ANTIBODY AND CYTOKINE DEVELOPMENTS AND SERVICES. Zsolt Keresztessy, PhD MBA TUDOMÁNYOS HÉTFŐ

Transcription

1 ANTIBODY AND CYTOKINE DEVELOPMENTS AND SERVICES AT DEBRECEN CLINICAL GENOMICS CENTER DEPT BIOCHEMISTRY AND MOLECULAR BIOLOGY Zsolt Keresztessy, PhD MBA TUDOMÁNYOS HÉTFŐ

2 MISSION CUTTING EDGE TECHNOLOGY INTRODUCE MOST ADVANCED TECHNOLOGIES DEVELOP OWN TECHNOLOGIES TO SUPPORT MARKET ENTRY HIGH QUALITY CORE FACILITY SERVICES CONCENTRATE KNOWLEDGE AND EXPERTIES TO ENABLE HI QUALITY RESEARCH AT THE UNIVERSITY BIOTECHNOLOGY BUSINESS DEVELOPMENT PROPRIETARY TECHNOLOGY PLATFORMS AND BIOTECH PRODUCTS/SERVICES FOR THE BIOPHARMA/BIOTECH INDUSTRY PLATFORM FOR COLLABORATIVE R&D AND IP DEVELOPMENT COLLABORATIVE RESEARCH POSSIBILITIES AND IP-CO-DEVELOPMENT CAPACITY

3 WHO WE ARE GENERAL THE LABORATORY OF PROTEIN EXPRESSION AND CELL ENGINEERING OPERATESAS A RESEARCH AND DEVELOPMENT LAB AS WELL AS A CORE FACILITY. LOCATION LAB 3.26 (60 m ) OFFICE in the brand new IVDI BUILDING SHORT INFRASTRUCTURE GENERAL MOLECULAR BIOLOGY CELL CULTURE AND ANALYSIS PROTEIN EXPRESSION CELLINE/HYPERFLASK BIOREACTORS PROTEIN PURIFICATION AKTA + PROFINIA, FULL RANGE AND SCALE PROTEIN CHARACTERISATION IMMUNO, BIOLOGICAL ACCESS TO CELL SORTING, NGS, HTS, LARGE SCALE FERMENTATION STAFF HEAD OF LAB: ZSOLT KERESZTESSY Project Coordinator/Leader MOLECULAR BIOLOGISTS: ÉVA NAGY Pre-Doc ZOLTÁN DORÓ Pre-Doc RESEARCH ASSISTANT: JÓZSEF HORVÁTH MSc Student (Mol. Biol.)

4 PROTEIN PURIFICATION

5 WE HAVE BEEN BUSY FROM THE BEGINNING ACHIEVMENTS ( ) R&D Projects INNANTet, EPIGEN, BIOSIM Cloned vector constructs ptriex4neo, pim.rag1, pim.lp2 Transgenic stable monoclonal cell lines HEK AD 293, Jurkat, YT, MM6 Recombinant cytokines IL-4, IL-2, GM-CSF, LIF Numbers 9 (4) 20 (8) 19 (6) 6 (3) Monoclonal antibodies (3-6) SMRT, RXR, RAR Innovative epitope predictions SMRT, RXR, RAR, cfos, PPAR Recombinant proteins produced Cytokines, SMRT, PNGase F Products at prototype phase Cytokines, anti-smrt Services established Gene-to-Protein, Cell line development IP development mabs, epigenetics (5)

6 BRIEF DESCRIPTIONS OF CORE SERVICE POTENTIALS Gene-to-Protein Services We deliver full service from custom DNA sequence to purified recombinant protein. Individually available service components are cloning, expression, purification, and characterisation of your protein of interest. We provide advanced Cytokine protein Development expression services Servicesusing optimised synthetic If genes you need and a cytokine wide range at an of affordable host price organisms (bacterial, for yeast, your research insect, mammalian) -we produce for it small for you. The and large scale protein workflow production, includes and gene equipped design for and state- of the-art protein optimisation, purification and gene biochemical/biological synthesis, Monoclonal cloning antibody into development characterisation expression vectors, expression, You provide purification a custom target sequence we and biochemical/biological produce characterisation specific mouse of monoclonal your recombinant cytokines. antibodies. Epitope design, peptide synthesis, Recombinant immunisation, proteins hybridoma generation and We offer screening, special antibody services production, and consultation purification in Antibodies and cytokines at great prices the field and of characterisation. custom recombinant protein Cytokine products: development, engineering, design, and human IL-4, IL-2, GM-CSF, LIF evolution, including mutagenesis, library mouse IL-4, GM-CSF generation, directed molecular evolution, Coming soon: FLT-3 Ligand, IL-9, IL-15 phage display technology, HTS screening.

7 CYTOKINES WORKFLOW TARGET SELECTION CLONING EXPRESSION TESTING PRODUCTION PURIFY CHARACTER. LABELLING PRODUCTS IL-4 GM-CSF LIF FLT3-LIGAND Gene Design Gene synthesis Constructs E. COLI Rosetta INSECT Sf9 SDS/PAGE Western Blot ELISA Biological act. Transfection Selection FACS sorting Stable celllines PROFINIA IMAC Q ion-exchange CHT ion-exch SDS/PAGE Western blot ELISA Biological act. Fluorescent Q-Dot CYTOKINES LABELLED CYTOKINES IL-2 IL-9 MAMMALIAN HEK, CHO Cell supernat. FERMENTATION Desalting Storage MS CD IL-15

8 TARGET SELECTION CHARACTERISE PURIFICATION PRODUCTION GENE DESIGN OPTIMISATION GENE SYNTHESIS PRODUCTS EXPRESSION TESTING rhil-4 rhil-4 rhlif rhlif PROFINIA Transient Stable CELLine HyperFlask HEK/CHO TF-1 K6 SC DC Endotox HEK/CHO INSECT E.COLI SDS/PAGE Western ELISA BIOLOGICAL Medium K6 SC CYTOKINES PIPELINE rhil-2 rhil-2 rhgm-csf rhgm-csf rmil-4 rmil-4 rhlif rhlif rmgm-csf rmgm-csf FLT3-lig FLT3-lig IL-9 IL-9 IL-15 IL-15

9 CYTOKINES

10 CYTOKINES

11 CYTOKINES

12 IL-4 production in HEK cells Transient transfection

13 IL-4 production in HEK cells

14 IL-4 production in HEK cells Purification on PROFINIA IMAC Ni-affinity + Desalting

15 IL-4 production in HEK cells SDS-PAGE Coomassie anti-il-4 Western blot A B (A) Ni-affinity purification: 1. conditioned medium, 2. flowthrough, 3. purified IL4. (B) mouse monoclonal anti-human IL-4 antibody Western.

16 Rekombinant human IL-4 biological activity Human MPh differentiation from monocytes - CCL18, IRF4, CCL22 qpcr Prof. Dr. László Nagy, Attila Papp, Zsolt Czimmerer, NLAB - Dept Biochemistry and Molecular Biology

17 Rekombinant human IL-4 biological activity Human MPh differentiation from monocytes - CCL18, IRF4, CCL22 qpcr Prof. Dr. László Nagy, Attila Papp, Zsolt Czimmerer, NLAB - Dept Biochemistry and Molecular Biology

18 Rekombinant human IL-4 biological activity Human MPh differentiation from monocytes - CCL18, IRF4, CCL22 qpcr Prof. Dr. László Nagy, Attila Papp, Zsolt Czimmerer, NLAB - Dept Biochemistry and Molecular Biology

19 Rekombinant human IL-4 biological activity Human MPh differentiation from monocytes - CCL18, IRF4, CCL22 qpcr Prof. Dr. László Nagy, Attila Papp, Zsolt Czimmerer, NLAB - Dept Biochemistry and Molecular Biology (C) Dr. Keresztessy

20 Rekombinant human IL-4 biological activity Human dendritic cell differentiation and LPS test Prof. Éva Rajnavölgyi, Csilla Fekete, Dept Immunology 100 CD1a - LPS polyi:c 100 CD209 - LPS polyi:c % % Peprotech IL-4 HEK IL-4 10X HEK IL-4 20X 0 Peprotech IL-4 HEK IL-4 10X HEK IL-4 20X Monocyte derived human dendritic cells differentiated in the presence of rhil-4 containing medium express cell surface markers, CD209 and CD1, at similar ratios to those obtained in the presence of Peprotech IL-4

21 Rekombinant human IL-4 biological activity Human dendritic cell differentiation and LPS test Prof. Éva Rajnavölgyi, Csilla Fekete, Dept Immunology IL-6 Peprotech IL-4 d.0.lps HEK IL-4 10X HEK IL-4 20X RE h 1h 3h Early stimulation of dendritic cells with low dose LPS is capable of inhibiting later activation by high dose LPS

22

23 Rekombinant mouse IL-4 biological activity L929 cell differentiationn YM1, Fizz1 qpcr Prof. Dr. László Nagy, Attila Papp, NLAB - Dept Biochemistry and Molecular Biology M-CSF (L929 sn) M-CSF (L929 sn) + IL4 sn 5% M-CSF (L929 sn) + IL4 rec. 5ng/ml M-CSF (L929 sn) + IL4 sn 10% M-CSF (L929 sn) + IL4 sn 20%

24 Rekombinant mouse IL-4 biological activity L929 cell differentiation YM1, Fizz1 qpcr Prof. Dr. László Nagy, Attila Papp NLAB - Dept Biochemistry and Molecular Biology YM1 ( mouse IL4 sn test) 0.60 YM1/cyclo M-CSF M+IL4 rec. 5ng/ml M+IL4 sn. 5% M+IL4 sn. 10% M+IL4 sn. 20% The two IL4 regulated target genes show that even the 5% mil4 supernatant can induce the expression of these genes at the same level as recombinant IL4 (5 ng/ml) can do Fizz1 ( mouse IL4 sn test) It is interesting, that 10% IL4 sn could not, but 20% IL4 sn could further increase the gene expression. Fizz1/cyclo M-CSF M+IL4 rec. 5ng/ml M+IL4 sn. 5% M+IL4 sn. 10% M+IL4 sn. 20%

25 Rekombinant human LIF biological activity M1 cell differentiation assay IL-6 qpcr Bálint L. Bálint, Lilla Ozgyn, Genomics

26 PRODUCT: Rekombinant human IL-4 containing culture medium (transiently transfected HEK293T cell supernatant) Product description Recombinant human IL-4 is produced in HEK293T cells (DMEM, 10% FBS) by secreting the protein into the medium. The protein is 17 kda, glycosylated, contains affinity and epitope tags, specific cleavage sites. Biological activity The biological activity of the product is tested in TF-1 proliferation assay, IL-4 responsive gene expression assay, and Thp-1 STAT6 phosphorilation assay. Suggested application Dilution of the product to be used is application dependent, suggested dilution range to be tested by the customer is 1:10 1:1000. Packaging Product is marketed sterile filtered, in 5 ml aliquots in cryotubes, boxed (405 ml), stored at -20 oc. Quality Control Batch testing TF-1 proliferation assay, ELISA, Western.

27 AN ORDER FORM...

28 EXAMPLE WORKFLOW

29 COLLABORATIONS Funkcionális genomika Epigenetika Antitest fejlesztés Rekombináns fehérje termelés Rekombináns fehérje termelés

30 ACKNOWLEDGEMENTS LÁSZLÓ NAGY Director DCGC Bálint L Bálint Gábor Zahuczky Éva Rajnavölgyi István Szatmári Éva Nagy Zoltán Doró Attila Papp Zsolt Czimmerer Lilla Ozgyn József Horváth Ádám Pallér Csilla Fekete Lab Head CEO Director Head DCGC UD-GenoMed Immunology Stem Cell facility DCGC BAROSS GÁBOR PROGRAM NLAB DCGC Immunology (REG_EA_KFI_09-INNANTet)