Hilde Revets

Efficient Cancer Therapy with a Nanobody-Based Conjugate

Nanobodies are the smallest fragments of naturally occurring single-domain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies are strictly monomeric, very stable, and highly soluble entities. We identified a nanobody with subnanomolar affinity for the human tumor-associated carcinoembryonic antigen. This nanobody was conjugated to Enterobacter cloacae beta-lactamase, and its site-selective anticancer prodrug activation capacity was evaluated. The conjugate was readily purified in high yields without aggregation or loss of functionality of the constituents. In vitro experiments showed that the nanobody-enzyme conjugate effectively activated the release of phenylenediamine mustard from the cephalosporin nitrogen mustard prodrug 7-(4-carboxybutanamido) cephalosporin mustard at the surface of carcinoembryonic antigen-expressing LS174T cancer cells. In vivo studies demonstrated that the conjugate had an excellent biodistribution profile and induced regressions and cures of established tumor xenografts. The easy generation and manufacturing yield of nanobody-based conjugates together with their potent antitumor activity make nanobodies promising vehicles for new generation cancer therapeutics.

Camelid immunoglobulins and nanobody technology.

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.

Efficient inhibition of EGFR signaling and of tumour growth by antagonistic anti-EFGR Nanobodies.

The development of a number of different solid tumours is associated with over-expression of ErbB1, or the epidermal growth factor receptor (EGFR), and this over-expression is often correlated with poor prognosis of patients. Therefore, this receptor tyrosine kinase is considered to be an attractive target for antibody-based therapy. Indeed, antibodies to the EGFR have already proven their value for the treatment of several solid tumours, especially in combination with chemotherapeutic treatment regimens. Variable domains of camelid heavy chain-only antibodies (called Nanobodies™) have superior properties compared with classical antibodies in that they are small, very stable, easy to produce in large quantities and easy to re-format into multi-valent or multi-specific proteins. Furthermore, they can specifically be selected for a desired function by phage antibody display. In this report, we describe the successful selection and the characterisation of antagonistic anti-EGFR Nanobodies. By using a functional selection strategy, Nanobodies that specifically competed for EGF binding to the EGFR were isolated from ‘immune’ phage Nanobody repertoires. The selected antibody fragments were found to efficiently inhibit EGF binding to the EGFR without acting as receptor agonists themselves. In addition, they blocked EGF-mediated signalling and EGF-induced cell proliferation. In an in vivo murine xenograft model, the Nanobodies were effective in delaying the outgrowth of A431-derived solid tumours. This is the first report describing the successful use of untagged Nanobodies for the in vivo treatment of solid tumours. The results show that functional phage antibody selection, coupled to the rational design of Nanobodies, permits the rapid development of novel anti-cancer antibody-based therapeutics.

Efficient tumor targeting by single‐domain antibody fragments of camels

The variable domain of functional heavy chain antibodies (VHH) devoid of light chains, found in camels, constitute the smallest intact antigen‐binding domain fragment. Two camel single‐domain fragments, cAb‐Lys2 and cAb‐Lys3, recognizing an overlapping epitope of lysozyme with a dissociation constant of 2 nM and 65 nM, respectively, and a bivalent cAb‐Lys3 were investigated for their ability to target transgenic tumors expressing lysozyme on their membrane. Biodistribution studies revealed that these non‐immunogenic monomeric and bivalent camel single‐domain antigen binders specifically target lysozyme‐expressing tumors and metastatic lesions. The excess of antibody is rapidly eliminated from the blood circulation and no cAb retention was observed in normal organs. The tumor to organ cAb‐ratios at 2 and 8 hr were in the (2.1–10.8):1 and (6.2–23.7):1 range, respectively. The degree and specificity of tumor retention is independent of the affinity of the recombinant camel single‐domain fragments for their antigen and from their univalent monomeric (15 kDa) or bivalent format (33 kDa). This study demonstrates the successful and specific in vivo targeting of tumors by camel single‐domain fragments. It may open perspectives for their future use as tumor‐targeting vehicle, due to their small size, soluble behaviour and because they are non‐immunogenic and interact with epitopes that are less antigenic for conventional antibodies.

Nanobodies as novel agents for cancer therapy.

Nanobodies are the smallest fragments of naturally occurring heavy-chain antibodies that have evolved to be fully functional in the absence of a light chain. As such, the cloning and selection of antigen-specific nanobodies obviate the need for construction and screening of large libraries, and for lengthy and unpredictable invitro affinity maturation steps. The unique and well-characterised properties enable nanobodies to excel conventional therapeutic antibodies in terms of recognising uncommon or hidden epitopes, binding into cavities or active sites of protein targets, tailoring of half-life, drug format flexibility, low immunogenic potential and ease of manufacture. Moreover, the favourable biophysical and pharmacological properties of nanobodies, together with the ease of formatting them into multifunctional protein therapeutics, leaves them ideally placed as a new generation of antibody-based therapeutics. This review describes the state of the art on nanobodies and illustrates their potential as cancer therapeutic agents.

Comparison of the Biodistribution and Tumor Targeting of Two 99mTc-Labeled Anti-EGFR Nanobodies in Mice, Using Pinhole SPECT/Micro-CT

Camelidae possess an unusual class of antibodies devoid of light chains. Nanobodies are intact antigen-binding fragments that are stable, easily generated against different targets, and fully functional. Their rapid clearance from the blood circulation favors their use as imaging agents. We compared the in vivo tumor uptake and biodistribution of 2 anti–epidermal growth factor receptor (anti-EGFR) Nanobodies, 99mTc-7C12 and 99mTc-7D12. Methods: Nanobodies were labeled via their hexahistidine tail with 99mTc-tricarbonyl (99mTc(CO)3) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFR-negative) xenografts were intravenously injected with 99mTc-7C12 and 99mTc-7D12 on separate days. Pinhole SPECT/micro-CT images were acquired at 1 h after administration to assess noninvasively the biodistribution and tumor targeting of the labeled compounds. Pinhole SPECT and micro-CT images from the same mouse were automatically fused on the basis of a mathematic rigid-body-transformation algorithm using six 57Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm3) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either 99mTc-7C12 or 99mTc-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g). Results: Binding of both tracers was receptor-specific. Image analysis showed high and similar tumor uptake values for both 99mTc-7C12 and 99mTc-7D12 (4.55 ± 0.24 %IA/cm3 and 4.62 ± 0.36 %IA/cm3, respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16 ± 0.14 for 99mTc-7C12 and 1.49 ± 0.60 for 99mTc-7D12). 99mTc-7C12 showed significantly higher kidney uptake (63.48 ± 2.36 vs. 56.25 ± 2.46 %IA/cm3) and lower liver uptake (2.55 ± 0.26 vs. 4.88 ± 0.86 %IA/cm3) than did 99mTc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-to-background ratio; however, 99mTc-7C12 showed higher tumor uptake (9.11 ± 1.12 %IA/g) than did 99mTc-7D12 (6.09 ± 0.77 %IA/g). 99mTc-7D12 demonstrated significantly higher blood activity than did 99mTc-7C12, but both showed short plasma half-lives (<10 min).Conclusion: The Nanobody fragments used here show high tumor uptake, low liver uptake, and rapid blood clearance. Nanobodies are promising probes for noninvasive radioimmunodetection of specific targets early after administration. On the basis of its favorable biodistribution, 99mTc-7C12 was selected for further studies.

Lipoprotein I, a TLR2/4 ligand modulates Th2-driven allergic immune responses

Asthma is an inflammatory lung disease that is initiated and directed by Th2 and inhibited by Th1 cytokines. Microbial infections have been shown to prevent allergic responses by inducing the secretion of the Th1 cytokines IL-12 and IFN-γ. In this study, we examined whether administration of lipoprotein I (OprI) from Pseudomonas aeruginosa could prevent the inflammatory and physiological manifestations of asthma in a murine model of OVA-induced allergic asthma. OprI triggered dendritic cells to make IL-12 and TNF-α, with subsequent IFN-γ production from T cells. OprI stimulation of dendritic cells involved both TLR2 and TLR4. Intranasal coadministration of OprI with OVA allergen resulted in a significant decrease in airway eosinophilia and Th2 (IL-4 and IL-13) cytokines and this effect was sustained after repeated allergen challenge. The immediate suppressive effect of OprI (within 2 days of administration) was accompanied by an increase in Th1 cytokine IFN-γ production and a significant, but transient infiltration of neutrophils. OprI did not redirect the immune system toward a Th1 response since no increased activation of locally recruited Th1 cells could be observed upon repeated challenge with allergen. Our data show for the first time that a bacterial lipoprotein can modulate allergen-specific Th2 effector cells in an allergic response in vivo for a prolonged period via stimulation of the TLR2/4 signaling pathway.

Secretory Leukocyte Protease Inhibitor in Cancer Development

Abstract: Secretory leukocyte protease inhibitor (SLPI), an epithelial‐specific serine protease inhibitor of the whey acidic protein (WAP) family, exerts broad tissue‐protective functions: on the one hand, it counteracts inflammatory responses and invading pathogens, and on the other hand, it repairs the damaged tissue. Here, we report that subcutaneous in vivo passage of low‐malignant 3LL‐S cells increases the malignancy of these cancer cells. Applying the subtraction suppressive hybridization method to this cancer model, we identify SLPI as one of the genes whose level of expression directly correlates with the malignancy of the cancer cells. Using transfection experiments, we demonstrate that overexpression of mouse or human SLPI in 3LL‐S cells is sufficient to enhance their malignant behavior, and that this activity of SLPI is related to its ability to inhibit serine proteases. Furthermore, using cDNA dot‐blot hybridization, we show that in human gynecological cancer tissue SLPI expression is frequently increased as compared with normal tissue. This study underscores the promalignant role of SLPI in the development of cancer and its potential usefulness as a biomarker for gynecological cancer.

Targeting breast carcinoma with radioiodinated anti-HER2 Nanobody

INTRODUCTION With a molecular weight an order of magnitude lower than antibodies but possessing comparable affinities, Nanobodies (Nbs) are attractive as targeting agents for cancer diagnosis and therapy. An anti-HER2 Nb could be utilized to determine HER2 status in breast cancer patients prior to trastuzumab treatment. This provided motivation for the generation of HER2-specific 5F7GGC Nb, its radioiodination and evaluation for targeting HER2 expressing tumors. METHODS 5F7GGC Nb was radioiodinated with ¹²⁵I using Iodogen and with ¹³¹I using the residualizing agent N(ɛ)-(3-[¹³¹I]iodobenzoyl)-Lys⁵-N(α)-maleimido-Gly¹-GEEEK ([¹³¹I]IB-Mal-D-GEEEK) used previously successfully with intact antibodies. Paired-label internalization assays using BT474M1 cells and tissue distribution experiments in athymic mice bearing BT474M1 xenografts were performed to compare the two labeled Nb preparations. RESULTS The radiochemical yields for Iodogen and [¹³¹I]IB-Mal-D-GEEEK labeling were 83.6±5.0% (n=10) and 59.6±9.4% (n=15), respectively. The immunoreactivity of labeled proteins was preserved as confirmed by in vitro and in vivo binding to tumor cells. Biodistribution studies showed that Nb radiolabeled using [¹³¹I]IB-Mal-D-GEEEK, compared with the directly labeled Nb, had a higher tumor uptake (4.65±0.61% ID/g vs. 2.92±0.24% ID/g at 8h), faster blood clearance, lower accumulation in non-target organs except kidneys, and as a result, higher concomitant tumor-to-blood and tumor-to-tissue ratios. CONCLUSIONS Taken together, these results demonstrate that 5F7GGC anti-HER2 Nb labeled with residualizing [¹³¹I]IB-Mal-D-GEEEK had better tumor targeting properties compared to the directly labeled Nb suggesting the potential utility of this Nb conjugate for SPECT (¹²⁹I) and PET imaging (¹²⁴I) of patients with HER2-expressing tumors.

Bacterial lipoprotein-based vaccines induce tumor necrosis factor-dependent type 1 protective immunity against Leishmania major.

ABSTRACT Immunity against Leishmania major requires rapid induction of a type 1 immune response in which tumor necrosis factor alpha (TNF-α) plays an essential role. Hence, vaccination strategies that simulate the protective immune response found in hosts that have recovered from natural infection provide a rational approach to combat leishmaniasis. One method for optimizing the qualitative and quantitative immune responses after vaccination is to use an adjuvant. In this study we demonstrate that the OprI lipoprotein (L-OprI) from Pseudomonas aeruginosa induces a long-term cellular (gamma interferon [IFN-γ]) and humoral (immunoglobulin G2a) type 1 immune response against a truncated 32-kDa version (COOHgp63) of the 63-kDa major cell surface glycoprotein gp63. By contrast, immunization with COOHgp63 either fused to OprI nonlipoprotein or with no adjuvant did not result in the induction of type 1 immune responses. The adjuvanticity of L-OprI is strongly dependent on its capacity to induce TNF-α, since generation of type 1 immune responses is clearly delayed and impaired in TNF-α−/− mice. Vaccination with L-OprICOOHgp63 fusion protein protected BALB/c mice against L. major infection for at least 19 weeks. Vaccinated mice were largely free of lesions or clearly controlled lesion size on termination of the experiment. The control of disease progression in mice vaccinated with L-OprICOOHgp63 was associated with enhancement of antigen-specific IFN-γ production. These data indicate that bacterial lipoproteins constitute appropriate adjuvants to include in vaccines against diseases in which type 1 immune responses are important for protection.