Monoclonal antibodies (mAbs) are artificially produced antibodies that are all identical and recognize the same epitope. This document summarizes the history and production of mAbs, from early discoveries in the late 19th century to modern techniques. It describes the key breakthrough of hybridoma technology by Köhler and Milstein in 1975 that allowed mass production of mAbs by fusing antibody-producing cells with myeloma cells. Various approaches to mAb production are also summarized, including recombinant techniques, phage display, plantibodies, bacterial display, and yeast display. The document concludes with discussion of applications, clinical trials, and future innovations using mAb technology.

2. SEMINAR-Ι
RADHIKA D. HEGDE
PGS10AGR6520

3. Contents:
 Introduction about
 Immune system
 Antibody, structure
 Polyclonal Abs
 Monoclonal Abs
 History
 Different approaches to mAb production
 Application
 Release of mAb
 Recent challenges
 Future innovations

4. Immune System:
• Innate- General • Adaptive-Specific

5. Contd..
 Antibody
 Antigen
 Epitope
 Paratope

6. Basic Structure of Antibody
• Fab region
• Fc region
• Heavy Chain
• Light chain
• Paratope
• Hinge region

7. Immunoglo
bulin
% in
serum
Half life
(days)
Location Function
IgG 75 21-24 Blood
Lymph
Intestine
Enhances phagocytosis
Neutralizes toxins
Protects new born
IgM 10 5-10 Blood
Lymph
Intestine
Effective against microbes
IgA 1-4 4-7 Secretions
Blood ,lymph. Protection in secretary surface
IgD 2-8 0.2 B cell surface
Blood ,lymph Initiate immune response
IgE 1-5 0.004 Mast cell,
Basophils, through
out body
Against allergic reaction ,
in lysis of worms
Classification of Immunoglobulins

8. Variation in Antibodies

9. Development of Artificial Antibodies:

10. HISTORY
 Emil Von Behring & Kitasato Shibasaburo(1890)- Effective serum
against diphtheria
 Paul Ehrlich (1891) – First time used antibody word
 John Marrack (1930s) – Biochemical property of antigen –
antibody binding
 Elin A.Kabat (1939)- Heterogeneity of antibodies
 Astrid Fagraeus (1948) - Plasma B-cell specifically involve in
antibody production
 Gustav Nossal - B-cell produce only one antibody
 Rodney Porter - Antibody binding region of IgG
 Susumu Tonegawa (1976) - Rearrangement of genes

11. Polyclonal Antibodies:
 Antibody secreted - different B-cell lineage
 Each Ab identify different epitope
 Normal humoral immune response - many plasma cells
 Overlap in antibody binding
 Ideal for disease eradication
Science and
medicine, this
creates inexactness
and imprecision
Single clone products
provide precision and
reproducibility
mAbs

12. Monoclonal Antibodies:
Polyclonal Monoclonal
Single type of antibody → against one specific epitope
Clones of unique parent

13. Hybridoma cell Production
Recombinant techniques
Phage
display
Different Approaches to mAbs Production:
bacterial
display
Plantibodies
Yeast
display
Ribosomal
display
Ascites method
Tissue culture
method

14. Hybridoma cell Production
 Köhler and Milstein(1975)
 Antibodies-short life span
 Myelomas- propagate indefinitely
 Fused-murine (mouse) antibody secreting plasma cells and
immortal murine myeloma cells- hybridoma
 Hybridoma cells - secrete monoclonal antibodies

15. Hybridoma Technology:

16. Review committee recommendations to IACUC:
American Anti-Vivisection Society
NIH→NRC→ Review committee
 When ascites method used-effort made to minimize pain or
distress, including frequent observation, limiting the numbers of
taps, and prompt euthanasia if signs of distress appear
 mAb now being commercially produced by the mouse ascites
method should continue, but industry should move toward
tissue-culture methods

17. Recombinant Techniques
 Avoid or minimize pain and suffering by animals
 Growing knowledge of antibody, gene structure and regulation
has made possible…
Vector library

18. Phage display:
 Phage display was first described by George P. Smith - 1985
 Displayed- peptides on filamentous phage by fusing gene of
interest to coat protein coding gene
 T4, λ,M13,T7

19.  M13 filamentous phage
 pIII & pVIII gene
 Encode minor & major coat protein

20. General Steps in Phage Display

21. (A) Antigen is immobilised onto plastic tubes. (B) Library/outputs are incubated with the
antigen surface. (C) Unbound phage are removed and the surface is washed. (D) Phage
are eluted. (E) E. coli cells are infected with eluted phage. (F) Cells are plated onto
selective agar plates. (G) Selection output is amplified. (H) Helper phage superinfect cells
containing phagemid vector. (I) Phage replication and assembly provides population of
enriched phage for next round of selection
Biopanning

22. Plantibodies
 Antibody produced by plants (Biolex)
 Concept- Hiatt et al., (1983)
 MAb directed against genital herpes
(Horn et al., 2004)
 GE Corn can produce up to 1 kg antibody/acre and can be stored
at RT for up to 5 years.
(Humphreys et al., 2001)

23. Plantibody Production:

24. Expression in Tobacco of a Functional Monoclonal
Antibody Specific to Stylet Secretions of the Root
Knot-Nematode : (Hiatt et al., 1996)
 Gene for the heavy and light protein chains of a monoclonal
antibody(6D4) specific to stylet secretions of Meloidogyne incognita
were cloned as cDNA containing native leader sequence
 Heavy and light chains constructs under control of the cauliflower
mosaic virus 35S promoter were transferred independently into
Xanthi tobacco
 Transgenic plants producing antibody(plantibody) identical to
hybridoma cell line

25. Expression Study
(Tissue printing)
A. Transgenic plant expression
B. Wild type tobacco spiked with
6D4 culture filtrate.
C. wild type tobacco

27. Bacterial display:
 Freudl et al.,& Charbit et al.,-1986
Surface Display of a Functional Single-Chain Fv Antibody on
Staphylococci : (Elin Gunneriusson-1995)
 Gene fragment encoding a mouse hybridoma-derived single chain
Fv reactive with human IgE (antigen) was amplified
 Gene fragment encoding ScFv inserted into two general
expression vectors pSEaIgEABPXM and pSPPaIgEABPXM
designed for surface display on S. xylosus and S. carnosus,
respectively
 Lysostaphin treatment to release cell wall-bound proteins
 Analyzed by SDS-PAGE

28. Ribosomal display
 Mattheakis., Bhatt and Dower-1994
Steps Involved:
 Large DNA library that encodes the polypeptide of interest
fused to C-terminal tether region
 Transcribed to mRNA
 Resulting modified mRNA used as template for translation-
folds into a 3D structure
 Purification

29. T. Kanamori et al.,2014

30. Pure system components
T. Kanamori et al.,2014

31. Yeast display:
 Boder and K.Dane Wittrup-1997
 Yeast –microbe & eukaryotic
 Aga1p & Aga2p cell wall protein stably expressed The
expression of both under the control galactose-inducible GAL1
promoter
 Heterogeneous immunoglobulin gene cloned into tyeast display
plasmid
 Expressed as fusion products with the Aga2p protein
 Human antibody fragments typically fused to the C-terminus of
the Aga2p subunit

34. Nomenclature of Monoclonal Antibody :
 United States Adopted Name (USAN) Council has provided
guidelines for nomenclature,( 2011)
 The name for a monoclonal antibody is formatted as PREFIX –
TARGET - SOURCE SPECIES
 SUFFIX Prefix:
 Product Target:
 Disease or target Source species:
 Suffix:
 Ex: (Rituximab) ri = unique prefix
 tu = tumor
 xi = chimeric
 mab = monoclonal antibody

35. Application of Monoclonal Antibodies:
 As diagnostic and research reagents:
ELISA, Immunofluorescence
 As marker in proteins, nucleic acids- identification
 Against teliospores of two Tilletia species revealed some
quantitative differences in the two fungi.
(Banowetz, 1984)
 Differentiate pathogenic and non-pathogenic strains of a fungus

36. Contd..
 Blood grouping
 Diagnosis in cancer
 Imaging
 Purification of antigen

37. Release of mAbs
IND, investigational new drug application = request for authorization to test a new
compound in humans from the US Food and Drug Administration (FDA); NDA,
new drug application = request for marketing approval from the FDA.

38. Preclinical Research:
Safety and immunotoxicity assessment of mAbs
 General toxicity of mAbs
 Immunopharmacology and Immunotoxicity of mAbs
 Adverse effects of immunosuppression
 Assessment of Risk of Immunotoxicity in Humans
 General Approach to Safety Testing of mAbs
 Assessing Potential Immunotoxicity Concerns of mAbs by
Evaluating the Biology and Expression of the Target and the
Intended Clinical Population
 In Vivo Studies with Immunomodulatory mAbs Species Selection
and Qualification

39. Need of Safety and immunotoxicity assessment
 Select a pharmacologically-relevant species
 To understand the limitations of the chosen animal species and
whether in vivo safety data should be supplemented with in vitro
assays with human cells
 To try and predict the immunological response and the risk of
adverse immunotoxicological events occurring in human
 To select a safe human starting dose for FIH clinical studies based
on the minimum anticipated biological effect level (MABEL)

40. Clinical Trials

41. Strategies to select the best targets
 Functionally validated targets
 Experimentally validated targets
 Clinically validated targets
Strategies to optimize structures
 Improving pharmaceutical properties
 Second- and third-generation antibody–drug conjugates
 Improving antibody functions
 Polyclonal or oligoclonal antibodies
Strategies to provide affordable treatments
 Decreasing production and processing costs
 Biomarker identification and selection of patients
(Alain Beck et al.,)
 Advancement in plant disease management

42. Monoclonal antibodies marketed for therapeutic use (approved
by FDA)

43. Future Innovations:
 Advancement of new technologies
 Treatment of cancer
 Designer mAbs as drug
 Bispecific and bifunctional single chain recombinant antibodies
 Dual-variable domain (DVD-Ig) technology -Increased binding of
an epitope.

44. CONCLUSION: