This document discusses site-specific drug delivery using monoclonal antibodies (MAbs). It describes how MAbs are produced through hybridoma technology, fusing antibody-producing cells with myeloma cells. MAbs can then be conjugated to drugs, toxins, or radioisotopes for targeted delivery to cells expressing the antigen recognized by the MAb. Several factors must be considered for effective drug-MAb conjugates, including antigen expression levels, internalization of the antigen-antibody complex, and clearance of the MAb from the bloodstream. Current research highlights include MAbs conjugated to drugs for cancer treatment and interleukins for conditions like AIDS and septic shock.

2. Site-specific drug delivery utilizing
monoclonal antibodies
Contents
I. Introduction
A. Chemistry
B. Polyclonals vs. monoclonals
C. Conjugation of antibodies
II. Production of monoclonal antibodies
III. Drug-monoclonal antibody conjugates for drug targeting
A. Principles
B. Drug antibody bonding
IV. A. Highlights of current research

3. Introduction
In 1976, Kohler and Milstein employed a
method of somatic-cell hybridization in order
to successfully generate a continuous
“hybridoma” cell line capable of producing
monoclonal antibody (MAb) of a defined
specificity. Subsequently, several MAbs have
exhibited specificity for target sites. It is this
property of MAbs that makes them excellent
candidates as carriers of therapeutic agents for
delivery to specific sites.

4. Chemistry
Antibodies are complex proteins, consisting of
multiple polypeptide chains that contain a variety
of reactive chemical groups, such as amino,
carboxyl, hydroxyl, and sulfhydryl. Functionally,
MAbs possess a molecular polarity based on the
joining of an antigen-binding fragment (Fab) to a
complement- fixing fragment (Fc). The Fab
fragment is responsible for specific antigen
binding, whereas the Fc fragment binds to
effector cells, fixes complements, and elicits other
in vivo biological responses.

6. Polyclonals vs. monoclonals
Polyclonal antibodies Monoclonal antibodies
Produces large amounts of non specific
antibodies
Can produce large amounts of specific
antibodies but may be too specific
Recognizes multiple epitopes on any one
antigen
Recognizes only one epitope on an antigen
Inexpensive to produce Expensive to produce
Technology required is low High technology required
Skills required are low Training is required for the technology use
Time scale is short Time scale is long for hybridomas
Can be batch to batch variability
Once a hybridoma is made it is a constant
and renewable source and all batches will
be identical

7. Conjugation of antibodies
1. Radioisotope conjugates: 131-I labeled Mab
2.Toxin conjugates: Ricin immunoconjugates
3.Drug conjugates: Conjugates of methotrexate
with Mab

8. Production of monoclonal antibodies
Monoclonal antibodies are antibodies which
have a single, selected, specificity and which are
continuously secreted by "immortalised"
hybridoma cells. A hybridoma is a biologically
constructed hybrid between an antibody-
producing, mortal, lymphoid cell and a
malignant, or "immortal", myeloma cell.
Hybridomas utilizing hybridoma technology are
routinely made in stepwise conventional small-
scale culture procedures.

9. HAT Selection
Immunize mouse
Isolate spleen cells Myeloma
cells(HGPRT-)
Non fused spleen cells(s)
Non fused myeloma cells (M)
Fused (s-s)
Fused(m-m)
Fused(s-m)
HAT
medium
s-m groW & divide
M&M_M; s& s –s can not
divide

11. Production of monoclonal antibodies
1. The antigen, a foreign substance, such as a
lung cancer cell, is injected into a mouse. The
spleen is then removed, and the antibody
producing cells are collected.
2. Myeloma cells are isolated from a mouse
tumor.
3. Spleen and tumor cells are fused together to
form hybridomas. The surviving have the
spleen cell’s ability to produce antibodies and
the tumor cell’s ability to reproduce.
“hybridomas.”

12. Production of monoclonal antibodies
4. Each hybridoma is isolated and allowed to grow
into a large colony of cells that produce a single
MAb.
5. Each MAb is screened for its ability to attack the
original cancer cells, and the hybridomas producing
the desired antibody are kept.
6. The desired hybridoma cells are injected into a
mouse where they form a tumor that produces
large amounts of concentrated antibody. The first
critical step in generating a therapeutic or
diagnostic MAb— after initial isolation — is to
produce the antibody product.

13. Drug-monoclonal antibody conjugates for
drug targeting
Use of MAbs in targeting cytotoxic drugs to specific
tissues on cancer therapy has been studied
considering the followings:
• Number of antigen molecules per cell surface
• Number of cells expressing the reactive antigen in
the tumor mass
• Size of the tumor mass
• Fate of the antigen-antibody complex (stability on
cell surface, internalization, capping, shedding)

14. Drug-monoclonal antibody conjugates for
drug targeting
• Degree of tumor vascularization
• Degree of tumor mass infiltration and necrosis;
presence and reactivity of circulating antigen in
the blood
• Duration of MAb binding to cell surface
• Isotype of immunoglobulin (IgG subtypes or
IgM)
• Species of immunoglobulin (murine, human, or
chimeric recombinant)

16. Drug-monoclonal antibody conjugates for
drug targeting
• Whole immunoglobulin or fragments (Fab,
Fab’, F(ab’)2)
• Clearance of Mab from blood, excretion, or
reticuloendothelial system
• Dose of MAb used
• Route of inoculation of MAb (intravenous,
intraperitoneal, intralymphatic, or
intraarterial)

17. Drug-monoclonal antibody conjugates for
drug targeting
• Development of a human immune response to
the administered MAb.

19. Requirements of antibody-drug conjugates
• The antibody has to be humanized to avoid an
immune response
• It has to show selectivity for an antigen which
is over expressed in cancer cells
• It needs to be internalized into the cell by
receptor-mediated endocytosis
• The link between the antibody & the drug
should be stable

22. Highlights of current research
Corporation Product Usage
Biogen CD4 agent In AIDS
NeoRx Oncotrac Detect spread of
melanomas
Genentech Oncogene Breast and ovarian cancer
Cetus Prolenkin (interleukin-2) Anticancer
Xoma E5 (Xomen) Septic shock

23. Questions……?????

24. THANK YOU ALL