Cancer and Metastasis Reviews 18: 421–425, 1999.

© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

Transgenic mice as a source of fully human antibodies for

the treatment of cancer

C. Geoffrey Davis, Michael L. Gallo and Jose R.F. Corvalan

Abgenix, Inc., Fremont, CA, USA

Key words: cancer, epidermal growth factor receptor, human antibodies, transgenic

Abstract

The last two years have seen a renaissance of monoclonal antibodies for the treatment of disease. Of the eight
antibodies currently approved for human therapy, two are for the treatment of cancer. In large part, the revival of
antibodies has been driven by technology developments geared toward making antibodies less likely to elicit an anti-
antibody response in humans. The development of transgenic mice, XenoMouseTM animals, capable of making fully
human antibodies offers new opportunities for generating antibodies of therapeutic quality. Recently, this technology
has been applied to the generation of a fully human antibody to the epidermal growth factor receptor. A description
of the development of this antibody serves to illustrate the power and ease of use of XenoMouse technology.

The status of monoclonal antibodies as

therapeutics

Ever since the development of hybridoma technology

a quarter century ago, considerable effort has been
expended in adding monoclonal antibodies (MAbs)
to the armamentarium of cancer therapeutics. Only
recently have these efforts proven successful. Of the
eight therapeutic MAbs now on the market, six have
been approved in the last two years. Two of these,
Rituxan and Herceptin, are for the treatment of can-
cer. Rituxan targets the CD20 molecule on B lympho-
cytes and is used to treat non-Hodgkin’s lymphoma [1].
Herceptin targets Her2-neu, a member of the epidermal
growth factor receptor family, and is approved for the
treatment of breast cancer [2]. Many more MAbs are
currently in clinical trials. In fact, within the realm of
biologics, MAbs are second only to vaccines in terms
of the number of drugs currently being developed.
For the most part, this recent success of MAbs
can be attributed to advances in antibody technolo-
gies (Figure 1). The first products of hybridoma
technology were murine antibodies. The usefulness
of these antibodies as therapeutics was limited by
their immunogenicity. Patients treated with murine
antibodies rapidly mounted an immune response to
Figure 1. The evolution of monoclonal antibody technologies.
these foreign proteins, a response known as a HAMA,
for human anti-mouse antibody [3]. In the face of the
HAMA response, chronic treatment with murine anti-
bodies was not possible. In recent years, however, there
has been considerable progress in gene engineering
techniques to make murine antibodies more humanlike.
In one approach, the DNA sequences encoding the
antigen binding domains (the variable regions) of the
murine antibody are spliced to the sequences encoding
the effector domain (the constant region) of a human
antibody [4]. This results in a ‘chimeric’ antibody, the
composition of which is about one-third mouse and
two-thirds human. Four of the eight antibodies now
approved, including Rituxan, are chimeric antibodies.
422
A more sophisticated strategy is CDR grafting [5].
With this technique, only the DNA sequences encod-
ing the actual antigen binding pocket of the original
murine antibody, the CDR sequences, are retained.
These sequences are inserted into the genes encoding a
carefully selected human antibody. ‘Humanized’ anti-
bodies produced in this fashion retain only about five
percent murine protein sequence and are considerably
less immunogenic in humans. Three of the approved
antibodies, including Herceptin, are products of CDR
grafting.
A further advance in antibody technology is the
development of a transgenic mouse strain, XenoMouse
animals, that has been engineered in such a way
that it now produces fully human antibodies rather
than murine antibodies when immunized [6]. Using
XenoMouse animals to produce monoclonal antibod-
ies avoids the need for any engineering of the anti-
body genes, since the products are already one hundred
percent human protein. XenoMouse animals are fully
compatible with standard hybridoma technology and
can be readily adopted by laboratories experienced in
monoclonal antibody production.
Origin and properties of XenoMouse animals
The first step in developing XenoMouse animals was
to inactivate the endogenous mouse antibody genes
(Figure 2). This was accomplished by using gene
knockout technology to delete specific segments of the
mouse immunoglobulin heavy chain and kappa light
chain loci required for recombination. As a result, the
gene rearrangements required for mouse antibody gen-
eration can no longer occur in XenoMouse animals.
Consequently, XenoMouse animals are incapable of
making mouse antibodies.
The more challenging step was to introduce into
the mouse germline the genes encoding for the human
immunoglobulin heavy chain and kappa light chain.
From the outset, the objective was to retain as much of
the complexity of the human antibody loci as possible.
This was deemed important as a means of ensuring that
XenoMouse animals would retain a spectrum of anti-
gen recognition comparable to that of humans. To this
end, yeast artificial chromosomes (YACs) were used to
clone megabase size segments of the loci in germline
configuration. Consequently, XenoMouse animals bear
in their germline the great majority of the human anti-
body sequences required for diversity. With respect to
the heavy chain locus, XenoMouse animals possess
Figure 2. Development of the XenoMouse. The endogenous
mouse immunoglobulin heavy chain and kappa light chain loci
were inactivated using gene knockout technology to remove the
JH genes and the kappa constant region gene. In parallel, the
majority of the human heavy and kappa light chain loci were
cloned into YACs, which were then used to generate transgenic
mice. Thus, in XenoMouse animals the murine antibody genes
are functionally replaced by human antibody genes.
34 functional V genes, all of the D genes, and all of
the J genes, and the locus extends through the Cµ
and Cδ constant region genes in germline configura-
tion. With respect to the kappa light chain locus, the
XenoMouse animals possess 18 functional V genes, all
of the J genes, and the Cκ constant region gene.
While antigen binding is clearly a defining feature of
a therapeutic antibody, equally important is the effec-
tor function inherent in the constant region. Within the
IgG class of human antibodies, there are four subclasses
or isotypes – IgG1, IgG2, IgG3, and IgG4. These iso-
types differ in their ability to bind to Fc receptors on
effector cells (such as macrophages and natural killer
cells) and to fix complement. Thus, if the desired mode
of action of an antibody is to directly kill the target
cell, such as a tumor cell, the IgG1 isotype, which both
binds to Fc receptors and fixes complement efficiently,
would be preferred. In contrast, if the mode of action
is to only block the binding of a ligand to its recep-
tor, an IgG4 or IgG2 isotype would be preferred. With
the intent of facilitating the generation of MAbs of the
preferred isotype, multiple strains of XenoMouse have
been developed. While these strains each possess an
identical complement of variable genes, they differ in
that each will produce a different IgG isotype, namely
IgG1, IgG2, or IgG4.
Because the only human proteins expressed in
XenoMouse animals are the antibodies themselves,
XenoMouse animals recognize all other human

proteins as foreign and mount vigorous humoral
immune responses. Furthermore, the human antibody
genes appear to be fully compatible with the mouse
enzymes mediating both class switching and somatic
hypermutation. As a result, antibodies of the IgG class
with high affinities are readily obtained. Typically, pan-
els of antibodies of the desired specificity and with
affinities in the 10–1000 pM range can be obtained
within two to four months.
Application of XenoMouse technology to
human therapy
The power of the XenoMouse technology lies in its
ability to produce high affinity human antibodies of
therapeutic quality in a relatively short period of time.
The anticipation is that these antibodies will have long
serum half-lives and will not be immunogenic. Further,
XenoMouse technology offers an additional advantage
in that the antibodies can be manufactured in hybrido-
mas. This obviates the need (but does not preclude
the choice) to clone the antibody genes into traditional
manufacturing cell lines, thereby accelerating develop-
ment timelines subsequent to the initial identification
of the antibody.
The first human antibody derived from transgenic
mouse technologies to enter clinical trials was an anti-
body to interleukin-8 (IL-8) [7]. This antibody, ABX-
IL8, was generated in XenoMouse animals. In a Phase
I trial in patients with moderate to severe psoriasis, this
antibody was found to have a serum half-life equiv-
alent to that of normal serum immunoglobulin, i.e.
about three weeks. Also significant was the finding that
none of the patients in this trial developed an immune
response to the human antibody. Thus, in this initial
trial, a therapeutic antibody derived from XenoMouse
animals has met expectations.
Generation of a XenoMouse-derived antibody
for the treatment of cancer
The second antibody derived from XenoMouse animals
to enter clinical trials is an antibody specific for the epi-
dermal growth factor receptor (EGFr). The results from
many studies suggest that EGFr is overexpressed on a
significant proportion of many types of human tumors,
including lung, colon, breast, prostate, brain, head and
neck, pancreatic, renal, and ovarian cancers. Typically,
overexpression of the EGFr is also accompanied by
423
overproduction of one or both of the EGFr ligands,
EGF or TGFα, suggesting autocrine growth control.
This suggests that blocking binding of the ligands to
EGFr may be effective in arresting tumor growth. In
nude mouse xenograft models in which human tumor
cells were injected subcutaneously and anti-EGFr anti-
bodies were administered systemically, the antibodies
have indeed been shown to profoundly inhibit tumor
growth.
An account of the generation of this antibody serves
to further illustrate the efficiency and facility of using
the XenoMouse to derive antibody candidates for ther-
apy. In this case, XenoMouse-G2 animals were immu-
nized with intact cells, A431 cells. A431 is a cell line
derived from a human epidermoid cervical carcinoma.
Each cell bears approximately one million receptors on
its surface. The mice were first immunized with 2×107
cells in complete Freunds adjuvant. Subsequently, they
received an additional 3–5 injections of the same num-
ber of cells in incomplete Freunds adjuvant. For three
of the four fusions, the cells were injected intraperi-
toneally (Table 1). For the fourth fusion, the cells were
injected subcutaneously at the base of the tail.
With the mice that received IP injections, for each
fusion spleens were harvested from four mice. With
mice that received SC injections, the inguinal and the
internal medial iliac lymph nodes were harvested from
ten mice. In either case, fusion of the lymphocytes with
myeloma cells and selection of hybridomas was per-
formed using standard techniques as described previ-
ously [8].
To detect hybridomas producing anti-EGFr anti-
bodies, the hybridoma supernatants were screened by
ELISA for binding to solubilized receptor immobilized
on microtiter plates. As shown in Table 1, these analy-
ses produced a total of 70 anti-EGFr hybridomas. The
Table 1. Anti-EGFr MAbs derived from XenoMouse animals
Mice Route of Injections Anti-EGFr Neutralizing
immuni- hybridomas MAbs/MAbs
zation tested
4 IP 4 13 4/13
4 IP 6 4 3/4
4 IP 6 11 6/11
10 BOT 4 42 2/10
XenoMouse animals were immunized with A431 cells by either
intraperitoneal (IP) or base of tail (BOT) injections. In four sepa-
rate fusions, lymphoid cells were fused with myeloma cells using
standard procedures. Seventy hybridomas producing anti-EGFr
MAbs were generated. A subset of these antibodies was tested
in neutralization assays, which yielded 15 neutralizing MAbs.
424

Table 2. Affinities of neutralizing anti-EGFr MAbs lymph node fusion yielded over half of the reactive
Hybridoma Dissociation constant KD (M)
clones. Of the 70 antibodies, 38 were evaluated in neu-
tralization assays. Fifteen of the 38 monoclonal anti-
E1.1 7.60 × 10−11
bodies blocked binding of EGF to the receptor.
E2.5 1.61 × 10−11
E2.11 1.63 × 10−10 In the course of further evaluation of these anti-EGFr
E2.3.1 8.98 × 10−10 antibodies, seven of them were subjected to affinity
E2.4 3.53 × 10−11 measurements, and the results are shown in Table 2. As
E7.5.2 3.55 × 10−9 can be seen, the affinities of six of the seven antibodies
E7.6.3 5.74 × 10−11 are subnanomolar with four in the range of 10–100 pM.
Affinities were determined using a BIAcore 2000 (Phar- The transcripts encoding eleven of the neutralizing
macia). Purified membrane-derived EGFr was immobi- antibodies were amplified by RT-PCR and subjected to
lized on the sensor chip at low density (303 RU). DNA sequencing. Strikingly, eight of the eleven anti-
bodies were very similar in their gene composition
(Table 3). Each utilized either VH 4-31 or VH 4-61, two
Table 3. Gene usage of anti-EGFr MAbs
closely related and highly homologous members of the
MAb VH D JH VK JK VH4 family. It should be noted that both of these VH
E1.1 4-31 4-23 JH5 018 JK4 genes are relatively under-represented in the expressed
E2.4 4-31 4-17 JH3 018 JK4 human antibody repertoire [9]. The fact that the VH
E2.5 4-31 2-21/3-9 JH4 018 JK2 genes are recombined with different D and J genes
E2.11 4-61 3-9 JH4 018 JK4 indicates that these eight antibodies are independently
E6.2 4-31 3-10 JH6 018 JK1
E6.3 4-61 1-26 JH4 018 JK4
derived. It was also determined that seven of these eight
E6.4 4-31 4-4/4-11 JH4 012 JK2 antibodies used the same VK gene, 018. The light chain
E7.6.3 4-61 2-21/3-9 JH3 018 JK4 sequence of the eighth antibody aligned best with VK
E7.7 1-2 4-17 JH4 018 JK4 012 but also aligned very closely with 018. Additional
E20.8.1 3-33 4-23 JH4 L5 JK1 analysis of the heavy chain sequences revealed even
E20.11.2 3-33 4-23 JH4 L5 JKl more striking similarities in that all eight antibodies

Figure 3. Sequences of heavy chain variable regions from anti-EGFr antibodies. Sequences of five variable regions containing VH 4-31
and three sequences containing VH 4-61 are aligned with the germline sequences. Mutations resulting in amino acid substitutions are
indicated in the single letter amino acid code. An aspartate residue (D) has been introduced as a mutation from germline into CDR1 of
all eight sequences.

bore an aspartate residue in CDR1 that was the result
of somatic mutation (Figure 3). Thus, there appear to
be rigorous structural requirements for antibodies that
bind effectively to the ligand binding site on the EGFr.
Of the remaining three antibodies, two used VH 3-33
and VK L5 and differed from one another by only two
amino acids, suggesting that they derived from the same
germinal center. The other antibody used VH 1-2 and,
again, VK 018.
To select a candidate for moving forward into prod-
uct development, the relative potencies and productiv-
ities of the hybridomas in culture were assayed. The
clone E7.6.3 was found to be highly potent as well as
to have a productivity in serum-containing medium of
5 pg/cell/day. After adaptation to serum free medium,
the productivity increased to l2 pg/cell/day. In a fed
spinner flask, the accumulated antibody concentration
was approximately 400 mg/L.
The E7.6.3 antibody has now been evaluated in an
extensive series of mouse xenograft tumor models and
has been found to be effective not only in inhibiting
tumor growth but also in actually clearing established
tumors as large as l.2 cm3 [l0]. Based on these results,
the antibody has been advanced into a Phase I clini-
cal trial in multiple cancer indications. The antibody
for these studies is manufactured from hybridoma cells
cultured in airlift fermenters.
Conclusion
XenoMouse technology offers a rapid path to the
development of fully human monoclonal antibodies
for human therapy. Large panels of neutralizing anti-
bodies specific for a given target antigen are readily
obtained using standard hybridoma technology. Pre-
liminary results from a clinical trial indicate that the
first antibody produced from XenoMouse animals to
be tested in humans has a pharmacokinetic profile sim-
ilar to normal human immunoglobulin and is nonim-
munogenic. In the example described herein, a high
affinity (50 pM) monoclonal antibody specific for the
EGF receptor was selected from a panel of seventy
anti-EGFr MAbs generated from four fusions. This
antibody has now advanced to clinical trials for the
treatment of cancer. It is anticipated that the application
425
of XenoMouse technology to other target antigens
will yield additional candidate antibodies for cancer
therapy.
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Address for offprints: C. Geoffrey Davis, Abgenix, Inc.,
7601 Dumbarton Circle, Fremont, CA 94555, USA; e-mail:
davis g@abgenix.com