Chap. 5 Production of Monoclonal Antibodies Introduction

1. Chap. 5 Production of Monoclonal Antibodies Introduction Expression and Purification of Mab in an economically viable manner a) production system requirement i. form of Ab required ii. applications --- scale of production specification of Ab purification Expression of Ab in mammalian cells a) Hybridoma cells for mouse and rat Mab expression [productivity / stability / adventitious agents (viruses)] unstable or low productivity of the hybridomas --- recombinant Ab expression in other systems such as myelomas or non-lymphoid cell b) Mammalian cells for large scale production of intact monoclonal Ab appropriate cellular machinery [assembly, secretion, post-translational modifications] c) transient expression (cloned DNA into the nuclei of mammalian cells) stable expression (low-frequency integration selected with an appropriate marker gene) [insertion into random sites within the genome]

2. [1] Transient expression system • a) COS cells from monkey kidney cell line • --- CV1 transformed with an origin-deficient SV-40 virus (CV1-Origin-deficient SV40) • SV40 T antigen expression --- replication of plasmid containing the SV40 origin (104-105 copies/cell within a few days) i. L-chain and H-chain on separate plasmids (each with the SV40 origin) ii. strong promoter and enhancer adenovirus major late promoter and SV40 enhancer MIE (major immediate early) promoter/enhancer from hCMV 0.1 – 10 mg/ml b) 293 cells from human embryo hCMV-MIE promoter in an adenovirus transformed cell line containing a Tx enhancing factor (E1a) c) CHO (chinese hamster ovary) cells with the adenovirus protein E1a gene incorporated into the genome [2] Stable expression system Myeloma lines (SP2/0 and NS0) --- fusion partners used in the hybridoma production secreting large amounts of Ab and grow well in suspension culture in fermentors CHO cells readily transfectable, grow well in both attached and suspension culture, efficient expression systems based on vector amplification

3. a) Development of stable cell line (by using selectable marker to select cell) Bacterial genes in the vector with mammalian Tx signals i. gpt gene --- xanthine-guanine phophoribosyl transferase (E. coli) resistance to mycophenolic acid [Salvage pathway without De novo synthesis of GMP] ii. neo gene --- resistance to the antibiotic G418 which blocks protein synthesis [neomycin, kanamycin, gentamycin] iii. hph gene --- resistance to hygromycin b) Selection of cells i. two vectors in combination (one for light and the other for heavy chain) light chain: prior introduction to H-chain gene (H-chain accumulation in ER with H-chain binding protein grp78) ii. vector containing both L- and H-chain genes Light gene --- Tx terminator --- Heavy gene balanced level of H- and L-chains c) Expression level control i. immunoglobulin promoters and enhancers (V-region) --- Ab expression in myeloma low expression level (additional enhancer requirement for both H- and L-chains) introduction of hCMV-MIE and mouse metallothionein promoter ii. target gene integration into highly transcribed region of host DNA homologous recombination

4. d) Productive cell line 생산 ---- gene amplification 이용 Selection via marker enzymes inhibited using a selective inhibitor i. DHFR (dihydrofolate reductase) nucleotide biosynthesis and inhibited by methotrexate CHO cell --- transfection with vector containing both DHFR gene and Ab gene --- addition of methotrexate --- selection of resistant colonies (cf) Myeloma cell containing the endogenous enzyme DHFR gene and stronger promoter ii. GS (glutamine synthetase) Glu + NH3 Gln GS is an essential enzyme without Gln supplied. GS is inhibited by methionine sulfoximine (MSX). CHO cell (sufficient GS) / Myeloma cell (no endogenous GS) e) Vectors with selectable, amplifiable marker confirmation of integration of vectors into active transcriptional sites f) Fed-batch fermentation GS amplified NSO cells > DHFR amplified CHO cells cf) glycosylation on Ab --- cell type dependence hIgG from Myeloma cell (no bisecting residue) hIgG from CHO cell (bisecting residue) [Microheterogeneity could affect the function of Ab.]

5. [3] Expression of Ab fragments in mammalian cells a) co-expression of L-chain with Fd or Fd’ --- secretion of Fab or Fab’ fragments b) oxidation of Fab’ produced in CHO cells --- F(ab’)2 production c) Fv and scFv expression in mammalian cells --- low yields scFv-based fusion proteins --- mammalian cell expression [structurally complex protein, glycosylation, etc.]

6. Expression in E. coli E. coli for gene expression [advantage] i. availability of various expression vectors ii. transformation iii. easy and quick growth [disadvantage] i. no post-translational modification ii. inclusion body formation (protein solubilization/refolding and protein secretion via signal sequence) E. coli as an expression system for Ab fragments soluble, functional Ab fragments in high yields and more economical manufacturing [1] Intracellular expression of antibody fragments in E. coli rapid accumulation of large amounts of insoluble protein --- Fv, scFv, Fab (solubilization and refolding to fully functional state --- limits of Ab fragment production) Nevertheless, useful system for i. Ab fragments for which the secretion route fails ii. toxic fusion proteins to the cell in native form

7. • Protein Expression in E. coli a) cDNA insertion into the expression cassette of an expression vector (plasmid origin of replication/antibiotic selectable marker/expression cassette) b) expression of foreign protein --- a considerable strain on the cells selection pressure for the cell i. segregational instability (losing the expression vector) ii. structural instability (deletion in the expression cassette) c) transcriptional regulation (strong promoters: ptac, lpL, T7) lac based promoter: IPTG, lactose as alternative carbon source d) translation ribosomal RNA complementary binding site --- spacing of 6-10 bp --- initiation codon • Inclusion bodies a) cell lysis and centrifugation b) solubilization with high conc. of denaturants in the presence of reducing agents c) refolding i. solubilization agent, pH, redox conditions, protein purity and conc., temp. ii. rate of change to native conditions iii. prevent the reaggregation of partially unfolded protein d) evaluation of the functionality of Ab fragments scFv and IgG: loss of Ag binding activity e) refolding efficiency

8. [2] Secretion of Ab fragments from E. coli secretion of Fv, scFv, Fab, and other Ab fragments --- E. coli periplasm or leaked into the medium signal sequence and Ab gene fusion: pelB, ompA, phoA, stl1 aggregation of folding intermediates: small fragments --- less prone to aggregate sequence dependence • Improving the expression of soluble Ab fragments a) switching Fab constant region b) engineered scFv (eliminating the hydrophobic patch) c) CDR grafting of an Ab to the well expressed soluble humanized Fab’ fragment d) control of cell growth parameters growth at low temp. / addition of non-metabolizable sugars e) rate of protein synthesis (Tx and Tl control) reduced rate of protein synthesis lower inducer conc. expression for a prolonged period f) suitable promoters (regulation of Tx rate) lac promoter/operator, lacUV5, tac promoter (IPTG or Carbon source) phoA (phosphate starvation), araB (arabinose), trpE (temp) g) coexpression of proteins for assisting protein folding h) high cell density growth in fermentation

9. Expression in other microbial system • Gram+ bacterium --- Bacillus subtilis • svFv expression • secretion directly into the growth medium (with extracellular proteases) • multiple protease deficient strains developed! • Yeast Saccharomyces cerevisiae • functional IgM expression, but inefficient secretion (assembled Ab inside the cell) • strong phophoglycerate kinase promoter (H and L chains from different plasmids) • glycosylated H-chain • chimeric IgG and Fab --- secretion • IgG --- ADCC but no complement activation (Yeast type CHO) • c) Methylotrophic Yeast Pichia pastoria • fast growth properties with eukaryotic protein expression and secretion • AOX1 (alcohol oxidase gene) promoter is regulated by methanol. • Filamentous fungus Trichoderma reesi • secretion of Ab fragments • cellobiohydrolase (cbh1) promoter • Fab fragment expression • cbh1-Fd fusion protein coexpressed with L-chain (cleavage by T. reesei protease)

10. Expression in plants ‘plantibody’ from transgenic plants --- most economical system a) murine IgG production Ti plasmid of Agrobacterium tumefaciens --- plant cell infection (Nicotiana) [H and L-chain cDNA with signal segments --- separate introduction to leaf segments --- mature plant --- crossing --- progeny --- assembled antibody --- intracellular space] scFv and Fab’ fragment secretory IgA in tobacco plants four protein chains --- separate plants --- crossing --- assembled sIgA Dental caries as edible plant tissue (the mouth or gastrointestinal tract) b) seeds to express scFv long term storage of scFv w/o loss of Ag-binding affinity c) CHO of plant-produced Ab different glycosylation compared to mammalian cell derived Ab Fab or scFv expression (not normally glycosylated) no animal viruses or other animal derived agents Production in transgenic animals Mab production in the milk of transgenic animals (sheep, goats, cows) [gene --- microinjection --- fertilized egg --- transgenic animals]

11. Expression in insect cells Sf9 insect cell line + baculovirus expression vector (promoter from baculovirus polyhedrin gene) signal peptide cleavage, glycosylation, efficient secretion low level of production due to lytic infection no mammlian viruses, no mammalian DNA encoding oncogenes H- and L-chains in two recombinant virues in a dual transfer vector containing the two Tx units in opposite orientation insect cell specific CHO --- effector function not always maintained Production of monoclonal antibodies --- cell culture a) cell culture in small scale --- attached or suspension culture b) growing hybridoma cells as ascites tumors in mice or rats risk of introducing adventitious agents into the Ab prep. c) in vitro cell culture techniques i. attached cells in roller bottles ii. shake-flask or spinner cultures of cells in suspension iii. bioreactor (hollow fibre perfusion system and air-lift fermentors) d) productivity of a cell line in fermentation optimization of culture media, nutrient supplementation, environmental parameters the best process at the lowest cost and the time available

12. Purification of monoclonal antibodies [1] Purification of IgG a) sample preparation (primary recovery) i. centrifugation and filtration ii. (NH4)2SO4 precipitation iii. conditioning before purification (pH or ionic strength adjustments) b) purification i. affinity chromatography with the bacterial Ig binding proteins Protein A (Staphylococcus aureus) and Protein G (group C and G Streptococci) Fc (CH2 and CH3 interface) of IgG (Table 5-1) clarified culture supernatant --- preconditioning --- elution w/ pH and chaotropic agents elimination of bovine IgG in bovine serum (cell culture media) Ab from ascitic fluid (high speed ctg. and contamination of host mouse IgG) affinity chromatography using immobilized Ag alternative methods (Ab stability and cost) ii. ion-exchange chromatography (pI of Mab = 4.5 – 8.5) anion-exchanger (2nd step to remove DNA, 1st step usage ?) cation-exchanger (extensive preconditioning) high resolution anion exchange materials mixed-mode ion-exchanger (Bakerbond Abx) hydroxyapatite (calcium hydroxyphosphate: Ca10(PO4)6(OH)2)

13. iii. hydrophobic interaction chromatography high salt conc. for sample loading (Ab ppt. or loss of Ag binding property) iv. gel-filtration chromatography final clean-up step (removing antibody aggregates) v. thiophilic adsorption (mercaptoethanol to divinylsulfone activated agarose) Ab binding at high salt conc. and elution by reducing the salt conc. vi. expanded bed chromatography (ion exchanger or protein A) fluidized dense solid phase --- Ab binding --- packing and elution [2] Purification of IgM IgM --- lower stability --- difficult to purify i. ion-exchange chromatography (mixed mode ion exchanger, Abx) ii. gel filtration (900 kDa IgM) iii. selective precipitation (water dialysis or ammonium sulfate ppt) iv. affinity chromatography mannose binding protein (MBP) --- IgM binding, no IgG binding protein A and protein L --- Fab binding ex) human Mab IgM COU-1 [3] Purification of monoclonal antibody fragments (a) proteolyzed Ab fragments (IgG  Ab fragments) i. removal of proteases (immobilized proteases) ii. negative purification (Ab fragments in the flow-through) iii. ion-exchange and gel-filtration chromatography

14. b) purification of recombinant Ab fragments i. Fab or Fab’ with protein A and G protein A (Fab’ via VH3 interaction) protein G (Fab via CH1 interaction) protein L (Peptostreptococcus magnus) ii. scFv --- VH3 gene family H-chain domain (protein A) iii. fusion protein between G and L --- protein LG iv. expanded bed mode purification protein A (Fab’), ion-exchanger (Fab’ and other Ab fragments) v. ion-exchange chromatography combination with other chromatography vi. affinity chromatography with immobilized antigen vii. immunopurification using an antibody directed against the Ab fragments c) scFv or other small Ag-binding fragments (where Ag is not available) purification tags i. hexa-histidine ---- metal ion affinity chromatography immobilized chelator: iminodiacetic acid metals: nickel, copper, zinc elution with imidazole ii. strep-tag ---- biotin mimetic peptide bound to immobilized streptavidin iii. FLAG system

15. [4] Purification for therapeutic use safety [high purity and absence of potentially harmful contaminants] Removal of protein contaminants, DNA, and endotoxins i. ion-exchanger for the removal of DNA and endotoxins ii. virus inactivation by low pH iii. filtration iv. small-scale replica exp. Good manufacturing practice (GMP) preparation of high quality material (reproduction from batch to batch) Final characterization of purified material i. antibody identity ii. purity and activity iii. contaminants

16. Chap. 6 Prospects for engineered antibodies in biotechnology Gene therapy [1] intracellular Ab a) Ab to interfere with cellular processes inside the cell (intracellular targeting) scFv > intact IgG scFv targeting to various compartments i. KDEL at the C-terminus of scFV --- ER retention ii. removal of the signal sequence --- cytosol localization (Fab>scFv) iii. nucleus via nuclear localization signal b) inhibition of expression of cell surface molecules analysis of the role of cell surface protein therapeutic effects through down-regulation of cell surface receptors ex) ER-located scFv against erb-b2 receptor tyrosine kinase ER-located scFv against IL-2 receptor a-chain inhibition of cytosolic oncoproteins (p21ras) intracellular Ab, antisense RNA, RNAi, gene disruption --- tools for functional analysis of cellular proteins c) therapeutics against infectious agents HIV --- Ab against gp120 (virus assembly), rev (reverse Txase), tat (matrix protein, p17) [limiting factor: availability of effective gene transfer methods]

17. [2] Other applications of Mab in gene therapy a) vector development (critical for gene therapy or intracellular Ab production) vectors [engineered virus & non-viral systems] Ab to target the vector (binding, internalization, delivery of the genes) i. engineered viruses ii. liposomes encapsulating DNA iii. polycationic substances (DNA binding) iv. DNA (direct binding) ex) Fab’-protamine fusion protein (binding to plasmid DNA encoding a toxin gene) b) gene therapy using the specificity of antibody variable regions scFv-intracellular signaling domains of receptor i. gene transfer of TcR-scFv (tumor-associated Ag) into Tc cell ----- Tc cell expression and lysis of tumor cell ii. TcR z subunit-scFv for p185HER2 iii. IgFc receptor g subunit-scFv for p185HER2 iv. FceRI receptor g chain-scFv for renal carcinoma Ag

18. Applications of Ab in plants a) resistance to viral attact (or plant pathogens) scFv against artichoke mottled crinkle virus intact IgG --- tobacco mosaic virus b) controlling action of plant hormones scFv to abscisic acid Catalytic antobodies Mab against structural analoques of the transition state --- catalytic activity (abzymes) [low catalytic efficiency x 103-105 rate enhancement (>1011 for enzyme)] catalytic Ab i. reaction for selective enantiomers (organic chemistry) ii. converting prodrug to drug (cancer therapy) iii. inactivation of cocaine (in vivo) iv. biosensors (in vitro) Curiosity? Any practical applications possible?

19. Towards drug design Ab to design low m.w. organic drugs CDR based peptides (analogues based on modelling and structural information) i. drug ii. in vivo imaging applications CDR3 of anti-platelet Ab --- vascular thrombi in animal models CDRH3 of Ab of tumor-associated Ag --- breast cancer site iii. gathering structural information about a target molecule (peptidomimetics) Ab1 against drug action site of receptor, enzyme, or virus Anti-idiotype Ab (Ab2) --- positive image of the original molecules [development of organic mimetic compounds] relatively weak binding of CDR peptides high affinity single domain Ab (three CDR instead of six CDR) camels --- single V-domains of H-chain (camelized VH domain) Improving affinity mutations based on modelling and structural information phage display Eventual designing of Ab variable region to an Ag without preexisting Ab

20. Summary and Prospects a) antibody-based molecules for specific applications b) Mab generation techniques [phage display] c) production methods of Mab i. recombinant cells or transgenic organisms ii. genetic engineering and chemical modifications d) design of molecules with optimal properties [new reagents for diagnostic and therapeutics] e) therapeutic applications --- targeting property of Ab i. reducing immunogenicity ii. targeting novel effector function iii. pharmacokinetic properties f) Ab at lower cost and in larger quantities !