The main idea of the project is to obtain mouse hybridoma cell lines that produce monoclonal antibodies against recombinant fragment of the human cytotoxic T-lymphocyte receptor (CTLA-4). In accordance with the methodology for obtaining humanized antibodies, mouse hybridoma cell lines are the basis for the development of medicines for targeted therapy of cancer. The implementation of the project will make it possible in the future to develop a domestic technology for the production of medicines for targeted therapy of oncological diseases.

Relevance

Cancer therapy by blocking immune system checkpoints is currently becoming widespread. The resulting biologically active molecules that block immune checkpoints are used to develop effective methods for treating and diagnosing cancer and to study the functioning of the immune system in the microenvironment of tumor cells. The PD-1 T-lymphocyte receptor is a control point in the signaling pathway aimed at destroying T-lymphocytes, including the CTLA-4 receptor. Consequently, monoclonal antibodies to these T-lymphocyte receptors will block the signaling pathway and enhance the formation of immunity.

The scientific novelty of the project lies in the creation of a strain of microorganisms that produces a recombinant extracellular fragment of the CTLA-4 receptor, as well as the production of monoclonal antibodies to a wider range of epitopes of the CTLA-4 protein. The possibility of their use to enhance antitumor immunity will be studied under in vitro conditions and in experimental models on laboratory animals.The resulting strains of microorganisms producing a recombinant extracellular fragment of the CTLA-4 receptor and anti-CTLA-4 monoclonal antibodies will contribute to the further expansion of scientific research to increase antitumor immunity. The resulting hybridoma cell lines, producers of monoclonal antibodies, will also create the prerequisites for creating the production of anti-oncological biological products.

Goal

Obtaining hybridoma cell lines producing monoclonal antibodies against the extracellular fragment of the human cytotoxic T-lymphocyte receptor CTLA-4.

Expected results

As a result of the research, a microorganism strain producing recombinant extracellular fragment of the human CTLA-4 receptor will be obtained. Hybridoma cell lines producing monoclonal antibodies against the human CTLA-4 receptor will be obtained and their immunochemical properties will be studied. The anticancer activity of anti-CTLA-4 monoclonal antibodies will be studied. The social, economic, scientific and technical effect is to create a platform for the production of high-tech domestic biological products to increase antitumor immunity. The implementation of the project will contribute to the scientific substantiation of the use of recombinant proteins and monoclonal antibodies for the treatment of cancer.

Project Manager

Mukanov Kasym Kasenovich, project leader, Doctor of Veterinary Sciences, Professor, h-index 3 (Author ID in Scopus – 56340590800).

Research team members

Borovikov S.N., Candidate of Biological Sciences, Associate Professor, h-index 2, profile (http://orcid.org/0000-0002-9721-9732).

Tursunov K.A., PhD, h-index 3 (Author ID Scopus -57193579180, Researcher ID Web of Science N-6319-2017).

Adish Zh., PhD doctoral student majoring in biology, Researcher, National Center for Biotechnology.

Kaukabaeva G., master, Junior Researcher, National Center for Biotechnology.

Publications and documents of protection of the Project manager and members of the research group concerning the topic of the project

1. Sotnikov, D.V., Barshevskaya, L.V., Zherdev, A.V., Eskendirova, S.Z., Mukanov, K.K.,Mukantayev, K.K., Ramankulov, Y.M., Dzantiev, B.B. Immunochromatographic system for serodiagnostics of cattle brucellosis using gold nanoparticles and signal amplification with quantum dots (2020) Applied Sciences (Switzerland), 10 (3). https://www.scopus.com/inward/record.uri?eid=2-s2.0-85081584093&doi=10.3390%2fapp 10030738&partnerID=40&md5= c18d312fdcd6eae186bafa 9cb8b8c62c. DOI: 10.3390/app10030738
2. Mukantayev, K., Kairova, Z., Tursunov, K., Shustov, A., Zhumabekova, S., Ramankulov, E., Mukanov, K. Recombinant expression and purification of a pathogen-specific murein hydrolase lysin from γ-bacteriophage of Bacillus anthracis (2019) Current Topics in Peptide and Protein Research, 20, P. 41-49. https://www.scopus.com/inward/record.uri?eid=2-s0-85080894201&partnerID=40&md5=1337be9c346e87a8efb3f74e5062273b
3. Mukanov, K.K., Adish, Z.B., Mukantayev, K.N., Tursunov, K.A., Kairova, Z.K., Kaukabayeva, G.K., Kulyyassov, A.T., Tarlykov, P.V. Recombinant expression and purification of adenocarcinoma gpr161 receptor (2019) Asia-Pacific Journal of Molecular Biology and Biotechnology, 27 (4), P. 85-95. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85074484552&doi=10.35118%2fapjmbb.2019.027.4.10&partnerID=40&md5=bb97d2dd250cd7d2e68fcb7c8bf7faeb. DOI: 10.35118/apjmbb.2019.027.4.10
4. Sotnikov, D.V., Berlina, A.N., Zherdev, A.V., Eskendirova, S.Z., Mukanov, K.K., Ramankulov, Y.M., Mukantayev, K.N., Dzantiev, B.B. Immunochromatographicserodiagnosis of brucellosis in cattle using gold nanoparticles and quantum dots (2019) International Journal of Veterinary Science, 8 (1), P. 28-34. https://www.scopus.com/inward/record.uri?eid=2-s0-85063189960&partnerID=40&md5=c9ba666b2eb9772380eff393ec05e6fd
5. Barshevskaya, L.V., Sotnikov, D.V., Zherdev, A.V., Khassenov, B.B., Baltin, K.K., Eskendirova, S.Z., Mukanov, K.K., Mukantayev, K.K., Dzantiev, B.B. Triple immunochromatographic system for simultaneous serodiagnosis of bovine brucellosis, tuberculosis, and leukemia (2019) Biosensors, 9 (4). https://www.scopus.com/inward/record.uri?eid=2-s2.0-85072779454&doi=10.3390%2fbios 9040115&partnerID=40&md5=a8180d6dbd30827509d2ac8675c99751 DOI: 10.3390/bios90401152.
6. Sotnikov D.V., Byzova N.A., Zherdev A.V., Eskendirova S.Z., Baltin K.K., Mukanov K.K., Ramankulov E.M., Sadykhov E.G., Dzantiev B.B. Express immunochromatographic detection of antibodies against brucella abortus in cattle sera based on quantitative photometric registration and modulated cut-off level. Journal of Immunoassay and Immunochemistry, Issue 1, 2015; IF – 0,727.
7. Shevtsov, E. Ramanculov, E.Shevtsova, A.Kairzhanova, P.Tarlykov, M.Filipenko, M. Dymova, G. Abisheva, A.Jailbekova, D.Kamalova, A.Chsherbakov, S. Tulegenov, A. Akhmetova, I. Sytnik, T. Karibaev, K. Mukanov. Genetic diversity of Brucella abortus and Brucella melitensis in Kazakhstan using MLVA-16. Infection, Genetics and Evolution 34 (2015) P.173-180. IF- 2,8.
8. Shevtsova, A. Shevtsov, K. Mukanov, M. Filipenko, D. Kamalova, I. Sytnik, M. Syzdykov, A Kuznetsov, A. Akhmetova, M. Zharova, T. Karibaev, P. Tarlykov, E. Ramanculov. Epidemiology of Brucellosis and Genetic Diversity of Brucella abortus in Kazakhstan. PLOSONE | DOI:10.1371/journal.pone.0167496 December 1, 2016, P.1-16. IF 3.54
9. Escherichia coli microorganism strain B834/pET15/3A is a producer of the recombinant non-structural protein 3A of the foot-and-mouth disease virus. Innovative patent No. 25095. Mukantaev K.N., Tursunov K., Lazarev V.N., Levitsky S.A., Kharlampieva D.D., Kushcheva N.A., Baltin K.K., Mukanov K.K., Ramankulov E.M.
10. Hybrid strain of cultured animal cells Mus Musculus L., producer of monoclonal antibodies to the recombinant antigen p24 of the bovine leukemia virus. Copyright certificate No. 73446. Mukantaev K.N., Bakirova G.A., Belyalova A.R., Zhylkibaev A.A., Baltin K.K., Mukanov K.K., Ramankulov E.M.
11. Escherichia coli microorganism strain B834/pET32/TpN17 is a producer of the recombinant membrane lipoprotein Treponema pallidum TpN17. Copyright certificate No. 73335. Mukantaev K.N., Bakirova G.A., Lazarev V.N., Levitsky S.A., Shkarupeta M.M., Mukanov K.K., Ramankulov E.M.
12. Escherichia coli microorganism strain B834/pET32/TpN47 is a producer of the recombinant membrane lipoprotein Treponema pallidum TpN47. Copyright certificate No. 73328. Mukantaev K.N., Bakirova G.A., Lazarev V.N., Levitsky S.A., Shkarupeta M.M., Mukanov K.K., Ramankulov E.M.
13. Escherichia coli microorganism strain B834/pET32/TpN15 is a producer of the recombinant membrane lipoprotein Treponema pallidum TpN15. Author’s certificate No. 73314 1 Mukantaev K.N., Bakirova G.A., Lazarev V.N., Levitsky S.A., Shkarupeta M.M., Mukanov K.K., Ramankulov E.M.
14. Escherichia coli microorganism strain B834/pET32/TprK is a producer of the recombinant membrane lipoprotein Treponema pallidum TprK. Copyright certificate No. 73307. Mukantaev K.N., Bakirova G.A., Lazarev V.N., Levitsky S.A., Shkarupeta M.M., Mukanov K.K., Ramankulov E.M.
15. Escherichia coli microorganism strain B834/pET32/Tp0453 is a producer of the recombinant membrane lipoprotein Treponema pallidum Tp0453. Copyright certificate No. 73321. Mukantaev K.N., Bakirova G.A., Lazarev V.N., Levitsky S.A., Shkarupeta M.M., Mukanov K.K., Ramankulov E.M.
16. The microorganism strain Escherichia coli BL21/pET32/VP1 Asia produces the recombinant VP1 antigen of the foot-and-mouth disease virus type Asia. Copyright certificate No. 88014. Mukanov K.K., Ramankulov E.M., Shustov A.V., Mukantaev K.N., Baidosova Sh.
17. Escherichia coli microorganism strain BL21/pET32/VP1 O producer of recombinant VP1 antigen of foot-and-mouth disease virus type O. Author’s certificate No. 88019. Mukanov K.K., Ramankulov E.M., Shustov A.V., Mukantaev K.N., Baidosova Sh.
18. Escherichia coli microorganism strain BL21/pET32/VP1 A producer of recombinant VP1 antigen of foot-and-mouth disease virus type A. Author’s certificate No. 90924. Mukanov K.K., Ramankulov E.M., Shustov A.V., Mukantaev K.N., Tursunov K., Begalieva A.
19. Escherichia coli microorganism strain BL21/E3.pET22/gp51 is a producer of the recombinant gp51 antigen of the bovine leukemia virus. Patent for invention No. 30998. Mukanov K.K., Ramankulov E.M., Shustov A.V., Mukantaev K.N., Tursunov K., Begalieva A.
20. Escherichia coli microorganism strain BL21(DE3)/pET32/MPRV is a producer of recombinant matrix protein of the rabies virus. Author’s ID No. 106743. Mukantaev K.N., Shustov A.V., Tursunov K.A., Inirbay B., Adish Zh., Ramankulov E.M., Mukanov K.K.
21. Escherichia coli microorganism strain BL21(DE3)/pET32/NPRV is a producer of recombinant rabies virus nucleoprotein. Author’s ID No. 101822. Mukantaev K.N., Shustov A.V., Tursunov K.A., Inirbay B., Adish Zh., Ramankulov E.M., Mukanov K.K.
22. Tursunov K., Begaliyeva A., Ingirbay B., Mukanov K., Ramanculov E., Shustov A., Mukantayev K. Cloning and expression of fragment of the rabies virus nucleoprotein gene in Escherichia coli and evaluation of antigenicity of the expression product//Iranian Journal of Veterinary Research. – 2017. –Vol.18, No. 1, Ser. No. 58, — P.36-42. PMID: 28588631.Q4.
23. Bulashev, T. Jakubowski, K. Mukantayev, Tursunov, V. Kiyan, A. Zhumalin. Using combined recombinant protein in the diagnosis of bovine brucellosis//Med. Weter. 2018, 74 (3), 193-198. DOI: dx.doi.org/10.21521/mw.6079. Q3.
24. Kanatbek Mukantayev, Kanat Tursunov, Guljan Raimbek, Alexander Shustov, Asem Begaliyeva, Bakhytkali Ingirbay, Kasym Mukanov, Erlan Ramanculov. Immunochromatographic assay for diagnosis of bovine leukaemia virus infection in cows using the recombinant protein gp51//ISSN 1392-2130. VETERINARIJA IR ZOOTECHNIKA (Vet Med Zoot). T. 76 (98). 2018, p. 34-40. https://vetzoo.lsmuni.lt/data/vols/2018/76/pdf/mukantayev.pdf. Q4.
25. Mukantayev, K., Tursunov, B. Ingirbay, Z. Adish, M. Azhibayeva, Z. Kairova, E. Ramankulov, K. Mukanov and A. Shustov. Immunochromatographic assay for the foot-and-mouth disease utilizing recombinant nonstructural proteins 2C, 3A, 3B and 3D// Bulgarian Journal Agricultural Science, 2018, 24 (3): 489–496. https://www.agrojournal.org/24/03-21.html. Q4.
26. Mukantaev KN, Kairova Zh., Tursunov KA, Zhumabekova SH., Ramankulov EM, Mukanov K.K. Recombinant expression and purification of a pathogen-specific murein hydrolase lysin from γ-bacteriophage of Bacillus anthracis//Current Topics in Peptide and Protein Research. – 2019. – Vol.20. http://www.researchtrends.net/tia/article_pdf.asp?in=0&vn=20&tid=26&aid=6375. Q4.
27. Mukanov, K.K., Adish, Z.B., Mukantayev, K.N., Kulyyassov, A.T., Tarlykov, P.V.Recombinant expression and purification of adenocarcinoma gpr161 receptor//Asia-Pacific Journal of Molecular Biology and Biotechnology. -2019. 27(4), P. 85-95. DOI: 10.35118/apjmbb.2019.027.4.10. Q4.
28. Adish Zhansaya, Mukantayev Kanatbek, Tursunov Kanat, Ingirbay Bakhytkali, Kanayev Darkhan, Kulyyassov Arman, Tarlykov Pavel, Mukanov Kasym, Ramankulov Yerlan. Recombinant Expression and Purification of Extracellular Domain of the Programmed Cell Death Protein Receptor//Reports of Biochemistry & Molecular Biology.Vol.8, No.4, 2020. http://rbmb.net/article-1-391-en.pdf3, Q3.
29. Bulashev, A., Jakubowski, T., Tursunov, K., Kiyan, V., &Zhumalin, A. (2018). Immunogenicity and antigenicity of Brucella recombinant outer membrane proteins. VeterinarijaIrZootechnika, 76(98), 17–24.
30. Borovikov S., Syzdykova A.,Scharmyschova M., Kiyan V. Thesynthesis of the immunoactive components of the Сampylobacter fetus antigen and the immunization of the laboratory animals for the purposes of synthesis of the specific antibodies. International Journal of Pharmacy & Technology IJPT.-2016.Vol. 8, 27097-27108 P.Q4.
31. Bulashev A.K., Borovikov S.N.,Serikova S.S., Suranshiev Z.A., Kiyan V.S., Eskendirova S.Z. 2016: Development of an ELISA using anti-idiotypic antibody for diagnosis of opisthorchiasis. Folia Parasitol. 63: 025; DOI:10.14411/fp.2016.025; Q3
32. Borovikov S., Aitmagambetova M. S. Results of cattle examination for campylobacteriosis using the polymerase chain reaction method. EEC – EM – Ecology, Environment and Conservation. VOL. 25 (1): 2019. –Р.456–459. Q4.
33. Borovikov S.N. Оbtaining monoclonal antibodies to cardiac markers I & Т. Joint Conference: German Symposium on Zoonoses Research 2014 and 7th International Conference on Emerging Zoonoses. — Berlin, Germany, October 16-17, 2014.- P.82.
34. Innovative patent No. 30961 Republic of Kazakhstan, MPKG01N 33/53, A61K 39/40. Strain of hybrid cultured animal cells MusmusculusL. – used to obtain monoclonal antibodies to the drug cardiac troponin I/ Borovikov S.N., Kuibagarov M.A., Syzdykova A.S., Kartabaeva G.O.: applicant and patent holder JSC “Kazakh Agrotechnical University named after. S. Seifullina.” publ. 15.03. 2016, Bull. No. 3.

Results achieved

2021

The amino acid and nucleotide sequence of the human CTLA4 receptor was analyzed. The extracellular domain of the human CTLA4 receptor, 125 amino acid residues in size, containing the sequence MYPPPYYLG was selected. The nucleotide sequence of the receptor gene was optimized for efficient expression in E. coli. Oligonucleotides were obtained for the synthesis of CTLA-4 under de novo conditions. A pair of primers was selected to amplify the human CTLA-4 receptor gene for expression in P. pastoris. Schemes of genetic constructs based on pET28, pET32 and pPICZα vectors carrying the CTLA4 receptor gene have been developed. The CTLA4 receptor gene was synthesized and amplified under de novo conditions for bacterial expression. Expression vectors pET28/CTLA-4 and pET32/CTLA-4 and E. coli BL21 cells producing rCTLA-4 and rTrx/CTLA-4 proteins were obtained. The conditions for the expression and purification of recombinant CTLA-4 were developed.

2022

The immunochemical and immunological characteristics of the recombinant rCTLA-4 protein were studied. As a result of the analysis of the most probable proteins corresponding to the combined MS/MS spectra, the highest score (4950) corresponded to only one CTLA-4 protein. Recombinant rCTLA-4 protein with a high degree of binding was reacted with a commercial preparation of recombinant B7-1Fc protein. As a result of hybridization, five clones were obtained (C9, A5, H8, A4, E9), producing IgG class antibodies and having an affinity from 2×10⁷M^-1 to 3×10⁸M^-1. The productivity of hybridoma cell lines was from 20 to 30 μg/ml in vitro and from 8 to 16 mg/ml in vivo. Purified monoclonal antibody preparations were obtained using affinity chromatography.

2023

In the immunofluorescence reaction, monoclonal antibodies reacted specifically with the MCF-7 cell line. Treatment of macrophages with rCTLA-4, rPD-L1 and rPD-1 proteins in ex vivo conditions, followed by intraperitoneal administration to mice, activates the production of antibodies that react with homogeneous receptors on cancer cells. The results showed that macrophages process proteins into forms suitable for recognition by T and B cells and activate the production of antibodies that react with homogeneous receptors on cancer cells. Analysis of the effect of protein concentration when treating macrophages on antibody titer in mice showed a difference in the results between rPD-1/rPD-L1 and rCTLA-4 proteins. The difference in anti-rPD-1/anti-rPD-L1 antibody titers in mice was insignificant despite significant differences in protein concentrations in the culture medium. In contrast to the rPD-1/rPD-L1 proteins, the antibody titer against rCTLA-4 highly depended on the protein concentration when macrophages were treated in vitro.