Jennifer Richardson

Epidemiology, molecular virology and diagnostics of Schmallenberg virus, an emerging orthobunyavirus in Europe

After the unexpected emergence of Bluetongue virus serotype 8 (BTV-8) in northern Europe in 2006, another arbovirus, Schmallenberg virus (SBV), emerged in Europe in 2011 causing a new economically important disease in ruminants. The virus, belonging to the Orthobunyavirus genus in the Bunyaviridae family, was first detected in Germany, in The Netherlands and in Belgium in 2011 and soon after in the United Kingdom, France, Italy, Luxembourg, Spain, Denmark and Switzerland. This review describes the current knowledge on the emergence, epidemiology, clinical signs, molecular virology and diagnosis of SBV infection.

Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index.

A proteolytically activatable EGFR Probody demonstrates antitumor efficacy while alleviating toxicity. Seek and Destroy One of the main problems with current cancer therapies is lack of specificity: Traditional chemotherapeutics target all dividing cells, and even more restricted drugs, like monoclonal antibodies, may have on-target but off-tumor side effects. But what if you had a drug that was only turned on in the presence of the tumor? Desnoyers et al. now report the development of a Probody that targets epidermal growth factor receptor (EGFR) only in the presence of tumor. Cetuximab is a Food and Drug Administration–approved EGFR-targeting antibody used to treat metastatic colorectal cancer and head and neck cancer, but therapy often results in dose-limiting skin rash. The authors modified cetuximab to form a Probody (PB1)—where the antigen-binding sites are masked until the antibody is activated by proteases commonly found in the tumor microenvironment. The authors found that PB1 was largely inert while in circulation in mice, but that it had comparable efficacy to cetuximab in the presence of tumor. In nonhuman primates, PB1 demonstrated safety and decreased toxicity at higher doses than cetuximab. What’s more, ex vivo human primary tumor samples were sufficient to activate PB1. If these data hold true in human trials and for other antibodies, Probodies could be used to target cancer while minimizing treatment side effects. Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)–directed Probody therapeutic—an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab. In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of empowered antibody formats such as antibody-drug conjugates and bispecifics.

Borna Disease Virus Infects Human Neural Progenitor Cells and Impairs Neurogenesis

ABSTRACT Understanding the complex mechanisms by which infectious agents can disrupt behavior represents a major challenge. The Borna disease virus (BDV), a potential human pathogen, provides a unique model to study such mechanisms. Because BDV induces neurodegeneration in brain areas that are still undergoing maturation at the time of infection, we tested the hypothesis that BDV interferes with neurogenesis. We showed that human neural stem/progenitor cells are highly permissive to BDV, although infection does not alter their survival or undifferentiated phenotype. In contrast, upon the induction of differentiation, BDV is capable of severely impairing neurogenesis by interfering with the survival of newly generated neurons. Such impairment was specific to neurogenesis, since astrogliogenesis was unaltered. In conclusion, we demonstrate a new mechanism by which BDV might impair neural function and brain plasticity in infected individuals. These results may contribute to a better understanding of behavioral disorders associated with BDV infection.

Antigen encoded by vaccine vectors derived from human adenovirus serotype 5 is preferentially presented to CD8+ T lymphocytes by the CD8α+ dendritic cell subset

Different subsets of dendritic cells (DC) elicit qualitatively different immune responses. In mice, two lymphoid tissue-resident subsets, CD8α(+) and CD8α(-), have been implicated in the induction of T helper 1 (Th1) or Th2 responses, respectively. Moreover, CD8α(+) DC appear to play a major role in priming CD8(+) T lymphocyte responses to viral antigens in the course of diverse viral infections. These considerations have been less extensively explored for vaccine vectors derived from viruses. Despite inefficient ex vivo transduction of DC, vectored vaccines derived from human adenoviruses of serotype 5 (Ad5) elicit robust immune responses, predominantly of the Th1 orientation, in humans and mice. At present it is unknown whether Ad5 interacts with DC subsets in a differential manner, thereby influencing the quality of the elicited IR. To address this issue, successive steps (attachment, transgene expression, MHC class I antigen presentation and activation of antigen-specific T lymphocytes) involved in induction of immune responses by Ad5-based vectors have been examined in CD8α(+) and CD8α(-) murine DC subsets. Although in both ex vivo and in vivo experiments CD8α(+) and CD8α(-) DC subsets captured an Ad5-based vector to a similar extent, transgene expression and subsequent MHC class I display of a transgene-encoded antigen were more efficient in CD8α(+) DC. Moreover, following in vivo and ex vivo transduction with an Ad5-based vaccine, antigen-specific CD8(+) T lymphocytes were more efficiently activated by CD8α(+) DC than by CD8α(-) DC. Thus, superior antigen expression and MHC class I display in CD8α(+) DC may contribute to preferred priming of antigen-specific CD8(+) lymphocytes by Ad5-transduced CD8α(+) DC.

Canine adenoviruses elicit both humoral and cell-mediated immune responses against rabies following immunisation of sheep

Safe and efficient vaccination is important for rabies prevention in domestic animals. Replicative vectors expressing the rabies virus glycoprotein, derived from canine adenovirus have been reported to be promising vaccines in various animal models. In this paper we compare the potential of a replicative and a non-replicative vector, both based on canine adenovirus type 2 and expressing the rabies glycoprotein. Upon inoculation in sheep, immune responses against the rabies virus protein elicited by recombinant vectors were monitored. All immunised sheep produced a rapid and potent neutralizing antibody response against rabies virus after a single inoculation of either replicative or non-replicative recombinant canine adenovirus type 2. In addition, the non-replicative vector expressing the rabies glycoprotein stimulated antigen-specific CD4(+) and CD8(+) lymphocyte proliferation as well as IFN-γ production. These results suggest that vectors derived from canine adenovirus 2 could be considered for the development of promising vaccines in the ruminant species.

IS-98-ST1 West Nile Virus Derived from an Infectious cDNA Clone Retains Neuroinvasiveness and Neurovirulence Properties of the Original Virus

Infectious clones of West Nile virus (WNV) have previously been generated and used to decipher the role of viral proteins in WNV virulence. The majority of molecular clones obtained to date have been derived from North American, Australian, or African isolates. Here, we describe the construction of an infectious cDNA clone of a Mediterranean WNV strain, IS-98-ST1. We characterized the biological properties of the recovered recombinant virus in cell culture and in mice. The growth kinetics of recombinant and parental WNV were similar in Vero cells. Moreover, the phenotype of recombinant and parental WNV was indistinguishable as regards viremia, viral load in the brain, and mortality in susceptible and resistant mice. Finally, the pathobiology of the infectious clone was examined in embryonated chicken eggs. The capacity of different WNV strains to replicate in embryonated chicken eggs closely paralleled their ability to replicate in mice, suggesting that inoculation of embryonated chicken eggs could provide a practical in vivo model for the study of WNV pathogenesis. In conclusion, the IS-98-ST1 infectious clone will allow assessment of the impact of selected mutations and novel genomic changes appearing in emerging European strains pathogenicity and endemic or epidemic potential. This will be invaluable in the context of an increasing number of outbreaks and enhanced severity of infections in the Mediterranean basin and Eastern Europe.

Expression of VP7, a Bluetongue Virus Group Specific Antigen by Viral Vectors: Analysis of the Induced Immune Responses and Evaluation of Protective Potential in Sheep

Bluetongue virus (BTV) is an economically important Orbivirus transmitted by biting midges to domestic and wild ruminants. The need for new vaccines has been highlighted by the occurrence of repeated outbreaks caused by different BTV serotypes since 1998. The major group-reactive antigen of BTV, VP7, is conserved in the 26 serotypes described so far, and its role in the induction of protective immunity has been proposed. Viral-based vectors as antigen delivery systems display considerable promise as veterinary vaccine candidates. In this paper we have evaluated the capacity of the BTV-2 serotype VP7 core protein expressed by either a non-replicative canine adenovirus type 2 (Cav-VP7 R0) or a leporipoxvirus (SG33-VP7), to induce immune responses in sheep. Humoral responses were elicited against VP7 in almost all animals that received the recombinant vectors. Both Cav-VP7 R0 and SG33-VP7 stimulated an antigen-specific CD4+ response and Cav-VP7 R0 stimulated substantial proliferation of antigen-specific CD8+ lymphocytes. Encouraged by the results obtained with the Cav-VP7 R0 vaccine vector, immunized animals were challenged with either the homologous BTV-2 or the heterologous BTV-8 serotype and viral burden in plasma was followed by real-time RT-PCR. The immune responses triggered by Cav-VP7 R0 were insufficient to afford protective immunity against BTV infection, despite partial protection obtained against homologous challenge. This work underscores the need to further characterize the role of BTV proteins in cross-protective immunity.

Abstract C158: Tumor-specific inhibition of jagged-dependent notch signaling using a Probody™ therapeutic.

Probodies represent a unique class of antibody-based therapeutics that specifically target activity to diseased tissues including cancer. Probodies are designed to be inactive in circulation and in healthy tissue and activated only within the tumor microenvironment by dysregulated protease activity. The Probody approach enables promising therapeutic targets not druggable with conventional antibodies due to toxicity. Inhibition of Notch signaling has revealed the therapeutic potential of targeting this pathway in cancer; however, systemic inhibition results in severe toxicities (e.g. gastrointestinal and cardiac) in preclinical and clinical studies limiting the development of Notch-targeting therapeutics. We have developed a fully human IgG1 monoclonal antibody that binds and inhibits the activity of both human and mouse Jagged1 and Jagged2 (Jagged1/2). In vitro cell based assays and in vivo ectopic xenograft mouse tumor models (BxPC3 and H292) show dose-dependent inhibition of Notch signaling and anti-tumor activity, respectively. However, the antibody also shows profound toxicities in mice, including weight loss, alopecia, hyperkeratosis, and athymia, due to on-target inhibition of Jagged1/2 in healthy tissue. To address these toxicities, we developed a fully recombinant anti-Jagged1/2 Probody, CTX-033, comprised of the same antibody but with a unique peptide mask that blocks the antigen binding site linked to the light chain of the antibody by a linker containing a substrate that is cleavable by one or more proteases upregulated in cancer. CTX-033 had similar anti-tumor activity in the mouse xenograft models as compared to the antibody. However, despite a two-fold higher systemic exposure to CTX-033 as compared to the antibody, Probody treated mice did not exhibit the toxicities associated with the antibody treatment. Furthermore, CTX-033 shows additive anti-tumor efficacy when combined with gemcitabine in the BxPC3 model. Notably, the Probody plus gemcitabine combination lacks the significant toxicity associated with the antibody plus gemcitabine combination treatment. To validate both Jagged expression and protease activity in patient tumor samples, we used a novel in situ assay, immunofluorescent Probody zymography. The results reveal broad expression of the Jagged ligands and specific activation and binding of CTX-033 in patient pancreatic adenocarcinoma tissue samples. The data described here with the anti-Jagged Probody, CTX-033, demonstrates (1) the potential of the Probody platform to enable the safe targeting of two key ligands in the Notch signaling pathway not possible with a traditional antibody format and (2) the presence of both Jagged1/2 and proteases capable of activating CTX-033 in patient tumor samples. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C158. Citation Format: Jason Sagert, James West, Olga Vasiljeva, Jennifer Richardson, Luc Desnoyers, Shouchun Liu, Annie Yang, Chihunt Wong, Elizabeth Menendez, Krishna Polu, Henry Lowman. Tumor-specific inhibition of jagged-dependent notch signaling using a Probody™ therapeutic. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C158.

Abstract 2665: Transforming Notch ligands into tumor-antigen targets: A Probody-Drug Conjugate (PDC) targeting Jagged 1 and Jagged 2

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The development of antibody drug conjugates (ADC) holds significant promise for improving outcomes in patients with cancer. However, toxicity can limit the number of accessible targets for these highly potent and empowered antibody formats due to expression in healthy tissue. Probody formatted ADCs enable opening up the therapeutic window for high potential but previously inaccessible targets, such as the Jagged ligands in the Notch pathway. Probodies are fully recombinant biotherapeutics comprised of a monoclonal antibody whose binding to target antigen is blocked by a masking peptide. Upon cleavage of a specific substrate-linker by tumor-specific proteases, the activated Probody binds its target, resulting in tumor-localized activity. Jagged expression is observed in a wide variety of patient tumors including multiple myeloma, pancreatic cancer, breast cancer, and prostate cancer. We previously described a novel anti-Jagged 1/2 antibody that is efficacious in slowing tumor growth in mouse in-vivo tumor models but results in systemic toxicity. A corresponding Probody mitigates systemic toxicities associated with inhibition of Jagged-induced Notch signaling while maintaining anti-tumor efficacy. Here we show that the Notch ligands Jagged 1 and Jagged 2 have properties that could also enable an antibody-drug conjugate (ADC) approach because the ligands are both expressed on the cell surface and can internalize an anti-Jagged antibody. Using FACS we have shown that Jagged 1/2 are expressed on several human cancer cell lines and by fluorescent IHC staining, the expression of Jagged 1/2 is maintained in the corresponding xenograft tumors. To further explore the potential of Jagged as an ADC target, we engineered a Probody Drug Conjugate (PDC) conjugated to the microtubule inhibitor MMAE. This PDC is efficacious in a pancreatic xenograft tumor model BxPC3. Importantly, in the BxPC3 model the PDC shows equivalent in vivo efficacy to the corresponding ADC without causing the systemic toxicity associated with ADC treatment. Supportive of the potential clinical benefit of an anti-Jagged PDC, more than 75% of lung, pancreatic, and breast cancer patient tumor samples evaluated show moderate to high Jagged 1/2 expression as measured by IHC staining. These data demonstrate that the ProbodyTM platform has potential to enable the use of drug conjugates to target Jagged ligands in the Notch pathway. Citation Format: Jason Sagert, Jim West, Chihunt Wong, Luc Desnoyers, Olga Vasiljeva, Jennifer Richardson, Krishna Polu, Henry Lowman. Transforming Notch ligands into tumor-antigen targets: A Probody-Drug Conjugate (PDC) targeting Jagged 1 and Jagged 2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2665. doi:10.1158/1538-7445.AM2014-2665

Abstract A203: CD3-EGFR bispecific Probody™ therapeutics induced tumor regressions and increased therapeutic window in preclinical studies

T cell-engaging bispecific antibodies (TCBs) represent a highly potent modality to direct the activity of cytotoxic T cells to tumors. TCBs are designed to bind both a surface tumor antigen and the CD3e subunit of the T cell receptor, thereby enabling T cells to kill tumor cells. Because T cell bispecifics bypass TCR antigen recognition and co-stimulatory requirements for T cell activation, they obviate the need for tumor-specific immunity and overcome many impediments faced by T cells in the tumor-microenvironment. TCBs have shown clinical activity in hematologic malignancies as evidenced by the recent approval of blinatumomab for the treatment of relapsed refractory acute lymphoblastic leukemia based on its ability to direct T cells against CD19+ cells. Development of TCBs for non-hematologic cancers is proving more challenging, however, due to the high potency of the modality and its inability to discriminate between cells expressing target antigen in the tumor and those in healthy tissue. Because antigens with highly restricted tumor expression are rare, toxicity directed against healthy tissues has limited the therapeutic utility of TCBs for most solid tumor targets. In addition, because TCBs are administered at very low doses, preferential distribution to circulating T cells or to healthy tissues can limit drug exposure in tumors. Therefore new methods are needed that enable the potent anti-tumor activity of TCBs without on-target damage to normal tissues. Here we describe a T cell-engaging Bispecific Probody therapeutic (TCBP) targeting CD3 and Epidermal Growth Factor Receptor (EGFR). Probody therapeutics are recombinant, proteolytically-activated antibody prodrugs designed to widen therapeutic window by minimizing interaction with normal tissues and maximizing interactions with tumors. A Probody therapeutic is an antibody in which the antigen-binding site is blocked by a masking peptide extension from the amino terminus of the light chain. The peptide mask, joined to the antibody by a protease-cleavable linker, can be removed by proteases that are more active in the tumor microenvironment than in normal tissue, allowing for tumor-localized activity. We have previously demonstrated that an EGFR-targeted Probody therapeutic limits interaction with EGFR-expressing tissue while preserving anti-tumor activity. To demonstrate that Probody technology can address the limitations of bispecific antibodies, we compared CD3-EGFR TCBPs to the corresponding unmasked TCB for their ability to direct T cell-mediated cytotoxicity of EGFR+ tumor cells in vitro. While the unmasked CD3-EGFR TCB demonstrated potent T cell- and EGFR-dependent killing at single-digit pM concentrations, the CD3-EGFR TCBP exhibited reduced targeted cytotoxicity by up to 3000-fold. In a tumor model utilizing engraftment of human T cells in NSG mice, both TCB Probody therapeutic and its parental TCB effectively eliminated established EGFR+ HT-29 colorectal tumors at equivalent doses. In contrast, an ongoing study in non-human primates has shown at least a 10-fold higher maximum tolerated dose for the CD3-EGFR TCBP relative to its corresponding CD3-EGFR TCB. As a result, Probody technology has the potential to increase therapeutic window and may enable potent bispecific therapies for solid tumors that are limited by on-target toxicities in healthy tissue. Citation Format: Sherry L. LaPorte, Daniel R. Hostetter, Laurie Wong, Jennifer Razo, Linnea Diep, Clayton W. White, Jennifer H. Richardson, W. Michael Kavanaugh, Bryan A. Irving. CD3-EGFR bispecific Probody™ therapeutics induced tumor regressions and increased therapeutic window in preclinical studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A203.