monoclonal antibodies

1. Monoclonal Antibodies
MONOCLONAL ANTIBODIES
Most antigens such as a bacterial cell possess multiple epitopes and multiple
copies of each of those epitopes. Based on this fact, upon injection one would
expect a variety of B cells to interact with the various epitopes.
The resulting antiserum would be a heterogeneous mixture of antibodies each
specific for one epitope among the various epitopes = a polyclonal response.
A polyclonal response has advantages for host in that it facilitates the
localization, phagocytosis, and complement mediated lysis of antigen.
However in experimental situations, antibody heterogeneity often clouds
the results and reduces the antiserum’s efficiency.
For most research, diagnostic, and therapeutic purposes a monoclonal
antibody is needed-that is, an antibody derived from a single clone and
thus specific for a single epitope is preferable.
Monoclonal antibody production by somatic cell fusion or hybridoma
technology was introduced by Kolher and Milstein in 1975 (got Nobel prize in
1984). Overall: The technique involves fusing a normal antibody producing B cell
with a myeloma cell to produce a hybrid cell or hybridoma. The hybridoma
would possess the immortal growth properties of the myeloma cell while
secreting the antibody produced by the B cell. The resulting hybridoma could be
cultured indefinitely thus providing large amounts of homogeneous antibody for
research purposes.
The usefulness of monoclonal antibodies stems from 3 characteristics-
their specificity of binding,
their homogeneity, and
their ability to be produced in unlimited quantities.
*Additionally, one unique advantage of hybridoma production is that impure
antigens can be used to produce specific antibodies. This is based on the fact that
you screen with the pure antigen for the antibody of choice and that Ab is
produced by isolating a single cell clone!

2. MONOCLONAL PRODUCTION
First mice are injected with an Ag+adjuvant mixture- mice generally
receive a primary injection followed by two boosts (14 to 28 days later).
Why?
After the final boost the mice are euthanized 3 to 5 days later and the
spleen is harvested for cell fusion.
A single cell suspension from the spleen is made and mixed with the
myeloma partner. A fusing agent such as polyethylene glycol is then
added. PEG fuses the plasma membranes of adjacent cells forming a
single cell with two or more nuclei.
This heterokaryon retains these nuclei until the nuclear
membranes dissolve prior to mitosis. During mitosis and
further rounds of division, the individual chromosomes are
segregated into daughter cells. Often chromosomes are lost.
Even in the most efficient hybridoma fusions, only about 1%
of the starting cells are fused, and only about 1 in 105 form
viable hybrids. This leaves a large number of unfused cells
still in the culture. The cells from the immunized animal do
not continue to grow in tissue culture because they are
terminally differentiated cells and thus only capable of
limited growth, and so do not confuse further work.
However, the myeloma cells are well adapted to tissue
culture and must be killed. This is usually done by drug
selection.
DRUG SELECTION
Cells have two pathways for the synthesis of nucleotides, the de novo and
salvage pathways. Since cells in tissue culture can survive by using either of
these pathways, mutations in the enzymes responsible for nucleotide synthesis
have become common and readily manipulated targets for mutagenesis of
mammalian cells. The most common target is the enzyme hypoxanthine-guanine
phosphoribosyl transferase (HGPRT).
HPGRT performs one of the essential steps in the salvage pathway
Mutations in the HPGRT gene can be selected by growing cells in the
presence of purine analogs such as 8-azoguanine (8-AG). HPGRT will
recognize 8-AG as a substrate and convert it to the monophosphate

3. nucleotide. The 8-AG-containing nucleotide is then processed further and
incorporated into DNA and RNA, where it is toxic substitution. Therefore,
cells with a functional HPGRT enzyme grown in the presence of 8-AG will
die.
However, because the HPGRT enzyme is part of a nonessential pathway
(the de novo pathway is still available) cells harboring a mutant HPGRT
gene can continue to grow.
Therefore, selection with 8-AG will kill cells with a wild-type HPGRT but
will not affect cells with a mutant HPGRT. The normal rate of mutagenesis
is sufficient for treating cells with 8-AG and the surviving cells are
HPGRT deficient.
SP2/0 myeloma cell line from the ATCC (american type culture collection)
The myeloma cell line does not synthesize or secrete any immunoglobulin
chains, and is HGPRT-.
HAT Selection for Hybridoma
Drugs like aminopterin block de novo synthesis of nucleotides. Both
purine and pyrimidine synthesis are inhibited so both need to be
produced by the salvage pathway and precursors for these pathways
(such as hypoxanthine and thymidine) must be included in the selection
media.
Myeloma fusion partners are deficient in the enzyme required for the
salvage pathway of nucleotide synthesis. These cells will die in HAT
containing medium because aminopterin blocks normal nucleotide
synthesis and the enzyme deficiency blocks utilization of the salvage
pathway.
If the myeloma and B cell fuse, the resulting hybridoma will live
indefinitely in culture because the normal cell supplies the missing
enzyme and the myeloma immortalizes the cell line. Unfused normal cells
will only survive in tissue culture for approximately 1 week before they
die off.
After 7-10 days, most of the wells contain dead cells but a few contain
clusters of viable cells - each cluster represents a clonal expansion of a
hybridoma!

4. At this point it is necessary to screen your fusion. We use the ELISA
(enzyme linked immunosorbent assay) method. Screening at this time
point is important because non-Ab producing hybrids will overgrow the
slower growing Ab producers!
ELISA
Coat 96 well plates with antigen at 1 ug/ml in carbonate buffer pH 9.6
(add 100ul per well). Incubate overnight at 4oC. (Polystyrene binds protein
noncovalently).
Remove the contents of the wells and block plate with 3% NFDM in PBS
to block nonspecific antibody binding. Incubate. (Blocking must be
performed to prevent nonspecific binding of Igs to any sites on the plate).
Wash plate with PBS-tween.
Add 100ul of (antibody containing) supernatant from Fusion into Elisa
plates. Incubate.
Wash plate and add 100ul of anti-Ig labeled with horseradish peroxidase.
Incubate then wash plate.
Add 100ul of substrate solution to each well. Incubate.
Evaluate wells for positives- If an Ag+Ab+enzyme labeled Ab + enzyme
substrate is in the well there will be a color reaction!
Positive wells contain hybrids making specific Ab to your test Ag!
Once clones have been identified, the positive wells need to be expanded
and cloned by limiting dilution.
You can’t be sure that the positive well arose from only one hybrid clone
so the cells have to undergo limiting dilution- you dilute out the cells in
one well into a series of wells until only one cell is in the well to insure
clonality.
After each cloning the cells are again screened by ELISA to insure Abs are
still being produced.
Because hybridoma cells have a very low plating effiency, single cell
cloning is normally done in the presence of feeder cells. We use a single

5. cell suspension of spleen cells-these cells produce growth factors and also
provide cell contact for your hybridoma.
We usually do limiting dilution 3 consecutive times to one clone to assure
clonality. ELISA screening is performed each time.
At this point, once you have a clone the cells can be expanded for
antibody production and experimental use!
Cells can be frozen in a DMSO-FCS mixture and stored in liquid nitrogen
for years. * Cells should be frozen down at each step of the cloning
procedure in case of contamination. If you do get contamination-you don’t
have to start all over- you simply retrieve the cell line from the previous
cloning and start at that point!
CLINICAL USE- Wagner et al. Hybridoma 16(1):33-40 1997
Generated an IgG1 murine monoclonal Ab -anti-idiotypic Ab (Ab2) designated
ACA125 which mimics an epitope on the tumor associated antigen designated
CA125. This antigen is expressed by most malignant ovarian tumors.
Used ACA125 Ab2 for vaccine therapy in 16 patients with advanced
epithelial ovarian cancer. Patients with CA125 positive tumors are
immunologically tolerant to CA125 -in other words they don’t produce an
immune response to it.
Tolerance - a state in which lymphocytes are present that would
normally respond but are somehow prevented from doing so.
It affects both the T and B lymphocytes.
When the antigen is cleared, and with production of new
lymphocytes - you eventually regain response ability- so tolerance
is a temporary state.
Certain conditions favor tolerance induction:
state of the lymphoid system- its easier to induce with
immature immune systems or immune systems damaged by
irradiation, drugs, ect
physiochemical properties of the Ag-size (small, soluble Ags are
harder to capture from circulation by the phagocytic system,
chemical nature (d-amino acids are toleragens whereas l-

6. amino acids are immunogens) , epitope density on molecule
(too many induces tolerance)
route of Ag administration- determines accessibility of Ag to
MOs
Ag dose- very low and very high doses induce tolerance.
Does the tumor display a very low or very high epitope density to
induce tolerance? Does it flood the system with soluble Ag?
The patients received 3 to 19 injections of 2mg/inj of the Ab2.
Patients showed development of specific humoral and cellular immune
responses to an otherwise nonimmunogenic tumor antigen. They
developed Ab3 (anti-anti-id) and had an increase in IFNg production.
(*Remember an increase in IFNg results in an increase of TNF).
9 patients produced the Ab3 with few side effects and displayed a median
progression free survival for 11 +/-5.6 (5.4-16.6) months compared to 8+/-
4.2 (3.8-12.2) months in the Ab3 negative group.
Human Hybridomas
When mouse monoclonals are introduced into humans they are recognized as
foreign and evoke an Ab response. The induced human anti-mouse antibodies
quickly reduce the effectiveness of the mouse monoclonal by clearing it from the
bloodstream. Also, circulating immune complexes of mouse + human Abs can
cause allergic reactions or you can get a buildup of complexes on the basement
filter membranes of the kidney- glomerulonephritis. One area of development
over the last 5 years has been the development of systems for the production of
human hybridomas. Human monoclonal antibodies can be used for clinical
applications.
The progress has been slow because of the lack of a suitable myeloma
partner but several line have been isolated and are now in use.
The use of EBV transformed Ab secreting cells has solved some of the
problems but these transformants seldom secrete large amounts of Abs.
This has been overcome in some cases by fusing the EBV transformed cell
with a mouse myeloma cell line to increase Ab secretion- a
heterohybridoma.

7. Also there is the problem of obtaining Ag primed B cells in humans-can’t
take their spleens! So human hybridomas have to be prepared from B cells
in human peripheral blood. Lymphocytes only make up about 20-30% of
your WBC population and most (~90%) of the lymphocytes in circulation
are T cells.
Plus human volunteers can not be just immunized with a range of
antigens- have tried in vitro Ag priming of the B cells but conditions are
not optimal so only see low affinity IgM production.
Immunotoxins
Tumor specific monoclonal antibodies with lethal toxins coupled to it are
potentially valuable therapeutic reagents. Toxins such as ricin and shigella are so
toxic that one molecule can kill a cell. These toxins are composed of two chains-
one is the toxin chain and the other is a binding chain that interacts with
receptors on the cell surface. Without this binding chain the toxin is harmless
since it cannot enter the cell. In theory: the monoclonal Ab with the attached
toxin chain will bind specifically to a tumor cell where it will be endocytosed and
the toxin will cause death by inhibiting protein synthesis.