monoclonal antibodies and engineered antibodies

7/29/2019 Monoclonal antibodies and engineered antibodies

1/24


2/24

Introduction

Advantages and disadvantages

Production methods

Problems associated

Applications

Engineering antibodies

Conclusion

References 2


3/24

Antibody(Ab) is a protein used by the immune system to identify and

neutralize foreign objects like bacteria and viruses.

An antibody is called monoclonal (mAb)

when each immunoglobulin is produced

by a single clone of cells and hence is

identical to every other molecule in the

preparation, in terms of heavy as well as

light chain structure.

Polyclonal antibodies (pAb) are

produced by B-lymphocyte which

respond to many epitopes of antigen.

3


4/24

4

Polyclonal antibodies(Polyclonal antiserum)

B B B B B B B B

Harvest Ab

Monoclonal antibodies


5/24

Specificity.Affinity.

Potential to generate large quantities of Abs under precisely controlled

conditions.

In vivo and in vitro production is possible with high production rate.

Immortal cell lines.

5

mAb cant differentiate between two antigens if the body is directed to an

epitope common to antigens.mAb to viral strains are so specific that they dont react to other minor strains of

same virus.

pAb have potential for co-operatively binding to respective antigens, so

stabilising the overall affinity and binding forming precipitating complexes

while mAbs form network with antigens.


6/24

6


7/24

7


8/24

8

Hybridoma cells are injected into pristane primed rodents, the cell line

proliferates and are stored in ascitic fluid.

10-50 mL of fluid is collected containing several mg/mL of antibody.

mAbs produced this way are considered unsuitable because of viralcontamination.

Widely used in research applications.

The inherent variability in animals can result

in lack of consistency, while some linesproduce solid rather than diffuse tumors,

and some produce none or

kill the host.


9/24

9

Fermentation is most widely employed for production of monoclonal

antibodies because of the problems associated with above listed

methods.No contamination with normal mouse immunoglobulin is seen with

fermentation.

Apart from this, bacterial cell cultures, transgenic animals and

transgenic plants are also used for production of monoclonal antibodies

but to a very limited extent.


10/24

10

The main problem is the mouse antibodies are recognised by human

immune system as foreign material and the patients develop a immune

response against them, producing HAMA (Human Anti-Mouse

Antibodies).

The doses of monoclonal antibodies to treat chronic diseases are

typically large.

Stability issues concerned with oxidation, deamidation, aggregation,

fragmentation and other forms of chemical modification with alter orprobably nullify the antibody function.


11/24

11

Freeze dried and again recoveredfor moisture uptake.

Carbohydrates are added

Ex- Trehalose, sucrose etc.,

Monitoring

Spray dried Ab added topolylactide-co-glycolide (PLGA)

to produce microspheres.

Characterization.


12/24

12

Identification of tumors as they express specific membrane proteins.

Measurement of circulation steroid harmones and in differentiating viral

strains.

Bacterial infections and STD.

Drug immunoconjugates.

Radioisotopes anchored mAbs.

RIA and ELISA.

Purification of proteins.


13/24

13


14/24

14


15/24

15

Antibodies exhibit four main effector functions: antibody-dependent

cellular cytotoxicity (ADCC), phagocytosis, complement-dependent

cytotoxicity (CDC), and half-life/clearance rate.

Each of these effector functions is mediated through interaction with a

specific set of receptors and cell types: ADCC and phagocytosis throughinteraction of cell-bound mAbs with Fc gamma receptors (Fc R), CDC

through interaction of cell-bound mAbs with the series of soluble blood

proteins that constitute the complement system (e.g. C1q, C3, C4, etc.),

and half-life/clearance rate through binding of antibodies to the neonatal

Fc receptor (FcRn).C1q binding and complement activation.

FcR binding and ADCC.

FcRn and half-life/clearance rate.


16/24

16

Chimeric antibody- As HAMA response of patients is due to Fc

portion of murine Abs, murine Fv region was fused with human Fc to

produce Chimeric genes.

Ex- Infliximab, rituximab, and abciximab

Humanised antibody- This consists of fusion of hyper variable region,aminao acids responsible for antigen binding with human antibody thus

replacing its own hyper variable region.

Ex-Mylotarg, Herceptin, Xolair

Chimeric antiody

Humanised antiody


17/24

17

mAb + Calcichemicin

Antibody portion of molecule targets CD33 (a cell surface molecule), abundanton the surface of acute myeloid leukemia cells (AML) & absent from normalblood stem cells.

AML cells accumulate in the bone marrow and prevent normal bone marrowfrom growing an functioning properly.

Calcichemicin is a potent anti-cancer drug which intercalates into DNA andbreaks it, because of which cells undergo apoptosis.

Antibody targets calcichemicin to AML cell specifically through CD33 cellsurface molecule

Calcichemicin kills AML cells.


18/24

Calcichemicin intercalates in DNA

18


19/24

19


20/24

20


21/24

21

Successful clinical application of these novel agents requires the development

of stable formulations that can be used for specific delivery methods.

Antibodies, because of their endogenous nature, have built-in features that may

pose problems for stability as bio-therapeutics.

Lyophilization and pH dependent modification.

Several modes of targeting is achieved successfully.

Linking up the technology to gene therapy will ensure highly specific treatment

Examples of such an approach include studies performed using an antibody

directed against the Her2/neu antigen to delivery liposomes containing achemotherapeutic have been described, antibodies used to deliver cationic

liposomes for the administration of nucleic acid material for gene therapy.


22/24

22

L.G. Presta, Engineering antibodies for therapy, Curr. Pharm. Biotechnol. 3(2002) 237256.

J.W. Park, K. Hong, P. Carter, H. Asgari, L.Y. Guo, G.A. Keller, C. Wirth, R.

Shalaby, C. Kotts, W.I. Wood, et al., Development of anti-p185HER2

immunoliposomes for cancer therapy, Proc. Natl. Acad. Sci. U. S. A. 92 (1995)

13271331.A.L. Daugherty, R.J. Mrsny, Formulation and delivery issues for monoclonal

antibody therapeutics, Advanced Drug Delivery Reviews. 58 (2006) 686706.

Leonard G. Presta, Engineering therapeutic antibodies to minimize

immunogenicity and optimize function, Advanced Drug Delivery Reviews 58

(2006) 640656.

Lee K. Tan and Sherie L. Morrison, Antibody structure and antibody

engineering, Advanced Drug Delivery Reviews, 2 (1998) 129-142.


23/24

23

http://www.youtube.com/watch?v=48VSU4AZ-L0

http://www.biology.arizona.edu/immunology/tutorials/antibody/structur

e.html

http://www.accessexcellence.org/RC/VL/GG/monoclonal.php

http://www.antibodyresource.com/educational.html
http://www.youtube.com/watch?v=48VSU4AZ-L0http://www.biology.arizona.edu/immunology/tutorials/antibody/structure.htmlhttp://www.biology.arizona.edu/immunology/tutorials/antibody/structure.htmlhttp://www.accessexcellence.org/RC/VL/GG/monoclonal.phphttp://www.antibodyresource.com/educational.htmlhttp://www.antibodyresource.com/educational.htmlhttp://www.accessexcellence.org/RC/VL/GG/monoclonal.phphttp://www.biology.arizona.edu/immunology/tutorials/antibody/structure.htmlhttp://www.biology.arizona.edu/immunology/tutorials/antibody/structure.htmlhttp://www.youtube.com/watch?v=48VSU4AZ-L0http://www.youtube.com/watch?v=48VSU4AZ-L0http://www.youtube.com/watch?v=48VSU4AZ-L0


24/24

24

monoclonal antibodies and engineered antibodies

Documents