We recently demonstrated that a human recombinant scFv, L19, reacting with the ED-B domain of fibronectin, a marker of angiogenesis, selectively targets tumoral vasculature in vivo. Using the variable regions of L19, we constructed and expressed a human "small immunoprotein" (SIP) and a complete human IgG1 and performed biodistribution studies in tumor-bearing mice to compare the blood clearance rate, in vivo stability and performance in tumor targeting of the 3 L19 formats [dimeric scFv (scFv) 2 , SIP and IgG1]. The accumulation of the different antibody formats in the tumors studied was a consequence of the clearance rate and in vivo stability of the molecules. Using the SIP, the %ID/g in tumors was 2-5 times higher than that of the (scFv) 2 , reaching a maximum 4 -6 hr after injection. By contrast, the accumulation of IgG1 in tumors constantly rose during the experiments. However, due to its slow clearance, the tumor-blood ratio of the %ID/g after 144 hr was only about 3 compared to a ratio of 10 for the (scFv) 2 and 70 for the SIP after the same period of time. The different in vivo behavior of these 3 completely human L19 formats could be exploited for different diagnostic and/or therapeutic purposes, depending on clinical needs and disease. Furthermore, the fact that ED-B is 100% homologous in human and mouse, which ensures that L19 reacts equally well with the human and the murine antigen, should expedite the transfer of these reagents to clinical trials. © 2002 Wiley-Liss, Inc. Key words: antibody formats; tumor vasculature; tumor targeting; clinical applications; cancer diagnosis and therapyDespite their enormous potential as therapeutic agents, monoclonal antibodies (mAbs) of nonhuman origin have not performed as well as expected in clinical trials as a result of their immunogenicity, 1,2 poor pharmacokinetic properties 3,4 and inefficiency in recruiting effector functions. 5,6 The recent prospect of isolating human antibody fragments from phage display libraries 7-10 transcends these problems, revitalizing studies and rekindling hopes of using these reagents to treat major diseases. Indeed, these molecules should serve as ideal building blocks for novel diagnostic and therapeutic tools. 11,12 Furthermore, these antibodies can be "matured" to reach affinities in the picomolar range, 13 desirable, if not necessary, for their clinical use. 14,15 Clinical applications of human antibody fragments for the selective delivery of diagnostic or therapeutic agents nonetheless require highly specific targets. In the case of tumors, the most popular targets are cell-surface antigens, which are usually neither abundant nor stable. On the other hand, during tumor progression the microenvironment surrounding tumor cells undergoes extensive modification that generates a "tumoral environment" that could ultimately represent a suitable target for antibody-based tumor therapy. 16 In fact, the concept that the altered tumor microenvironment is itself a carcinogen that can be targeted is increasingly gaining consensus. Mol...

Recombinant single domain antibody fragments (VHHs) that derive from the unusual camelid heavy chain only IgG class (HCAbs) have many favourable properties compared with single-chain antibodies prepared from conventional IgG. As a result, VHHs have become widely used as binding reagents and are beginning to show potential as therapeutic agents. To date, the source of VHH genetic material has been camels and llamas despite their large size and limited availability. Here we demonstrate that the smaller, more tractable and widely available alpaca is an excellent source of VHH coding DNA. Alpaca sera IgG consists of about 50% HCAbs, mostly of the short-hinge variety. Sequencing of DNA encoding more than 50 random VHH and hinge domains permitted the design of PCR primers that will amplify virtually all alpaca VHH coding DNAs for phage display library construction. Alpacas were immunized with ovine tumour necrosis factor alpha (TNFalpha) and a VHH phage display library was prepared from a lymph node that drains the sites of immunizations and successfully employed in the isolation of VHHs that bind and neutralize ovine TNFalpha.

Botulinum neurotoxins (BoNTs) function by delivering a protease to neuronal cells that cleave SNARE proteins and inactivate neurotransmitter exocytosis. Small (14 kDa) binding domains specific for the protease of BoNT serotypes A or B were selected from libraries of heavy chain only antibody domains (VHHs or nanobodies) cloned from immunized alpacas. Several VHHs bind the BoNT proteases with high affinity (KD near 1 nM) and include potent inhibitors of BoNT/A protease activity (Ki near 1 nM). The VHHs retain their binding specificity and inhibitory functions when expressed within mammalian neuronal cells as intrabodies. A VHH inhibitor of BoNT/A protease was able to protect neuronal cell SNAP25 protein from cleavage following intoxication with BoNT/A holotoxin. These results demonstrate that VHH domains have potential as components of therapeutic agents for reversal of botulism intoxication.

Antitoxins for botulinum neurotoxins (BoNTs) and other toxins are needed that can be produced economically with improved safety and shelf-life properties compared to conventional therapeutics with large-animal antisera. Here we show that protection from BoNT lethality and rapid BoNT clearance through the liver can be elicited in mice by administration of a pool of epitope-tagged small protein binding agents together with a single anti-tag monoclonal antibody (MAb). The protein binding agents used in this study were single-chain Fv domains (scFvs) with high affinity for BoNT serotype A (BoNT/A). The addition of increasing numbers of differently tagged scFvs synergistically increased the level of protection against BoNT/A. It was not necessary that any of the BoNT/A binding agents possess toxin-neutralizing activity. Mice were protected from a dose equivalent to 1,000 to 10,000 50% lethal doses (LD 50 ) of BoNT/A when given three or four different anti-BoNT scFvs, each fused to an E-tag peptide, and an anti-E-tag IgG1 MAb. Toxin protection was enhanced when an scFv contained two copies of the E tag. Pharmacokinetic studies demonstrated that BoNT/A was rapidly cleared from the sera of mice given a pool of anti-BoNT/A scFvs and an anti-tag MAb but not from the sera of mice given scFvs alone or anti-tag MAb alone. The scFv pool and anti-tag MAb protected mice from lethality when administered up to 2 h following exposure of mice to a dose equivalent to 10 LD 50 of BoNT/A. These results suggest that it will be possible to rapidly and economically develop and produce therapeutic antitoxins consisting of pools of tagged binding agents that are administered with a single, stockpiled anti-tag MAb.

Improvement of the strategy to target tumor Ags to dendritic cells (DCs) for immunotherapy requires the identification of the most appropriate ligand/receptor pairing. We screened a library of Ab fragments on mouse DCs to isolate new potential Abs capable of inducing protective immune responses. The screening identified a high-affinity Ab against CD36, a multi-ligand scavenger receptor primarily expressed by the CD8α+ subset of conventional DCs. The Ab variable regions were genetically linked to the model Ag OVA and tested in Ag presentation assays in vitro and in vivo. Anti-CD36-OVA was capable of delivering exogenous Ags to the MHC class I and MHC class II processing pathways. In vivo, immunization with anti-CD36-OVA induced robust activation of naive CD4+ and CD8+ Ag-specific T lymphocytes and the differentiation of primed CD8+ T cells into long-term effector CTLs. Vaccination with anti-CD36-OVA elicited humoral and cell-mediated protection from the growth of an Ag-specific tumor. Notably, the relative efficacy of targeting CD11c/CD8α+ via CD36 or DEC205 was qualitatively different. Anti-DEC205-OVA was more efficient than anti-CD36-OVA in inducing early events of naive CD8+ T cell activation. In contrast, long-term persistence of effector CTLs was stronger following immunization with anti-CD36-OVA and did not require the addition of exogenous maturation stimuli. The results identify CD36 as a novel potential target for immunotherapy and indicate that the outcome of the immune responses vary by targeting different receptors on CD8α+ DCs.

Acetobacter diazotrophicus is a nitrogen‐fixing endophytic bacterium, originally isolated from sugarcane. Its colonizing ability was evaluated in high and low N‐fertilized sugarcane plants by inoculating stem‐cuts with a β‐glucuronidase marked A. diazotrophicus strain. Bacterial quantification by the most probable number technique showed a severe decrease of A. diazotrophicus cells in plants fertilized with high levels of nitrogen. The inoculated strain was detected inside low N‐fertilized sugarcane plants by histochemical staining of β‐glucuronidase and scanning electron microscopy. A. diazotrophicus was found mainly inside cortical cells of stems and inside xylem vessels. No β‐glucuronidase activity was observed in non‐inoculated plants. High nitrogen fertilization of fields might be a threat to maintaining naturally occurring endophytic associations.

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Introduction of double-stranded RNA (dsRNA) into a wide variety of cells and organisms results in post-transcriptional depletion of the homologue endogenous mRNA. This well-preserved phenomenon known as RNA interference (RNAi) is present in evolutionarily diverse organisms such as plants, fungi, insects, metazoans, and mammals. Because the identification of the targeted mRNA by the RNAi machinery depends upon WatsonCrick base-pairing interactions, RNAi can be exquisitely specific. We took advantage of this powerful and flexible technique to demonstrate that selective silencing of genes essential for viral propagation prevents in vitro and in vivo viral infection. Using the baculovirus Autographa californica, a rapidly replicating and highly cytolytic double-stranded DNA virus that infects many different insect species, we show for the first time that introduction of dsRNA from gp64 and ie1, two genes essential for baculovirus propagation, results in prevention of viral infection in vitro and in vivo. This is the first report demonstrating the use of RNAi to inhibit a viral infection in animals. This inhibition was specific, because dsRNA from the polyhedrin promoter (used as control) or unrelated dsRNAs did not affect the time course of viral infection. The most relevant consequences from the present study are: 1) RNAi offers a rapid and efficient way to interfere with viral genes to assess the role of specific proteins in viral function and 2) using RNAi to interfere with viral genes essential for cell infection may provide a powerful therapeutic tool for the treatment of viral infections.Introduction of double-stranded RNA (dsRNA) 1 into a wide variety of cells and organisms results in post-transcriptional depletion of the homologue endogenous mRNA (1). This wellpreserved phenomenon known as RNA interference (RNAi) is present in evolutionarily diverse organisms such as plants,