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for Biomedical


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9-6 Biotechnology for Biomedical Engineers

and V
genes are also relatively conserved so as to enable the design of universal “upstream” primers as

well. These primers were then used to establish a repertoire of antibody variable-region genes. Second,
the successful expression of functional antigen-binding fragments in bacteria using a periplasmic secretion
strategy enabled the direct screening of libraries of cloned antibody genes for antigen binding [Better
et al, 1988; Skerra and Pluckthun, 1988].

The first attempt at repertoire cloning resulted in the establishment of diverse libraries of VH genes
from spleen genomic DNA of mice immunized with either lysozyme or keyhole-limpet hemocyanin
(KLH). From these libraries, VH domains were expressed and secreted in E. coli [Ward et al., 1989].
Binding activities were detected against both antigens in both libraries; the first library, immunized
against lysozyme, yielded 21 clones with lysozyme activity and 2 with KLH activity, while the second
library, immunized against KLH, yielded 14 clones with KLH activity and 2 with lysozyme activity. Two
VH domains were characterized with affinities for lysozyme in the 20 nM range. The complete sequences
of 48 VH gene clones were determined and shown to be unique. The problems associated with this
singledomain approach are (1) isolated VH domains suffer from several drawbacks such as lower selectivity
and poor solubility and (2) an important source of diversity arising from the combination of the heavy
and light chains is lost.

In the so-called combinatorial approach, Huse et al. [1989] used a novel bacteriophage l, vector
system (l-ZAP technology) to express in E. coli a combinatorial library of Fab fragments of the murine
antibody repertoire. The combinatorial expression library was constructed from spleen mRNA isolated
from a mouse immunized with KLH-coupled p-nitrophenyl phosphonamidate (NPN) antigen in two
steps. In the first step, separate heavy-chain (Fd) and light-chain (K) libraries were constructed. These
two libraries were then combined randomly, resulting in the combinatorial library in which each clone
potentially coexpresses a heavy and a light chain. In this case, they obtained 25 million clones in the
library, approximately 60% of which coexpressed both chains. One million of these were subsequently
screened for antigen binding, resulting in approximately 100 antibody-producing, antigen-binding
clones. The light- and heavy-chain libraries, when expressed individually, did not show any binding
activity. In addition, the vector systems used also permitted the excision of a phagemid containing the
Fab genes; when grown in E. coli, these permitted the production of functional Fab fragments in the
periplasmic supernatants. While the study did not address the overall diversity of the library, it did
establish repertoire cloning as a potential alternative to conventional hybridoma technology.

Repertoire cloning via the l-ZAP technology (now commercially available as the ImmunoZap kit
from Stratacyte) has been used to generate antibodies to influenza virus hemagglutinin (HA) starting
with mRNA from immunized mice [Caton and Kaprowski, 1990]. A total of 10 antigen-binding clones
was obtained by screening 125,000 clones from the combinatorial library consisting of 25 million
members. Partial sequence analysis of the VH and VK regions of five of the HA-positive recombinants
revealed that all the HA-specific antibodies generated by repertoire cloning utilized a VH region derived
from members of a single B-cell clone in conjunction with one of two light-chain variable regions. A
majority of the HA-specific antibodies exhibited a common heavy-light-chain combination that was
very similar to one previously identified among HA-specific hybridoma monoclonal antibodies. The
relative representation of these sequences and the overall diversity of the library also were studied via
hybridization studies and sequence analysis of randomly selected clones. It was determined that a
single functional VH sequence was present at a frequency of 1 in 50, while the more commonly
occurring light-chain sequence was present at a frequency of 1 in 275. This indicates that the overall
diversity of the gene family representation in the library is fairly limited.

The l-ZAP technology also has been used to produce high-affinity human monoclonal antibodies
specific for tetanus toxoid [Mullinax et al, 1990; Persson et al, 1991]. The source of the mRNA in these
studies was peripheral blood lymphocytes (PBLs) from human donors previously immunized with
tetanus toxoid and boosted with the antigen. Mullinax et al. [1990] estimated that the frequency of
positive clones in their library was about 1 in 500, and their affinity constants ranged from 106 to 109 M
(Molar)-1. However, the presence of a naturally occurring SacI site (one of the restriction enzymes used
to force clone PCR-amplified light-chain genes) in the gene for human CK may have resulted in a

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9-7Monoclonal Antibodies and Their Engineered Fragments

reduction in the frequency of positive clones. Persson et al. [1991] constructed three different
combinatorial libraries using untreated PBLs, antigen-stimulated PBLs (cells cultured in the presence of
the antigen), and antigen-panned PBLs (cells that were selected for binding to the antigen). Positive
clones were obtained from all three libraries with frequencies of 1 in 6000, 1 in 5000, and 1 in 4000,
respectively. Apparently binding constants were estimated to be in the range of 107 to 109 M-1. Sequence
analysis of a limited number of clones isolated from the antigen-stimulated cell library indicated a greater
diversity than that described for HA or NPN. For example, of the eight heavy-chain CDR3 sequences
examined, only two pairs appeared to be clonally related. The l-ZAP technology also has been used to
rescue a functional human antirhesus D Fab from an EBV-transformed cell line [Burton, 1991].

In principle, repertoire cloning would allow for the rapid and easy identification of monoclonal
antibody fragments in a form suitable for genetic manipulation. It also provides for a much better
survey of the immunologic repertoire than conventional hybridoma technology. However, repertoire
cloning is not without its disadvantages. First, it allows for the production of only antibody fragments.
This limitation can be overcome by mounting the repertoire cloned variable domains onto constant
domains that possess the desired effector functions and using transfectoma technology to express the
intact immunoglobulin genes in a variety of host systems. This has been demonstrated for the case of
a human Fab fragment to tetanus toxoid, where the Fab gene fragment obtained via repertoire cloning
was linked to an Fc fragment gene and successfully expressed in a CHO cell line [Bender et al., 1993].
The second limitation to the use of “immunized” repertoires, which has serious implications in the
applicability of this technology for the production of human monoclonal antibodies. The studies
reviewed above have all used spleen cells or PBLs from immunized donors. This has resulted in relatively
high frequency of positive clones, eliminating the need for extensive screening. Generating monoclonal
antibodies from “naive” donors (who have not had any exposure to the antigen) would require the
screening of very large libraries. Third, the actual diversity of these libraries is still unclear. The studies
reported above show a wide spectrum ranging from very limited (the HA studies) to moderate (NPN)
to fairly marked diversity (tetanus toxoid). Finally, the combinatorial approach is disadvantageous in that
it destroys the original pairing of the heavy and light chains selected for by immunization. Strategies for
overcoming some of these limitations have already been developed and are reviewed below.

Phage Display Technology
A critical aspect of the repertoire cloning approach is the ability to screen large libraries rapidly for
clones that possess desired binding properties, e.g., binding affinity or specificity, catalysis, etc. This is
especially the case for “naive” human repertoires, wherein the host has not been immunized against the
antigen of interest for ethical and/or safety reasons. In order to facilitate screening of large libraries of
antibody genes, phage display of functional antibody fragments has been developed, which has resulted
in an enormous increase in the utility of repertoire cloning technology. In phage display technology,
functional antibody fragments (such as the sFv and Fab) are expressed on the surface of filamentous
bacteriophages, which facilitates the selection of specific binders (or any other property such as
catalysis, etc.) from a large pool of irrelevant antibody fragments. Typically, several hundreds of millions
of phage particles (in a small volume of 50 to 100 ml) can be tested for specific binders by allowing
them to bind to the antigen of interest immobilized to a solid matrix, washing away the nonbinders,
and eluting the binders using a suitable elution protocol.

Phage display of antibody fragments is accomplished by coupling of the antibody fragment to a
coat protein of the bacteriophage. Two different coat proteins have been used for this purpose, namely,
the major coat protein encoded for by gene VIII and the adsorption protein encoded for by gene III.
The system based on gene VIII displays several copies of the antibody fragment (theoretically there are
2000 copies of gene VIII product per phage) and is used for the selection of low-affinity binders. The
gene III product is, on the other hand, present at approximately four copies per phage particle and
leads to the selection of high-affinity binders. However, since the native gene III product is required for
infectivity, at least one copy on the phage has to be a native one.

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physiology, 5-6
type I , 5 -8

curve, way of presenting, 7-14

maneuver, 7-12
Splanchnic circulation, 1-9
Spleen cells, immunoglobulins expressed by, 9-5
Split vaccines, 12-9
Spodoptera frugipeeda, 12-11

nonspecific, 12-10
pharmaceutical, 12-10
specific, 12-10

Standard temperature and pressure, dry (STPD), 7-9
Stellate cells, 5-8
Stereopsis, 4-6
Steroid hormones, production of, 2-2
Steroidogenesis, 2-4

enhanced response to, 3-3
peaks, locator of, 3-6

anatomical features, 6-6
canine, electrical rhythmic activity in, 6-5
electrogastrogram, 6–7
gastric ECA, 6-6

STPD, see Standard temperature and pressure, dry
Strabismus, 4-6
Stretch error, calculation of, 3-3
Stroke volume (SV), 1-5
STS content mapping, see Sequence-tagged site content
Subunit antigens, 12-1, 12-10, 12-11
Superior colliculus, 4-3
Superior olivary complex (SOC), 5-1, 5-10
SV, see Stroke volume
Systemic circulation, 1-4


T3, see Triiodothyronine
T4, see Thyroxine
Taylor’s series expansions, 3-6
Testosterone, 2-3
Tetanus toxins, 12-6
Thermus aquaticus, 11-5
Threonine synthesis, reduction in, 15-3
Thrombocytes, 1-2
Thyrotropin (TSH), 2-3, 2-5
Thyrotropin-releasing hormone (TRH), 2-3, 2-5
Thyroxine (T4), 2-3, 2-7
Tidal breathing loop, 7-14
Tidal volume, definition of, 7-5

-affecting hormones, 2-2
resistance, 7-10

TLC, see Total lung capacity
Tonic receptors, 3-2
Total lung capacity (TLC), 7-6, 7-12
Toxoid(s), 12-7

diphtheria, 12-11
tetanus, 12-11

Tracheobronchial receptors, 7-11
Transfectoma technology, 9-10
Transitory airways, 7-4
TRH, see Thyrotropin-releasing hormone
Trichoplusia ni, 12-11
Trichromacy, 4-7
Triiodothyronine (T3), 2-3, 2-6, 2-7
Tropic hormones, 2-2
TSH, see Thyrotropin

growth, restart of, 10-11
necrosis factor-a, 9-8
sites, homing of cytotoxic lymphocytes to, 14-6

Tunica adventitia, 1-6
Tunica intima, 1-6
Typhoid fever vaccine, 12-10
Tyrosine, 2-2


Ulcerative colitis, 10-13
Urea cycle disorders, gene therapy of, 13-2


bacterial, cultivation, 12-4
C. diphtheria, 12-8
cultivation, most recent technique in, 12-4
hepatitis B, 12-9
live-organism, 12-1, 12-13
passive, 12-1
polio, 12-10
split, 12-9
subunit antigen, 12-1
typhoid fever, 12-10
whole-cell, 12-3

Vaccine production, 12-1 to 16
antigen cultivation, 12-2 to 6

microbial cultivation, 12-2 to 4
virus cultivation, 12-4 to 6

downstream processing, 12-6 to 9
purification examples, 12-7 to 9
purification principles, 12-6 to 7

formulation and delivery, 12-9 to 11
live organisms, 12-9 to 10
subunit antigens, 12-10 to 11

future trends, 12-11 to 13
downstream processing, 12-12
vaccine adjuvants and formulation, 12-12 to 13
vaccine cultivation, 12-11 to 12

Varicella zoster, 12-8
Vascular endothelial growth factor (VEGF), 13-7, 13-11
Vascular-graft polymer, immobilization of amino acid
sequence on, 14-4
Vasopressin, 2-3, 2-5
VEGF, see Vascular endothelial growth factor

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Index-12 Biotechnology for Biomedical Engineers

Venous system, 1-8
Ventilatory obstruction, 7-14
Vesicular stomatitis virus (VSV), 13-6
Viral diseases, 10-10 to 11

cancer, 10-11
human immunodeficiency virus, 10-10 to 11

adeno-associated, 13-3, 13-9
canarypox, 13-13
cell-associated, 12-8
chickenpox, 12-8

ex vivo, 12-4
in vivo, 12-4

herpes simplex, 13-13
-inactivation procedure, 9-16
production kinetics, 12-5
Rous sarcoma, 10-1
vaccinia, 13-13
vesicular stomatitis, 13-6

Vision system, 4-1 to 9
fundamentals of vision research, 4-1
modular view of vision system, 4-1 to 8

area Vl, 4-4 to 6
color, 4-7
eyes, 4-1 to 2
higher cortical centers, 4-7 to 8

LGN, 4-4
optic chiasm, 4-3
retina, 4-2 to 3
superior colliculus, 4-3 to 4

Visual field, topographic mapping of, 4-6
Volume velocity, definition of, 5-1
VSV, see Vesicular stomatitis virus


Wall-eye, 4-6
Watson-crick base-pairing interactions, 10-1, 10-2
Whole-cell vaccines, 12-3


Xenopus oocytes, expression of fibronectin mRNA in, 10-8


artificial chromosome, 11-7
fermentation, 9-11

Young-Helmholtz model, 4–7

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