C hapter 7 H ybridoma Technology for the Generation
of Monoclonal Antibodies
C honghui Z hang
A bstract
H ybridoma technology has long been a remarkable and indispensable platform for generating high-quality monoclonal antibodies (mAbs). Hybridoma-derived mAbs have not only served as powerful tool reagents but also have emerged as the most rapidly expanding class of therapeutic biologics. With the establishment of mAb humanization and with the development of transgenic-humanized mice, hybridoma technology has opened new avenues for effectively generating humanized or fully human mAbs as therapeutics. In this chapter, an overview of hybridoma technology and the laboratory procedures used routinely for hybri-doma generation are discussed and detailed in the following sections: cell fusion for hybridoma generation, antibody screening and characterization, hybridoma subcloning and mAb isotyping, as well as production of mAbs from hybridoma cells.
K ey words:C ell fusion ,E LISA ,F low cytometry ,H ybridoma technology ,I mmunohistochemistry ,
I mmunization ,M onoclonal antibody ,M yeloma cells ,T herapeutic antibody ,S creening
1.I ntroduction
T he invention of hybridoma technology by Georges Köhler and
César Milstein in 1975 is a signi fic ant milestone in immunology
and biomedicine  (1). This technology has enabled scientists for the
fir st time to generate unlimited quantities of pure, monospeci fic
antibodies directed against virtually any antigen. A monoclonal
antibody (mAb) is a highly speci fic and homogeneous species of
immunoglobulin molecule produced by a single hybridoma clone
that has been generated by the fusion of a myeloma cell with a B
lymphocyte from a donor or from an immunized animal. Hybridoma
technology has thus revolutionized discovery research and thera-
peutic development in such diverse  fie lds as immunology, biology,
oncology, and infectious diseases  (2–4). The mAbs generated from Gabriele Proetzel and Hilmar Ebersbach (eds.), Antibody Methods and Protocols,Methods in Molecular Biology, vol. 901,
DOI 10.1007/978-1-61779-931-0_7, © Springer Science+Business Media, LLC 2012
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118  C. Zhang
this technology have served as reagents for the identi fic ation and
characterization of cell surface antigens  (5, 6), for classi fic ation and
isolation of hematopoietic cell subsets  (7–9), and for the develop-
ment of biomarkers to distinguish aberrant or cancerous cells from
normal cells  (10–13). Hybridoma technology has long been a
powerful tool for investigators to make discoveries in the biological
sciences and has led to many important advances in medicine.
W ith the breakthrough in molecular engineering and antibody
humanization  (14, 15), mAbs have emerged as the most rapidly
expanding category of biopharmaceuticals for a large variety of
clinical scenarios. For example, mAbs have been used to aid in suc-
cessful organ transplantation  (16, 17), as well as being used to treat
in fla mmatory diseases  (18, 19), cancer, and infectious diseases
(20–22). Based on published data, nearly 30 FDA-approved anti-
body drugs are on the US market today (Fig.  1) and it is estimated
that hundreds of mAbs are currently in various phases of clinical
trials worldwide  (23).
m Abs can be produced from an immune or nonimmune
resource using a range of recently developed antibody technologies,
including methods such as display technologies  (24, 25)or memory
B-cell immortalization and cloning  (26, 27). However, since hybri-
doma technology is so well established, it will continue to provide a
powerful and indispensable platform for generating high-quality
mAbs to meet unmet needs. It is important to note that mAbs gen-
erated from immune hosts by the hybridoma approach often exhibit
good binding af fin ity due to in vivo secondary immune responses.
These mAbs routinely obviate the requirements for subsequent
in vitro af fin ity maturation or other modi fic ations to improve anti-
body potency by additional technologies  (28, 29). Furthermore,
the primary production of the whole Ig molecule from hybridomas
allows investigators to screen directly for the desired biological
function of mAbs from the very beginning. Therefore, it is not sur-
prising that 26 out of the 28 therapeutic mAbs that have been
approved by the FDA in the United States today have originated
from hybridomas, with or without chimerization or humanization
(Fig.  1). With the recent development of transgenic humanized
mouse strains that are capable of natural recombination and af fin ity
maturation in vivo and which have a large repertoire of high-af fin ity
antibodies to any antigen  (30–32), the “old-fashioned” hybridoma
technology will open up new avenues for more effectively generat-
generated
ing large panels of high-quality and fully human mAbs. These fully
human mAbs generated from transgenic humanized mice will accel-
erate the development and application of mAbs as therapeutics for
human cancer and disease  (29, 33).
H ybridoma technology is composed of several technical aspects,
including antigen preparation, animal immunization, cell fusion,
hybridoma screening and subcloning, as well as characterization
and production of speci fic antibodies (Fig.  2). mAb generation by
119
7 Hybridoma-Derived mAbs the hybridoma approach requires knowledge of multiple disciplines
and practice of versatile technical skills, ranging from animal han-
dling (immunization and sample collection), immunology (immu-
noassays and antibody characterization) to cellular and molecular
biology (cell fusion for hybridoma generation, protein sequencing
analysis for antigen preparation, and  fl o w cytometry or other cell-
based assays for screening hybridomas). Generation and
identi fi c ation of high-quality hybridoma clones is a comprehensive
and labor-intensive process, and requires months of work during
the time frame from immunization to speci fi c  hybridoma
identi fi c ation. The key aspect of hybridoma generation is the
screening procedure used to identify and select the desired hybri-
doma clones from the fusion plates. As shown in Fig.
3 , cell fusion Human mAb (-umab)2Other
Approach
(2 mAbs)Murine mAb (-omab)3
Chimeric mAb (-ximab)5Humanized mAb (-zumab)11Human mAb (-umab)7
RITUXAN®/MABTHERA®rituximab (1997)SIMULECT®basiliximab (1998)REOPRO®abciximab
(1994)
REMICADE®infliximab (1998)ERBITUX®cetuximab (2004)ORTHOCLONE OKT3®muromonab -CD3(1986)BEXXAR®tositumomab (2003)ZEVALIN®ibritumomab tiuxetan (2002)ZENAPAX®daclizumab
(1997)SYNAGIS®palivizumab (1998)HERCEPTIN®trastuzumab
(1998)
CAMPATH®MABCAMPAT®alemtuzumab (2001)XOLAIR®omalizumab (2003)AVASTIN®bevacizumab
(2004)
TYSABRI®/ANTEGREN®natalizumab (2004)LUCENTIS®ranibizumab
(2006)SOLIRIS ®eculizumab (2007)Actemra®Tocilizumab
(2010)
CIMZIA®certolizumab
pegol (2008)Hybridoma Origin (26 mAbs)VECTIBIX®panitumumab
(2006)
SIMPONI®golimumab (2009)STELARA®ustekinumab (2009)ARZERRA®ofatumumab (2009)PROLIA®/XGEVA ®Denosumab (2010)YERVOY®ipilimumab (2011)ILARIS®canakinumab (2
009)BENLYSTA®belimumab
(2011)HUMIRA®/TRUDEXA®adalimumab (2002)
F ig. 1.  A  list of FDA-approved therapeutic mAbs currently on the market. Over 30 therapeutic mAbs have been approved by the FDA for marketing in the United States to date, whereas a small number of the mAb drugs, such as Mylotarg (
G umtuzumab ozogamicin  ) and Raptiva ( E falizumab  ), have been withdrawn from the market due to their side effects and/or poor clinical bene fi t s. Most of the FDA-approved therapeutic mAbs currently on the market have originated from hybri-domas and are in the full-length antibody molecular format, including the murine (suf fi x ed with - o mab  ), chimeric (- x imab ),humanized (- z umab  ), and human (- u mab  ) antibody category. All human mAbs of hybridoma origin are generated from the
XenoMouse ® or HuMAb-Mouse ® transgenic strain, both of which have nearly the entire human Ig loci introduced into the germ line with inactivation of the mouse Ig machinery. For each antibody drug, its trade name, generic mAb name and the year of FDA approval are indicated in the  fi g ure. The digit shown represents the number of therapeutic mAbs in the antibody category.
120  C. Zhang
Unfused
Cells Homokaryotic
Hybrids
Heterokaryotic
Hybrids (SC-MC) Die naturally in culture
MC
SC SC-SC MC-MC Die in selective HAT medium Die naturally in culture
Survive in HAT
culture medium
Non producer Non-specific
Ab producer Specific Ab producer
Splenocyte
(SC)
Myeloma cell
(MC)Cell fusion
F ig. 3.  M ultiple cell types generated from fusion of splenocytes (SC) and myeloma cells (MC). PEG-mediated cell fusion is likely to result in a mixed population of cells consisting of nonproducing hybridomas, antibody-producing hybridomas and unfused cells. In the presence of aminopterin in HAT-selective medium, cells are dependent on another pathway that needs the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT) for survival. Under this culture condition, unfused myeloma cells or hybrids of myeloma cells with myeloma cells will die because of the absence of HGPRT, whereas unfused splenocytes or hybrids of splenocytes with splenocytes also die because of their lack of immortal growth potential. Only hybridomas from fusion of splenocytes with myeloma cells will inherit the HGPRT gene from splenocytes and the immortal growth property from myeloma cells, and can thus grow in HAT medium. By hybridoma screening and subcloning, speci fi c  hybridoma clones will be identi fi e d and isolated from nonspeci fi c
antibody producers or nonproducers of myeloma-splenocyte hybridomas.
F ig. 2.  A  diagram of mAb generation by the hybridoma approach. Generation and identi fi c ation of high-quality mAbs by the hybridoma approach requires months of work during the time frame from immunization to establishment of speci fi c  hybri-doma clones. The work involves stages of antigen preparation, animal immunization, cell fusion for hybridoma generation, hybridoma screening and subcloning, as well as characterization and production of speci fi c  mAbs.
7 Hybridoma-Derived mAbs
121
mediated either by PEG or electrofusion typically generates a mix-ture of cells within the culture, which is composed of unfused sple-nocytes or myeloma cells, heterokaryotic hybrids (hybridomas) of splenocytes and myelomas with or without the secretion of speci fic antibodies, and the homokaryotic hybrids of either myeloma–myeloma cells or splenocytes–splenocytes. However, only the hybridomas from the fusion between splenocytes and myeloma cells are able to  s urvive in the HAT medium. It is important to note that the myeloma–splenocyte hybridoma cells may turn out to be a speci fic antibody producer, nonspeci fic antibody producer or nonproducer. Development of appropriate antibody screening assays is thus required to ef fic iently identify the subpopulation of hy
bridoma cells in the fusion plates. The screening assays of choice should be speci fic, reliable, and effective. In general, the identi fic ation and selection process of antibody-secreting hybrido-mas comprises an initial screening of antibodies in polyclonal cul-tures and a secondary, more sophisticated characterization of mAbs afterwards. With the initial screening, antibody-secreting hybrido-mas are identi fie d from the well of fusion plates, of which positive hybridomas are selected and then subcloned into monoclonals.
A more sophisticated characterization of the mAbs generated will further determine their speci fic ity, binding af fin ity, molecular fea-tures, and the functional activity of the mAbs, if any. Culture super-natants from the fusion plates are initially screened for positive hybridoma clones by a number of different immunoassays. While immuno flu orescence  flo w cytometry is often applied to particulate antigens such as whole cells, an enzyme-linked immunosorbent assay (ELISA) is used for soluble antigens such as proteins or poly-peptides, and immunohistochemistry (IHC) is developed for tissue antigens. Lastly, the hybridoma clones selected from the initial screens often require more testing for biochemical features of the mAb by immunoprecipitation and/or immunoblots, and further testing for biological activity by in vitro functional assays, such as blocking of the ligand binding to its receptor, detection of protein phosphorylation or signaling pathway, mediating agonistic or antagonistic activity,
inhibiting cell proliferation, or interfering with the potency to mediate cell killing  (34–36). In general, the functional screening assays are complex to perform and construe, and therefore are only carried out as necessary.
I n this chapter, the strategy and laboratory methods for hybri-doma generation are described and detailed in the following sec-tions: cell fusion for hybridoma generation, antibody screening and characterization, hybridoma subcloning, cryopreservation and antibody isotyping, as well as production and puri fic ation of mAbs from hybridoma cells.

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