Chap. 2 Antibody Engineering: Design for Specific Applications Factors need to be considered in the design of Ab molecules Specificity and affinity of the binding site Valency of Ab Sizes Requirement for effector function Attachment of therapeutic effector or reporter molecules Designing in vivo properties Cost of production Isolation of variable region genes Prerequisite for Ab engineering – production of mAb and cloning of the variable region genes Cloning Phage display From hybridoma cells a) genomic DNA cloning b) cDNA cloning c) PCR cloning

Overcoming Immunogenicity Chimeric or fully humanized Ab Chemical modification with polymers such as PEG Use of Ab fragments Immunosuppressant therapy Chimeric and humanized Antibodies (1) Chimeric antibody (murine variable regions + human constant regions) a. cloning with genomic or cDNA b. homologous recombination in hybridoma cells c. transgenic mouse d. primatized antibody (monkey Ab + human Ab) (2) Fully humanized antibody (murine CDR + human framework regions) a. CDR grafting (reshaping, hyperchimerization, civilization) b. veneering or resurfacing (framework exchange) c. assembly of the humanized antibody variable domain Antibody fragments to reduce immunogenicity Fab, F(ab’)2, Fv (reduced HAMA response) chimeric and humanized Ab fragments

Chemical modification to reduce immunogenicity PEG (& dextran) attachment to Ab [reduced immunogenicity, resistance to proteolysis, increased circulation half-life] PEG attachment (Fig. 2-2) a. cyanuric chloride b. N-hydroxysuccinimide ester c. tresyl chloride d. 2-iminothiolane (Traut’s reagent) and thiol-specific PEG-maleimide Tolerogens (IgG-PEG conjugates) specific immunosuppression of HAMA against the specific antigen Immunosuppressive Therapy Cyclosporin A

a. building blocks of carriers to a desired Ag Antibody Fragments a. building blocks of carriers to a desired Ag b. in vivo applications c. Fc removal – no effector function

1. Antibody fragments via proteolysis of IgG

2. Recombinant antibody fragments [F(ab’)2, Fab’, Fab, Fv] direct expression of recombinant Ab fragments with monoclonal Ab specificity

2. Recombinant antibody fragments [F(ab’)2, Fab’, Fab, Fv] a. Fab-based fragments preparation of the bivalent molecule [direct expression of F(ab’)2, in vitro reoxidation of Fab’, chemically crosslinked F(ab’)2]

2. Recombinant antibody fragments [F(ab’)2, Fab’, Fab, Fv] b. Fv-based fragments stabilizing strategies (mutation on VH and VL to have cysteine residue, single chain Fv) Dimer of scFv’ – diabodies with two different binding specificity Single domain antibody -- dAbs

Recombinant antibody fragments c. multivalent antibody fragments [multivalent Fv fragments with a relatively small size to increase the avidity] (1) direct expression of scFv (2) peptide linker between two scFv fragments (3) cysteine residues (4) fusion of Fv fragments with peptides that naturally form dimeric structure (5) fusion of Fv fragments via IgG CH3 domain (6) higher avidity antigen-binding proteins (tetrameric scFv’s) (7) crosslinking of Fab’ or scFv’

Avidin(streptavidin)-Biotin interaction

Antibodies with multiple specificities 1. Quadromas 2. Chemical crosslinking of two IgG molecules 3. Protein engineering techniques – bispecific IgG with ‘knob’ and ‘hole’ 4. Bispecific F(ab’)2 5. Dimerization domain 6. Bispecific scFv molecules 7. Bispecific diabodies 8. Tetravalent bispecific molecules

Engineering effector function therapeutics and diagnostics [1] Engineering natural effector functions a) different human isotype constant regions – different biological properties (IgG1&G3/IgG2&G4) b) to remove the effector function (Leu235Glu mutation within the Leu234-Ser329) c) flexibility of the hinge region (increased avidity of chimeric IgG1) d) IgG4: antibody with few effector function (no interheavy chain S-S bond) e) production of recombinant antibodies of different classes – Fc receptor binding f) manipulation of antibody CHO structure (w/o CHO – no effector function) g) production of polymeric IgG antibodies – Fc clustering just like IgM (chemical crosslinking, Ser444Cys, IgG with a tail piece from IgM m chain) h) bispecific antibody fragments – redirecting effector function

[2] Attachment of diagnostic or therapeutic agents Chemical conjugates a) chemical conjugation시 고려사항 (degree of substitution, stability of the linkage, biological activity of the resulting conjugates, heterogeneous nature of the conjugates) Crosslinker selection a) cleavability b) photosensitivity c) homo- versus heterobifunctionality d) hydrophobicity or hydrophilicity e) reactive group specificity (primary amines, sulfhydryls, carbonyls, carbohydrates, carboxylic acids, nonselective reaction) f) molecular dimension of the reagent

Reactive group specificity Primary amine reactive – imidoesters, N-hydroxysuccinimidyl (NHS) Other lysine reactive groups – fluorescein, rhodamine isothiocyanates, PEGylation strategies Sulfhydryl reactive – maleimides, aryl or alkyl halides, a-haloacyls, pyridyl disulfides, vinyl sulfone Thiol-group introduction by modifying lysine – 2-iminothiolane, SATA, SPDP Nonselective – azide, aldehydes Mixed type – carbodiimide (carboxyl and amine reactive)

(a) Imidoester (b) N-hydroxysuccinimide esters (c) Maleimides

(d) Haloacetyls (e) Pyridyl disulfides (f) Carbodiimide

(g) Arylazide (h) Carbonyl specific X-linkers

(i) Mixed function X-linking strategy

(2) Site-specific attachment a. modification of hinge cysteine residues b. modification of Fc carbohydrate c. introduction of specific attachment sites onto antibodies d. reverse proteolysis to attach reagents to the C-terminus of antibody fragments

(3) Fusion proteins [direct expression of fusion proteins (Table 2.6)] a. choice of antibody form (overall size, pharmacokinetics, valency) b. spacer peptide between antibody and fused protein c. location of the fusion (C- or N-terminus of the antibody chain) d. fusion with cleavable linker e. fusion with proteins f. fusion with nonproteinaceous materials

Engineering pharmacokinetics and biodistribution Conditions for target antigen binding in vivo a. affinity or avidity of Ab b. accessibility of Ag (targets in the vascular or extravascular compartment) c. antibody concentrations (Ab conc. and the rate of clearance) d. time (duration) of Ab exposure Antibody clearance from the blood a. a–phase: short distribution phase (t½ a) b. b–phase: elimination or clearance phase (t½ b) Pharmacokinetics of IgG a. IgG > IgM (long circulating half-life in human) b. IgG (half-life  1 / Ab conc.) c. receptor-mediated event i. receptor protection of IgG ii. Fc region interaction (the interface region between CH2 and CH3 domains) -- protein A binding site iii. neonatal Fc receptor (FcRn) iv. FcRn-mediated pathway for maintaining high IgG – pH dependence

d. t ½ b of chimeric or humanized Ab is higher than murine Ab half-life e. Long half-life of humanized antibodies i. protection against infection and long term neutralization of a cytokine ii. unacceptable toxicity during delivery of diagnostic or therapeutic agents iii. ideal molecule: target the toxic agent to the tumor cells and clear rapidly from the rest of the body -- short in vivo half-life f. Manipulation of Ab pharmacokinetics

(2) Pharmacokinetics of Ab fragments Ab fragments such as F(ab’)2 : rapid clearance rapid distribution and penetration into tissues (pharmacokinetics: IgG >> F(ab’)2 > Fab) a. F(ab’)2: g2 > g1, g4 (disulfide bonds – increased half-life) b. Fv & scFv: very rapid clearance c. controlling half-life of Ab fragments i. PEGylation (half-life 증가) ii. removal of Ab CHO (Asn297) (half-life 감소) iii. subtle variation in CHO structure (high mannose – half-life 감소) iv. mutations on the FcRn binding region (high affinity for FcRn) v. removal of CH2 domain (short half-life) d. bispecific diabodies targeted for serum Ig – increased half-life (dissociation of diabody from IgG at low pH) (3) Clearance [to remove Ab from circulation] Clearance strategies for an anti-tumor antibody a. second Ab specific for the anti-tumor antibody b. high affinity between Ab-biotin and avidin (streptavidin) cf) complex and expensive procedure / liver damage by the large complex -- extracorporeal immunoadsorption

(4) Chemical modification Galactosylation of Ab – rapid clearance via asialoglycoprotein receptor in liver a. co-administration of asialo-bovine submaxillary mucin and the galactosylated Ab b. rapid deposition of Ab in the liver (nontoxic therapeutic agents to the liver) c. radioiodinated Ab (rapid metabolism in the liver) d. use as second Ab for rapid removal of the first Ab (5) Fc region to extend half-life Ab half-life  Fc region CD4 to control HIV infection a. immunoadhesins i. fusion product between CD4 and Fc – increased half-life ii. CD4 and CH2-domain fusion