Monoclonal antibodies

Presented by: Richa Arora Apoorva Gupta

Introduction
Antibodies Antigen binding glycoproteins produced by Bcells
Antigen surface - large no. of antigenic determinants or epitopes (MAbs)specific to single

Monoclonal Antibodies determinant.

Evolution of MAbs
Antigenic exposure- activation of multiple B-cell- formation of mixture of MAbs specific for each epitope- known as polyclonal antibodies
Disadvantages of polyclonal antibodies - Problem of cross reactivity - Limits the use of Polyclonal Abs For research, diagnostic and therapeutic purposes- highly specific MAbs are preferred

 MAbs B- cells generating antibodies to one specific epitopeisolated from spleen of immunized animals Limited life span of B cells- unfavourable for large scale production of MAbs In 1975, Kohler and Milstein- Hybridoma Technology myeloma B cells fused with lymphocytes

Hybridoma Technology

Structure of Antibody

Antibodies are made up of: 2 light chains (identical) ~25 kda 2 heavy chains (identical) ~50 kda

Each light chain bound to heavy chain by disulfide (H-L) Heavy chain bound to heavy chain (H-H) Heavy chains 1 VH and either 3 or 4 CH(CH1, CH2, CH3, CH4) Light chains 1 VL and 1 CL

 Variable region - Divided into 2 parts

- Hypervariable regions - CDR (Complementarity Determining Regions) - Ag (epitope) Binding site - Frame work regions: Relatively constant region

Light Chains: Amino acid sequence of CL- k or Humans k- 60%, 40%, Mice: k- 95% , - 5% Heavy Chains: Amino acid sequence of CH ,, , ,

Types of Antibody

Antibody Engineering
Murine Abs - isolated from mouse Due to Human Anti-mouse Antibody (HAMA) response- lower therapeutic effeciency- allergic reactions Attempts to overcome this problem- Reconstructing rodent antibodies by genetic engineering , primatised antibody and generating human MAbs

Chimeric Antibody: Mouse variable genes into human constantregion genes Humanized Antibody: Mouse complementarity-determining regions (CDRs) onto human constant and variable domain frameworks

 Human Antibody: Selection of human antibody fragments from in vitro libraries and its expression in different host systems Antibody fragments : Fab, F(ab)2 and Fv - proteolytic digestion or by recombinant means Antibody fragments usually results in the accumulation of the expressed protein in a denatured form as an insoluble inclusion body which requires solubilisation and refolding to obtain active material- expensive and low yielding procedure
Thus mostly Fv fragments in the form of sFv (Single chain Fv fragments) are expressed- which results in secretion of the antibody fragment

Problems with conventional MAb production

Problems with Hybridoma technology : - Sensitive to environmental changes - Animal welfare concerns associated with hybridoma-based antibody production methods - Sensitive to shear and bubble damage in bioreactors complicates their large-scale culture - No control over the epitopes to which antibodies are formed - Antibodies must be screened extensively after they are created in the hope that one has been created with characteristics that are desirable to the investigator - Toxic antigens may kill the host animal before antibodies are produced - Hybridoma derived antibodies cannot be improved until they are first converted into recombinant antibodies - Hybridoma derived antibodies can take between 4 and 6 months to create

Genetic method for Mab production

Bacteriophage display library system

Transfection of gene into host cells

Bacteriophage display library system

Engineering of phage genome displays antibody like proteins on surface encoded by DNA present in phage

Selection of highest affinity antibody recovery of gene encoding that Ab

After selection of gene Expression in a suitable host

Key components :
- A repertoire of antibody genes, typically derived from PCR - A method for producing a stable library - A method for selecting the highest affinity antibody - An affinity maturation strategy - An expression system for large-scale production

 Antibody Isolation: - Paramagnetic beads, fluorescence-activated cell sorting (FACS),and/or Enzyme-Linked Immunosorbent Assays (ELISAs) - The antigen of interest is incubated with the antibody-displaying microorganisms and the rAbs that do not stick to the antigen are discarded - Bound rAbs are removed from the antigen and screened for desirable characteristics. (Panning Process) Modification: - Selection process to enrich for the highest affinity antibodies - The antibodies can be "matured" through random or rational mutagenesis methods - Molecular biology techniques such as site-directed mutagenesis, error prone PCR, DNA shuffling, or mutator strains of bacteria can be used to mutate selected residues of a given antibody fragment creating a whole new library that can be tested for increased function

Fig.: A typical construct for the expression of Fab in phages and plasmids

 Antibody Expression: - The genes for the antibody are transferred via expression vectors into an expression systembacteria, yeast, or mammalian cell lines The choice of vector and expression system depends on the type of antibody that is to be produced.

Antibodies from antibodies libraries are usually monovalent fragments that contain only one antigen binding site and no constant region Full length antibody expression is most frequently done in yeast and mammalian expression systems. Lonza's CHOK1SV, Percivia's PERC.6 and HEK293 - mammalian cell lines currently in use for the production of whole recombinant antibodies

Steps in Mab production using mammalian cells

Transfection of the gene of interest into the cells, selection of clones, adaptation to different culture conditions, culture in bioreactors and scaleup to industrial level Expression system : (i) support high-level product expression over long periods of time maintaining high viable cell density and genetic stability; (ii) be scalable; (iii) have appropriate abilities for post-translational processing and (iv) allow appropriate characterization for human safety Transfection : - Expression vector three main features : i) expression levels independent from the site of integration in the genome, ii) expression levels correlated with the number of integrated transgene copies and iii) maintenance of expression efciency over time

Expression vector : i) Promoters/enhancers (e.g. SV40, CMV) ; ii) Elements that stabilize and enhance translation of primary product (e.g. polyadenylation signals, Kozak sequence and intervening sequences)

Transfection methods : i) Stable v/s transient gene transfection - Gene markers : Dihydrofolate reductase (DHFR) and glutamine synthetase (GS) ii) DNA delivery systems : calciumphosphate precipitation, electroporation, lipofection and polymer-mediated gene transfer

Selection : - Survival and growth of cell clones expressing the marker gene product - These clones are transferred as single cells to a second cultivation vessel, where the cultures are expanded to produce clonal populations - Clones are then evaluated in terms of growth and product (mAb) titer, and the highest producers are selected for another round(s) of cultivation and analysis, to conrm whether the levels of productivity are maintained

Other host cells

E.coli - Advantages : easy and quick to grow, wide range of expression vectors can be introduced. - Disadvantages : No post-translational modifications, protein accumulates in inclusion bodies - cDNA for the antibody fragment is inserted into the expression cassette of an expression vector Bacillus subtilis can express scFv can secrete protein Yeast can synthesize, process and secrete heavy and light chains Pichia pastoris methylotropic yeast fast growth coupled with protein expression and secretion can express scFv

 Trichoderma reesei filamentous fungi for Fab fragments

Transgenic plants Introducing genes in Ti plasmid heavy and light chain cDNAs introduced - Also scFv fragment production in seed protein

Transgenic animals Genes introduced into fertilized egg by microinjection Heterologous proteins expressed in milk of lactating transgenic animals e.g. in sheep, goats and cows

Insect cells use of baculovirus vector production of murine, chimeric, humanized IgG and scFv

Purification of MAbs
By chromatography Affinity chromatography Combination of affinity and ion-exchange chromatography Final polishing step gel filtration chromatography

Advantages of Recombinant Antibodies Non-animal technology Speed Control Isotype Conversion - Nucleic acid sequence of rAb is easily accessible for further manipulation Recombinant antibodies from antibody gene libraries require less purified antigen to produce Eliminate constraints on the types of antigens that can be used Antibodies to highly toxic or non-immunogenic antigens can be created using library methods The affinity of antibodies derived from libraries can be increased to levels unobtainable by an animal's natural immune system. Recombinant antibodies from antibody gene libraries are also amenable to high-throughput production rAb fragments can be produced cheaply in bacterial and yeast expression systems

Impediments to Recombinant Antibody Use Intellectual property: commercial interests have restricted access to improved methods of generating rAbs to protect intellectual property. Initial Investment: High-throughput equipment to automate selection procedures can be expensive Technical Expertise required : Selection and screening of antibodies from antibody libraries requires technical expertise in molecular biology especially if phage-displayed libraries are used False positives : Phages themselves are inherently sticky and may bind antigens regardless of whether or not they bear an antibody corresponding to the antigen multiple rounds of selection needed Maturation and Structure Conversion : Affinity matured antibody fragments selected from an antibody library may also need to be converted to divalent or full-length antibodies to increase their utility in specific applications

Applications
a. Clinical diagnosis and research Radioimmunoassay Immunoradiometric assay Non-isotopic immunoassays Immunosensors Immunocytochemistry Flow cytometry and cell sorting Immunopurification Radioimmunodetection of tumour antigens Blood testing company, Phadia, incorporated non-animal antibodies into its Varelisa and EliA products that test for autoimmune disorders. Proteomic initiatives including the ProteomeBinders Consortium and the Antibody Factory use phage display technology in an attempt to create antibodies against all the proteins encoded in the human proteome. Antibody vendors such as AbDSerotec and Axxora.com have begun to offer non animal, recombinant antibodies from phage-display platforms alongside the traditional hybridoma-based antibodies

b. Therapeutics - Cancer : Bispecific antibodies, Antibody-cytokine fusion proteins, Immunotoxins - Infectious diseases - Cardiovascular disease - Disorders of the immune system/ inflammatory diseases
In 2002 the FDA approved the first fully human mAb derived from phage display technology, Humira. Humira (generic name Adalimumab ) was initially approved to treat patients with moderate to severe rheumatoid arthritis. Since then, the drug has been approved to relieve symptoms associated with other autoimmune disorders including Crohns disease and psoriatic arthritis

Some commercial MAbs

Antibody Digibind Herceptin Zenapax Simulect Rituxan Panorex Reopro Synagis Mylotarg Campath Zevalin Humira Xolair Bexxar Raptiva Avastin Erbitux Target Digoxin HER- 2 IL2 R-a IL2 R-a CD20 CA17-1A Platelet RSV CD33 CD52 CD20 TNF IgE CD20 CD11a VEGF EGFR Indication Digoxin poisoning Metastatic.breast cancer Kindney transplantation rejection Acute organ rejection Non-Hodgkins lymphoma Colorectal cancer Ischemic cardiac complications RSV infection Relapsed CD33-positive AML (calicheamin) B cell CLL Non-Hodgkins lymphoma (I111 & Y90) Rhematoid arthritis Asthma Non-Hodgkins lymphoma (I131) Mod.. severe plaque psoriasis, psoriatic arthritis Metastatic carcinoma of the colon or rectum EGFR-expressing, metastatic colorectal cancer Producer (Glaxo/Welcome) (Genentech/MedImmune) (Protein Design Labs/Roche) (Norvatis) (Genentech/Roche) (GlaxoSmithKline / Centocor) (Centocor/Eli Lilly) * (MedImmune) (Celltech/Wyeth-Ayerst) (Millenium Ph/Schering AG) (IDEC) (Abbot) (Genentech/Novartis) (Corixa/Glaxo-SmithKline) (Xoma/Genentech) (Genetech) (Brystol-Myer-quibb,Imclone)

Chimeric Antibody Humanized Antibody

References
Construction, Screening and Expression of Recombinant Antibodies R.A. Irving, P.J. Hudson, J.W. Goding Guidelines to cell engineering for monoclonal antibody production - A.Rita Costa, M. Elisa Rodrigues, Mariana Henriques, Joana Azeredo, Rosrio Oliveira www.alttox.org Antibody phage display Methods and protocols - Philippa M. OBrien And Robert Aitken Concepts in antibody phage display Sara Carmen and Lutz Jermutus