Antibody (immunoglobulin) Serum protein electrophoresis (SEP) Basic structure and function of immunoglobulin. Monoclonal antibody, its modification and application

Serum protein electrophoresis

Buffer pH 8.6

Serum electrophoresis

Immunoglobulin (antibody, BCR): universal adaptor

典型抗體分子的構造(1) BCR and Ab N 端 構造 功能 異 C端序列 作用機制 同 免疫球蛋白 辨識抗原 C 端

Proteolytic digestion of IgG Variable region Fab: papain digest F(ab')2: pepsin digest Constant region (Fc)

Different domains have different functions

抗體分子與專一性抗原之交互作用(1) 局部區域的高度變異序列形成抗原結合位 Hypervariable region Framework region CDR (complementarity-determining region)

General structures of five classes of antibody

Structure and formation of secretory IgA

Antigenic determinants of immunoglobulin Isotypes: all variants present in serum of normal individual types: heavy and light chain classes: IgG, IgA, IgM, IgD, IgE subclasses: IgG1, IgG2, IgG3, IgG4 subgroups: VHI, VHII, VHIII Allotypes: genetically controlled alternative forms Idiotypes: individually specific to each immunoglobulin molecule

Immunoglobulin superfamily

Generation of diversity (G.O.D.) VDJ rearrangement of heavy chain VJ rearrangement of light chain random combination of heavy and light chain Imprecision of rearrangement

V-region genes are constructed from gene segments

Overview of B cell development

Model of allelic exclusion

Immunoglobulin concentration changes along the age

Serum immunoglobulin vary among different healthy persons IgG: 800-1600 mg/dl IgA: 140-400 mg/dl IgM: 50-200mg/dl IgD: 0-40 mg/dl

Antibody response Clonal selection Primary and secondary antibody response Isotype switch: IgM----->IgG Affinity maturation: Somatic mutation

Monoclonal antibody Myeloma (HGPRT-) Spleen from immunized mice HAT medium Selection Limiting dilution and cloning

Method Immunization with antigen Spleen and myeloma cell fusion Polyethylene glycol (PEG): polymer of ethylene oxide HAT medium: selection of hybridoma Screening Cloning by limiting dilution: Poisson distribution Large scale production and storage: can be purified by protein A affinity column cell line (10-50 mg/ml) ascites (1-10 mg/ml)

Main pathway (de novo) Secondary Pathway ~~~~~~~~~~~~~~~~~~~~~~ DNA Aminopterin Block DNA NUCLEOTIDES RNA HGPRT TK Hypoxanthine Thymidine Secondary Pathway Enzyme-deficient (HGPRT-) myeloma cell Hybrid cell Normal splenocyle spontaneous death HAT-dependent death ACTIVE GROWTH

Application of monoclonal antibodies: universal adaptor Antigen purification: affinity column Diagnosis: detect Ag or Ab pathogen cell surface marker soluble antigens e.g. AFP cancer diagnosis: immunoimaging Immunotherapy Cancer therapy Autoimmune disease treatment Allergy prevention Tissue transplantation

Application of monoclonal antibody

Application of mAb as immunotherapeutic drugs

Monoclonal antibodies that recognize tumor-specific antigens might be used in a variety of ways to help eliminate tumors

MAT: the magic bullet Therapeutic results were disappointing until recently. Two major hurdles were encountered. Biologic activity in humans severely limited due to the host’s immune response to foreign mAbs (production of human anti-mouse Abs). Causes neutralization of activity and toxicity. Large-scale production

Mouse mAb may cause serum sickness in vivo

Approaches to reduce or eliminate human anti-mouse antibodies. Chimeric Antibodies Cloned antibody genes are generated in which the variable regions from the original mouse mAb are combined with human antibody-constant regions. Highly effective in reducing human anti-mouse antibodies (HAMA). However, the variable (Ag-recognition) domains are still foreign; HAMA often still occurs neutralizing efficacy.

Generation of chimeric mouse-human mAb

Antibodies can be engineered to reduce their immunogenicity in humans humanized chimaetic Red: mouse White: human

Chimeric Antibodies

Humanized mAbs All portions of the mAb not required for antigen binding, including framework residues in the variable region, are replaced with human sequences. Gene engineering, expression, evaluation < 10% of optimized mAb retains mouse sequence Highly-effective (but time-consuming) approach

抗體分子與專一性抗原之交互作用 局部區域的高度變異序列形成抗原結合位 Hypervariable region Framework region CDR (complementarity-determining region)

Humanized mAb CDR: complementary determination region

Antibodies can be engineered to reduce their immunogenicity in humans humanized chimaetic Red: mouse White: human

Modification of mouse mAb

Immunotoxin: magic bullet

Antibodies in action

MAT in Organ Transplantation Rejection of allografts is an immunological response exerted primarily by T-cells. mAb approaches against T-cell antigens has proven effective. CD25 (IL2-Receptor a chain). Highly effective approach for the prevention of graft rejection episodes and graft loss (e.g., renal). basiliximab (chimeric) dacliximab (humanized)

MAT in Coronary Artery Disease Atherosclerotic lesions (plaques) involve platelet aggregation followed by coagulation and thrombus formation. Platelet receptors involved in early steps of plaque formation. basis of “one-aspirin-a-day” concept limited efficacy in preventing platelet aggregation Abciximab (chimeric mAb) has been developed/approved as an effective treatment for the prevention of thrombosis and plaque formation in high risk patients. Targets the platelet receptors (integrins) Prevents platelet aggregation

MAT in Autoimmune Diseases TNFa is a proinflammatory cytokine that has been implicated as a causal factor in a variety of inflammatory diseases including Rheumatoid Arthritis. Infliximab is a chimeric mAb targeted to TNFa. has displayed remarkable activity against Rheumatoid Arthritis and other inflammatory diseases (Crohn’s Disease). Approved in 1998. First for Crohn’s Disease; now R.A.

Antibodies can be used to alleviate and suppress autoimmune disease Anti-inflammatory effects of anti-TNF-a therapy in rheumatoid arthritis

MAT in Cancer (I) Most intensively studied area (25 years of research). Cancer poses the highest hurdles. Also being evaluated in modified versions involving the conjugation to radio-ionizing particles, immunotoxins, immunoliposomes.

MAT in Cancer (II) Rituximab (chimeric mAb) Targets CD20; a cell surface protein expressed exclusively on B cells. Approved for the treatment of B cell malignancies (NHL, CLL, ALL). Trastuzumab (humanized mAb) Her2 is a form of the EGF-Receptor that undergoes amplification in approximately 25% of breast cancers. 1st identified as a rat oncogene called neu (HER2/neu) Patients with amplified HER2 expression suffer a very poor prognosis. Trastuzumab (Herceptin) has displayed impressive activity against HER2+ breast cancer both alone and in combination.

Summary Antibodies are universal adaptors which can bind to any specific target both in vitro and in vivo. Humanized monoclonal antibody reduce its immunogenecity without change its specificity. MAT provides another way to develop new therapy against cancer, autoimmune disease…etc. (serum therapy)

New approaches under development Phage-display libraries expressing human VH and VL chains are assembled into libraries and expressed as combinatorial matrices. Assayed for immunoreactivity against antigen of interest. Transgenic Mouse Approach A procedure in which the endogenous mouse Ig gene loci is replaced by homologous recombination with their human homologs. Immunization of transgenic mouse followed by standard hybridoma procedures produces a fully humanized mAb.

Advantages of phage display More efficiently than through conventional hybridoma system. Cheaper to produce recombinant antibodies using bacteria, rather than mammalian cell line. Easier to maintain and grow bacterial cultures for recombinant antibody production. Bypass immunization in antibody selection. Bypass the use of animal cells for production of antibodies. Producing the combinatorial library (ideally with 108 to 109 members) of functional antibodies to generate a larger repertoire of antibodies than those available through conventional hybridoma technology. Easy isolation and expression of the cloned gene in a bacterial host. Excellent potential to further improve binding properties of the selected antibody by protein engineering techniques.

PNAS 101:2536, 2004 Feb.

Selection of phage library and screening of phage antibodies Two human nonimmune single-chain variable region fragment (scFv) libraries (a total of 2.7x1010 members) constructed from B cells of 57 unimmunized donors were used for selection of scFvs against the purified S1-C9

Generation human mAb by transgenic mice