Laetitia Fend

Therapeutic Effects of Anti-CD115 Monoclonal Antibody in Mouse Cancer Models through Dual Inhibition of Tumor-Associated Macrophages and Osteoclasts

Tumor progression is promoted by Tumor-Associated Macrophages (TAMs) and metastasis-induced bone destruction by osteoclasts. Both myeloid cell types depend on the CD115-CSF-1 pathway for their differentiation and function. We used 3 different mouse cancer models to study the effects of targeting cancer host myeloid cells with a monoclonal antibody (mAb) capable of blocking CSF-1 binding to murine CD115. In mice bearing sub-cutaneous EL4 tumors, which are CD115-negative, the anti-CD115 mAb depleted F4/80+ CD163+ M2-type TAMs and reduced tumor growth, resulting in prolonged survival. In the MMTV-PyMT mouse model, the spontaneous appearance of palpable mammary tumors was delayed when the anti-CD115 mAb was administered before malignant transition and tumors became palpable only after termination of the immunotherapy. When administered to mice already bearing established PyMT tumors, anti-CD115 treatment prolonged their survival and potentiated the effect of chemotherapy with Paclitaxel. As shown by immunohistochemistry, this therapeutic effect correlated with the depletion of F4/80+CD163+ M2-polarized TAMs. In a breast cancer model of bone metastasis, the anti-CD115 mAb potently blocked the differentiation of osteoclasts and their bone destruction activity. This resulted in the inhibition of cancer-induced weight loss. CD115 thus represents a promising target for cancer immunotherapy, since a specific blocking antibody may not only inhibit the growth of a primary tumor through TAM depletion, but also metastasis-induced bone destruction through osteoclast inhibition.

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.

Immune checkpoint blockade, immunogenic chemotherapy or IFN-α blockade boost the local and abscopal effects of oncolytic virotherapy

Athough the clinical efficacy of oncolytic viruses has been demonstrated for local treatment, the ability to induce immune-mediated regression of distant metastases is still poorly documented. We report here that the engineered oncolytic vaccinia virus VVWR-TK-RR--Fcu1 can induce immunogenic cell death and generate a systemic immune response. Effects on tumor growth and survival was largely driven by CD8+ T cells, and immune cell infiltrate in the tumor could be reprogrammed toward a higher ratio of effector T cells to regulatory CD4+ T cells. The key role of type 1 IFN pathway in oncolytic virotherapy was also highlighted, as we observed a strong abscopal response in Ifnar-/- tumors. In this model, single administration of virus directly into the tumors on one flank led to regression in the contralateral flank. Moreover, these effects were further enhanced when oncolytic treatment was combined with immunogenic chemotherapy or with immune checkpoint blockade. Taken together, our results suggest how to safely improve the efficacy of local oncolytic virotherapy in patients whose tumors are characterized by dysregulated IFNα signaling. Cancer Res; 77(15); 4146-57. ©2017 AACR.

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.

Oncolytic virotherapy with an armed vaccinia virus in an orthotopic model of renal carcinoma is associated with modification of the tumor microenvironment

ABSTRACT Oncolytic virotherapy is an emergent promising therapeutic approach for the treatment of cancer. We have constructed a vaccinia virus (WR strain) deleted for thymidine kinase (TK) and ribonucleotide reductase (RR) genes that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. We evaluated this construct (VV-FCU1) in the orthotopic model of renal carcinoma (RenCa). Systemic administration of VV-FCU1 resulted in orthotopic tumor growth inhibition, despite temporary expression of viral proteins. VV-FCU1 treatment was associated with an infiltration of tumors by CD8+ T lymphocytes and a decrease in the proportion of infiltrating Tregs, thus modifying the ratio of CD8+/CD4+ Treg in favor of CD8+cytotoxic T cells. We demonstrated that VV-FCU1 treatment prolonged survival of animals implanted with RenCa cells in kidney. Depletion of CD8+ T cells abolished the therapeutic effect of VV-FCU1 while depletion of CD4+ T cells enhanced its protective activity. Administration of the prodrug 5-fluorocytosine (5-FC) resulted in a sustained control of tumor growth but did not extend survival. This study shows the importance of CD4+ and CD8+ T cells in vaccinia virus-mediated oncolytic virotherapy and suggests that this approach may be evaluated for the treatment of human renal cell carcinoma.

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.

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.

TNFR2/BIRC3-TRAF1 signaling pathway as a novel NK cell immune checkpoint in cancer

ABSTRACT Natural Killer (NK) cells control metastatic dissemination of murine tumors and are an important prognostic factor in several human malignancies. However, tumor cells hijack many of the NK cell functional features compromising their tumoricidal activity. Here, we show a deleterious role of the TNFα/TNFR2/BIRC3/TRAF1 signaling cascade in NK cells from the tumor microenvironment (TME). TNFα induces BIRC3/cIAP2 transcripts and reduces NKp46/NCR1 transcription and surface expression on NK cells, promoting metastases dissemination in mice and poor prognosis in GIST patients. NKp30 engagement, by promoting the release of TNFα, also contributes to BIRC3 upregulation, and more so in patients expressing predominantly NKp30C isoforms. These findings reveal that in the absence of IL-12 or a Th1-geared TME, TNFα can be considered as a negative regulatory cytokine for innate effectors.

Abstract 4563: Local and abscopal effects in oncolytic virotherapy are boosted by immune checkpoint blockade, immunogenic chemotherapy, or IFNAR blockade

Athough the clinical efficacy of oncolytic viruses has been demonstrated for local treatment, the ability to induce immune-mediated regression of distant metastases is still poorly documented. We here report that an engineered oncolytic Vaccinia Virus, VVWR-TK-RR--Fcu1, is able to induce an immunogenic cell death and thus to generate a systemic immune response. Effect on tumor growth and survival is largely driven by CD8+ T-cells, and we could demonstrate that the immune cell infiltrate in the tumor could be reprogrammed towards a higher ratio of effector T-cells to regulatory CD4+ T-cells. The key role of the type 1-IFN pathway in oncolytic virotherapy was also highlighted, and we could show a strong abscopal response in Ifnar-/- tumors. In this model, the single administration of the virus directly into the tumors, on one flank, led to regression in the contralateral flank (i.e. opposite to the virus injection site). Moreover, we observed that these effects are further enhanced when the oncolytic treatment is combined with either immunogenic chemotherapy such as oxaliplatin, or with immune checkpoint blockers such as anti-PD-1 or anti-CTLA-4. Altogether, these data suggest that local oncolytic virotherapy in patients with tumors altered in IFNAR signaling could increase immune-mediated abscopal regression of distant metastases. Citation Format: Laetitia Fend, Takahiro Yamazaki, Xavier Preville, Eric Quemeneur, Oliver Kepp, Julien Adam, Aurelien Marabelle, Jonathan Pitt, Guido Kroemer, Laurence Zitvogel. Local and abscopal effects in oncolytic virotherapy are boosted by immune checkpoint blockade, immunogenic chemotherapy, or IFNAR blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4563. doi:10.1158/1538-7445.AM2017-4563

Abstract 2352: Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allow their intratumoral delivery and an improved tumor-growth inhibition

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 in 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 3 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 1900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e. without tumor) confirming the virus tropism for tumoral cells and/or that tumoral microenvironment allows virus escape from immune surveillance. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mPD-1, than after IT administration of 10 μg of J43. Interestingly, in the MCA 205 tumor model, WR-mPD-1 (both mAb and scFv) induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that provided by an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs. New generation of oncolytic vaccinia virus that will express several transgenes simultaneously may also be designed with the goal of providing to the patients enhanced therapeutic/toxicity ratio. Citation Format: Jean-Baptiste Marchand, Patricia Kleinpeter, Laetitia Fend, Christine Thioudellet, Michel Geist, Nathalie Sfrontato, Veronique Koerper, Renee Brandely, Dominique Villeval, Karola Rittner, Nathalie Silvestre, Philippe Erbs, Laurence Zitvogel, Eric Quemeneur, Xavier Preville. Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allow their intratumoral delivery and an improved tumor-growth inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2352.