Development of a bifunctional CD1d-anti-HER2 scFv fusion molecule to redirect the iNKT cell-mediated immune response to the tumor site

Abstract : Invariant natural killer T lymphocytes (iNKT) are a unique subpopulation of T lymphocytes recognizing glycolipid antigens in the context of the MHC class I-like molecule CD1d. Upon activation with the high affinity ligand α-galactosylceramide (αGalCer), iNKT cells rapidly produce large amounts of the pro-inflammatory cytokine interferon gamma (IFN-γ) and potently activate cells of the innate and adaptive immune response, such as dendritic cells (DCs), NK and T cells. In this context, iNKT cells have been shown to efficiently mediate antitumor activity, and recent research has focused on the manipulation of these cells for antitumor therapies. However, a major drawback of αGalCer as a free drug is that a single injection of this ligand leads to a short-lived iNKT cell activation followed by a long-term anergy, limiting its therapeutic use. In contrast, we demonstrate here that when αGalCer is loaded on a recombinant soluble CD1d molecule (αGalCer/sCD1d), repeated injections lead to a sustained iNKT and NK cell activation associated with IFN-γ secretion as well as with DC maturation. Most importantly, when the αGalCer/sCD1d is fused to an anti-HER2 scFv antibody fragment, potent inhibition of experimental lung metastasis and established subcutaneous tumors is obtained when systemic treatment is started two to seven days after the injection of HER2-expressing B16 melanoma cells, whereas at this time free αGalCer has no effect. The antitumor activity of the sCD1d-anti-HER2 fusion protein is associated with HER2-specific tumor localization and accumulation of iNKT, NK and T cells at the tumor site. Importantly, active T cell immunization combined with the sCD1d-anti-HER2 treatment leads to the accumulation of antigen-specific CD8 T cells exclusively in HER2-expressing tumors, resulting in potent tumor inhibition. In conclusion, sustained activation and tumor targeting of iNKT cells by recombinant αGalCer/sCD1d molecules thus may promote a combined innate and adaptive immune response at the tumor site that may prove to be effective in cancer immunotherapy. RESUME : Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines du complexe majeur d'histocompatibilité de classe I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. En revanche, l'étude présentée ici démontre que, si l'αGalCer est chargé sur des molécules récombinantes soluble CD1d (αGalCer/sCDld), des injections répétées aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si on fusionne la molécule αGalCer/sCD1d avec un fragment single-chain (scFv) de l'anticorps anti-HER2, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. De plus, une immunisation active combinée avec le traitement sCD1d-anti-HER2 aboutit à une accumulation des lymphocytes T CD8 spécifiques de l'antigène d'immunisation, ceci exclusivement dans des tumeurs qui expriment l'antigène HER2. Cette combinaison résulte dans une activité anti-tumeur accrue. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules recombinantes αGalCer/sCDld conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer. RESUME POUR UN LARGE PUBLIC : Le cancer est une cause majeure de décès dans le monde. Sur un total de 58 millions de décès enregistrés au niveau mondial en 2005, 7,6 millions (soit 13%) étaient dus au cancer. Les principaux traitements de nombreux cancers sont la chirurgie, en association avec la radiothérapie et la chimiothérapie. Néanmoins, ces traitements nuisent aussi aux cellules normales de notre corps et parfois, ils ne suffisent pas pour éliminer définitivement une tumeur. L'immunothérapie est l'une des nouvelles approches pour la lutte contre le cancer et elle vise à exploiter la spécificité du système immunitaire qui peut distinguer des cellules normales et tumorales. Une cellule exprimant un marqueur tumoral (antigène) peut être reconnue par le système immunitaire humoral (anticorps) et/ou cellulaire, induisant une réponse spécifique contre la tumeur. L'immunothérapie peut s'appuyer alors sur la perfusion d'anticorps monoclonaux dirigés contre des antigènes tumoraux, par exemple les anticorps dirigés contre les protéines oncogéniques Her-2/neu dans le cancer du sein. Ces anticorps ont le grand avantage de spécifiquement se localiser à la tumeur et d'induire la lyse ou d'inhiber la prolifération des cellules tumorales exprimant l'antigène. Aujourd'hui, six anticorps monoclonaux non-conjugés sont approuvés en clinique. Cependant l'efficacité de ces anticorps contre des tumeurs solides reste limitée et les traitements sont souvent combinés avec de la chimiothérapie. L'immunothérapie spécifique peut également être cellulaire et exploiter par immunisation active le développement de lymphocytes T cytotoxiques (CTL) capables de détruire spécifiquement les cellules malignes. De telles «vaccinations »sont actuellement testées en clinique, mais jusqu'à présent elles n'ont pas abouti aux résultats satisfaisants. Pour obtenir une réponse lymphocytaire T cytotoxique antitumorale, la cellule T doit reconnaître un antigène associé à la tumeur, présenté sous forme de peptide dans un complexe majeur d'histocompatibilité de classe I (CHM I). Cependant les cellules tumorales sont peu efficace dans la présentation d'antigène, car souvent elles se caractérisent par une diminution ou une absence d'expression des molécules d'histocompatibilité de classe I, et expriment peu ou pas de molécules d'adhésion et de cytokines costimulatrices. C'est en partie pourquoi, malgré l'induction de fortes réponses CTL spécifiquement dirigés contre des antigènes tumoraux, les régressions tumorales obtenus grâce à ces vaccinations sont relativement rares. Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines CMH I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. Notre groupe de recherche a donc eu l'idée de développer une nouvelle approche thérapeutique où la réponse immunitaire des cellules iNKT serait prolongée et redirigée vers la tumeur par des anticorps monoclonaux. Concrètement, nous avons produit des molécules récombinantes soluble CD1d (sCD1d) qui, si elles sont chargés avec l'αGalCer (αGalCer/sCDld), aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si la molécule αGalCer/sCD1d est fusionnée avec un fragment single-chain (scFv) de l'anticorps anti-HER2, la réponse immunitaire est redirigée à la tumeur pour autant que les cellules cancéreuses expriment l'antigène HER2. Les molécules αGalCer/sCDld ainsi présentées activent les lymphocytes iNKT. Avec cette stratégie, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées, même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules récombinantes αGalCer/sCD1d conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer.

EFNS guidelines on diagnosis and management of neuromyelitis optica.

BACKGROUND AND PURPOSE: Neuromyelitis optica (NMO) or Devic's disease is a rare inflammatory and demyelinating autoimmune disorder of the central nervous system (CNS) characterized by recurrent attacks of optic neuritis (ON) and longitudinally extensive transverse myelitis (LETM), which is distinct from multiple sclerosis (MS). The guidelines are designed to provide guidance for best clinical practice based on the current state of clinical and scientific knowledge. SEARCH STRATEGY: Evidence for this guideline was collected by searches for original articles, case reports and meta-analyses in the MEDLINE and Cochrane databases. In addition, clinical practice guidelines of professional neurological and rheumatological organizations were studied. RESULTS: Different diagnostic criteria for NMO diagnosis [Wingerchuk et al. Revised NMO criteria, 2006 and Miller et al. National Multiple Sclerosis Society (NMSS) task force criteria, 2008] and features potentially indicative of NMO facilitate the diagnosis. In addition, guidance for the work-up and diagnosis of spatially limited NMO spectrum disorders is provided by the task force. Due to lack of studies fulfilling requirement for the highest levels of evidence, the task force suggests concepts for treatment of acute exacerbations and attack prevention based on expert opinion. CONCLUSIONS: Studies on diagnosis and management of NMO fulfilling requirements for the highest levels of evidence (class I-III rating) are limited, and diagnostic and therapeutic concepts based on expert opinion and consensus of the task force members were assembled for this guideline.

Outcomes of disconnective surgery in intractable pediatric hemispheric and subhemispheric epilepsy.

Objectives: To study the outcome of disconnective epilepsy surgery for intractable hemispheric and sub-hemispheric pediatric epilepsy. Methods: A retrospective analysis of the epilepsy surgery database was done in all children (age <18 years) who underwent a peri-insular hemispherotomy (PIH) or a peri-insular posterior quadrantectomy (PIPQ) from April 2000 to March 2011. All patients underwent a detailed pre surgical evaluation. Seizure outcome was assessed by the Engel's classification and cognitive skills by appropriate measures of intelligence that were repeated annually. Results: There were 34 patients in all. Epilepsy was due to Rasmussen's encephalitis (RE), Infantile hemiplegia seizure syndrome (IHSS), Hemimegalencephaly (HM), Sturge Weber syndrome (SWS) and due to post encephalitic sequelae (PES). Twenty seven (79.4%) patients underwent PIH and seven (20.6%) underwent PIPQ. The mean follow up was 30.5 months. At the last follow up, 31 (91.1%) were seizure free. The age of seizure onset and etiology of the disease causing epilepsy were predictors of a Class I seizure outcome. Conclusions: There is an excellent seizure outcome following disconnective epilepsy surgery for intractable hemispheric and subhemispheric pediatric epilepsy. An older age of seizure onset, RE, SWS and PES were good predictors of a Class I seizure outcome.

Transcriptional profiling of CD4 T cells identifies distinct subgroups of HIV-1 elite controllers.

Human immunodeficiency virus type 1 (HIV-1) elite controllers maintain undetectable levels of viral replication in the absence of antiretroviral therapy (ART), but their underlying immunological and virological characteristics may vary. Here, we used a whole-genome transcriptional profiling approach to characterize gene expression signatures of CD4 T cells from an unselected cohort of elite controllers. The transcriptional profiles for the majority of elite controllers were similar to those of ART-treated patients but different from those of HIV-1-negative persons. Yet, a smaller proportion of elite controllers showed an alternative gene expression pattern that was indistinguishable from that of HIV-1-negative persons but different from that of highly active antiretroviral therapy (HAART)-treated individuals. Elite controllers with the latter gene expression signature had significantly higher CD4 T cell counts and lower levels of HIV-1-specific CD8(+) T cell responses but did not significantly differ from other elite controllers in terms of HLA class I alleles, HIV-1 viral loads determined by ultrasensitive single-copy PCR assays, or chemokine receptor polymorphisms. Thus, these data identify a specific subgroup of elite controllers whose immunological and gene expression characteristics approximate those of HIV-1-negative persons.

Fine structural variations of alphabetaTCRs selected by vaccination with natural versus altered self-antigen in melanoma patients.

Immunotherapy of cancer is often performed with altered "analog" peptide Ags optimized for HLA class I binding, resulting in enhanced immunogenicity, but the induced T cell responses require further evaluation. Recently, we demonstrated fine specificity differences and enhanced recognition of naturally presented Ag by T cells after vaccination with natural Melan-A/MART-1 peptide, as compared with analog peptide. In this study, we compared the TCR primary structures of 1489 HLA-A*0201/Melan-A(26-35)-specific CD8 T cells derived from both cohorts of patients. Although a strong preference for TRAV12-2 segment usage was present in nearly all patients, usage of particular TRAJ gene segments and CDR3alpha composition differed slightly after vaccination with natural vs analog peptide. Moreover, TCR beta-chain repertoires were broader after natural than analog peptide vaccination. In all patients, we observed a marked conservation of the CDR3beta amino acid composition with recurrent sequences centered on a glycyl-leucyl/valyl/alanyl-glycyl motif. In contrast to viral-specific TCR repertoires, such "public" motifs were primarily expressed by nondominant T cell clonotypes, which contrasted with "private" CDR3beta signatures frequently found in T cell clonotypes that dominated repertoires of individual patients. Interestingly, no differences in functional avidity were observed between public and private T cell clonotypes. Collectively, our data indicate that T cell repertoires generated against natural or analog Melan-A peptide exhibited slightly distinct but otherwise overlapping and structurally conserved TCR features, suggesting that the differences in binding affinity/avidity of TCRs toward pMHC observed in the two cohorts of patients are caused by subtle structural TCR variations.

Prospective validation of the Pulmonary Embolism Severity Index. A clinical prognostic model for pulmonary embolism

Practice guidelines recommend outpatient care for selected patients with non-massive pulmonary embolism (PE), but fail to specify how these low-risk patients should be identified. Using data from U.S. patients, we previously derived the Pulmonary Embolism Severity Index (PESI), a prediction rule that risk stratifies patients with PE. We sought to validate the PESI in a European patient cohort. We prospectively validated the PESI in patients with PE diagnosed at six emergency departments in three European countries. We used baseline data for the rule's 11 prognostic variables to stratify patients into five risk classes (I-V) of increasing probability of mortality. The outcome was overall mortality at 90 days after presentation. To assess the accuracy of the PESI to predict mortality, we estimated the sensitivity, specificity, and predictive values for low- (risk classes I/II) versus higher-risk patients (risk classes III-V), and the discriminatory power using the area under the receiver operating characteristic (ROC) curve. Among 357 patients with PE, overall mortality was 5.9%, ranging from 0% in class I to 17.9% in class V. The 186 (52%) low-risk patients had an overall mortality of 1.1% (95% confidence interval [CI]: 0.1-3.8%) compared to 11.1% (95% CI: 6.8-16.8%) in the 171 (48%) higher-risk patients. The PESI had a high sensitivity (91%, 95% CI: 71-97%) and a negative predictive value (99%, 95% CI: 96-100%) for predicting mortality. The area under the ROC curve was 0.78 (95% CI: 0.70-0.86). The PESI reliably identifies patients with PE who are at low risk of death and who are potential candidates for outpatient care. The PESI may help physicians make more rational decisions about hospitalization for patients with PE.

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.

Cutting edge: NKT cell development is selectively impaired in Fyn- deficient mice.

Most NK1.1+ T (NKT) cells express a biased TCRalphabeta repertoire that is positively selected by the monomorphic MHC class I-like molecule CD1d. The development of CD1d-dependent NKT cells is thymus dependent but, in contrast to conventional T cells, requires positive selection by cells of hemopoietic origin. Here, we show that the Src protein tyrosine kinase Fyn is required for development of CD1d-dependent NKT cells but not for the development of conventional T cells. In contrast, another Src kinase, Lck, is required for the development of both NKT and T cells. Impaired NKT cell development in Fyn-deficient mice cannot be rescued by transgenic expression of CD8, which is believed to increase the avidity of CD1d recognition by NKT cells. Taken together, our data reveal a selective and nonredundant role for Fyn in NKT cell development.

Virulence and the immune response in malaria

Many factors determine the virulence of a malaria infection. These include host innate resistance mechanisms and, with Plasmodium falciparum, the ability to cytoadhere to endothelial cells, form rosetts, and induce release of cytokines. The effect on virulence of acquired immune responses can be determined by Class I and Class II MHC-antigens; levels of immunological responsiveness may be determined too in other ways. The structure of parasite surface antigens and their great diversity modulate the immune response and influence parasite survival and hence virulence, and transmission to the vector.

Mono-allelic Ly49 NK cell receptor expression.

Each cell is equipped with two copies (alleles) of each autosomal gene. While the vast majority use both alleles, occasional genes are expressed from a single allele. The reason for mono-allelic expression is not always evident and can serve distinct purposes. First, it may facilitate the tight control over the dosage of certain gene products such as some growth factors and their receptors or X-linked genes. Second, the differential usage of the two parental alleles may reflect the mechanisms that ensure mono-specificity, e.g. olfactory receptors, T and B cell receptors. The context of allele-specific expression of the murine Ly49 natural killer (NK) cell receptor genes suggests that their allele-specific expression reflects a process that generates clonal variability.

Degree of host-parasite compatibility between Schistosoma mansoni and their intermediate molluscan hosts in Brazil

The compatibility of Biomphalaria tenagophila, B. straminea and B. glabrata from Minas Gerais with different strains of Schistosoma mansoni was evaluated using the method of Frandsen (1979b) in standardized experiments. One hundred and fifty of each species of snail were individually exposed in the laboratory to 50 miracidia of S. mansoni lines LE, SJ and AL. The cercariae from the infected snails were counted and used to calculate TCP/100 indices, which were compared with those of Frandsen (1979b). For B. tenagophila the TCP/100 indices varied from 37,996 to 74,266 (class II and III). The snail was poorly compatible with LE (class II) and compatible with SJ and AL (class III). For B. straminea the indices varied from 9,484 to 20,508. The snail was not very compatible with SJ (class I) and poorly compatible with LE and AL (class II). For B. glabrata the indices varied from 588,828 to 1,039,065. The snails was extremely compatible (class VI) with the three lines of S. mansoni. These results confirm the epidemiological importance of B. glabrata in Brazil followed by B. tenagophila and B. straminea.

Natural killer cell mediated missing-self recognition can protect mice from primary chronic myeloid leukemia in vivo.

Background: Natural Killer (NK) cells are thought to protect from residual leukemic cells in patients receiving stem cell transplantation. However, multiple retrospective analyses of patient data have yielded conflicting conclusions regarding a putative role of NK cells and the essential NK cell recognition events mediating a protective effect against leukemia. Further, a NK cell mediated protective effect against primary leukemia in vivo has not been shown directly.Methodology/Principal Findings: Here we addressed whether NK cells have the potential to control chronic myeloid leukemia (CML) arising based on the transplantation of BCR-ABL1 oncogene expressing primary bone marrow precursor cells into lethally irradiated recipient mice. These analyses identified missing-self recognition as the only NK cell-mediated recognition strategy, which is able to significantly protect from the development of CML disease in vivo.Conclusion: Our data provide a proof of principle that NK cells can control primary leukemic cells in vivo. Since the presence of NK cells reduced the abundance of leukemia propagating cancer stem cells, the data raise the possibility that NK cell recognition has the potential to cure CML, which may be difficult using small molecule BCR-ABL1 inhibitors. Finally, our findings validate approaches to treat leukemia using antibody-based blockade of self-specific inhibitory MHC class I receptors.

A role for the src family kinase Fyn in NK cell activation and the formation of the repertoire of Ly49 receptors.

NK cell function is regulated by a dual receptor system, which integrates signals from triggering receptors and MHC class I-specific inhibitory receptors. We show here that the src family kinase Fyn is required for efficient, NK cell-mediated lysis of target cells, which lack both self-MHC class I molecules and ligands for NKG2D, an activating NK cell receptor. In contrast, NK cell inhibition by the MHC class I-specific receptor Ly49A was independent of Fyn, suggesting that Fyn is specifically required for NK cell activation via non-MHC receptor(s). Compared to wild type, significantly fewer Fyn-deficient NK cells expressed the inhibitory Ly49A receptor. The presence of a transgenic Ly49A receptor together with its H-2(d) ligand strongly reduced the usage of endogenous Ly49 receptors in Fyn-deficient mice. These data suggest a model in which the repertoire of inhibitory Ly49 receptors is formed under the influenced of Fyn-dependent NK cell activation as well as the respective MHC class I environment. NK cells may acquire Ly49 receptors until they generate sufficient inhibitory signals to balance their activation levels. Such a process would ensure the induction of NK cell self-tolerance.

MHC-II-independent CD4+ T cells induce colitis in immunodeficient RAG-/- hosts.

CD4(+) alpha beta T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice engrafted into congenic immunodeficient RAG1(-/-) B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4(+) T cells from CD1d(-/-) knockout (KO) B6 donor mice but not those from MHC-I(-/-) (homozygous transgenic mice deficient for beta(2)-microglobulin) KO B6 mice induced a colitis in RAG(-/-) hosts. Abundant numbers of in vivo activated (CD69(high)CD44(high)CD28(high)) NK1(+) and NK1(-) CD4(+) T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-alpha and IFN-gamma but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4(+) T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice. cLP CD4(+) T cell populations from homozygous transgenic mice deficient for beta(2)-microglobulin KO B6 donor mice engrafted into RAG(-/-) hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4(+) T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.