NEWS AND VIEWS
Two heads are better than one Sherie L Morrison
© 2007 Nature Publishing Group http://www.nature.com/naturebiotechnology
A new design for bispecific antibodies enables efficient production of stable molecules with good pharmacodynamic properties. Antibody-based therapeutics have shown remarkable success in the treatment of conditions such as cancer, inflammation and infectious disease. Yet the clinical efficacy of this class of therapeutic agent is likely to be improved even further through research on antibody design. A report by Wu et al.1 in this issue explores one especially promising avenue: the generation of bispecific antibodies that simultaneously recognize two different molecules. The ease with which the new method produces bispecific, tetravalent antibodies of many different binding specificities suggests that such agents may soon prove clinically important. The biological and structural properties of the antibody make it an attractive therapeutic (Fig. 1a). Antibodies are assembled from two heavy (H) and two light (L) chains. Both the heavy and light chains are divided into variable and constant regions. The variable regions of the heavy (VH) and the light (VL) chain associate to form the binding site of the antibody and together are called the Fv fragment. Flexible linkers can be used to covalently join a VH and a VL chain, forming a single-chain Fv (scFv) that retains the binding specificity of the original antibody. Because of the considerable diversity of the variable regions, it is possible to produce antibodies that recognize virtually any molecule. In naturally occurring antibodies there are two binding sites, and simultaneous binding by two variable regions increases the avidity of the interactions, making the antibody more effective. The constant regions of the antibody primarily determine its functional properties, including its long in vivo half-life and its ability to directly kill cells or microorganisms through complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity. Initially, bispecific antibodies were made by fusing two cell lines, each producing a single monoclonal antibody2. Although the resulting hybrid hybridoma or quadroma did produce bispecific antibodies, they
Sherie L. Morrison is in the Department of Microbiology, Immunology and Molecular Genetics and the Molecular Biology Institute, University of California Los Angeles, 405 Hilgard Avenue, Los Angeles, California 90095, USA. e-mail:
[email protected]
Wu et al. describe a new approach for producing complete antibody molecules with two different binding specificities, which they name dual–variable domain immunoglobulin or DVD-Ig (Fig. 1g). Each light and heavy chain contains two different variable regions joined by short linker sequences. The N-terminal variable regions of the heavy and light chains are of one binding specificity (Fig. 1g; green), and the adjacent variable regions of the same heavy and light chains are of a different specificity (Fig. 1g; red). These extended heavy and light chains are synthesized and assembled into covalent molecules containing two heavy chains and two light chains. Importantly, the authors provide examples of several different DVD-Igs. They also used variable regions obtained from both kappa and lambda light chains, suggesting that the approach can be widely applied. One of the DVD-Igs that they characterized was specific for IL-12 and IL-18. For this protein, they were able to establish Chinese hamster ovary cells that produced it at levels comparable to those seen for a conventional human IgG1. Thus the problems of poor expression frequently encountered with complex molecules such as bispecific
were only a minor population, and extensive purification was required to isolate the desired antibody (Fig. 1b). With the advent of recombinant DNA technology, genetic modifications were made in the heavy chains to facilitate their heterodimerization and to produce greater yields of bispecific antibodies3. However, yields never reached 100%. Recombinant DNA technology has also been used to produce bispecific antibody fragments of many different forms (Fig. 1c–f), including diabodies, in which two scFvs (Fig. 1c) of differing specificities are linked4 (Fig. 1d), and miniantibodies, in which a variety of self-associating secondary structures, such as helix bundles or coiled coils, are used to bring about dimerization of scFv fragments5,6 (Fig. 1e). These have the advantage that they can be expressed at high levels in bacteria, and their small size enhances their penetration into tumors. They also clear rapidly; this can be advantageous for some applications but is a problem if persisting high levels of the therapeutic are required. A major limitation of all these antibody fragments is that they lack the constant region of the antibody with its associated functional properties.
a
Fv
b
H chain VH
L chain
VL
c
d
VH
scFv
IgG
VH VL
VH VL
Diabody VH
e
f VL
VH
VL
VL VH Dimerization motif
Bispecific miniantibodies
VL VH
VL
g
VH
VL VL
VH
Bispecific, tetravalent single-chain antibody
Bispecific, tetravalent DVD-Ig
Figure 1 Schematic representation of several mono- and bispecific antibodies. (a) IgG. (b) All possible antibodies produced by fusing two hybridomas. The circled antibody represents the bispecific molecule of interest. (c) scFv fragment. (d) Diabody. (e) Bispecific miniantibodies. (f) Bispecific, tetravalent single-chain antibody. (g) Bispecific, tetravalent dual–variable domain IgG.
NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 11 NOVEMBER 2007
1233
© 2007 Nature Publishing Group http://www.nature.com/naturebiotechnology
NEWS AND VIEWS antibodies were not seen with proteins of this structure, making it feasible to pursue more extensive preclinical and clinical testing. The half-life in rats of the IL-12/IL-18 bispecific antibody was similar to that of the original IgG1, consistent with its containing an intact constant region. The protein showed bivalent binding to both cytokines, binding each with an affinity similar to that of the original monospecific antibody. In a severe combined immunodeficient mouse model engrafted with human peripheral blood mononuclear cells, the IL-12/IL-18 bispecific antibody was as effective at inhibiting IFN-γ production induced by Staphylococcus aureus dried cells as was a combination of the two original anti-IL-12 and anti-IL-18 monospecific IgG1s. A second IL1α/IL-1β bispecific molecule was more effective than a single antibody in recognizing both IL-1α and IL-1β in a mouse collagen-induced arthritis model of rheumatoid arthritis. The bispecific antibody format described by the authors shares many properties with a singlechain bispecific antibody described by Natsume et al.7 (Fig. 1f). In this molecule, tandem singlechain Fvs specific for tumor-associated glycoprotein (TAG)-72 and MUC1 mucin were linked to the Fc of a human IgG1 heavy chain. The bispecific antibody was effectively produced in Chinese hamster ovary cells, retained the ability to recognize both TAG-72 and MUC1 and—in the presence of the appropriate carbohydrate on the constant region—triggered antibody-dependent cellular cytotoxicity in cells expressing TAG-72 or MUC1.
The report by Wu et al. provides convincing evidence that it will now be possible to produce bispecific antibodies that can simultaneously bind and neutralize two soluble proteins. However, the question of their immunogenicity remains and can only be answered by examining the immune response in treated patients. It is also unclear whether DVD-Igs will be capable of linking two different populations of cells. Early on it was hypothesized that bispecific antibodies recognizing two cell surface proteins on two different cell types could bring the two populations into close proximity. Such antibodies might be used to redirect cytotoxic immune cells to destroy pathogenic target cells, including tumors or virally infected cells, or to bring two cell populations together for activation. Wu et al. have shown that a bispecific protein recognizing both CD3 and CD20 can be made, but it remains to be determined whether it can be used to target CD3-expressing T cells to CD20-expressing B cells. However, the expression levels and homogeneity of the product made in the isolated cell lines suggest that it will soon be possible to answer these important questions in preclinical experiments. 1. Wu, C.Y.H., Grinnell, C. & Bryant, S. Nat. Biotechnol. 25, 1290–1297 (2007). 2. Milstein, C. & Cuello, A.C. Nature 305, 537–540 (1983). 3. Carter, P. J. Immunol. Methods 248, 7–15 (2001). 4. Holliger, P., Prospero, T. & Winter, G. Proc. Natl. Acad. Sci. USA 90, 6444–6448 (1993). 5. Pack, P., Muller, K., Zahn, R. & Pluckthun, A. J. Mol. Biol. 246, 28–34 (1995). 6. Pack, P. & Pluckthun, A. Biochemistry 31, 1579–1584 (1992). 7. Natsume, A. et al. J. Biochem. 140, 359–368 (2006).
Fluorescence nanoscopy goes multicolor Andreas Schönle & Stefan W Hell Photoswitchable fluorescent labels allow simultaneous optical imaging of multiple target complexes inside fixed cells with nanometer spatial resolution. Shades of gray might readily capture the scene, but without color we may miss the essential. This basic fact holds not only for appreciating the sight of turning leaves in the fall but also for unraveling the distribution of Andreas Schönle and Stefan W. Hell are in the Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37070 Göttingen, Germany. e-mail:
[email protected] or
[email protected].
1234
biomolecules in a cell using fluorescence. In a recent report in Science, Bates et al.1 introduced a powerful technique of multi-color labeling in fluorescence nanoscopy. Imaging molecules represented by different hues with a microscope reveals their spatial correlation. But standard microscopy techniques are limited in resolution by diffraction, implying that densely packed biomolecules cannot be located more precisely than within ~200 nm. Although tight prox-
imities (
