ISSN 1022-7954, Russian Journal of Genetics, 2008, Vol. 44, No. 8, pp. 890–894. © Pleiades Publishing, Inc., 2008.
Original Russian Text © O.L. Polanovsky, S.V. Lukash, O.A. Stremovskiy, D.V. Karpenko, S.M. Deyev, 2008, published in Genetika, 2008, Vol. 44, No. 8, pp. 1023–1028.
Advanced technologies for obtaining chimerical
and “humanized” antibodies, which are widely adopted
in clinical practice for in situ diagnostics and therapy,
have been actively developed in the past years [1–3].
These approaches are underlain by antibody recogni-
tion of the markers overexpressed in cancer cells. To be
clinically applicable, these antibodies must meet sev-
eral requirements, including high afﬁnity to the target,
low immunogenicity and high selectivity of binding.
Thus, the strategy for design and construction of mon-
oclonal humanized antibodies, selectively targeting
cancer cell markers, has emerged [4–7].
The fundamentally different approach is the design
and use of vector systems carrying constructs for in situ
expression of full-length antibodies intended for target
cells recognition. The HER-2/neu cell surface onco-
gene inducing the uncontrolled cell proliferation is
overexpressed by many carcinomas. Humanized anti-
HER-2/neu antibodies are used to treat patients with
metastatic breast cancer .
In this study, we developed recombinant genes
expressing full-length single-chain humanized antibod-
ies of IgE and IgG1 isotypes capable of cancer cell
HER-2/neu receptor selective recognition. It is note-
worthy that IgE isotype in some cases is more effective
than IgG for the treatment of cancer . Previously, we
have analyzed the expression of the chimeric IgE gene
in different mouse tissues using ballistic transfection
. The Humanized single-chain tandem VL-VH
(ScFv fragment 4D5) [11, 12] capable of HER-2/neu
receptor recognition was used to generate the expres-
sion constructs. The tandem was linked to human IgE
or IgG constant genes through the linker peptide-
encoding oligonucleotide. The primary objectives of
the study were to estimate the efﬁciency of expression
of recombinant genes, to identify the antibodies pro-
duced and to detect an interaction between these anti-
bodies and antigen.
MATERIALS AND METHODS
DNA ampliﬁcation primers and templates.
step overlap extension PCR was used for generating the
full-length humanized genes of IgE and IgG isotypes. The
following primers were used: 1) forward, 5'-taagaatgcggc-
cgcgccatggatatcgttatgacccagtctc-3'; 2) reverse, 5'-cctggag-
cagacgccaccactgctcccggg-3'; 3) forward, 5'-gcagtggtggcgtct-
gctccagggacttca-3'; 4) reverse, 5'-caggtcgcggcgctcagt-
gatggtgatggtggtgtttaccgggattta-cagacacc-3'; 5) reverse,
5'-gtcaggagatttgggctcggaagaaacggtaacggtggta-3'; 6) for-
7) reverse, 5'-caggtgcggccgctcagtgatggtgatggtggtgttta-
The plasmids 4D5 scFv-barnase-
the mini-antibody 4D5 ,
ing the entire constant region (including introns) of IgE
 carrying the human IgG1 con-
stant region genes were used as PCR templates.
Expression of Anti-Tumor Recombinant IgG-
and IgE-Like Genes in Eukaryotic Cells
O. L. Polanovsky
, S. V. Lukash
, O. A. Stremovskiy
, D.V. Karpenko
, and S. M. Deyev
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences,
Moscow, 119991 Russia; e-mail: email@example.com
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
Moscow, 117997 Russia
Received September 6, 2007
—The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high afﬁn-
ity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human
tumors) was attached through a ﬂexible linker to the second exon of human antibodies of IgG or IgE isotypes
constant gene. The humanized construct of IgE isotype was generated for the ﬁrst time. Genes of the recombi-
nant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) pro-
moter. Transfected HEK-293 cells efﬁciently produced antibodies of the corresponding isotypes IgE and IgG1.
The results of Western blotting conﬁrmed homogeneity of the expressed antibodies, which had the predicted
molecular weight and speciﬁcally interacted with the HER-2/neu. The attachment of leader peptide to the 5'-end
of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These
results indicate that de novo constructed humanized immunoglobulin genes express functionally active, single-
chain recombinant antibodies in eukaryotic cells.