Tumor growth inhibition and immune system activation following treatment with thorium-227 conjugates and PD-1 check-point inhibition in the MC-38 murine model.

Targeted thorium-227 conjugates comprise the combination of a monoclonal antibody with specificity for a tumor cell antigen and a 3,2-HOPO chelator enabling complexation of thorium-227 (Th-227). The radiolabeled conjugate functions as an effective delivery system of alpha-particle radiation to the surface of the tumor cell inducing difficult to repair complex DNA damage and cell death. In addition, the mechanism of action of targeted alpha therapy (TAT) appears to involve a significant component linked to stimulation of the immune system. We report herein evidence of immune activation and long-lasting immune protection of a TAT in a syngeneic model using the MC-38 murine cell line. Firstly, MC-38 cells were irradiated ex vivo with the thorium labeled antibody before subcutaneous implantation into mice. These mice were then rechallenged with MC-38 cells contra-laterally. In the group receiving irradiated cells, 9 out of 10 animals had no measurable tumor growth compared to aggressive tumor growth in the control group. Secondly, in an efficacy study, 500 kBq/kg of thorium labeled antibody alone or in combination with PD-1 checkpoint inhibitor gave statistically significant tumor growth inhibition compared to vehicle control. Animals with no measurable tumors were once again rechallenged contra-laterally with MC-38 cells. The re-growth of tumors was significantly delayed (approx. 60 days) in the treatment group compared to age-matched controls (approx. 30 days) in the monotherapy group. Interestingly, in the TAT/ PD-1 combination group no re-growth was observed demonstrating the potential of combining a TAT with checkpoint inhibition therapy. Finally, tumors were excised from treated mice and analyzed by flow cytometry and immunohistochemistry (IHC). Analysis revealed significant infiltration of CD8+ T-cells and mature dendritic cells compared to vehicle controls. Together these results indicated that an ongoing immune response from treatment with alpha radiation could be enhanced by check-point inhibition.

Immunostimulatory effects of targeted thorium-227 conjugates as single agent and in combination with anti-PD-L1 therapy.

BACKGROUND: Targeted thorium-227 conjugates (TTCs) are an emerging class of targeted alpha therapies (TATs). Their unique mode of action (MoA) is the induction of difficult-to-repair clustered DNA double-strand breaks. However, thus far, their effects on the immune system are largely unknown. Here, we investigated the immunostimulatory effects of the mesothelin-targeted thorium-227 conjugate (MSLN-TTC) in vitro and in vivo in monotherapy and in combination with an inhibitor of the immune checkpoint programmed death receptor ligand 1 (PD-L1) in immunocompetent mice. METHODS: The murine cell line MC38 was transfected with the human gene encoding for MSLN (hMSLN) to enable binding of the non-cross-reactive MSLN-TTC. The immunostimulatory effects of MSLN-TTC were studied in vitro on human cancer cell lines and MC38-hMSLN cells. The efficacy and MoA of MSLN-TTC were studied in vivo as monotherapy or in combination with anti-PD-L1 in MC38-hMSLN tumor-bearing immunocompetent C57BL/6 mice. Experiments were supported by RNA sequencing, flow cytometry, immunohistochemistry, mesoscale, and TaqMan PCR analyses to study the underlying immunostimulatory effects. In vivo depletion of CD8+ T cells and studies with Rag2/Il2Rg double knockout C57BL/6 mice were conducted to investigate the importance of immune cells to the efficacy of MSLN-TTC. RESULTS: MSLN-TTC treatment induced upregulation of DNA sensing pathway transcripts (IL-6, CCL20, CXCL10, and stimulator of interferon genes (STING)-related genes) in vitro as determined by RNASeq analysis. The results, including phospho-STING activation, were confirmed on the protein level. Danger-associated molecular pattern molecules were upregulated in parallel, leading to dendritic cell (DC) activation in vitro. MSLN-TTC showed strong antitumor activity (T:C 0.38, p<0.05) as a single agent in human MSLN-expressing MC38 tumor-bearing immunocompetent mice. Combining MSLN-TTC with anti-PD-L1 further enhanced the efficacy (T:C 0.08, p<0.001) as evidenced by the increased number of tumor-free surviving animals. MSLN-TTC monotherapy caused migration of CD103+ cDC1 DCs and infiltration of CD8+ T cells into tumors, which was enhanced on combination with anti-PD-L1. Intriguingly, CD8+ T-cell depletion decreased antitumor efficacy. CONCLUSIONS: These in vitro and in vivo data on MSLN-TTC demonstrate that the MoA of TTCs involves activation of the immune system. The findings are of relevance for other targeted radiotherapies and may guide clinical combination strategies.

Exploring 5'-Biotinylation of the Sense Strand to Improve siRNA Specificity and Potency.

Although RNA interference is widely used for gene silencing, unintended gene modulation generated by off-target effects represents a major barrier to its applications in biology and medicine. Off-targeting can be induced by both the sense and antisense siRNA strands. An approach to minimizing off-target gene silencing by the sense strand would be the blockade of the 5'-end phosphorylation, thereby impeding its entry into the RNA-induced silencing complex (RISC). In this chapter, a biotin group at the 5'-end of the sense strand was used to inhibit its incorporation into RISC, thereby facilitating the antisense strand selection and enhancing siRNA cleavage potency. Biotin is a naturally occurring compound, and its presence in siRNA sequences will not induce additional side effects.

Mesothelin-Targeted Thorium-227 Conjugate (MSLN-TTC): Preclinical Evaluation of a New Targeted Alpha Therapy for Mesothelin-Positive Cancers.

PURPOSE: Targeted thorium-227 conjugates (TTC) represent a new class of molecules for targeted alpha therapy (TAT). Covalent attachment of a 3,2-HOPO chelator to an antibody enables specific complexation and delivery of the alpha particle emitter thorium-227 to tumor cells. Because of the high energy and short penetration range, TAT efficiently induces double-strand DNA breaks (DSB) preferentially in the tumor cell with limited damage to the surrounding tissue. We present herein the preclinical evaluation of a mesothelin (MSLN)-targeted thorium-227 conjugate, BAY 2287411. MSLN is a GPI-anchored membrane glycoprotein overexpressed in mesothelioma, ovarian, pancreatic, lung, and breast cancers with limited expression in healthy tissue. EXPERIMENTAL DESIGN: The binding activity and radiostability of BAY 2287411 were confirmed bioanalytically. The mode-of-action and antitumor potency of BAY 2287411 were investigated in vitro and in vivo in cell line and patient-derived xenograft models of breast, colorectal, lung, ovarian, and pancreatic cancer. RESULTS: BAY 2287411 induced DSBs, apoptotic markers, and oxidative stress, leading to reduced cellular viability. Furthermore, upregulation of immunogenic cell death markers was observed. BAY 2287411 was well-tolerated and demonstrated significant antitumor efficacy when administered via single or multiple dosing regimens in vivo. In addition, significant survival benefit was observed in a disseminated lung cancer model. Biodistribution studies showed specific uptake and retention of BAY 2287411 in tumors and enabled the development of a mechanistic pharmacokinetic/pharmacodynamic model to describe the preclinical data. CONCLUSIONS: These promising preclinical results supported the transition of BAY 2287411 into a clinical phase I program in mesothelioma and ovarian cancer patients (NCT03507452).

Cancer cell-binding peptide fused Fc domain activates immune effector cells and blocks tumor growth.

Therapeutic strategies aiming at mobilizing immune effector cells to kill tumor cells independent of tumor mutational load and MHC expression status are expected to benefit cancer patients. Recently, we engineered various peptide-Fc fusion proteins for directing Fcg receptor-bearing immune cells toward tumor cells. Here, we investigated the immunostimulatory and anti-tumor effects of one of the engineered Fc fusion proteins (WN-Fc). In contrast to the Fc control, soluble WN-Fc-1 fusion protein activated innate immune cells (e.g. monocytes, macrophages, dendritic cells, NK cells), resulting in cytokine production and surface display of the lytic granule marker CD107a on NK cells. An engineered Fc-fusion variant carrying two peptide sequences (WN-Fc-2) also activated immune cells and bound to various cancer cell types with high affinity, including the murine 4T1 breast carcinoma cells. When injected into 4T1 tumor-bearing BALB/c mice, both peptide-Fc fusions accumulated in tumor tissues as compared to other organs such as the lungs. Moreover, treatment of 4T1 tumor-bearing BALB/c mice by means of two intravenous injections of the WN-Fc fusion proteins inhibited tumor growth with WN-Fc-2 being more effective than WN-Fc-1. Treatment resulted in tumor infiltration by T cells and NK cells. These new engineered WN-Fc fusion proteins may be a promising alternative to existing immunotherapies for cancer.

Diversification of Antitumour Immunity in a Patient with Metastatic Melanoma Treated with Ipilimumab and an IDO-Silenced Dendritic Cell Vaccine.

Indoleamine 2,3-dioxygenase (IDO) expression in dendritic cells (DCs) inhibits T-cell activation and promotes T-cell differentiation into regulatory T-cells. Moreover, IDO expression promotes resistance to immunotherapies targeting immune checkpoints such as the cytotoxic T lymphocyte antigen-4 (CTLA-4). Here, a patient with metastatic melanoma pretreated with ipilimumab, an anti-CTLA-4 blocking antibody, was vaccinated with IDO-silenced DCs cotransfected with mRNA for survivin or hTERT tumour antigens. During vaccination, T-cell responses to survivin and hTERT tumour antigens were generated, and a certain degree of clinical benefit was achieved, with a significant reduction in lung, liver, and skin metastases, along with a better performance status. T-cell responses against MART-1 and NY-ESO-1 tumour antigens were also detected in the peripheral blood. The patient also mounted an antibody response to several melanoma proteins, indicating diversification of the antitumour immunity in this patient. The identification of such serum antibody-reacting proteins could facilitate the discovery of tumour neoantigens.

Generation of new peptide-Fc fusion proteins that mediate antibody-dependent cellular cytotoxicity against different types of cancer cells.

Antibody-dependent cellular cytotoxicity (ADCC), a key effector function for the clinical effectiveness of monoclonal antibodies, is triggered by the engagement of the antibody Fc domain with the Fcγ receptors expressed by innate immune cells such as natural killer (NK) cells and macrophages. Here, we fused cancer cell-binding peptides to the Fc domain of human IgG1 to engineer novel peptide-Fc fusion proteins with ADCC activity. The designed fusion proteins were expressed in human embryonic kidney 293T cells, followed by purification and characterization by western blots. One of the engineered variants (WN-Fc), bound with high affinity to a wide range of solid tumor cell lines (e.g., colon, lung, prostate, skin, ovarian, and mammary tumors). Treatment of cancer cells with the engineered peptide-Fc fusions in the presence of effector NK cells potentially enhanced cytotoxicity, degranulation, and interferon-γ production by NK cells when compared to cells treated with the Fc control. The presence of competing peptides inhibited NK cell activation. Furthermore, a bispecific peptide-Fc fusion protein activated NK cells against HER-1- and/or HER-2-expressing cancer cells. Collectively, the engineered peptide-Fc fusions constitute a new promising strategy to recruit and activate NK cells against tumor cells, a primary goal of cancer immunotherapy.

Recombinant Lactobacillus plantarum induces immune responses to cancer testis antigen NY-ESO-1 and maturation of dendritic cells.

Given their safe use in humans and inherent adjuvanticity, Lactic Acid Bacteria may offer several advantages over other mucosal delivery strategies for cancer vaccines. The objective of this study is to evaluate the immune responses in mice after oral immunization with Lactobacillus (L) plantarum WCFS1 expressing a cell-wall anchored tumor antigen NY-ESO-1. And to investigate the immunostimulatory potency of this new candidate vaccine on human dendritic cells (DCs). L. plantarum displaying NY-ESO-1 induced NY-ESO-1 specific antibodies and T-cell responses in mice. By contrast, L. plantarum displaying conserved proteins such as heat shock protein-27 and galectin-1, did not induce immunity, suggesting that immune tolerance to self-proteins cannot be broken by oral administration of L. plantarum. With respect to immunomodulation, immature DCs incubated with wild type or L. plantarum-NY-ESO-1 upregulated the expression of co-stimulatory molecules and secreted a large amount of interleukin (IL)-12, TNF-α, but not IL-4. Moreover, they upregulated the expression of immunosuppressive factors such as IL-10 and indoleamine 2,3-dioxygenase. Although L. plantarum-matured DCs expressed inhibitory molecules, they stimulated allogeneic T cells in-vitro. Collectively, the data indicate that L. plantarum-NY-ESO-1 can evoke antigen-specific immunity upon oral administration and induce DC maturation, raising the potential of its use in cancer immunotherapies.

Immunosuppressive factor blockade in dendritic cells via siRNAs results in objective clinical responses.

Over the past decade, immunotherapy has emerged as a promising new form of cancer treatment with the potential to eradicate tumor metastasis. However, its curative potential is in general limited by the existence of negative feedback mechanisms that control dendritic cells (DCs) and T-cell activation. For clinically effective immunity, there is a need of inhibiting the expression of these immune suppressors. This could enhance the activation of DCs, T cells, and natural killer cells, and might be beneficial for cancer immunotherapy. Among the immune inhibitory molecules expressed by DCs is indoleamine 2,3-dioxygenase (IDO), an enzyme that conveys immunosuppressive effects by degrading tryptophan, an essential amino acid required for T-cell proliferation and survival. Depletion of tryptophan by IDO-positive DCs induces T-cell apoptosis and the conversion of naïve CD4+ T cells into regulatory T cells that further suppress antitumor immunity. Herein, we describe a protocol for in vitro synthesis of small interfering RNA against IDO and other immunosuppressive factors such as interleukin-10 and programmed cell death-1 ligands in order to reverse immune suppression mediated by DCs. Vaccination with IDO-silenced DC vaccines enhanced immune responses and antitumor immunity in cancer patients.

A facile method for interfering with off-target silencing mediated by the sense strand.

Although siRNA duplexes are widely used for gene silencing, several unwanted effects such as activation of innate immunity and off-target gene silencing can limit their therapeutic use. Off-targeting can be identified for both the sense and antisense siRNA strands. Some avenues of obstructing the incorporation of the sense strand into the RNA-induced silencing complex (RISC) are currently being pursued. Herein, a biotin group at the 5'-end of the sense strand was used to inhibit its incorporation into the RISC complex. In contrast to chemical modifications, biotin is a naturally occurring compound and its presence in siRNA sequences will not induce side effects.

Exosome-derived miRNAs and cellular miRNAs activate innate immunity.

Circulating exosome-containing small RNAs have been demonstrated in vitro to be taken up by recipient cells and to alter gene expression through RNA interference. Here, we show that exosomes purified from various cancer cell lines as well as gel-purified exosomal and cellular miRNAs can induce pro-inflammatory cytokine expression in human peripheral blood mononuclear cells. Thus, circulating miRNAs may trigger innate immunity via pathogen recognition receptors, a new miRNA-activated pathway that merits some consideration.

A novel peptide carrier for efficient targeting of antigens and nucleic acids to dendritic cells.

Dendritic cells (DCs) initiate host immune responses by presenting captured antigens to naive T cells. Hence, DC-binding peptides may be used for antigen targeting to boost naive and memory immune responses. By biopanning peptide phage libraries on human monocyte-derived DCs, we identified novel DC-binding peptides. One of the selected phages, displaying the NW peptide (NWYLPWLGTNDW), bound DCs with high affinity, and its binding was inhibited by the corresponding synthetic peptide. Antigenic peptides or proteins conjugated to the NW peptide bound to DCs and were internalized without negative effects on DC phenotype and function. Ex vivo targeted delivery of CMV-pp65 peptides to DCs via the NW peptide increased T-cell responses in HLA-A2(+)/CMV(+) donors compared to untargeted peptides (P<0.001). Stimulation of CD45RO-depleted peripheral blood mononuclear cells from CMV(-) donors with the NW-pp65 fusion peptides expanded pp65-specific precursor T cells. Moreover, the NW peptide mediated small interfering RNA delivery to DCs, and a significant gene silencing was obtained. Collectively, the data reveal that proteins and nucleic acids can be directed to DCs through the NW peptide, enabling effective uptake and functional effects such as T-cell activation in the context of MHC class I and II molecules.

Silencing of indoleamine 2,3-dioxygenase enhances dendritic cell immunogenicity and antitumour immunity in cancer patients.

Dendritic cells (DCs) are being explored as a therapeutic vaccine for cancers. However, their immunogenic potential is limited by the presence of immunosuppressive factors. Among these factors is the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO). In this study, we have investigated the safety, immunogenicity and clinical response of IDO-silenced DC vaccine in four patients with gynecological cancers. DCs were transfected with IDO small interfering RNA and mRNA encoding human telomerase reverse transcriptase (hTERT) or survivin, two universal tumour antigens. Silencing of IDO in DCs did not affect the expression of the co-stimulatory molecules CD80 and CD86, but enhanced the expression of the CCR7 and CD40 molecules. IDO-silenced DCs showed superior potency to activate allogeneic T cells compared to their IDO-positive counterparts. The immunisation with this novel DC cancer vaccine was well tolerated and all patients developed delayed-type hypersensitivity skin reaction and specific T-cell response against hTERT and survivin tumour antigens. Perhaps most importantly, the immune response seen in the patients was related to objective clinical response. Thus, IDO silencing can enhance the immunogenic function of DCs in vitro and in vivo. Overall, the data provide proof-of-principle that immunisation with IDO-silenced DC vaccine is safe and effective in inducing antitumour immunity.

Selective killing of cancer cells by peptide-targeted delivery of an anti-microbial peptide.

Antimicrobial peptides selectively kill bacteria while maintaining low mammalian cell cytotoxicity. However, they become cytotoxic subsequent to internalization. Here we have conjugated the lytic peptide (KLAKLAK)(2) to either a cancer-cell binding peptide (LTVSPWY) selected from peptide libraries or to a gastrin-releasing peptide (GNHWAVGHLM) in order to direct the lytic peptide to cancer cells. Peptide cytotoxicity was tested in breast MCF-7 and MDA-MB-231 cancer cells. The fusion peptides were internalized by cancer cells, disintegrated the cell membrane and induced rapid killing of the cells with IC50 values as low as 4-7 µM. Peptide cytotoxicity was dependent on the targeting receptor. Indeed, addition of free targeting peptide reduced cell killing. Blood lymphocytes and normal human mammary epithelial cells were less sensitive to the fusion peptides. Although most of the cells were killed by necrosis, fusion peptides branched with DNA oligonucleotides induced apoptosis as assayed by annexin V staining and activation of caspase 3. Therefore, the new designed drug peptides might provide a potent and selective anticancer therapy.

Efficient siRNA targeted delivery into cancer cells by gastrin-releasing peptides.

Small interfering RNAs (siRNAs) have displayed considerable promise for the treatment of cancer. However, their delivery to the desired cell population remains a challenging task. Here we have covalently conjugated a siRNA against survivin to gastrin-releasing peptides (GRPs) to direct siRNA molecules to cancer cells that express the GRP receptor. The cellular uptake of the peptide-siRNA conjugates was tested in breast MDA-MB 231 cancer cells, which express the GRP receptor. Fluorescein-tagged GRP-siRNA conjugates were taken up by cancer cells but not normal mammary epithelial cells or human blood monocytes. By 120 min of incubation, most of the cells have taken up the conjugates. Excess free peptide inhibited uptake, implying dependence of uptake on GRP receptor. Moreover, bitargeting of siRNA molecules by GR and luteinizing hormone-releasing peptides accelerated the uptake kinetics by MDA-MB 231 cells when compared to monotargeted siRNAs. Peptide-siRNA conjugates, but not free siRNAs, inhibited the expression of survivin, an endogenous gene involved in cancer cell survival. None of the peptide-siRNA conjugates induced the expression of inflammatory cytokines or interferon α in human blood leukocytes. Overall, the data demonstrate the feasibility of GRP receptor-mediated targeted delivery of siRNAs to cancer cells, an important step for RNA interference therapy in humans.

Galectin-1 and -3 gene silencing in immature and mature dendritic cells enhances T cell activation and interferon-γ production.

DCs are specialized APCs capable of inducing T cell activation as well as promoting tolerance. Although Gal, a family of ß-galactoside-binding proteins, were found to affect immunity, little is known about the contribution of DC-expressed Gal on T cell activation. Here, we show that human imDCs and mDCs constitutively express Gal-1, Gal-3, Gal-8, and Gal-9 at mRNA and protein levels. Two of the most abundant Gal-Gal-1 and Gal-3-were highly expressed and detected on the cell surface of DCs. In contrast to Gal-8, knockdown of Gal-1 or Gal-3 in DCs enhanced allogeneic T cell responses. This was observed with imDCs and mDCs, but the effects were more pronounced with imDCs. Furthermore, allogeneic CD4(+) T cells incubated with Gal-1 or Gal-3 knockdown DCs produced more IFN-γ and less IL-10 than did control cells. The percentage of apoptotic T cells was significantly higher in cultures with control DCs than that with Gal-1 or Gal-3 knockdown DCs. Collectively, the data indicate that DC-expressed Gal-1 and Gal-3 are regulatory molecules that favor the inhibition of T cell activation. Furthermore, the data provide a new mechanism for the poor capacity of imDCs to stimulate T cells.

Mesenchymal stem cell-mediated T cell suppression occurs through secreted galectins.

Human galectins are involved in a variety of biological and pathological processes including cell adhesion, apoptosis, differentiation, immune regulation and tumour evasion. Previously, we identified galectin-3 as the first human lectin involved in the modulation of the immunosuppressive potential of mesenchymal stem cells (MSCs). In this study, we report on the expression profiles and potential activities of other galectins expressed in these cells. The data show that MSCs constitutively express galectins-1, -3 and -8 at both the mRNA and protein levels. In contrast to galectin-8, galectins-1 and -3 are secreted and found on the cell surface. MSC-mediated T cell suppression was inhibited by galectin-1-specific siRNAs but not by galectin-8-specific siRNAs. The double knockdown of galectins-1 and -3 almost abolished the immunosuppressive capacity of MSCs. The use of a competitive inhibitor for galectin binding, ß lactose, restored alloresponsiveness, implying an extracellular mechanism of action of galectins. Collectively, the data highlight the involvement of secreted galectins-1 and -3 in MSC-mediated T cell suppression. The immunosuppression by MSC-secreted galectins should facilitate the use of recombinant galectin-1 and/or -3 as a novel therapy to alleviate inflammatory reactions such as those seen in graft versus host disease (GvHD) and autoimmune disorders.

Optimized protocols for siRNA delivery into monocytes and dendritic cells.

Over the past decade, immunotherapy has emerged as a promising alternative form of cancer treatment with the potential to eradicate tumour metastasis. Unfortunately, its curative potential is in general limited by immunosuppressive proteins that negatively regulate dendritic (DC) and/or T-cell function. The recent discovery of RNA interference (RNAi) has facilitated the study of gene function in immune cells and recent data indicate that DC maturation, function, and survival can be modulated by small interfering RNAs (siRNAs) targeting genes involved in immune suppression. This chapter describes detailed protocols for introducing siRNAs into human monocytes and dendritic cells using standard electroporation techniques.