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BackgroundTG6050 was designed as an improved oncolytic vector, combining the intrinsic properties of vaccinia virus to selectively replicate in tumors with the tumor-restricted expression of recombinant immune effectors to modify the tumor immune phenotype. These properties might be of particular interest for “cold” tumors, either poorly infiltrated or infiltrated with anergic T cells.Methods TG6050, an oncolytic vaccinia virus encodes single-chain human interleukin-12 (hIL-12) and full-length anti-cytotoxic T-lymphocyte-associated antigen-4 (@CTLA-4) monoclonal antibody. The relevant properties of TG6050 (replication, cytopathy, transgenes expression and functionality) were extensively characterized in vitro. The biodistribution and pharmacokinetics of the viral vector, @CTLA-4 and IL-12, as well as antitumoral activities (alone or combined with immune checkpoint inhibitors) were investigated in several “hot” (highly infiltrated) and “cold” (poorly infiltrated) syngeneic murine tumor models. The mechanism of action was deciphered by monitoring both systemic and intratumoral immune responses, and by tumor transcriptome analysis. The safety of TG6050 after repeated intravenous administrations was evaluated in cynomolgus monkeys, with a focus on the level of circulating IL-12.ResultsMultiplication and propagation of TG6050 in tumor cells in vitro and in vivo were associated with local expression of functional IL-12 and @CTLA-4. This dual mechanism translated into a strong antitumoral activity in both “cold” and “hot” tumor models (B16F10, LLC1 or EMT6, CT26, respectively) that was further amplified when combined with anti-programmed cell death protein-1. Analysis of changes in the tumor microenvironment (TME) after treatment with TG6050 showed increases in interferon-gamma, of CD8+T cells, and of M1/M2 macrophages ratio, as well as a drastic decrease of regulatory T cells. These local modifications were observed alongside bolstering a systemic and specific antitumor adaptive immune response. In toxicology studies, TG6050 did not display any observable adverse effects in cynomolgus monkeys.ConclusionsTG6050 effectively delivers functional IL-12 and @CTLA-4 into the tumor, resulting in strong antitumor activity. The shift towards an inflamed TME correlated with a boost in systemic antitumor T cells. The solid preclinical data and favorable benefit/risk ratio paved the way for the clinical evaluation of TG6050 in metastatic non-small cell lung cancer (NCT05788926 trial in progress).

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.

BackgroundImmune checkpoint blockade (ICB) is a clinically proven concept to treat cancer. Still, a majority of patients with cancer including those with poorly immune infiltrated ‘cold’ tumors are resistant to currently available ICB therapies. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is one of few clinically validated targets for ICB, but toxicities linked to efficacy in approved αCTLA-4 regimens have restricted their use and precluded full therapeutic dosing. At a mechanistic level, accumulating preclinical and clinical data indicate dual mechanisms for αCTLA-4; ICB and regulatory T cell (Treg) depletion are both thought to contribute efficacy and toxicity in available, systemic, αCTLA-4 regimens. Accordingly, strategies to deliver highly effective, yet safe αCTLA-4 therapies have been lacking. Here we assess and identify spatially restricted exposure to a novel strongly Treg-depleting, checkpoint-blocking, vectorized αCTLA-4, as a highly efficacious and potentially safe strategy to target CTLA-4.MethodsA novel human IgG1 CTLA-4 antibody (4-E03) was identified using function-first screening for monoclonal antibodies (mAbs) and targets associated with superior Treg-depleting activity. A tumor-selective oncolytic vaccinia vector was then engineered to encode this novel, strongly Treg-depleting, checkpoint-blocking, αCTLA-4 antibody or a matching surrogate antibody, and Granulocyte-macrophage colony-stimulating factor (GM-CSF) (VVGM-αCTLA-4).ResultsThe identified 4-E03 antibody showed significantly stronger Treg depletion, but equipotent checkpoint blockade, compared with clinically validated αCTLA-4 ipilimumab against CTLA-4-expressing Treg cells in a humanized mouse model in vivo. Intratumoral administration of VVGM-αCTLA-4 achieved tumor-restricted CTLA-4 receptor saturation and Treg depletion, which elicited antigen cross-presentation and stronger systemic expansion of tumor-specific CD8+ T cells and antitumor immunity compared with systemic αCTLA-4 antibody therapy. Efficacy correlated with FcγR-mediated intratumoral Treg depletion. Remarkably, in a clinically relevant mouse model resistant to systemic ICB, intratumoral VVGM-αCTLA-4 synergized with αPD-1 to reject cold tumors.ConclusionOur findings demonstrate in vivo proof of concept for spatial restriction of Treg depletion-optimized immune checkpoint blocking, vectorized αCTLA-4 as a highly effective and safe strategy to target CTLA-4. A clinical trial evaluating intratumoral VVGM-αhCTLA-4 (BT-001) alone and in combination with αPD-1 in metastatic or advanced solid tumors has commenced.

Arming oncolytic viruses with transgenes encoding immunomodulators improves their therapeutic efficacy by enhancing and/or sustaining the innate and adaptive anti-tumoral immune responses. We report here the isolation, selection, and vectorization of a blocking anti-human PDL1 single-domain antibody (sdAb) isolated from PDL1-immunized alpacas. Several formats of this sdAb were vectorized into the vaccinia virus (VV) and evaluated for their programmed cell death protein 1 (PD1)/PD1 ligand (PDL1) blocking activity in the culture medium of tumor cells infected in vitro . In those conditions, VV-encoded homodimeric sdAb generated superior PDL1 blocking activity compared to a benchmark virus encoding full-length avelumab. The sdAb was further used to design simple, secreted, and small tumor necrosis factor superfamily (TNFSF) fusions with the ability to engage their cognate receptors (TNFRSF) only in the presence of PDL1-positive cells. Finally, PDL1-independent alternatives of TNFRSF agonists were also constructed by fusing different variants of surfactant protein-D (SP-D) oligomerization domains with TNFSF ectodomains. An optimal SP-D–CD40L fusion with an SP-D collagen domain reduced by 80% was identified by screening with a transfection/infection method where poxvirus transfer plasmids and vaccinia virus were successively introduced into the same cell. However, once vectorized in VV, this construct had a much lower CD40 agonist activity compared to the SP-D–CD40L construct, which is completely devoid of the collagen domain that was finally selected. This latest result highlights the importance of working with recombinant viruses early in the payload selection process. Altogether, these results bring several complementary solutions to arm oncolytic vectors with powerful immunomodulators to improve their immune-based anti-tumoral activity.

BackgroundCheckpoint inhibitor antibodies have improved survival in a variety of cancers, however, a great unmet need remains since only a small fraction of patients responds. Reasons for lack of efficacy are believed to include lack of tumor infiltrating immune cells, a notion supported by improved efficacy observed following combined checkpoint blockade with tumor oncolytic virotherapy which promotes intratumoral T cell infiltration. Oncolytic vaccinia viruses (oVV) also allow genetic encoding of transgenes. This is of special interest for therapeutic proteins exhibiting toxicological limitation or pharmacokinetic issues. Here, BioInvent and Transgene present a potentially safe and more efficacious strategy to combine checkpoint inhibition in the context of oncolytic virotherapy.MethodsUsing the F.I.R.S.T™ discovery platform we have isolated a human recombinant Treg-depleting antibody that has been vectorized alongside GM-CSF into the Invir.IO® oVV. This product named BT-001 consists of a Copenhagen double deleted vaccinia virus encoding the human CTLA4-specific antibody 4-E03 IgG1, which shows improved Treg-depletion compared with ipilimumab in a human PBMC-based NOG/SCID-transfer model. BT-001 also encodes GM-CSF, the cytokine expressed in clinically approved products. A surrogate murine mAb was vectorized into the same oVV (mBT-1) allowing for functional and mechanistic in vivo studies.ResultsOur studies demonstrate that 4-E03 and GM-CSF were expressed as functional molecules after infection by BT-001 of human tumor cell lines in vitro. Moreover, following intratumoral administration in immune competent and immune deficient mice transplanted with mouse or human tumors, transgene expression was sustained at levels associated with receptor saturation for days to weeks. In contrast, and supporting the tumor-selective nature of oVV, blood concentrations of anti-CTLA4 mAb were lower compared to those observed following i.v. administration of therapeutic doses of mAb. The in vivo anti-tumor activity of mBT-1 was assessed in multiple syngeneic mouse tumor models including CT26, EMT6, A20 and C38. Murine surrogate mBT-1 conferred cures in the majority of challenged mice irrespective of tumor origin. The excellent anti-tumoral profile depends on anti-CTLA4 expression and could be boosted by co-administration of anti-PD-1 mAb. Intratumoral treatment with mBT-1 also induces abscopal anti-tumor responses and protects against tumor rechallenge demonstrating a long-lasting systemic anti-tumor activity.ConclusionsA clinical batch of BT-001 has been produced and toxicological evaluation is ongoing. Transgene and BioInvent have applied for a clinical trial targeting injectable superficial tumors. Here, the tumor-localized delivery of anti-CTLA4 may allow a better tolerated and more effective combination therapy with antibodies targeting the PD-1/PDL1 axis.

Anthracyclines can induce a type 1 interferon response in tumor cells that may predict clinical response to these drugs. Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell–mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN-α and IFN-β receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN–related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy.

BackgroundThere 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]. Preclinical research most often uses ectopic mouse tumor models for the readout fastness and easiness provided by such models. However, those models may not be the optimal way to address the issue of targeting effector T cells to a deep organ born tumor.MethodsWe set up an orthotopic preclinical model in which renal carcinoma cells (RenCa) expressing the human mucin 1 tumor-associated xeno-antigen are injected into the sub-capsular space of kidney in BALB/c mice. We then evaluated the impact of the route of injection on the therapeutic efficacy of a Modified Vaccinia virus Ankara expressing the human mucin 1 xeno-antigen (MVA-MUC1).ResultsWe show that intravenous administration of MVA-MUC1 is therapeutically superior to subcutaneous injection. Therapeutic efficacy of MVA-MUC1 can be further enhanced by intravenous administration of a TLR9 ligand (ODN1826). Substantial and necessary infiltration of tumor-bearing kidneys by CD8+ lymphocytes is associated with therapeutic protection. We observe that infiltration of CD8+ lymphocytes is not limited to tumor-bearing kidneys and is also detected in naive kidneys, liver and lungs. Kidney infiltrating lymphocytes are characterized by an effector memory phenotype and express PD-1 and Tim3 immune checkpoints molecules. Biodistribution experiments indicate that MVA encoded antigens quickly reach deep organs and in particular APC from the spleen upon i.v. injection of MVA in comparison to s.c. injection, resulting in a faster generation of MUC1 specific CD8+ T cells. Unexpectedly, addition of ODN1826 to MVA-MUC1 immunization does not result in an increase in the frequency of MUC1 specific T cells detected in the spleen. Instead, ODN1826 was associated with a more pronounced modification of the tumor microenvironment towards a TH1 type inflammatory response and a recruitment of activated lymphocytes.ConclusionsIntravenous injection of MVA-MUC1 is associated with therapeutic efficacy and deep organ infiltration by MUC1-specific CD8+ T cells in an orthotopic model of renal carcinoma. This therapeutic efficacy can be further increased through the use of ODN1826. This study supports the clinical evaluation of MVA-based immunotherapies via the intravenous route.

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.