Karola Rittner

Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition

ABSTRACT We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8+ and CD4+). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs.

TLR2 ligation protects effector T cells from regulatory T-cell mediated suppression and repolarizes T helper responses following MVA-based cancer immunotherapy

Cancer immunotherapy is hampered by the immunosuppression maintained by regulatory T cells (Tregs) in tumor-bearing hosts. Stimulation of the Toll-like receptor 2 (TLR2) by Pam3Cys is known to affect Treg-mediated suppression. We found that Pam3Cys increases the proliferation of both CD4+ effector T cells (Teffs) and Tregs co-cultured in vitro, but did not induce the proliferation of Tregs alone upon CD3 and CD28 stimulation. In a mouse model of RMA-MUC1 tumors, Pam3Cys was administered either alone or in combination with a modified vaccinia ankara (MVA)-based mucin 1 (MUC1) therapeutic vaccine. The combination of Pam3Cys with MVA-MUC1 (1) diminished splenic Treg/CD4+ T-cell ratios to those found in tumor-free mice, (2) stimulated a specific anti-MUC1 interferon γ (IFNγ) response and (3) had a significant therapeutic effect on tumor growth and mouse survival. When CD4+ Teffs and Tregs were isolated from Pam3Cys-treated mice, Teffs had become resistant to Treg-mediated suppression while upregulating the expression of BclL-xL. Tregs from Pam3Cys-treated mice were fully suppressive for Teffs from naïve mice. Bcl-xL was induced by Pam3Cys with different kinetics in Tregs and Teffs. Teff from Pam3Cys-treated mice produced increased levels of Th1 and Th2-type cytokines and an interleukin (IL)-6-dependent secretion of IL-17 was observed in Teff:Treg co-cultures, suggesting that TLR2 stimulation had skewed the immune response toward a Th17 profile. Our results show for the first time that in a tumor-bearing host, TLR2 stimulation with Pam3Cys affects both Tregs and Teffs, protects Teff from Treg-mediated suppression and has strong therapeutic effects when combined with an MVA-based antitumor vaccine.

Intravenous Injection of MVA Virus Targets CD8+ Lymphocytes to Tumors to Control Tumor Growth upon Combinatorial Treatment with a TLR9 Agonist

Fend and colleagues show in an orthotopic mouse model of renal carcinoma that intravenous and not subcutaneous injection of an MVA-MUC1 vaccine with TLR9 ligand (ODN1826) controls tumor growth by efficiently targeting tumor-specific effector/memory lymphocytes to the tumor. Effector T-cell access to tumor tissue is a limiting step for clinical efficacy of antigen-specific T cell–based immunotherapies. Ectopic mouse tumor models, in which a subcutaneously (s.c.) implanted tumor is treated with s.c. or intramuscular therapeutic immunization, may not be optimal for targeting effector T cells to an organ-borne tumor. We used an orthotopic renal carcinoma model to evaluate the impact of injection routes on therapeutic efficacy of a Modified Vaccinia virus Ankara viral vector expressing the human mucin 1 tumor–associated xeno-antigen (MVA-MUC1). We show that intravenous (i.v.) administration of MVA-MUC1 displayed enhanced efficacy when compared with s.c. injection. Therapeutic efficacy of MVA-MUC1 was further enhanced by i.v. injection of a TLR9 agonist. In all cases, infiltration of tumor-bearing kidney by CD8+ lymphocytes was associated with control of tumor growth. Biodistribution experiments indicate that, following i.v. injection, MVA-encoded antigens are quickly expressed in visceral organs and, in particular, in splenic antigen-presenting cells, compared with those following s.c. injection. This appears to result in a faster generation of MUC1-specific CD8+ T cells. Lymphocytes infiltrating tumor-bearing kidneys are characterized by an effector memory phenotype and express PD-1 and Tim3 immune checkpoint molecules. Therapeutic efficacy was associated with a modification of the tumor microenvironment toward a Th1-type immune response and recruitment of activated lymphocytes. This study supports the clinical evaluation of MVA-based immunotherapies via the i.v. route. Cancer Immunol Res; 2(12); 1163–74. ©2014 AACR.

Sequential administration of a MVA-based MUC1 cancer vaccine and the TLR9 ligand Litenimod (Li28) improves local immune defense against tumors

TG4010 is an immunotherapeutic vaccine based on Modified Vaccinia virus Ankara (MVA) encoding the human tumor-associated antigen MUC1 and human IL-2. In combination with first-line standard of care chemotherapy in advanced metastatic non-small-cell lung cancer (NSCLC), repeated subcutaneous injection of TG4010 improved progression-free survival in phase 2b clinical trials. In preclinical tumor models, MVATG9931, the research version of TG4010, conferred antigen-specific responses against the weak antigen human MUC1. The combination of a suboptimal dose of MVATG9931 and the type B TLR9 ligand Litenimod (Li28) markedly increased survival in a subcutaneous RMA-MUC1 tumor model compared to the treatment with MVATG9931 or Li28 alone. The requirements for this protection were (i) de novo synthesis of MUC1, (ii) Li28 delivered several hours after MVATG9931 at the same site, (iii) at least two vaccination cycles, and (iv) implantation of MUC1-positive tumor cells in the vicinity to the vaccination site. Subcutaneously injected MVATG9931 allowed transient local gene expression and induced the local accumulation of MCP-1, RANTES, M-CSF, IL-15/IL-15R and IP-10. After repeated injection, CD4+ and CD8+ T lymphocytes, B lymphocytes, NK cells, pDCs, neutrophils, and macrophages accumulated around the injection site, local RANTES levels remained high. Delayed injection of Li28 into this environment, led to further accumulation of macrophages, the secretion of IL-18 and IL-1 beta, and an increase of the percentage of activated CD69+ NK cell. Combination treatment augmented the number of activated CD86+ DCs in the draining lymph nodes and increased the percentage of KLRG1+ CD127-CD8+ T cells at the injection site. In vivo depletion of macrophages around the injection site by Clodronate liposomes reduced local IL-18 levels and diminished survival rates significantly. Thus, sequential administration of MVATG9931 and Li28 improves local innate and adaptive immune defense against tumors, arguing for intratumoral delivery of this peculiar sequential combination therapy.

Yeast Virus-Derived Stimulator of the Innate Immune System Augments the Efficacy of Virus Vector-Based Immunotherapy

ABSTRACT To identify novel stimulators of the innate immune system, we constructed a panel of eight HEK293 cell lines double positive for human Toll-like receptors (TLRs) and an NF-κB-inducible reporter gene. Screening of a large variety of compounds and cellular extracts detected a TLR3-activating compound in a microsomal yeast extract. Fractionation of this extract identified an RNA molecule of 4.6 kb, named nucleic acid band 2 (NAB2), that was sufficient to confer the activation of TLR3. Digests with single- and double-strand-specific RNases showed the double-strand nature of this RNA, and its sequence was found to be identical to that of the genome of the double-stranded RNA (dsRNA) L-BC virus of Saccharomyces cerevisiae. A large-scale process of production and purification of this RNA was established on the basis of chemical cell lysis and dsRNA-specific chromatography. NAB2 complexed with the cationic lipid Lipofectin but neither NAB2 nor Lipofectin alone induced the secretion of interleukin-12(p70) [IL-12(p70)], alpha interferon, gamma interferon-induced protein 10, macrophage inflammatory protein 1β, or IL-6 in human monocyte-derived dendritic cells. While NAB2 activated TLR3, Lipofectin-stabilized NAB2 also signaled via the cytoplasmic sensor for RNA recognition MDA-5. A significant increase of RMA-MUC1 tumor rejection and survival was observed in C57BL/6 mice after prophylactic vaccination with MUC1-encoding modified vaccinia virus Ankara (MVA) and NAB2-Lipofectin. This combination of immunotherapies strongly increased at the injection sites the percentage of infiltrating natural killer (NK) cells and plasmacytoid dendritic cells (pDCs), cell types which can modulate innate and adaptive immune responses. IMPORTANCE Virus-based cancer vaccines offer a good alternative to the treatment of cancer but could be improved. Starting from a screening approach, we have identified and characterized an unexplored biological molecule with immunomodulatory characteristics which augments the efficacy of an MVA-based immunotherapeutic agent. The immune modulator consists of the purified dsRNA genome isolated from a commercially used yeast strain, NAB2, mixed with a cationic lipid, Lipofectin. NAB2-Lipofectin stimulates the immune system via TLR3 and MDA-5. When it was injected at the MVA vaccination site, the immune modulator increased survival in a preclinical tumor model. We could demonstrate that NAB2-Lipofectin augments the MVA-induced infiltration of natural killer and plasmacytoid dendritic cells. We suggest indirect mechanisms of activation of these cell types by the influence of NAB2-Lipofectin on innate and adaptive immunity. Detailed analysis of cell migration at the vaccine injection site and the appropriate choice of an immune modulator should be considered to achieve the rational improvement of virus vector-based vaccination by immune modulators.

Shaping the tumor microenvironment with Modified Vaccinia Virus Ankara and TLR9 ligand

Our preclinical data demonstrate that an intravenous injection of Modified Vaccinia virus Ankara induces CD8+ lymphocytes to infiltrate organs to control the growth of orthotopic renal carcinoma upon combination with a toll-like receptor 9 agonist. Such shaping of the tumor microenvironment could constitute the basis of more effective clinical protocols of tumor immunotherapy.

Abstract 2497: Immune checkpoint inhibitors enhance benefits of modified vaccinia virus Ankara to improve survival in preclinical models of cancer

TG4010 is a Modified Vaccinia virus Ankara (MVA) expressing human interleukin 2 and the human mucin1 (MUC1) tumor associated antigen. TG4010 has demonstrated clinical benefit for advanced non small cell lung cancer patients in combination with standard of care chemotherapy in two phase 2 randomized and controlled clinical trials (NCT00415818 and NCT1383148). Immunotherapy based on the use of immune checkpoint blockers such as anti PD-1 and anti PD-L1 has also demonstrated efficacy in phase 2 trials. Hence, the combination of both approaches appears to be of great interest considering the high unmet medical need of this pathology. We developed experimental primary tumor and lung metastasis models to evaluate the rationale of combining both approaches at the preclinical level. Using two CT26 cell lines expressing different target antigens (β-galactosidase and MUC1) we evaluated the impact on overall survival of combined MVA-based and immune checkpoint-based immunotherapies. Synergistic increase in overall survival was observed in the therapeutic CT26-CL25 primary tumor and lung metastasis models upon treatment of mice with the combination of MVA-βgal and anti CTLA4 or anti PD-1 in comparison with either treatment alone. We provide evidence that TG4010 synergized with immune checkpoint inhibitors to increase overall survival in the therapeutic CT26-MUC1 tumor models in comparison with either treatment administrated independently. These observations were associated with an increase in the frequency and the quality of antigen-specific tumor infiltrating CD8+ T cells. These studies pave the way for the evaluation of combinatorial therapies including TG4010 and immune checkpoint blockers in the clinic. Citation Format: Karola Rittner, Christelle Remy-Ziller, Julie Hortelano, Isabelle Farine, Micael de Meyer, Virginie Nourtier, Murielle Gantzer, Christine Thioudellet, Philippe Slos, Xavier Preville. Immune checkpoint inhibitors enhance benefits of modified vaccinia virus Ankara to improve survival in preclinical models of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2497. doi:10.1158/1538-7445.AM2015-2497

Intravenous injection of a modified vaccinia ankara virus induces intratumoral CD8+ lymphocyte infiltration favoring tumor control in an orthotopic tumor model upon combinatorial treatment with a TLR9 agonist

Meeting abstracts There are many critical steps for the clinical efficacy of antigen-specific T cell-based immunotherapies among which traffic to and access to tumor beds of effector T cells, are the most limiting [[1][1]]. Preclinical research most often uses ectopic mouse tumor models for the

119. Studies on the Targeting of Adenoviral Vectors Carrying a Tumor-Cell Specific Peptide

Previously, we have described the identification of a tumor cell-specific peptide HEW (full sequence: HEWSYLAPYPWF) by panning of phage display libraries on the human colorectal cancer cell line WiDr (Rasmussen et al. 2002, Cancer Gene Therapy, 9:606). This peptide sequence was inserted into the HI loop of the wt fiber knob and of the CAR° fiber knob S408E of Ad5E1° vectors containing the b-galactosidase reporter gene. The in vitro infectivity of these viruses on various cell lines was tested. The HEW containing viruses showed a significantly increased infectivity on WiDr cells, as well as on the human breast cancer cell line MDA-MB435, but not on SW480, LoVo, CaCo2, HeLa and 293 indicating that the molecular target of the HEW peptide is not an ubiquitous receptor. Moreover, while the S408E mutation induced a 100-fold decrease of infectivity on CAR-positive cells (e.g. 293 and WiDr cells) the simultaneous insertion of the HEW peptide restored tumor cell-specific high infectivity on WiDr but not on 293 cells. Competion assays confirmed that the HEW receptor was an alternative entry pathway for HEW viruses (Schreiber V, PhD thesis 2003). With these studies we have obtained proof of concept for a de-and re-targeted Ad vector platform in vitro