Chap. 2 Antibody Engineering: Design for Specific Applications

Factors need to be considered in the design of Ab molecules1. Specificity and affinity of the binding site2. Valency of Ab3. Sizes4. Requirement for effector function5. Attachment of therapeutic effector or reporter molecules6. Designing in vivo properties7. Cost of production

Isolation of variable region genesPrerequisite for Ab engineering – production of mAb and cloning of the variable region genes Cloning1. Phage display2. From hybridoma cells

a) genomic DNA cloningb) cDNA cloningc) PCR cloning

Overcoming Immunogenicity

1. Chimeric or fully humanized Ab2. Chemical modification with polymers such as PEG3. Use of Ab fragments4. Immunosuppressant therapy

Chimeric and humanized Antibodies1. Chimeric antibody (murine variable regions + human constant regions)

a. cloning with genomic or cDNAb. homologous recombination in hybridoma cellsc. transgenic moused. 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 immunogenicityFab, 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 chlorideb. N-hydroxysuccinimide esterc. tresyl chlorided. 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

Antibody Fragments

a. building blocks of carriers to a desired Agb. in vivo applicationsc. 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 fragmentsstabilizing 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

2. 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. Quadromas2. Chemical crosslinking of two IgG molecules3. Protein engineering techniques – bispecific IgG with ‘knob’ and ‘hole’4. Bispecific F(ab’)2

5. Dimerization domain6. Bispecific scFv molecules7. Bispecific diabodies8. Tetravalent bispecific molecules

Engineering effector function

therapeutics and diagnostics

[1] Engineering natural effector functionsa) 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 bindingf) 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 chain)

h) bispecific antibody fragments – redirecting effector function

[2] Attachment of diagnostic or therapeutic agents

(1) Chemical conjugatesa) chemical conjugation 시 고려사항 (degree of substitution, stability of the linkage,

biological activity of the resulting conjugates, heterogeneous nature of the conjugates)

Crosslinker selectiona) cleavabilityb) photosensitivityc) homo- versus heterobifunctionality

d) hydrophobicity or hydrophilicitye) reactive group specificity (primary amines, sulfhydryls, carbonyls, carbohydrates,

carboxylic acids, nonselective reaction)f) molecular dimension of the reagent

Reactive group specificity

(1) Primary amine reactive – imidoesters, N-hydroxysuccinimidyl (NHS)(2) Other lysine reactive groups – fluorescein, rhodamine isothiocyanates, PEGylation strategies

(3) Sulfhydryl reactive – maleimides, aryl or alkyl halides, -haloacyls, pyridyl disulfides, vinyl sulfone

(4) Thiol-group introduction by modifying lysine – 2-iminothiolane, SATA, SPDP(5) Nonselective – azide, aldehydes(6) 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 residuesb. modification of Fc carbohydratec. introduction of specific attachment sites onto antibodiesd. 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 vivoa. affinity or avidity of Abb. 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. –phase: short distribution phase (t½ )b. –phase: elimination or clearance phase (t½ )

(1) 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 siteiii. neonatal Fc receptor (FcRn)iv. FcRn-mediated pathway for maintaining high IgG – pH dependence

d. t ½ 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 a cytokineii. unacceptable toxicity during delivery of diagnostic or therapeutic agentsiii. 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: 2 > 1, 4 (disulfide bonds – increased half-life)b. Fv & scFv: very rapid clearancec. 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 antibodya. second Ab specific for the anti-tumor antibodyb. 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 livera. co-administration of asialo-bovine submaxillary mucin and the galactosylated Abb. 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 regionCD4 to control HIV infection

a. immunoadhesins i. fusion product between CD4 and Fc – increased half-lifeii. CD4 and CH2-domain fusion