1. Monoclonal antibodies preparation and applications DEPARTMENT OF PHARMACEUTICS

2. ANTIBODY: Immunoglobulin synthesized by the body’s immune system in response to a foreign molecule(antigen).

3. Classification of Antibodies Antibodies are mainly classified into following types Polyclonal antibodiesMonoclonal antibodiesChimeric antibodiesHumanized antibodiesBispecific antibodiesImmunoconjugate.

4. These are the antibodies that are obtained from different B cell resources. They are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope.

5. These cultures of B-cells are called Monoclonal as they are derived from a single ancestral B-cell. These cells are used to harvest single kind of antibodies called as Monoclonal antibodies. The structure heavy and light chains of all Mo Ab’s will be identical. They are highly specific and offer more consistent efficacy. Monoclonal Antibodies: Monoclonal antibodies(MoAb’s) are specific antibodies produced by fusing B-cells(B- lymphocytes) derived from a single ancestral B-cell with a tumour (myeloma) cell.

6. Earliest MoAb’s were of non human origin which are immunogenic and exhibit human antimouse response(HAMA).To overcome this problem, antibody was cleaved into Fc and Fab fragments by papain digestion. Antibody Fragments: The Fab fragments are less immunogenic than intact antibodies,penetrate more into tumour cells, longer halflife. But, antigen binding capacity is lost up to some extent.

9. The resulting immunoconjugate may contain a spacer between drug and antibody (or) a polymer to increase the number of drug molecules that can be bound to each antibody. The drug can be incorporated noncovalently into a liposome or microsphere to which the targeting antibody is bound to surface is known as Immunoliposome or Immunomicrosphere. For MoAb’s targeted drug delivery, a drug is bound covalently to an antibody

11. Mice are immunized with an antigen that is prepared for injection either by emulsifying the antigen with Freund's adjuvant or other adjuvants or by homogenizing a gel slice that contains the antigen. Intact cells, whole membranes, and microorganisms are sometimes used as immunogens. In almost all laboratories, mice are used to produce the desired antibodies. In general, mice are immunized every 2-3 weeks but the immunization protocols vary among investigators. When a sufficient antibody titer is reached in serum, immunized mice are euthanized and the spleen removed to use as a source of cells for fusion with myeloma cells. Step 1: Immunization of Mice and Selection of Mouse Donors for Generation of Hybridoma Cells

12. After several weeks of immunization, blood samples are obtained from mice for measurement of serum antibodies. Several humane techniques have been developed for collection of small volumes of blood from mice. Serum antibody titer is determined with various techniques, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry. If the antibody titer is high, cell fusion can be performed. If the titer is too low, mice can be boosted until an adequate response is achieved, as determined by repeated blood sampling. When the antibody titer is high enough, mice are commonly boosted by injecting antigen without adjuvant intraperitoneally or intravenously (via the tail veins) 3 days before fusion but 2 weeks after the previous immunization. Then the mice are euthanized and their spleens removed for in vitro hybridoma cell production.

13. Step 3: Preparation of Myeloma Cells Fusing antibody-producing spleen cells, which have a limited life span, with cells derived from an immortal tumor of lymphocytes (myeloma) results in a hybridoma that is capable of unlimited growth. Myeloma cells are immortalized cells that are cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine-aminopterin- thymidine (HAT) selection medium used after cell fusion. A week before cell fusion, myeloma cells are grown in 8-azaguanine. Cells must have high viability and rapid growth. The HAT medium allows only the fused cells to survive in culture.

14. Step 4: Fusion of Myeloma Cells with Immune Spleen Cells

15. Transferred to 96 well plastic culture plates. Supernatant assayed for particular antigen specificity by  If a sample of supernatant contains the desired antibody.  It will bind to the antigen and will remain associated with the well and the unbound material is washed off. ELISA RIA

16. Single cells secreting the desired Ab are isolated from positive cultures and propagated into cell lines by two cloning techniques 1) Limiting dilution: C ells in the cultures are diluted and aliquoted into new wells so that only one cell found in any well. Cells are allowed to re grow and the procedure is repeated for several times to increase the probability that all cells in be a given well are monoclonal. 2)Soft agar: If the culture can be reliably dispersed into single cells then the visible colonies obtained which are picked out from the agar said to be monoclonal.

18. Commercial production of monoclonal antibodies

19. Antibody based drug delivery systems Preparation by an efficient method which gives high yield and capable of scale up. High stability of conjugate under shelf storage conditions and in circulation after injection. Retention of antigen binding ability of antibody while it is carrying the drug to target tissue. Upon reaching the target, the immuno- conjugate it self should show the desired pharmacological effect equivalent to free drug (or) must release the free drug or a derivative that is fully efficacious. Ideal Antibody directed delivery system characterstics:

20. The drug can be covalently bound to MoAb directly or through a short spacer or through a linker such as a water souble polymer. A carrier such as liposome or microsphere can be used where in the drug is entrapped in (or) bound to the carrier and the MoAb is bound to the surface of carrier. A variety of coupling reagents are available that aid in optimizing the properties of an immunoconjugate. Amino,sulfhydryl and carboxyl groups are the most common functional groups on the antibody,carrier and drug molecules used for coupling.

21. If the drug lacks the desired group, it may be possible to induce that group. Eg : Alcohol / Amine Succinicanhydide Carboxyl group. Amine 2-iminothiolane Sulfhydryl Classical protein cross linking reagents are used for linkage of drug to antibody. Eg: Carbodiimide reagents like Dicyclohexylcarbodiimide (DCC) and its water soluble analogs link amino groups with carboxyl group via amide bonds.

22. Dextran and similar carbohydrate linkers are used for conjugation of drugs and antibody. Recently, a number of bifunctional reagents have been developed that are more specific in forming linkages of antibody to drug. Eg: N-succimidyl-3-(pyridyldithio)propionate(SPDP) reagent is used to derivatize drug with a pyridyl disulfide group.This species react with the antibody containing free sulfhydryl groups,yeilding immunoconjugate. Combinations of classical coupling methods with bifunctional reagents have also been used for preparation of immunoconjugates. Eg: A 6 carbon spacer ending in a carboxyl group was introduced into dextran and then mitomycin-c was coupled to the spacer with a carbodiimide. Active ester method Carboxyl groups of drug are linked to N-hydroxysuccimide in the presence of a carbodiimide to form active ester derivative of drug.It then reacts with Amino group of antibody.

23. The remaining carboxyl groups of spacer were modified to amino groups and were then coupled to MoAb-A7 by means of SPDP,with a final MMC/ MoAb ratio of 40. The antibody activity of resulting conjugate was almost equivalent to native MoAb A 7 and release free MMC by chemical hydrolysis. Bifunctional reagents allow incorporation of a metabolizable linker into the immunoconjugate, releasing free drug or an active derivative at a predictable rate. A number of studies have explored coupling methods that allow control of the invivo rate of release of active drug. Eg: Free Doxorubicin was released from conjugated antibody linked by hydrazone with in 6 hours at 37 0 at pH 4.5. Daunomycin conjugates linked to MoAb-L6 via a polylysine and aconitate linkage, the conjugate releases free drug at pH 6.0.

24. The MoAb’s interact with growth factor receptors such as transferrin receptors show an anti tumour effect because transferrin is essential for the growth of cells and its receptors are predominantly present on proliferating cells

27. Cancer cells are heterogenous, so those cells that are not recognised by MoAb can escape and proliferate. Some tumours contain semidead cores with poor circulation and thus cannot be reached by monoclonals. MoAb’s can interact with circulating target antigens before reaching this target. Patients can experience possible immunogenic reactions. A MoAb is not as specific invivo as would be predicted from invitro studies.i.e, tumour antibodies may bind to normal cells as well as target cells. MoAb’s have poor penetration incase of solid tumors due to lack of vasculature. For these reasons, it is proven more effective to combine MoAb with standard chemotherapeutic agents. Problems associated with MoAb’s

28. Principle of targeting: MoAb’s are used for active targeting of immunotoxins and drugs(i.e,drug immunoconjugates). A number of anti tumour agents including chlorambucil, methotrexate, Daunomycin and Doxorubicin conjugated to tumour specific antibodies. Eg: Doxorubicin-BR96 immunoconjugate. It is a chimeric MoAb specific for lewis antigen found on the surface of tumour cells. CMA-676 which is a conjugate of an anti-CD33 MoAb and calicheamicin, an anticancer drug is 1000 times more potent than Doxorubicin. Bispecific MoAb’s composed of anti-CD3 or anti-CD2 MoAb, chemically conjugated to antitumour antibody and coated on lymphokine activated killer(LAK) cells have been clinically investigated for the treatment of malignant glioma,lymphoma and ovarian cancer. Applications of Monoclonal antibodies in Drug delivery

30. APPLICATIONS A.Diagnostic tests 1.Diagnostic imaging (a)Cardiovascular diseases (b)Sites of bacterial infection (c)Cell surface markers (d)Detection of circulating antigens (e)Cancer (f)Hormones (g)Immunometric assay procedures

31. B. Therapeutic applications 1.General (a)MAB itself (b) Radio isotope immuno conjugates (c) Toxin drug conjugates 2. Transplantation (a)Organ (b)Bone marrow 3. Infectious diseases (a) micro organisms (b) parasites 4.Enzyme and proteins 5.Cardiovascular disease 6.Auto immune disease 7.Cancer 8.Antidotes

32. C. Investigational and analytical applications 1.Lymphocyte phenotying 2.Purification of proteins 3.Radioimmuno assay D. Drug targeting 1.Immunotoxins 2.Supresser deletion 3. Site specific modification 4. Antibody enzyme conjugates E. Miscellaneous 1.Abzymes 2.Auto antibody finger printing

33. Pure one molecular species only Specificity for one antigenic determinant. Antibodies titer values obtained are very high Antibodies with avidity can be produced Invitro or invivo production is possible with high production rate Maintainance of farm/animals is not required for immunization and bleeding

34. Antiserum having identical antibody with an identical specificity and constant property can be obtained world wide High reproducibility with respect to specificity and avidity Producion of cell lines to individual components of a mixture is possible Radiolabelling and flourescent conjugation or enzyme marking of MAb’s are easy

35. Monoclonal antibodies (mAb) are important reagents used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer. As antibody-based drugs come to the forefront of therapeutics derived from biotechnology, they provide a range of new and exciting opportunities to treat a number of currently unmet medical needs CONCLUSION

36. A REPORT OF THE COMMITTEE ON METHODS OF PRODUCING MONOCLONAL ANTIBODIES NATIONAL RESEARCH COUNCIL. ENCYCLOPEDIA OF PHARMACEUTICAL TECHNOLOGY PHARMACEUTICAL BIOTECHNOLOGY S.P VYAS AND V.K DIXIT REFERENCES