We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This “crossover” retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible “CrossMab” formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab CH1-CL was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.

SWI2/SNF2 ATPases remodel chromatin or other DNA:protein complexes by a poorly understood mechanism that involves ATP-dependent DNA translocation and generation of superhelical torsion. Crystal structures of a dsDNA-translocating SWI2/SNF2 ATPase core from Sulfolobus solfataricus reveal two helical SWI2/SNF2 specific subdomains, fused to a DExx box helicase-related ATPase core. Fully base paired duplex DNA binds along a central cleft via both minor groove strands, indicating that SWI2/SNF2 ATPases travel along the dsDNA minor groove without strand separation. A structural switch, linking DNA binding and the active site DExx motif, may account for the stimulation of ATPase activity by dsDNA. Our results suggest that torque in remodeling processes is generated by an ATP-driven screw motion of DNA along the active site cleft. The structures also redefine SWI2/SNF2 functional motifs and uncover unexpected structural correlation of mutations in Cockayne and X-linked mental retardation syndromes.

The PHOT1 (NPH1) gene from Avena sativa specifies the blue light receptor for phototropism, phototropin, which comprises two FMN-binding LOV domains and a serine͞threonine protein kinase domain. Light exposure is conducive to autophosphorylation of the protein kinase domain. We have reconstituted a recombinant LOV2 domain of A. sativa phototropin with various 13 C͞ 15 N-labeled isotopomers of the cofactor, FMN. The reconstituted protein samples were analyzed by NMR spectroscopy under dark and light conditions. Blue light irradiation is shown to result in the addition of a thiol group (cysteine 450) to the 4a position of the FMN chromophore. The adduct reverts spontaneously in the dark by elimination. The light-driven flavin adduct formation results in conformational modification, which was diagnosed by 1 H and 31 P NMR spectroscopy. This conformational change is proposed to initiate the transmission of the light signal via conformational modulation of the protein kinase domain conducive to autophosphorylation of NPH1. N umerous phenomena in the life cycle of plants, including phototropism, stomatal opening, and circadian periodicity, are modulated by light. Photoreceptors responsive to UV, blue, red, and far red light have been reported. Together, they span the spectral range of about 280-800 nm.Blue light-responsive processes have been known for a long time, but cognate receptors have been characterized only recently. Thus, cryptochromes characterized by sequence similarity to DNA photolyases are now believed to be involved in the synchronization of the circadian clock (1-3). Phototropins are involved in phototropism (4, 5) and have no sequence similarity with the cryptochrome group. FAD and FMN serve as chromophores for cryptochromes and phototropins, respectively (6).Phototropin of Avena sativa is a protein with 923 amino acids specified by the NPH1 gene (ref. 7; for review, see also ref. 8).The protein comprises two FMN-binding LOV domains and a serine͞threonine protein kinase domain.The LOV domains of phototropin are similar to PAS domains involved in light, oxygen, or voltage sensing (4) in a variety of regulatory as well as sensor proteins exhibiting diverse functions (4, 9, 10). Recombinant LOV1 and LOV2 domains of phototropins from different plants have been shown to bind FMN (11). The crystal structure of the LOV2 domain of PHY3 protein of Adiantum capillus-veneris has been published recently (12).In this study, we reconstituted the LOV2 apoprotein from A. sativa with Experimental ProceduresMaterials. Stable isotope-labeled FMN samples were prepared by published procedures (13-15). Recombinant LOV2 Domain of Phototropin.A recombinant fusion protein comprising the calmodulin-binding domain from myosin light chain kinase and the LOV2 domain from NPH1 protein of A. sativa was prepared as described earlier (16,17). The fusion protein is subsequently designated recombinant LOV2 domain.Reconstitution of LOV2 Domain with Isotope-Labeled FMN. Recombinant LOV2 domain (fusion protein) was depleted of FMN and su...

Proteins with sequence similarity to the yeast Snf2 protein form a large family of ATPases that act to alter the structure of a diverse range of DNA–protein structures including chromatin. Snf2 family enzymes are related in sequence to DExx box helicases, yet they do not possess helicase activity. Recent biochemical and structural studies suggest that the mechanism by which these enzymes act involves ATP-dependent translocation on DNA. Crystal structures suggest that these enzymes travel along the minor groove, a process that can generate the torque or energy in remodelling processes. We review the recent structural and biochemical findings which suggest a common mechanistic basis underlies the action of many of both Snf2 family and DExx box helicases.

Remodelling protein nucleic acid interfaces is an important biological task, which is often carried out by nucleic acid stimulated ATPases of the Swi2/Snf2 superfamily. Here we study the mechano-chemical cycle of such an ATPase, namely the catalytic domain of the Sulfolobus solfataricus Rad54 homologue (SsoRad54cd), by means of fluorescence resonance energy transfer (FRET). The results of the FRET studies show that the enzyme can be found in (at least) two different possible conformations in solution. An open conformation, consistent with a recently reported crystal structure, is converted into a closed conformation after DNA binding. Upon subsequent binding of ATP no further change in conformation can be detected by the FRET measurements. Instead, a FRET detectable conformational change occurs after ATP hydrolysis and prior to ADP release, suggesting a powerstroke that is linked to phosphate release. Based on these data we will present a new model for the mechano-chemical cycle of this enzyme. This scheme in turn provides a working model for understanding the function of other members of the Swi2/Snf2 family.

Purpose: CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T-cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma. Experimental Design: FAP-CD40's in vitro activity and FAP specificity were validated by antigen-presenting cell (APC) activation and T-cell priming assays. In addition, FAP-CD40 was tested in subcutaneous MC38-FAP and KPC-4662-huCEA murine tumor models. Results: FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T-cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike nontargeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While nontargeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects. Conclusions: FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.

Hepsin is a type II transmembrane serine protease that is expressed in several human tissues. Overexpression of hepsin has been found to correlate with tumour progression and metastasis, which is so far best studied for prostate cancer, where more than 90% of such tumours show this characteristic. To enable improved future patient treatment, we have developed a monoclonal humanized antibody that selectively inhibits human hepsin and does not inhibit other related proteases. We found that our antibody, hH35, potently inhibits hepsin enzymatic activity at nanomolar concentrations. Kinetic characterization revealed non-linear, slow, tight-binding inhibition. This correlates with the crystal structure we obtained for the human hepsin-hH35 antibody Fab fragment complex, which showed that the antibody binds hepsin around α3-helix, located far from the active centre. The unique allosteric mode of inhibition of hH35 is distinct from the recently described HGFA (hepatocyte growth factor activator) allosteric antibody inhibition. We further explain how a small change in the antibody design induces dramatic structural rearrangements in the hepsin antigen upon binding, leading to complete enzyme inactivation.

Phototropins are a family of plant photoreceptors mediating blue light responses such as phototropism, leaf expansion, chloroplast relocation, and stomatal opening. Characteristic for phototropins are two LOV domains which, when expressed in heterologous systems, each carry a single flavin mononucleotide (FMN) chromophore. Here we describe removal of FMN from the LOV2 domain of Avena sativa using a hydrophobic matrix and successful incorporation of flavin adenine dinucleotide (FAD), riboflavin, and 5'-malonyl-riboflavin into the resulting apoprotein; 5-deaza-FMN was not incorporated under the applied conditions. The chromoproteins reconstituted with the various flavins showed absorption spectra and photocycle almost identical to those of the native LOV2 domain and that reconstituted with FMN except for the kinetics: LOV2-riboflavin and LOV2-5'-malonyl-riboflavin showed more rapid regeneration in the dark. LOV2-FAD can be hydrolyzed to LOV2-FMN with phosphodiesterase, indicating that the adenosine part extrudes from the protein. Together with the data from the X-ray structure (Crosson, S., and Moffat, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2995-3000), the results allow us to decide which of the chromophore-protein interactions are essential for the reconstitution process.