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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5992_Библиотеки_им_академика_М_И_Перельмана.pdf
INTRODUCTION
Antibodies are glycoprotein molecules present in the serum. They are produced in response to
antigens that are protein or polysaccharide molecules and may be foreign to the body. Antibodies
are secreted by a class of blood cells known as B lymphocytes, which form a clone of cells known as
plasma cells. The plasma cells produce proteinaceous molecules known as antibodies. Antibodies
are produced when the body comes in contact and is invaded by foreign particles. Different types
of antibodies may be synthesized depending upon different types of stimulants involved.
Antibodies can be classified into five major classes: (1) IgG, (2) IgM, (3) IgA, (4) IgE
and (5) IgD. Because of the multiple epitose on the antigen that could induce proliferation and
differentiation of a variety of B cell clones, the antibodies synthesized in response to an antigen are
usually heterogeneous. The heterogeneity of antibodies that increases immune protection in vivo
often reduces the efficacy of an antiserum for various in vitro uses. Alternatively, it is also possible
to generate pure clones of plasma cell in vitro from which monoclonal antibodies (mAbs) with a
single antigenic specificity may be isolated.
Antibodies are a part of the defence system to protect the body against the invading foreign
substances, namely antigens. In response to an antigen, B lymphocytes gear up and produce
different antibodies. These types of antibodies that can react with the same antigen are designated
as polyclonal antibodies (PoAbs). mAbs are a single type of antibody that is directed against a
specific antigenic determinant.
The evaluation of technology of mAbs is an important milestone in biotechnology, representing
cumulative research efforts in immunology and cell culture methodologies. G. Kohler and
C. Milstein (1975) were the first to report on the production of mAbs. The first production of mAbs
Hybridoma Technology and
Monoclonal Antibodies
11
CHAPTER
Introduction
Principle for Creation of Hybridoma Cells
Large-Scale Production of
Monoclonal Antibodies
Applications of Monoclonal Antibodies
Questions
Textbook of Pharmaceutical Biotechnology
156
is an amalgamation of immunochemistry, in vitro cultivation of cancer cells and the molecular
biology of malignant transformation, representing three disciplines of basic medical research.
A new era in the immunology was launched in 1975 with the discovery of the hybridoma
technique, a method of creating pure antibodies against a specific target ( antigen). Kohler and
Milstein (1975) succeeded in fusing a normal B cell with a myeloma cell, known as a hybridoma.
Hybridomas are the hybrid cells of myeloma (cancer) with antibody production (lymphocytes). This
fusion has an immunized donor (animal). The hybrid cells secrete immunoglobulins, which consist
of several types of polypeptides. Hybridoma is an important biotechnological tool and indicates
new path for achieving the goal of immunization of human body from infection.
PRINCIPLE FOR CREATION OF HYBRIDOMA CELLS
The myeloma cells used in hybridoma technology must not be capable of synthesizing their own
antibodies. The mammalian cells can synthesize nucleotides by two pathways de novo synthesis and
salvage pathway. The de novo synthesis of nucleotides requires tetrahydrofolate that is formed from
dihydrofolate. The formation of tetrahydrofolate can be blocked by the inhibitor aminopterin.
The establishment of hybridomas and production of mAbs involves the following steps:
1. Immunization
2. Cell fusion
3. Formation and selection of hybrid cells
4. Screening of products
5. Cloning and propagation
6. Characterization and storage
It can be explained as depicted in Figure 11.1.
Immunization
The very first step in hybridoma technology is to immunize an animal, with appropriate antigen.
The antigen along with an adjuvant like, Freund’s complete or incomplete adjuvant is injected
subcutaneously. When the serum concentration of the antibodies is optimal, the animal is killed. The
spleen is aseptically removed and disrupted by mechanical or enzymatic method to release the cells.
Cell Fusion
Splenocytes are then mixed with plasmocytoma cells in an appropriate medium. This mixture is
exposed to a high concentration of polyethylene glycol (PEG) for short time, and fusion is allowed
to take place over a period of time. Mouse is used in the production of mAbs secreting hybridoma
with splenocytes, because these animals are inexpensive, and several billion cells can be obtained
from a mice spleen. The use of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) cells
assures that only hybridomas are selected. After 10 days of culture in the hypoxanthine-aminopterin-
Hybridoma Technology and Monoclonal Antibodies
157
thymidine ( HAT) medium, most of the wells contain dead cells, but a few wells contain small
cultures of viable cells. The wells containing viable clusters are then screened for production of
antibody, and such positive clones are subcultured at low cell densities in order to ensure clonal
purity in each microwell.
Formation and Selection of Hybrid Cells
Important feature of heterokaryon research is its ability to select hybrids of two parent cell lines.
When the cells are cultured in HAT medium, only hybridoma cells grow, whereas the rest slowly
disappear. Selection of a single antibody-producing hybrid cells is very important. After fusion,
Protein of interest
Blood of mouse
Antibody-forming cells
(B cells)
Hybridoma
Tumour cell culture
Hybridomas screened for
protein-specific
antibody production
Protein-specific
monoclonal antibodies
isolated
Antibody-producing
hybridoma clones
Figure 11.1
Production of monoclonal antibodies.
Textbook of Pharmaceutical Biotechnology
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the hybrid cells must be separated from intact parent cells. When Sendai virus or PEG is used as
the fusing agent, only a small percentage of the cells actually fuse and some of the fused cells are
homogeneous, i.e. A–A or B–B cells rather than the desired A–B hybrid. This selection depends
upon the synthesis of nucleotides by mammalian cells.
Screening of Products
Hybridoma cells producing the desired antibody secreted by the hybrid cells are known as mAbs.
The techniques of enzyme-linked immunosorbent assay ( ELISA) and radioimmunoassay (RIA) are
commonly used for screening of hybridomas for secretion of the antibody of desired specificity.
Cloning and Propagation
The single hybrid cells producing the desired antibody are isolated and cloned. Two techniques are
commonly employed. In limiting dilution method, the suspension of hybridoma cells in culture is
enumerated, diluted and aliquoted into new wells so that only one cell is found in any well. The
cells are grown again, and the procedure is repeated several times to increase the probability that
all the cells in a given well are monoclonal.
In soft agar method, the hybridoma cells are cultured on soft agar. This method is based on the
fact that many malignant cells shall proliferate forming spherical colonies in a semisolid medium
containing low quantity of agar.
Characterization and Storage
The mAbs are subjected to biophysical and biochemical characterization for the desired specificity.
The stability of the cell lines and the mAbs are important features. The cells are required to be
characterized for their ability to withstand freezing and thawing.
LARGE-SCALE PRODUCTION OF MONOCLONAL ANTIBODIES
The production of culture bottles of mAbs is rather low (5–10 g/mL). The yield can be increased
by growing the hybrid cells as ascites in the peritoneal cavity of mice. In vitro techniques rather
than the use of animals are preferred, because several animals are required to be sacrificed to
produce mAbs in good concentration.
Human mAbs
The mAbs produced using mice are suitable for in vitro use. For human clinical trials, the use of
human mAbs is preferred avoiding any anti-isotype response. The technical difficulties faced in
production of human mAbs are as follows:
1. One cannot immunize a human volunteer with the range of antigen that is given to mice.
2. It is difficult to obtain antigen prone B cells in humans.
3. Proper method is selected for assaying mAbs in supernatant hybridomas.
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159
In vitro culturing of the cloned cell lines for production of antibodies and selecting proper method for
assaying mAbs in supernatant hybridomas are important prerequisites. The following evaluations
are required to be undertaken:
1. Enzyme-linked immunosorbent assay
2. Radioimmunoassay
3. Immunofluoroscence
4. Cytotoxicity
5. Flow cytometry
Advantages and Limitations of mAbs
The advantages and limitations of mAbs are given below.
Advantages
1. In vitro and in vivo production is possible.
2. High reproducibility.
3. One antigenic determinant.
4. High avidity can be produced.
5. Production of cell lines to individual components of a mixture is possible.
6. Determined to very high value of antiserum titre.
7. Cross-reaction can be determined.
8. Radiolabelling and conjugation fluorescent is possible.
9. The immunization and bleeding maintenance of farm or animals are not required.
Limitations
1. There are no suitable myeloma cells in human that can replace mouse myeloma cells.
2. The fused human lymphocytes mouse myeloma cells are very unstable.
3. The method is time consuming and initial cost involved is more.
4. The energy of binding to an antigen is slow in case of mAbs, and also average in case of
conventional antiserum.
5. It is not a standard double immunodiffusion method.
6. For ethical reason, humans cannot be immunized against antigens.
Fusion and Culture of Hybridomas
Various steps involved in fusion and culturing of hybridomas are briefly summarized as follows:
1. The mouse is boosted with an intravenous injection of antigen, 72 h before use.
2. On the day of fusion, spleen is collected and a free cell suspension is made by injecting and
flushing the spleen with sterile free medium.
3. The plasma cells are maintained in cell culture and fed into fresh flask medium for 16 h before
fusion. It ensures that they are in early phase of growth at the time of fusion.
4. Both myeloma and spleen cells are counted and then mixed in appropriate ratio.
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5. The cells are centrifuged into a loose pellet by spinning at 1000 rpm for 10 to 15 min.
6. The supernatant is removed and the pellet is overlayed with 1 mL of polyethylene glycol.
7. Following fusion, the cell is diluted with 30 mL of serum-free medium with the first 10 mL
medium being added and mixed at 1 mL per min (slow dilution reduces the risk of osmotic
disruption of the fused cells).
8. The cell is centrifuged and resuspended in complete medium containing HAT and then dispensed
into 96 well tissue culture plates, 10 plates for each 10
8
splenocytes used in the fusion.
9. Added 10
6
thymocytes to each well to serve as feeder cells.
10. The culture is incubated for 3–4 days in carbon dioxide incubator with high humidity.
11. Half of the medium is replaced with fresh HAT medium every fourth day.
12. After each test for antibody transfer positive cell lines to 24 well plates, 3–5×10
6
feeder cells
are added to each well to promote rapid cell growth.
13. The cells are maintained in static culture for a minimum of 2 weeks by removing 50–75% of
the hybrid cell after 2–3 days interval.
14. The duplicate cultures are made after 2 weeks and allowed the cells to overgrow and die.
15. The supernatant is collected and assayed for the presence of antibody.
Cloning of Preservation of Hybridomas
After preliminary selection of hybrids, following steps are involved for further processing and
preservation of antibodies:
1. The final supernatant is screened in detail to identify antibodies of immediate interest.
2. The parent cell lines are taken from the freezer and cloned by limiting dilution.
3. When viability is good, the cell lines are cloned immediately.
4. The remainders of the excess cells are cultured in a T75 flask for 1–2 days. After 12–14 days,
supernatant from the marked wells is assayed.
5. After noting which cloned lines are stable, 4–6 clones of each cell line are expanded into 6
well plates for cell preservation and production of antibody.
APPLICATIONS OF MONOCLONAL ANTIBODIES
mAbs with specificity and high purity have a wide range of applications, which can be broadly
categorized as follows:
1. Diagnostic applications
2. Investigational and analytical applications
3. Miscellaneous applications
Diagnostic Applications
mAbs are important diagnostic reagents used in biomedical research, microbiological research in
diagnosis of hepatitis, influenza, AIDS, herpes simplex, chlamydia infections and in treatment of
Hybridoma Technology and Monoclonal Antibodies
161
cancer and other infections. The worldwide clinical diagnostics industry is valued at approximately
US$19 billion, with a growth rate of nearly 5% per year. More than 100 different mAb diagnostic
products are currently available in the world market. mAb diagnostic kits are increasingly in use for
identification of communicable diseases including transfusion transmissible infections.
1. In cardiovascular disease: It is possible to detect the location and the degree of damage to
the heart by using radio labelled antimyosin mAb. The antimyosin mAb is specific for human
myosin and binds to intracellular myosins exposed as a consequence of myocardial necrosis.
Atherosclerosis mAb radiolabelled against platelets can be used to localize the atherosclerotic
lesions by imaging techniques.
2. Site of bacterial infection: In recent years, attempts are made to detect the sites of infection
by using mAb. This is made possible by directing mAb against bacterial antigens.
3. In cancer: mAbs with varying degrees of specificity have already been produced against
a variety of human and animal tumours. mAbs have important clinical applications in the
detection and early diagnosis of cancer, in staging procedures and in therapeutic trials. In
addition, many of these reagents have the capacity to distinguish between different types of
human tumours and shall be useful in the refinement of histopathologic classification scheme.
The mAbs against the antigens on surface of cancer cells are useful for the treatment of
cancer. This is brought out by antibody-dependent cell-mediated cytotoxicity, complement-
mediated cytotoxicity and phagocytosis of cancer cells by reticuloendothelial system.
Limitations
a. The mAbs produced in mice and directly used for therapeutic purpose may lead to the
development of anti-mouse antibodies and hypersensitivity reactions.
b. All the cancer cells may not carry the same antigen for which mAbs have been produced.
c. The free antigens present in the circulation may bind to mAbs and prevent them from
acting on the target cells.
4. In treatment of AIDS: Chang et al. (1994) identified a new herpes virus sequence in human
immunodeficiency virus (HIV). In addition to its occurrence predominantly in HIV-positive
patients, it is also occasionally present in elderly
HIV-negative patients. Immunosuppressant
is the hallmark of AIDS. This is caused by reduction in CD4 cells of T lymphocytes. The HIV
binds to specific receptor on CD4 cells by using surface membrane glycoprotein. Genetic
engineering has been successful to attach fragment crystallizable portion (Fc portion) of
mouse mAb to human CD4 molecules.
5. In malaria: Currently, malarial infection is diagnosed by microscopic examination of blood
smears, which is an effective, but labour-intensive and time-consuming process. Although,
immunological procedure for detection of plasmodium such as ELISA is rapid and amenable
to automation, it does not discriminate to detect antiplasmodium antibodies in the blood of
affected individuals. A diagnostic protocol using a DNA probe as a mean of detection is
developed for the detection of Plasmodium falciparum, parasite causing malaria.
6. In
diabetes: Hybridoma-produced mAb against insulin is useful for insulin assays because of
its specificity and plentiful supply. The spleen cells of male BALB/c mice immunized against
monocomponent porcine insulin are hybridized with mouse myeloma cells (P3-X63-Ag8-U1).
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The resulting anti-insulin antibody (Ab) is purified and characterized by RIA. The proper
concentration of this mAb for RIA is proved to be 1:15,00,000 and the smallest detectable
level of porcine insulin by RIA using this Ab is 0.5 ng/mL. These levels are similar to those
obtained with PoAb. The binding activity of this mAb to human insulin is quite similar to that
of porcine insulin.
7. Rheumatic arthritis: Arthritis is an autoimmune disease. Some success has been achieved
in trials of rheumatoid arthritis patients by using mAbs directed against T lymphocytes and
B lymphocytes.
Some important mAbs along with the names of their manufacturers and other details are given in
Table 11.1.
Table 11.2 includes the list of approved and investigational drugs as well as drugs that have
been withdrawn from market. Consequently, the column Use in Table 11.2 does not necessarily
indicate clinical usage.
Investigational and Analytical Applications
Identification and Isolation of Lymphocytes Phenotyping
mAbs specific for various lymphocytes subpopulation express characteristic pattern of
membrane protein. This characteristic can be utilized for identification of various stages of
lymphocytes differentiation, e.g. human and mice in both CD8 membranes expressed by
T helper cells.
Purification of Proteins
Antibodies can also be used in protein purification. When a purified antibody is added to a
crude mixture of proteins, the specific protein being sought selectively combines with the
antibody and gets precipitated from solution. High efficiency in purification technique can
be attained by using antibodies. It is possible to achieve more than 5000-fold purification of
interferon-_2.
.
Miscellaneous Applications
Application of Catalytic mAbs
Catalytic mAbs have wide-ranging applications in biology, medicine, chemistry and
biotechnology. In the last couple of years, mAbs with enzyme-like catalytic properties have
been successfully raised. They catalyse a variety of reactions ( hydrolysis of esters, amides
and carbonates, cyclization reactions, bimolecular reactions, elimination reactions, asymmetric
synthesis, etc., with rate enhancements of up to 6 × 10
6
-fold and high specificity, including
enantiomer specificity. Hybrid antibodies containing covalently-linked catalytic or probe
groups have been prepared and antibody-mediated peptide hydrolysis catalysed by metal–ion
complexes are reported.
Hybridoma Technology and Monoclonal Antibodies
163
Table 11.1 Generic name, brand name, year, manufacturer and indications of some mAbs
Generic name
Brand and year of FDA
approval
Manufacturer Indications
Infliximab Remicade (1998) Johnson & Johnson,
Merck
Crohn’s disease, ulcerative colitis,
ankylosing spondylitis (AS), rheumatoid
arthritis, psoriasis (PS), psoriatic
arthritis (PSA)
Bevacizumab Roche Colon mCRC/metastatic colorectal
cancer (MCRC), non–small-cell lung
cancer (NSCLC), metastatic breast
cancer (MBC), metastatic renal cell
carcinoma (mRCC), glioblastoma
Adalimumab Humira (2002) Abbott Rheumatoid arthritis, juvenile idiopathic
arthritis, psoriatic arthritis, psoriasis,
ankylosing spondylitis (AS), Crohn’s
disease
Trastuzumab Herceptin (1998) Roche Breast cancer
Ranibizumab Lucentis (2006) Novartis, Roche Macular degeneration
Palivizumab Synagis (1998) Astra Zeneca Coffee ringspot virus (CoRSV)
Cetuximab Erbitux (2004) Bristol Myers Squibb,
Merck
Head and neck cancer (HNC)
Omalizumab Xolair ( 2003) Roche, Novartis Allergic asthma
Abciximab ReoPro (1994) Johnson & Johnson, Lilly Thrombosis inhibitor
Panitumumab Vectibix (2006) Amgen Colon cancer
Certolizumab Cimzia (2008) Union Chimique Belge Crohn’s disease, rheumatoid arthritis
Golimumab Simponi (2009) Johnson & Johnson Rheumatoid arthritis, psoriatic arthritis,
ankylosing spondylitis
Canakinumab Ilaris (2009) Novartis Periodic syndrome
Ofatumumab Arzerra (2009) GlaxoSmithKline Chronic lymphocytic leukaemia
Tocilizumab Actemra EMA (2009),
FDA (2010)
Roche Rheumatoid arthritis
Rituximab CD20 Rituxan (1997) Roche Leukaemia, chronic lymphocytic
leukaemia
Natalizumab Tysabri (2004/2006) Biogen Idec, Elan Multiple sclerosis, Crohn’s disease
Ustekinumab Stelara (2008) Johnson & Johnson Psoriasis
mAbs in Drug Targeting
The objective of drug targeting is to deliver drugs to a specific site of action through a carrier system.
Of all the carrier systems available, mAbs are gaining importance because of their high specificity.
In cancer therapy, cytotoxic agents used to kill malignant cells also damage normal cells. mAbs
generated against specific antigens, when conjugated to cytotoxic drugs, can selectively deliver
drugs to cancer cells while minimizing damage to normal cells.
Textbook of Pharmaceutical Biotechnology
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Table 11.2 Generic name, trade name, source and clinical uses
Generic name Trade name Source Uses
Abciximab ReoPro Chimera Platelet aggregation inhibitor
Adalimumab Humira Human Rheumatoid arthritis
Alemtuzumab Campath,
MabCampath
Human Chronic lymphocytic leukaemia, cutaneous
T cell lymphoma (CTCL)
Altumomab
pentetate
Hybri-ceaker Mouse Colorectal cancer (diagnosis)
Arcitumomab CEA-Scan Mouse Gastrointestinal cancers (diagnosis)
Atlizumab Actemra Human Rheumatoid arthritis
Basiliximab Simulect Chimera Prevention of organ transplant rejections
Bectumomab LymphoScan Mouse Non-Hodgkin’s lymphoma (detection)
Belimumab Benlysta,
LymphoStat-B
Human Non-Hodgkn’s lymphoma
Besilesomab Scintimun Mouse Inflammatory lesions and metastases (detection)
Bevacizumab Avastin Human Metastatic cancer
Biciromab FibriScint Mouse Thromboembolism (diagnosis)
Canakinumab Ilaris Human Rheumatoid arthritis
Capromab pendetide ProstaScint Mouse Prostate cancer (detection)
Catumaxomab Removab Rat/mouse hybrid Ovarian cancer, malignant ascites, gastric cancer
Cetuximab Erbitux Chimera Metastatic colorectal cancer
Daclizumab Zenapax Human Prevention of organ transplant rejections
Denosumab Prolia Human Osteoporosis, bone metastases, etc.
Eculizumab Soliris Human Paroxysmal nocturnal haemoglobinuria
Edrecolomab Panorex Mouse Colorectal carcinoma
Efalizumab Raptiva Human Psoriasis (blocks T cell migration)
Ertumaxomab Rexomun Rat/mouse hybrid Breast cancer
Efungumab Mycograb Human
Invasive Candida infection
Etaracizumab Abegrin Human Melanoma, prostate cancer, ovarian cancer, etc.
Fanolesomab NeutroSpec Mouse Appendicitis (diagnosis)
Fontolizumab HuZAF Human Crohn’s disease
Gemtuzumab
ozogamicin
Mylotarg Human Acute myelogenous leukaemia
Golimumab Simponi Human Rheumatoid arthritis, psoriatic arthritis,
ankylosing spondylitis
Ibritumomab tiuxetan Zevalin Mouse Non-Hodgkin’s lymphoma
Igovomab Indimacis-125 Mouse Ovarian cancer (diagnosis)
Imciromab Myoscint Mouse Cardiac imaging
Infliximab Remicade Chimera Rheumatoid arthritis, ankylosing spondylitis,
psoriatic arthritis, psoriasis, Crohn’s disease,
ulcerative colitis