells at 12000 rpm./min for 10 minutes, was added sodium azide 0.05% of (wt./about.) and fucose (0.15 mol/l), divided into aliquots of 100 μl and stored at -80°C.

ELISA:

(1) Covering: purified CEA (R&D), Chimera rhCD28-Fc (R&D) and the above-described purified membrane antigen Jurkat was diluted in buffer for covering (1,36 g Na2CO3, 7,35 g NaHCO3, 1 liter, pH of 9.2) to a concentration of 1 µg/ml (CEA and Chimera rhCD28-Fc) or 10 µg/ml (membrane and is then Jurkat) and made of 100 µl/well in a 96 well plate for ELISA (Nunc). Tablet incubated at 37°C for 2 hours or at 4°C during the night.

(2) Blocking: coated tablet 1-2 times were washed in PBS was added in blocking buffer (S-1%BSA (bovine serum albumin, wt./about.) 200 µl/well. Tablet incubated 2 hours at 37°C.

(3) Add samples: locked tablet 3 times washed with PBS and were added diluted in PBS samples of 100 μl/well. Samples of CEA-scTsAb 6-fold serially diluted from the original solution with a concentration of 10 μg/ml Tablet incubated at 37°C for 2 hours.

(4) the addition of the first antibody: tablet after addition of the sample 3 times washed with PBS-T (PBS-0.05%, tween-20 (wt./about.) and added a mouse monoclonal antibody against cmyc-tag at 1/1000 dilution (Santa Crutz) no 100 µl/well. Tablet incubated at 37°C for 2 hours.

(5) adding a second antibody: tablet after addition of the first antibody 3 times washed with PBS-T and added conjugated with HRP (horseradish peroxidase) antibody goat against mouse IgG (Santa Crutz) at 1/1000 dilution 100 ál/well. Tablet incubated at 37°C for 2 hours.

(6) Visualization: the tablet after adding the second antibody 5 times washed with PBS-T and added to the solution to render containing 10 ml of buffer substrate (36,6 g of citric acid monohydrate, 113,5 g dibasic potassium phosphate, 1 liter, pH 6.0) and 4 mg OPD (orthophenylene-2 Hcl, Sigma) no 10 µg/well. The tablet was placed in the dark at room temperature for 20 minutes.

(7) Stopping reaction: to stop the reaction solution was added 1 mol/l Hcl at 100 µl/well.

(8) Measurement: the results of the optical density was recorded at 490 nm.

As shown in Fig, CEA-scTsAb very specific associated with three antigens: CEA expressed at high levels in Jurkat cells membrane antigen CD3 and Chimera rhCD28-Fc.

Example 6. Determination of binding specificity to tumor cells by FCM

Indirect method FCM was used to determine the binding of various tumor cells. The origin of these tumor cells is shown below

/tr>
DesignationOriginATS No.
AAdenocarcinoma of the lungCCL-185
MCF-7Adenocarcinoma of human breastNTV-22
SK-OV-3Adenocarcinoma of ovaryNTV-77
SW1116Adenocarcinoma colorectalCCL-233

The sequence of operations

(1) the Cultivation and collection of tumor cells: Three types of tumor cells (A, MCF-7, SK-OV-3) were cultured in medium RPMI 1640 (Gibco)containing 10% fetal calf serum (FCS) supplemented with antibiotics (100 units of penicillin) in the incubator in a humid environment with 5% CO2at 37°C. SW1116 were cultured in L15 medium (Gibco)containing 10% fetal calf serum (FCS) supplemented with antibiotics (100 units of penicillin) in the incubator in a humid environment with 5% CO2at 37°C. 5×105tumor cells in exponential growth phase were collected by centrifugation at 1000 g for 10 minutes and suspended in 100 µl PBS.

(2) Incubation of CEA-scTsAb with tumor cells: CEA-scTsAb was added to the PBS solution containing the above tumor cells at a final concentration of 10 μg/ml For each sample of tumor cells used control isotype. The cell suspensions were incubated at 4°C for 30 minutes.

(3) Incubation of the first antibody, mouse monoclonal antibodies against cmyc-tag (Santa Crutz), with tumor cells:

Unbound CEA-scTsAb removed, merging adosados after centrifugation at 1000 g for 10 minutes. Sediment cells suspended in 100 μl of PBS containing the first antibody in a dilution of 1/1000. Suspension cells were incubated at 4°C for 30 minutes.

(4) the second antibody Incubation, the con is whiremandogg with FITC antibodies goat against mouse IgG, with tumor cells: Unbound first antibody was removed, merging adosados after centrifugation at 1000 g for 10 minutes. Sediment cells suspended in 100 μl of PBS containing a second antibody in a dilution of 1/1000. The cell suspensions were incubated at 4°C for 30 minutes.

(5) FMC-analysis: Unbound second antibody was removed, merging adosados after centrifugation at 1000 g for 10 minutes.

Sediment cells suspended in 400 μl of PBS and analyzed using a FACS instrument Colibur (BD). The wavelength of excitation 488 nm. Every time collected 10000 cells.

As shown in Fig, CEA-scTsAb best associated with SW1116 and SK-OV-3; the binding of CEA-scTsAb with A is average; CEA-scTsAb is not associated with MCF-7.

Example 7. FMC-analysis of the specificity of binding of CEA-scTsAb with RVMS and Jurkat cells

The method of direct FMC-analysis was used to test the specificity of binding of CEA-scTsAb with RVMS and Jurkat cells.

Sequence of operations:

(1) conjugation of FITC (Sigma) with CEA-scTsAb: FITC conjugatively with CEA-scTsAb using the method of Clarke (Qui Fa-zhu et al., 2002).

(2) Obtaining RVMS: RWMS was obtained by centrifugation in a gradient of picola (Qui Fa-zhu et al., 2002) and were cultured in medium RPMI 1640 (Gibco)containing 10% fetal calf serum with added antibiotics (100 units of penicillin) in the incubator in a humid environment with 5% CO2at 37°C. After incubation for 4 hours su is pendiriannya cells, mainly lymphocytes, was transferred to a new flask. So remove attached cells.

(3) Culturing Jurkat cells: Jurkat cells were cultured in medium RPMI 1640 (Gibco)containing 10% fetal calf serum with added antibiotics (100 units of penicillin) in the incubator in a humid environment with 5% CO2at 37°C.

(4) Analysis of FMC: 5×105RVMS or Jurkat cells in exponential growth phase were collected by centrifugation at 1000 g for 10 minutes and suspended in 100 μl PBS containing 10 μg/ml conjugated with FITC CEA-scTsAb. The cell suspensions were incubated at 4°C for 30 minutes and then centrifuged at 1000 g for 10 minutes. Cellular precipitate resuspendable in 400 μl of PBS and analyzed using a FACS instrument Colibur (BD). The wavelength of excitation 488 nm. Every time collected 10000 cells.

As shown in Fig, CEA-scTsAb specific associated with RVMS and Jurkat cells.

Summarizing the results described in example 6 and example 7, it is seen that CEA-scTsAb can specific contact RVMS, Jurkat and some tumor cells, which is important for the induction of specific cytolysis of tumor cells.

Example 8. Identification of specific cytolysis of tumor cells carcinoma colorectal, SW1116, induced by CEA-scTsAb, in the presence of lymphocytes.

In the system analysis in vitro of specific cytolysis of tumor cells used is awali line of CEA-expressing tumor cells SW1116 as target cells (T) and lymphocytes from RVMS (mononuclear cells peripheral blood) as effector cells (E). After mixing target cells and effector cells in a ratio of E/T and adding CEA-scTsAb specific cytolysis of tumor cells induced by incubation at 37°C for 48 hours. The results were evaluated by MTT-assay.

(1) RWMS was obtained as described in example 7.

(2) SW1116 Cells were cultured and collected according to example 6.

(3) SW1116 Cells (1×105/ml) were first placed in 96-well plates (Nunc) at 100 μl/well. Then added effector cells (RWMS) at different ratios E/T (1, 5, 10) 100 μl/well. Added concentrated CEA-scTsAb 50 μl/well to achieve a final concentration of 1 μg/ml of the Mixture is incubated in an incubator with 5% CO2at 37°C for approximately 48 hours. For each concentration used in 4 wells. Negative controls: wells without CEA-scTsAb for each ratio E/T; wells containing only effector cells; wells containing only target cells; wells containing no cells.

(4) MTT analysis: Nadeshiko medium was removed by aspiration and the attached cells were once washed by PBS. To each well was added 200 μl of MTT solution (MTT: bromide (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium, 500 μg/ml, Sigma) and incubated for 4 hours at 37°C. the Tablet once washed in PBS and each well was added 200 μl of DMSO (Sigma). Continued to incubate at 37°C for 30 minutes. Optical the current density in each well was measured at a wavelength of 570 nm, subtracting the background at 620 nm.

(5) Specific cytolysis of tumor cells in percent was calculated according to the formula:

The percentage of specific cytolysis of tumor cells (%) = [A600(ET)600(ETA)]/[A600(ET)600(M)]×100%

And600(ET): the optical density in the wells with the negative control without addition of CEA-scTsAb;

And600(ETA): the optical density of the wells with the sample;

And600(M): optical density of the wells with a negative control containing no cells.

The influence of relationship E/T specific cytolysis of tumor cells, induced by CEA-scTsAb shown in Fig. We can conclude that U/T=5 is the optimal ratio at which specific cytolysis of tumor cells reaches 85%. This suggests that in addition to the ratio E/T there are other factors affecting specific cytolysis of tumor cells. The effect of the concentration of CEA-scTsAb, increasing from 0.4 ng/ml to 12 μg/ml, specific cytolysis of tumor cells at a constant ratio E/T, is equal to 5, shown in Fig. The curve shows the four step phase-specific cytolysis of tumor cells. In the first phase of 6 μg/ml to 12 μg/ml has a negative correlation to the effectiveness of specific cytolysis of tumor cells and the concentration of CEA-scTsAb, and the efficiency reaches a peak at 6 µg/ml In the second phase, from 750 ng/m is up to 6 µg/ml, there is a direct correlation, and the minimum efficiency is observed at 750 ng/ml In the third phase, from 24 ng/ml, 750 ng/ml, there is a return to negative correlation. In the fourth phase, from 24 ng/ml to zero, again manifested a direct correlation. In any case, there are two peaks of specific cytolysis of tumor cells: 85% at 6 mg/ml and 70% at 24 ng/ml On the basis of the above results we can conclude that a very effective specific cytolysis of tumor cells can be induced even at a lower ratio E/T or lower concentrations of CEA-scTsAb.

Example 9. Morphological study of tumor cells in the process of specific cytolysis of tumor cells

After mixing RUMS (effector cells) with SW1116 cells (target cells) in L15 medium (10% FBS) at a ratio of E/T, is equal to 5, and adding purified CEA-scTsAb at a concentration of 1 mg/ml and the mixture incubated at 37°C for 20-40 hours in an incubator with 5% CO2. Morphological changes of tumor cells and RVMS observed using a 40 x objective lens in an inverted microscope (OLYMPUS IMT-2 and recorded on the micrographs. As shown in Fig (A)-(G), there are four stages of morphological changes.

Example 10. Determination of proliferation of effector cells, inkubiruemykh with target cells and CEA-scTsAb.

The proliferation of EF is Echternach cells (mainly T lymphocytes), detected by MTT analysis, can be used to assess the activation of T-lymphocytes induced by joint incubation of tumor cells and CEA-scTsAb.

Sequence of operations:

(1) RWMS was obtained according to example 6.

(2) Tumor cells SW1116 were cultured and collected according to example 6.

(3) the Concentration of cells SW1116 drove about 106/ml in L15 medium containing mitomycin C (25 μg/ml, Sigma) and the cell mixture was incubated at 37°C in an incubator with 5% CO2within 20 minutes. After triple washing of tumor cells in PBS remaining mitomycin C was removed from the culture medium.

(4) the Concentration of cells SW1116 brought up to 105/ml and the concentration RVMS brought up to 5×105/ml in L15 medium. 100 ál of cells were planted in 96-well plate and cells were incubated at 37°C in an incubator with 5% CO2within 4 days. For each concentration of CEA-scTsAb used on 4 holes. Negative controls: wells without the addition of CEA-scTsAb for each ratio E/T; wells containing only effector cells; wells containing only target cells; wells containing no cells.

(5) the IMT-analysis: Adosados medium was removed by aspiration and the attached cells were once washed by PBS. To each well was added 200 μl of MTT solution (MTT: bromide (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium, 500 μg/ml, Sigma) and incubated in accordance with the s 4 hours at 37°C. The tablet once washed in PBS and each well was added 200 μl of DMSO (Sigma). Continued to incubate at 37°C for 30 minutes. The optical density in each well was measured at a wavelength of 570 nm, subtracting the background at 620 nm.

(6) the stimulation Index (SI) was calculated according to the following formula:

SI=[A600(ETA)/A600(ET)]

A600(ET): the optical density of the wells with negative control without addition of CEA-scTsAb;

And600(ETA): the optical density in the wells with samples.

As shown Fig, there is a three step phase change of the stimulation index (SI). In the first phase, from 750 ng/ml to 12 μg/ml, there is a direct correlation between SI and the concentration of CEA-scTsAb, and the smallest value is achieved at 750 ng/ml In the second phase, from 50 ng/ml to 6 μg/ml, there is a negative correlation, and the peak is reached at 50 ng/ml In the third phase, 50 ng/ml to zero, there is a return to the direct correlation. We can conclude that CEA-scTsAb has the ability to stimulate T cells even at very low concentrations. Also found that specific cytolysis of tumor cells, induced by CEA-scTsAb, corresponded to an activating condition together inkubiruemykh T-lymphocytes. Summarizing the results of examples 4-9, the authors believe that the function of CEA-scTsAb focus on two aspects: (1) targeting of effector cells to the environment swollen the left cells and (2) stimulation of effector cells, cause-specific death of target cells. As shown in Fig targeted cytotoxic T-lymphocytes (CTL) are activated to directly destroy tumor target cells; T-helper cells secrete cytokines, such as Il-2, IFN-γ and TNF-α, indirectly helping CTL and natural killer cells (NK-cells) to destroy the tumor target cells.

Example 11: a Mechanism-specific cytolysis of tumor cells induced by CEA-scTsAb

There are three ways in which the activated CTL destroy tumor cells in vivo (Kagi, Vignaux et al., 1994; Lowin, " Hahne " et al., 1994): the activated CTL secrete perforin making holes on the surface membrane of tumor cells, which are then destroyed and undergo necrosis; grimy, secreted activated CTL, can penetrate into tumor cells through the above openings and to induce apoptosis; activated CTL can be induced by the expression of Fas-ligand on the surface that interact with molecules Fas on tumor cells and induce apoptosis. In the present invention used FMC (fluorescent cytometry) double staining with PI/annexin-V-FITC and subsequent fluorescent photographs to distinguish necrosis from apoptosis of tumor cells in in vitro assays of specific cytolysis of tumor cells.

(1) RWMS was obtained in accordance with example 7.

(2) SW1116 Cells were cultured and collected in accordance with example 6.

(3) First sown in each well of a 48-hole tablet (Nunc) at 104of SW1116 cells in L15 medium. Then added effector cells (RVMS) in the ratio E/T, is equal to 5. Added concentrated CEA-scTsAb in L15 medium to a final concentration of 1 μg/ml of the Mixture were incubated at 37°C in an incubator with 5% CO2for approximately 20 hours. For each concentration used in 4 wells. Negative controls: wells without CEA-scTsAb for each ratio E/T; wells containing only effector cells; wells containing only target cells.

(4) suspended effector cells and some dead tumor cells were collected by aspiration of the medium from each well. Attached cells were treated with trypsin and collected. Merged cells in vials of 1.5 ml

(5) after a single wash in PBS was prepared sediment cells from each well by centrifugation at 1000 g for 10 minutes and then suspended in 100 μl of buffer for binding (BD) and incubated with 5 ál conjugate FITC and annexin V (BD) and 5 μl of the solution of PI (Sigma, 50 mg/ml). The mixture is incubated at room temperature in the dark for 10 minutes.

(6) the Results after adding 300 μg buffer for binding and photographed under a fluorescent microscope (Leica DMRA2) shown in Fi is 20.

(7) Diluted mixture of cells were directly analyzed using FCM (FACS Calibur, BD). The wavelength of excitation 488 nm. To each vial was collected 20000 cells.

As shown in Fig, using two dyes can be distinguished early apoptosis, late apoptosis and necrosis: cell at an early stage of apoptosis painted only green fluorescence (FITC conjugate and annexin V); necrotic cells stained only red fluorescence (PI); cells at the late stage of apoptosis and have green and red fluorescence.

On Fig four sectors are 4 status of tumor cells: the upper-left sector (UL) necrotic cells; upper right sector (UR) cells at the late stage of apoptosis; lower left sector - living cells; lower right sector cells at an early stage of apoptosis. Typical results are shown in Fig. Negative controls without the addition of CEA-scTsAb: UL -5,94%, UR - 2,33%, LL - 90,17%, LR - 1,66%. A sample: UL - 9,80%, UR - each holding 21.25%, LL - 52,83%, LR - 16,12%. We can conclude that induced by both type-specific cytolysis of tumor cells: necrosis and apoptosis. Compared with the negative control early apoptosis and late apoptosis increased 9-fold, whereas necrosis is increased in 2 times.

Links:

1. Linear single-stranded thespecification antibody (scTsAb) for the treatment of tumors expressing CEA consisting of antibodies against cancer-embryonic antigen (CEA), connecting peptide Fc, anti-CD3-antibodies connecting peptide serum albumin human (HSA) and VHanti-D28 antibodies.

2. scTsAb according to claim 1, in which with-ICC-tag and a (His)6-tag added alternatively or simultaneously to it-the end.

3. scTsAb no claim 1, wherein the anti-CEA-antibody, anti-D3-antibody and anti-D28-antibodies are single-chain fragment variable region (scFv), a Fab fragment or a variable heavy chain (VH) antibodies.

4. scTsAb according to claim 1, which successively merged from single-chain fragment variable region (scFv) anti-CEA antibodies, connecting peptide Fc, anti-CD3 scFv connecting peptide HSA and variable heavy chain (VH) anti-D28-antibodies and contains the amino acid sequence of SEQ ID NO: 4.

5. scTsAb according to claim 4, in which an and-CEA-scFv contains the amino acid sequence of SEQ ID NO: 1.

6. scTsAb according to claim 4, in which the anti-CD3 scFv contains the amino acid sequence of SEQ ID NO: 2.

7. The DNA sequence encoding scTsAb according to claim 4, containing the nucleic acid sequence SEQ ID NO.3.

8. Expressing the vector containing the DNA sequence according to claim 7.

9. Expressing the vector of claim 8, constructed by the cloning of anti-CEA single-chain thespecification antibody (CEA-scTsAb) pTRI.

10. A host cell E. coli expressing containing the vector of claim 8.



 

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SUBSTANCE: present invention relates to biotechnology. Description is given of a single-strand T-cell receptor (scTCR), containing an α segment, formed by a sequence of a variable region in a TCR chain, joined with the N end of the extracellular sequence with constant region in the TCR chain, a β segment, formed by a sequence of the variable region of the α TCR chain, joined with the N end of the extracellular sequence with constant region of the β TCR chain, and a linker sequence, joining the C end of the α segment with the N end of the β segment, or vice versa. Extracellular sequences of constant regions of α and β segments are joined by a disulphide bond. Extracellular sequences of constant regions can correspond to constant regions of α and β chains of native TCR, cut-off at their C ends such that, cysteic residues, which form the inter-chain native disulphide bond of the TCR, are excluded, or extracellular sequences of constant regions which are in the α and β segments, can correspond to constant regions of α and β chains of native TCR, in which cysteic residues, which form the native inter-chain disulphide bond, are replaced by another amino acid residue, or there is no uncoupled cysteic residue, which is in the β chain of the native TCR. This invention makes available a new class of alpha/beta analogues of scTCR, in which there is a disulphide bond between residues of a single amino acid, contributing to stability of the bond between the alpha and beta regions of the molecule.

EFFECT: such TCR are suitable for screening or for therapeutic purposes.

3 cl, 14 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: hybrid protein - human insulin precursor consists of N-end fragment of human gamma-interferon connected through peptide linker with amino acid sequence of human proinsulin. Recombinant human insulin is obtained by cultivation of Escherichia coli JM109/pHINS11 strain-producer, carrying plasmid pHINS11, isolation of inclusion bodies and their dissolving in buffer which contains urea and dithiotreitole. Then hybrid protein re-naturation, sedimentation of admixture compounds, purification of re-naturated hybrid protein by ion-exchanging chromatography, combined fermentative hydrolysis of hybrid protein with tripsin and carbopeptidase B are carried out. At the last stage insulin purification with cation-exchanging chromatography and method of highly efficient reverse phase liquid chromatography are carried out.

EFFECT: simplification of obtaining highly purified recombinant human insulin and increase of its output.

6 cl, 1 dwg, 4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: claimed is polynucleous succession, coding polypeptide of human papilloma virus, representing epitope of antigen E1 from HPV 6b, epitope from HPV 6b E2 and epitope HPV 11 E2. Also claimed are expression vector and pharmacological composition.

EFFECT: invention can be used in treatment and prevention of human papillomaviral infections.

18 cl, 18 dwg, 7 ex

FIELD: pharmacology; biotechnology.

SUBSTANCE: invention concerns biotechnology and represents a vector of carrying over of DNA which contains the sequence coding the p185neu fragment. The given vector is a part of a pharmaceutical composition for use in preventive or therapeutic treatment of patients with risk of development p185-positive tumours or the patients having primary tumours, metastasises or relapses p185neu-expressing tumours.

EFFECT: obtaining of the new more effective pharmaceutical compositions for preventive maintenance and treatment of p185neu-positive tumours.

11 cl, 14 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: hybrid protein - human insulin precursor consists of N-end fragment of human gamma-interferon connected through peptide linker with amino acid sequence of human proinsulin. Recombinant human insulin is obtained by cultivation of Escherichia coli JM109/pHINS11 strain-producer, carrying plasmid pHINS11, isolation of inclusion bodies and their dissolving in buffer which contains urea and dithiotreitole. Then hybrid protein re-naturation, sedimentation of admixture compounds, purification of re-naturated hybrid protein by ion-exchanging chromatography, combined fermentative hydrolysis of hybrid protein with tripsin and carbopeptidase B are carried out. At the last stage insulin purification with cation-exchanging chromatography and method of highly efficient reverse phase liquid chromatography are carried out.

EFFECT: simplification of obtaining highly purified recombinant human insulin and increase of its output.

6 cl, 1 dwg, 4 tbl, 5 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: current invention relates to the field of biotechnology and immunology. Proposed is an antibody, specific to the human ED-B. Antibody specified is a molecule in the form of either dimerizated mini-immunoglobulin or IgG1, whose variable region comes from the antibody L19. In case the mini-immunoglobulin variable region L19 is merged with εS2-CH4, then as in the case IgG1, the variable region L19 is merged with the constant domain of IgG1. Conjugates of antibodies with radioisotopes have been discovered. Described is the coding nucleic acid, carrying its host cell, capable of producing antibodies, and method of obtaining antibodies from cells. Discovered is a method of determining the degree of bonding of antibodies, also compositions based on antibodies. Described is the use of antibodies for preparing medicine for treating either damage related to angiogenesis, or for treating tumours. Utilisation of the invention provides antibodies, which possess high accumulating capacity to tumours, improved capability to bonding with radioactive labels and unexpectedly retains immunoreactivity in the plasma, in comparison to scFv L19. Antibody specified can be used in diagnostics and treatment of tumours.

EFFECT: obtaining antibodies which can be used in diagnostics and treatment of tumours.

22 cl, 13 dwg, 8 tbl

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to biotechnology and can be used in biomedicine for producing hyaluronan. Proposal is given of a method of producing hyaluronan, involving culturing Bacillus host cells in conditions, suitable for obtaining hyaluronan, and extraction of the target product from the culture medium. The Bacillus host cell contains a genetic structure, comprising a promoter, functionally active in the given cell, and encoding a region, consisting of a nucleotide sequence, encoding streptococcal hyaluronansynthase (hasA); sequence encoding UDP-glucose-6-dehydrogenase Bacillus (tuaD) or a similar enzyme of streptococcal origin (hasB), and a sequence, encoding bacterial or streptococcal UDP-glucose pyrophosphorylase (gtaB and hasC respectively). Use of the invented method provides for production of considerable quantities of hyaluronan with good examined, nonpathogenic cellular system.

EFFECT: obtaining considerable of hyaluronan with good examined, nonpathogenic cellular system.

15 cl, 45 dwg, 2 tbl, 20 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to genetic engineering, more specifically to chimeric polypeptides, containing an antagonist of growth hormone receptor. The invention can be used in medicine. The binding domain of the growth hormone is modified by substituting glycine amino acid residue in position 120 and is further modified in site 1, where at least one amino acid residue is substituted, which increases affinity of the growth hormone to its binding domain on the growth hormone receptor. The amino acid residue is then conjugated with the ligand-binding domain of the growth hormone receptor, through a peptide linker.

EFFECT: obtaining a highly effective antagonist of the growth hormone receptor with longer half-life, reduced immunogenesity and nontoxicity, compared to known mutant forms.

35 cl, 16 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: invention claims compositions which can include one or several mammary gland tumour proteins, their immunogenic parts or polynucleotides encoding such parts. Alternatively the therapeutic composition can include antigen-presenting cell expressing mammary gland tumour protein, or T-cell specific to cells expressing such protein. These compositions can be applied in prevention and treatment of such diseases as mammary gland cancer. Invention also claims diagnostic methods based on determination of mammary gland tumour protein or mRNA encoding such protein in sample.

EFFECT: use of peptides obtained from protein expressed from mammary gland by tumour in diagnostics and therapy of mammary gland cancer.

37 cl, 6 ex, 1 dwg

FIELD: medicine; pharmacology.

SUBSTANCE: immunogenic hybrid polypeptide includes mimetic peptide of V-cellular epitope of apolypoprotein B-100 in which C-end of mimetic peptide is merged with N-end of T-helper epitope. Amino acid sequences of polypeptide variants are presented in description. Described is method of specified polypeptide production providing application of host cell transformed with recombinant express vector including gene coding specified polypeptide. Besides, invention concerns vaccine composition including specified immunogenic hybrid polypeptide for obesity prevention or treatment, recombinant express vector and host cell.

EFFECT: excellent anti-obesity activity without induction of immune response or severe by-effects.

15 cl, 25 dwg, 4 tbl, 15 ex

FIELD: chemistry, immonology.

SUBSTANCE: hybrid protein includes 936 protein sequence from Neisseria meningitides or protein with sequence identical to the mentioned protein sequence by 90% or more, and 741 protein sequence from Neisseria meningitides or protein with sequence identical to the mentioned protein sequence by 90% or more. The sequences can be linked by N- and/or C-end to histidine labels, with or without a linker. Linker is selected out of group of polyglycine linker, histidine labels and GSGGGG linker. A nucleic acid encoding this protein is displayed. Invention also claims composition for treatment and/or prevention of disease caused by Neisseria meningitides bacterium, based on hybrid protein and one or more proteins of the following group: 287, 741, ORF46.1, 961, NH2-A-[X-L-]n-B-COOH, where n=2, X1=287, and X2 is selected out group of: 953, 919, 961, 741. Invention claims application of composition in production of medicine for treatment of disease caused by Neisseria.

EFFECT: efficient treatment and prevention of disease.

13 cl, 5 dwg, 28 tbl, 1 ex

FIELD: biology, gene engineering.

SUBSTANCE: invention can be used for marking of biological objects. The molecule of nucleic acid which codes the fluorescing protein chosen from fluorescing proteins of representatives of kind Phialidium sp. are both suborder Anthomedusae and fluorescing mutants of the specified proteins allocated. By means of the allocated nucleic acid are obtained cloning and expressing vectors, fluorescing protein, the protein of merge capable to fluorescence, and also the expressing cartridge. The cell and the stable cellular line, containing such express ionic cartridge, produce fluorescing fiber. The fluorescing protein, nucleic acid coding it and the express ionic genetic designs containing this nucleic acid, use in a set for marking of a biological molecule. Fluorescent protein is also used in methods of marking of a biological molecule, a cell or a cellular organella.

EFFECT: invention application allows dilating an arsenal of agents for marking of biological objects.

13 cl, 12 dwg,12 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to genetic engineering. New interferon-binding proteins, which modulate activity of different interferon-α subtypes, as well as interferon-β activity, are obtained. Described is cloning of DNA fragment, which codes interferon-α/β, binding protein IFNAB-BPI, and expression of obtained DNA fragment in host-cells both with formation of respective polypeptide and in form of fused proteins. Practical application of obtained proteins as components of pharmaceutical compositions for inhibiting activity of IFN-α or IFN-β is suggested. Invention can be applied in medicine for inhibiting undesirable impact of IFN-α or IFN-β.

EFFECT: obtaining new interferon-binding proteins, which can find application in medicine for inhibiting undesirable impact of IFN-α or IFN-β.

13 cl, 10 dwg, 6 tbl, 17 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. Claimed is therapeutically active fused protein with reduced immunogenicity. Protein consists of two proteins derived from human proteins connected through the fusion region. Connective region, which covers or surrounds fusion region within the limits from 1 to 25 amino acid residues, contains modification, which removes T-cell epitope, in norm absent in humans. Claimed is application of fused protein for obtaining pharmaceutical composition for tumour treatment. Claimed is nucleic acid coding fused protein. Method of reduction of fused protein immunogenicity by introduction of substitutes of corresponding amino acids is described. Application of the invention allows reducing ability of connective epitope of therapeutically active fused protein to bind with molecules of the main complex of hystocompatibility (MHC) of class II, which finally reduces interaction of epitope with receptors of T-cells and can find application in medicine for prevention of immunological disorders arising with introduction of therapeutically active protein non-modified in connective region.

EFFECT: reduction of interaction of epitope with receptors of T-cells, which can find application in medicine for prevention of immunological disorders arising with introduction of therapeutically active protein non-modified in connective region.

23 cl, 12 ex

FIELD: chemistry, medicine.

SUBSTANCE: novel antibodies and fragments of human antibodies are bound with GDF-8 in a specific way and inhibit its activity in vitro and/or in vivo. On the basis of said invention pharmaceutical composition is created, which can be used for diagnostics, prevention or treatment of degenerative dysfunctions of muscle or bone or disorders of insulin metabolism.

EFFECT: extending range of arsenal of technical means used in treatment of diseases related to muscular, bone tissue or insulin metabolism.

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