Chapter 3

3. Hybridoma Technology
3.1. Introduction to Hybridoma Technology
 What are antibodies?
 An antibody is a protein used by the immune system to identify
and neutralize foreign objects like bacteria and viruses. Each
antibody recognizes a specific antigen unique to its target.
 Monoclonal antibodies (mAb): homogeneous antibody
preparations produced in the laboratory. Consist of a single type
of antigen binding site, produced by a single B cell clone.
Polyclonal antibodies: antibody preparations from
immunized animals. Consist of complex mixtures of
different antibodies produced by many different B
cell clones
Clonal selection of B cell
Cont.,
1. Monoclonal Antibody
 Antibody from a single clone antibody producing B cell and
therefore only binds with one unique epitope
 B-cell is isolated and fused to an immortal myloma cell line so that
large quantities of identical antibodies can be generated.
 Are antibodies that are identical because they are produced by one
clones of a single parent cell.
 Basically produced by white blood cell which is called as plasma
cell.
 Is used for treatment of cancerous cells and as anti-venom( anti
snake venom)
 Advantages
 Can produce large quantities of identical antibody and have batch
to batch homogeneity.
 High specificity to a single epitope and Reduced probability of cross
reactivity.
 Can provide better results in assays requiring quantification of the
protein levels.
Disadvantages
 Significantly more expensive to produce
 Requires significantly more time to produce and develop the
hybridized clone
2. Polyclonal Antibody
 collection of antibodies from different B cells that recognize
multiple epitopes on the same antigen
 Each of these individual antibodies recognizes a unique epitope
that is located on that antigen.
 Advantages
 Inexpensive and quick to produce (Purified antibody ready to use
in under four months)
Higher overall antibody affinity against the antigen due to
recognition of multiple epitopes.
Offers greater sensitivity for detecting proteins that are present in
low quantities in a sample since multiple antibodies will bind to
multiple epitopes on the protein.
 Disadvantages
 Variability between different batches produced in
different animals at different times
FDC
 Higher potential for cross reactivity due to recognizing
multiple epitopes
 Affinity purification of the serum will typically be
required to minimize cross reactivity
 What Is Hybridoma Technology?
 Hybridoma Technology is a technique to generate
continuous supply of monoclonal antibodies.
 Hybridoma is a fusion of two cells – myeloma (plasma cell
tumor) cells and splenic B cells.
 Takes advantage of the unlimited growth capacity of
myeloma cells and cellular machinery to produce
antibodies of uniform antigenic specificity of B cells
 Hybrid Cells
 Normal splenic cells are fused with a cancerous cell line
 E.g. myeloma, lymphoma (NSO1)
 Fusion is accomplished with PEG (polyethylene glycol)
 The new hybrid cell exhibits properties of both cell
types:
 Unlimited growth (from myeloma cells)
 Secretes monoclonal antibody (from splenic cells)
 Or Secretes cytokines
 History of mAb development
 1964 Littlefield developed a way to isolate hybrid cells from 2 parent
cell lines using the hypoxanthine-aminopterin-thymidine (HAT)
selection media.
 1975 Kohler and Milstein provided the most outstanding proof of the
clonal selection theory by fusion of normal and malignant cells
 1990 Milstein produced the first monoclonal antibodies.
 Paul Ehrlich at the beginning of the 20th century theorized that a
cell under threat grew additional side-chains to bind the toxin, and
that these additional side chains broke off to become the antibodies
that are circulated through the body. It was these antibodies that
Ehrlich first described as "magic bullets" in search of toxins.
3.2. Generation of hybridomas:
Permanent cell lines secreting monoclonal antibodies
 Monoclonal Antibody Production technology was developed in 1975.
 Cell lines are produced by fusing B cells from the immunized
animal with the substance to be studied and myeloma cells
 The generation of mAb-producing cells requires the use of animals,
usually mice.
 The procedure yields a cell line capable of producing one type of
antibody protein for a long period.
 The fused product of a hybrid cell called a hybridoma and the
technology that produce those fused hybrid cells called hybridoma
technology
 To produce the desired mAb, the cells must be grown in either of
two ways
 injection into the peritoneal cavity of a suitably prepared mouse
 in vitro tissue culture.
 Procedure of production of monoclonal antibody
1. Immunization of specific animal which generate
hybridoma cell with spleen cell.
2. Isolation of myeloma cells.
3. Fusion between spleen cell and myeloma cell.
4. Selection of HAT medium.
5. Isolation of hybridoma cell.
6. Screening of hybridoma cell.
4. Selection of HAT medium
( Hypoxanthine, Aminopterin, Thymidine)
 Before multiplication of Anti-body, it has to synthesize new
copy of DNA and for that it require synthesis of nucleotide.
 For synthesis of nucleotide mainly two pathways are there:
1. Salvage pathway
2. De-novo Synthesis
 In 1 , Salvage pathway it requires degraded part of old
nucleotide to produce new nucleotide.
 In 2, De-novo synthesis it synthesized completely new
nucleotide by small molecules (sugar, amino-acid).
 So in HAT medium, Cells not synthesized by De-novo
synthesis due to presence of Aminopterin in HAT
medium which blocks Di-hydro follate enzyme which
is necessary for these synthesis.
 For synthesis in salvage pathway it must requires
HGPRT enzyme (Hypoxanthine Guanine Phospho-
Ribosyl Transferase).
 Where hypoxanthine and thymidine are used as
precursors.
 Fused myeloma and unfused myeloma didn’t have
HGPRT enzyme so, can’t survive in HAT medium.
 Fused plasma and unfused plasma have HGPRT
enzyme but didn’t have long-life.
 Hybrid cell has HGPRT enzyme from spleen cell as well
as they have the ability to multiply repeatedly as
myeloma cell.
 So, isolation of hybrid cell because is only cell which
survive in HAT medium.
6. Screening of hybridoma cell
 ELISA screening method which done by incubating
hybridoma culture in which secondary enzyme gets
conjugate and formation of colored product shows
positive hybridoma.
 ELISA (enzyme-linked immuno sorbent assay) is a highly
sensitive method for the detection of antibodies or soluble
antigens in sera and other body fluids.
 Used for multiplying the hybridoma cells
 In-vivo
 In-vitro
3.3. In vitro and in vivo production of monoclonal
antibody
I. In vivo production of
monoclonal antibody
 Mouse ascites method
 Hybridoma cells injected in mouse
 Produce ascites
 Fluid contains high concentration of Ab’s
 No further concentration required
 Purification required
 Easy and inexpensive
 Animal mortality
 Scientific Needs for Mouse Ascites Production of mAb
1. Some hybridoma cell lines do not adapt well to in vitro
conditions.
2. mAb from mouse ascitic fluids might be essential for
experiments in which mAb are used in mice.
3. Rat hybridoma cell lines do not generate ascites efficiently in rats,
usually adapt poorly to in vitro conditions, but usually generate
ascites in immunocompromised mice.
4. Downstream purification can lead to protein denaturation and
decreased antibody activity.
5. Serum-free or low-serum conditions cannot provide sufficient
amounts of mAb for some purposes, such as the evaluation of
new vaccines against infectious organisms.
6. Culture methods sometimes yield populations of IgG mAb
that are glycosylated at positions different from those
harvested from mouse ascites fluid, thereby influencing
antigen-binding capacity and important biologic functions.
7. When hybridoma cells producing mAb are contaminated with
infectious agents, such as yeasts or fungi, the cells often must
be passed through mice.
 Advantages of Mouse Ascites Method
 Usually produces very high mAb concentrations that often do
not require further concentration procedures that can denature
antibody and decrease effectiveness.
 The high concentration of the desired mAb in mouse ascites
fluid avoids the effects of contaminants in in vitro batch-culture
fluid when comparable quantities of mAb are used.
 Avoids the need to teach the antibody producer tissue-culture
methods.
 Relatively inexpensive and easy
 Disadvantages of Mouse Ascites Methods
 Involves the continued use of mice requiring daily observation.
 MAb produced by in vivo methods can contain various mouse
proteins and other contaminants that might require purification.
 Can be expensive if immuno deficient mice in a barrier facility
must be used.
 Can cause significant pain or distress in mice.
 There are ethical concerns with using animals.
II. In Vitro Production of Monoclonal Antibody
a) Batch tissue culture method:
 Grow hybridoma cells in batches
 Purify Mabs from the culture media
 Fetal bovine serum commonly used and contains bovine
immunoglobulin at about 50 mg/ml
 Low concentration
 Denaturation during concentration
 In most cases, hybridoma growing in 10% fetal calf serum(FCS)
can be adapted with in 8 – 12 days to grow in < 1% FCS or in FCS
free media. By this approach it yields concentrations that are
typically below 20µg/ml.
 The disadvantages of these methods are :
 Large volumes of tissue-culture media must be processed
 The mAb concentration achieved will be low (around a few
micrograms per milliliter), and
 Some mAb are denatured during concentration or purification.
 In fact, a random screen of mAb revealed that activity was
decreased in 42% by one or another of the standard
concentration or purification processes.
b) Semi permeable membrane based system :
 These devices are called semipermeable-membrane-based
systems.
 Isolate the cells and mAb produced in a small chamber
separated by a barrier from a larger compartment that contains
the culture media.
 A barrier – hollow fibre or a membrane , with a low-molecular-
weight cutoff (10,000-30,000 kD)
 Larger compartment containing culture media
 Smaller chamber to isolate cells and Mabs
 High concentrations
 Method of choice for large scale production
 Advantages of In Vitro Methods
 Reduce the use of mice at the antibody-production stage (but can
use mice as a source of feeder cells when antibody generation is
under way).
 Are usually the methods of choice for large-scale production by the
pharmaceutical industry because of the ease of culture for
production, compared with use of animals, and because of
economic considerations.
 Avoid the need to submit animal protocols to IACUCs.
 Avoid or decrease the need for laboratory personnel experienced in
animal handling.
 Using semipermeable-membrane-based systems produce mAb in
concentrations often as high as those found in ascitic fluid and are free of
mouse ascitic fluid contaminants.
 Disadvantages of In Vitro Methods
 Some hybridomas do not grow well in culture or are lost in
culture.
 Generally require the use of FCS, which limits some antibody
uses.
 The loss of proper glycosylation of the antibody (in contrast with
in vivo production) might make the antibody product unsuitable
for in vivo experiments because of increased immunogenicity,
reduced binding affinity, changes in biologic functions, or
accelerated clearance in vivo.
 In general, batch-culture supernatants contain less mAb
(typically 0.002-0.01) per milliliter of medium than the mouse
ascites method.
 In batch tissue-culture methods, mAb concentration tends to be
low in the supernatant; this necessitates concentrating steps that
can change antibody affinity, denature the antibody, and add
time and expense.
 Most batches of mAb produced by membrane-based in vitro
methods are contaminated with dead hybridoma cells and dead
hybridoma-cell products, thus requiring early and expensive
purification before study.
 MAb produced in vitro might yield poorer binding affinity than
those obtained by the ascites method.
 Are generally more expensive than the ascites method for small-
scale or medium-scale production of mAb.
3.4. Large-scale production of monoclonal antibodies
 The production MAbs in the culture bottles is rather low (5-10
mg/ml).
 The yield can be increased by growing the hybrid cells as ascites in
the peritoneal cavity of mice.
 The ascitic fluid contains about 5-20 mg of MAb/ml.
 This is far superior than the in vitro cultivation techniques.
 But collection of MAb from ascitic fluid is associated with the
heavy risk of contamination by pathogenic organisms of the
animal.
 In addition, several animals have to be sacrificed to produce MAb.
 Hence, many workers prefer in vitro techniques rather than the use
of animals.
 Large amounts of antibody can be produced using
animals
– Prime Balb/c animal with IFA or pristane
– Inject clone i.p
– Collect peritoneal ascites
 Alternatively bioreactors are used
– Cells are cultured in hollow fibers
– Fresh media and waste are recirculated
– High concentrations of Ab produced in cell compartment
– Collected at different time points
– Others: Roller bottle, Microcarrier, Membrane bound cell
culture processes
3.5. Types of monoclonal antibodies
 Origin First generation
 Murine, rabbit or rat proteins purified after immunisation with antigen
 Abs to these proteins (Ag) generated in patients: human antimurine
antibody (HAMA)
 Block effectiveness of therapy
 Adverse events serum sickness or anaphylaxis
 Origin Second generation
 DNA technology or genetic engineering used to construct hybrids
composed of human Abs regions with murine
 Chimeric Abs
 Humanized
 Human
Evolution Of Monoclonal Antibodies
1. Murine
 Derived from mice
 Patients treated with
murine mAbs develop a
human antimouse antibody
(HAMA) response
 Rapid clearance of the mAb
 Poor tumour penetration
 Hypersensitivity reactions
 90Y-ibritumomab
 131I -Tositumomab
 Whole of the antibody
is of murine origin
 Major problems
associated with
murine antibodies
include
 Reduced stimulation
of cytotoxicity
 Allergic reactions
 Anaphylactic shock
2 Chimeric Abs
 Antigen binding parts (variable region) of mouse with effector
parts (constant region) of human
 Infliximab
 Abciximab
 Rituximab
 Chimeric antibodies are those in which the Fc part of an
immunoglobulin is of a human sequence
 Antibodies are approximately 65% human.
 This reduces immunogenicity and thus increases serum half-life.
3. Humanized
 Human Ab with complimentary determining region (CDR) or
hypervariable region from non human source
 Daclizumab
 Trastuzumab
 Humanised antibodies contain segments from sources in the
complementary determining regions (CDR) interspersed among
human derived segments in constant regions
 This results in a molecule of approximately 95% human origin
4. Human Abs
 Recombinant DNA technology:
 Genes for variable Fab portion of human Abs is inserted in
genome of bacteriophages & replicated
 Mixed with Ag & complementary Ab producing phages selected
e.g. Adalimumab
 Human monoclonal antibodies are produced by transferring
human immunoglobulin genes into the murine genome, after
which the transgenic mouse is vaccinated against the desired
antigen, leading to the production of monoclonal antibodies
3.6. Application of monoclonal antibodies
 The application of monoclonal antibodies can be broadly
categorized as:
 Diagnostic Application
 Catalytic Mab (Abzymes)
 Therapeutic Application
1. Diagnostic Application
 The western blot test & immuno dot blot tests detect the protein
on a membrane.
 Useful in immunohistochemistry, which detect antigen in fixed
tissue sections.
 Immuno fluoresence test,which detect the substance in a frogen
tissue section or in live cells.
 MAbs are utilized in diagnostic kits for the diagnosis of various
infectious diseases, detecting pregnancy, monitoring drug
levels, matching histocompatibility antigen, detecting diabetes,
cancer and in immunoscintigraphy.
 FDA licensed a new diagnostic imaging agent that can
determine the extent of disease in patients diagnosed with
small cell lung cancer (SCLC). Because these agents can detect
tumor in different part of the body at one time, it can help
physician to advice certain patients with advanced forms of the
disease about treatment option without requiring further
diagnostic tests.
 The new agent, Nofetumomab, is a fragment of a monoclonal
antibody that when tagged with the radioisotope technique,
can detect a protein found on the surface of most small lung
cancer cells.
2. Catalytic Mab (Abzymes)
 Abzymes are usually raised in lab animals immunized against
synthetic haptens, but some natural abzymes can be found in
normal humans (anti-vasoactive intestinal peptide
autoantibodies) and in patients with autoimmune diseases such
as systemic lupus erythematosus, where they can bind to and
hydrolyze DNA.
 The antibodies are extremely efficient at binding ground states of
the target molecule while enzymes obtained their catalytic
efficiency from tight binding of the transition state for the
reaction.
 Thus antibodies can be made efficient catalysts if they are made
for reaction transition state.
3. Immunoconjugate
 These are antibodies conjugated (joined) to a second molecule,
usually a toxin, radioisotope or label.
 For mAB’s targeted drug delivery, a drug is bound covalently to
an antibody.
 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.
4. Therapeutic Application
 Improving the outcome of bone marrow transplantation by using
CD52 MAbs to prevent Graft-Versus-Host disease and Graft
rejection.
 Graft-versus-Host Disease (GVHD) is a major cause of mortality
and mobidity after allogenic bone marrow transplantation, but
can be avoided by removing T- lymphocytes from the donor bone
marrow.
 However, T-cell depletion increases the risk of graft rejection. This
study examined the use of CD52 MAb to eliminate T- cells from
both donor marrow and recipient to prevent both GVHD and
rejection.
 Alemtuzumab is the monoclonal antibody used for this purpose.
5. mAb In Tissue Transplantation
 Muromonoab-CD3:
 Muromonoab-CD3 is used for the treatment of acute organ
transplant rejection.
 It is effective in preventing graft rejection after kidney, heart or
liver transplantation.
 Muromonoab-CD3 is effective in patients who after acute
cardiac or liver allograft rejection do not respond to steroid
therapy.
6. Monoclonal Antibodies For Cancer
 Radio immunotherapy
 Antibody-directed enzyme prodrug therapy (ADEPT)
 Immunoliposomes ADEPT(antibody directed enzyme prodrug
therapy)
 Antibody Dependent Cell-mediated Cytotoxicity (ADCC)
 Complement Dependent Cytotoxicity (CDC)
 scFV(single-chain Fv fragment)
 Cancer therapy
 Rituximab is a chimeric mAb that targets the CD20
B-cell antigen – B cell lymphomas
 Trastuzumab (Herceptin) – anti breast cancer
(against HER2/neu (erbB2) receptor)
 Gemtuzumab ozogamicin (Mylotarg) -calicheamycin
conjugated mAb against acute myelogenous leukemia
(AML)
7. mAb In Auto Immune Diseases
 Monoclonal antibodies used for autoimmune diseases , which are
effective in rheumatoid arthritis, crohn’s disease and ulcerative
colitis (Infliximab, Limumab).
 Preventing acute rejection of kidney transplants
(Basiliximab,Daclizumab).
 Useful in moderate to severe allergic asthma (Omalizumab).
 Immunoliposomes are used for intracellular delivery of
compounds that intrinsically do not enter the diseased cells.
 Eg: Methotrexate-γ-aspartate.
3.7. Clinical applications Of Monoclonals In
Autoimmunity and Transplantation Medicine
 Zenapax
 Targeted to IL-2 Receptor alpha subunit on activated T-Cells (Anti-
TAC)
 Modulates acute kidney rejection
 Orthoclone OKT3
 Targeted to CD3 co-receptor on activated T-cells
 Controls rejection in liver, heart, and kidney transplants
 Remicade
 Targets Tumor Necrosis Factor mediator of inflammation
 Treatment of Autoimmune Rheumatoid Arthritis and Crohn’s
Disease
 Xolair
 Antibody to IgE (Antibody to an antibody)
 Type 1 Allergy Treatment
 mAb In Allergy
 Allergic disorders, including asthma, allergic rhino conjunctivitis,
atopic dermatitis, food allergies, urticaria and anaphylaxis have
significant impacts on our daily lives.
 Metabolic disorders
 mAbs targeting secreted fatty acid–binding protein
aP2 -new treatment for type 2 diabetes
 Evolocumab and Alirocumab substantially reduced
the LDL-C level by over 50%
 Infectious diseases
 Raxibazumab - for the treatment of infectious
inhalational anthrax
 Tocilizumab - anti IL-6 mAb used for treating
COVID-19