Induction Of Anti-tumour Immunity Research Articles

Practical clinical guide on the use of talimogene laherparepvec monotherapy in patients with unresectable melanoma in Europe

Talimogene laherparepvec, a herpes simplex virus type 1-based intralesional oncolytic immunotherapy, is approved in Europe for the treatment of adults with unresectable stage IIIB-IVM1a melanoma, with no bone, brain, lung or other visceral disease. It has direct oncolytic effects in injected lesions, leading to the release of tumour-derived antigens and systemic immune effects mediated by the induction of anti-tumour immunity, which is enhanced by the production of granulocyte macrophage colony-stimulating factor. Responses (which occur in >40% of stage IIIB-IVM1a patients) are often durable (>50% last ≥6months) and occur in injected and uninjected lesions (in stage IIIB-IVM1c patients, 64%/34% of evaluable injected/uninjected non-visceral lesions, respectively, decreased in size by ≥50%). As with other immunotherapies, responses may be delayed or can arise after pseudoprogression. The pattern of treatment-emergent adverse events is distinct, being mostly grade 1/2, easy to manage, and rarely leading to treatment discontinuation. Systemic therapy represents the backbone of care for many metastatic melanoma patients. Nonetheless, the potential for durable locoregional control with a locally injected agent may make talimogene laherparepvec suitable for selected patients with stage IIIB/C disease, for whom surgery is not possible (e.g. with in-transit metastases, multiple melanoma lesions at different body sites, or those relapsing rapidly after repeated rounds of surgery) and slowly progressing disease. Here, we discuss which patients could be suitable for talimogene laherparepvec monotherapy based on the European indication, review the patterns/timing of response, and discuss the incidence/management of adverse events. Its potential use combined with immune checkpoint inhibitors is also discussed.

Prospects for combining targeted and conventional cancer therapy with immunotherapy.

Over the past 25 years, research in cancer therapeutics has largely focused on two distinct lines of enquiry. In one approach, efforts to understand the underlying cell-autonomous, genetic drivers of tumorigenesis have led to the development of clinically important targeted agents that result in profound, but often not durable, tumour responses in genetically defined patient populations. In the second parallel approach, exploration of the mechanisms of protective tumour immunity has provided several therapeutic strategies - most notably the 'immune checkpoint' antibodies that reverse the negative regulators of T cell function - that accomplish durable clinical responses in subsets of patients with various tumour types. The integration of these potentially complementary research fields provides new opportunities to improve cancer treatments. Targeted and immune-based therapies have already transformed the standard-of-care for several malignancies. However, additional insights into the effects of targeted therapies, along with conventional chemotherapy and radiation therapy, on the induction of antitumour immunity will help to advance the design of combination strategies that increase the rate of complete and durable clinical response in patients.

Combined toll-like receptor 3/7/9 deficiency on host cells results in T-cell-dependent control of tumour growth

Toll-like receptors (TLRs) are located either on the cell surface or intracellularly in endosomes and their activation normally contributes to the induction of protective immune responses. However, in cancer their activation by endogenous ligands can modulate tumour progression. It is currently unknown how endosomal TLRs regulate endogenous anti-tumour immunity. Here we show that TLR3, 7 and 9 deficiencies on host cells, after initial tumour growth, result in complete tumour regression and induction of anti-tumour immunity. Tumour regression requires the combined absence of all three receptors, is dependent on both CD4 and CD8 T cells and protects the mice from subsequent tumour challenge. While tumours in control mice are infiltrated by higher numbers of regulatory T cells, tumour regression in TLR-deficient mice is paralleled by altered vascular structure and strongly induced influx of cytotoxic and cytokine-producing effector T cells. Thus, endosomal TLRs may represent a molecular link between the inflamed tumour cell phenotype, anti-tumour immunity and the regulation of T-cell activation.

MUC1 immunotherapy against a metastatic mammary adenocarcinoma model: Importance of IFN-gamma.

Immunotherapy using mucin 1 (MUC1) linked to oxidised mannan (MFP) was investigated in an aggressive MUC1+ metastatic tumour, DA3-MUC1 because, unlike many MUC1+ tumour models, DA3-MUC1 is not spontaneously rejected in mice making it an alternative model for immunotherapy studies. Further, DA3-MUC1 cells are resistant to lysis by anti-MUC1 cytotoxic T cells (CTLs). The inability of DA3-MUC1 tumours to be rejected in naïve mice as well as vaccination to MUC1 was attributed to a deficiency of expression of MHC class I molecules on the tumour cell surface. In vitro and in vivo analysis of subcutaneous tumours and lung metastases demonstrated that DA3-MUC1 tumour cells have a low expression (< 6%) of MHC class I which can be upregulated (> 90%) following culturing with IFN-γ. Results from flow cytometry analysis and immunoperoxidase staining indicated that the in vitro up-regulation of MHC class I could be maintained for up to seven days in vivo, without affecting the expression levels of MUC1 antigen. Interestingly, MUC1-specific CTL that lyse DA3-MUC1 targets in vitro were induced in MFP immunised mice but failed to protect mice from a DA3-MUC1 tumour challenge. These results highlight the importance of MHC class I molecules in the induction of anti-tumour immunity and the MFP immune response.

ITOC2 – 025. Transduction with C-C-chemokine receptor type 4 (CCR4) enhances tumour-specific migration of adoptively transferred T cells in a model of pancreatic cancer

<h3>Introduction</h3> Regulatory T cells selectively express C-C-chemokine receptor type 4 (CCR4) and are attracted by intratumoral CCL22 while CCR4 is absent from most cytotoxic T cells. We hypothesised that inducing forced expression of CCR4 on adoptively transferred cytotoxic T cells could enhance therapeutic efficacy. <h3>Methods</h3> CCR4 and a new non-functional CCR4-deletion mutant were cloned into the retroviral vector pMP71 and transduced into murine ovalbumin-specific T cells (OT-1). T cell migration and function in vitro were assessed using transwell assays. <h3>Results</h3> <i>In vitro</i>, CCR4- but not CCR4del-transduced OT-1 T cells specifically migrated towards recombinant CCL22 and tumour-induced CCL22 (25-fold increase). <i>In vivo</i>, CCR4-transduced OT-1 T cells cured 50 % of mice bearing established Panc-OVA tumours compared to 12 % in the CCR4del- or untransduced OT-1 T cell- treated mice (n=8 per group, <i>p</i><0.0001). Cured mice were protected from rechallenge, indicating the induction of anti-tumour immunity. Enhanced numbers of CCR4-transduced OT-1 T cells compared to CCR4del-transduced cells were found in the tumour, suggesting enhanced homing to the tumour site as the mode of action of CCR4-transduced T cells. <h3>Conclusions</h3> The transduction of cytotoxic T cells with CCR4 represents a new therapeutic approach for effectively guiding adoptively transferred T cells into pancreatic tumours and thereby inducing a potent antitumour immune response.

Simultaneous immunisation with a Wilms’ Tumour 1 epitope and its ubiquitin fusions results in enhanced cell mediated immunity and tumour rejection in C57BL/6 mice

Protein fusion to ubiquitin results in its targeting to proteasome and processing through MHC class I pathway. We used this approach to induce cytotoxic T lymphocyte (CTL) response against a MHC class I epitope. Therefore, two known proteasome targeting systems, “ubiquitin fusion degradation” (UFD) and “N-end rule”, were used to immunise C57BL/6 mice. Two plasmids encoding an epitope from Wilms’ Tumour 1 (WT1-126), fused N-terminally to ubiquitin, were constructed. They were designated as “pUbVVPT” and “pUbGRPT”, targeting the fused epitope to UFD and N-end pathways, respectively. A plasmid encoding WT1-126 without ubiquitin fusion (pPT) was also constructed as control. Three mice groups were immunised using these constructs (UGR, UVV and PT groups). Two other groups received mixed immunisations of pUbVVPT or pUbGRPT plus pPT plasmids (UVV+PT and UGR+PT). All mice received a WT1-126 peptide booster. Lymphoproliferative responses following stimulation with WT1-126 were observed in all immunisation groups, with mice receiving the mixture of plasmids eliciting the highest proliferation (UVV+PT>UGR+PT>PT). Moreover, In vivo cytotoxicity assay results revealed highest specific lysis of target cells in UVV+PT group. Tumour growth was decreased in all immunised groups, and was completely abrogated in UGR+PT group. In addition, TH1 type cytokines patterns were detected from all immunised groups and WT1-126-specific IFNγ producing lymphocytes were developed in them. These results suggest that the delivery of ubiquitin-fused epitopes along with epitopes alone can be used to optimise the effect of DNA vaccines on the induction of anti-tumour immunity.

Retraction: Enhanced Inhibition of Tumour Growth and Metastasis, and Induction of Antitumour Immunity by IL-2-IgG2b Fusion Protein

Retraction: Enhanced Inhibition of Tumour Growth and Metastasis, and Induction of Antitumour Immunity by IL-2-IgG2b Fusion Protein

Membrane‐bound HSP70‐engineered myeloma cell‐derived exosomes stimulate more efficient CD8+ CTL‐ and NK‐mediated antitumour immunity than exosomes released from heat‐shocked tumour cells expressing cytoplasmic HSP70

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour-derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T-cell immune responses. To enhance EXO-based antitumour immunity, we generated an engineered myeloma cell line J558HSP expressing endogenous P1A tumour antigen and transgenic form of membrane-bound HSP70 and heat-shocked J558HS expressing cytoplasmic HSP70, and purified EXOHSP and EXOHS from J558HSP and J558HS tumour cell culture supernatants by ultracentrifugation. We found that EXOHSP were able to more efficiently stimulate maturation of DCs with up-regulation of Iab, CD40, CD80 and inflammatory cytokines than EXOHS after overnight incubation of immature bone-marrow-derived DCs (5 × 106 cells) with EXO (100 μg), respectively. We also i.v. immunized BALB/c mice with EXO (30 μg/mouse) and assessed P1A-specific T-cell responses after immunization. We demonstrate that EXOHSP are able to stimulate type 1 CD4+ helper T (Th1) cell responses, and more efficient P1A-specific CD8+ cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXOHS. In addition, we further elucidate that EXOHSP-stimulated antitumour immunity is mediated by both P1A-specific CD8+ CTL and non-P1A-specific natural killer (NK) responses. Therefore, membrane-bound HSP70-expressing tumour cell-released EXO may represent a more effective EXO-based vaccine in induction of antitumour immunity.

Immunomodulation with dendritic cells and donor lymphocyte infusion converge to induce graft vs neuroblastoma reactions without GVHD after allogeneic bone marrow transplantation.

Background:Mounting evidence points to the efficacy of donor lymphocyte infusion (DLI) and immunisation with tumour-pulsed dendritic cells (DC) in generating graft vs leukaemia reactions after allogeneic bone marrow transplantation (BMT). We assessed the efficacy of DLI and DC in generating potent graft vs neuroblastoma tumour (GVT) reactions following allogeneic BMT.Methods:Mice bearing congenic (H2Ka) Neuro-2a tumours were grafted with allogeneic (H2Kb) T-cell-depleted bone marrow cells. Tumour-pulsed donor DC (DCNeuro2a) were inoculated (on day +7) in conjunction with donor (H2Kb) and haploidentical (H2Ka/b) lymphocytes.Results:Murine Neuro-2a cells elicit immune reactions as efficient as B lymphoma in major histocompatibility complex antigen-disparate mice. Lymphopenia induced by conditioning facilitates GVT, and transition to adaptive immunity is enhanced by simultaneous infusion of and DCNeuro2a and lymphocytes devoid of graft vs host (GVH) activity (H2Ka/b). In variance, the efficacy of DC-mediated immunomodulation was diminished by severe graft vs host disease (GVHD), showing mechanistic dissociation and antagonising potential to GVT.Conclsions:The GVHD is not a prerequisite to induce GVT reactivity after allogeneic BMT, but is rather detrimental to induction of anti-tumour immunity by DC-mediated immunomodulation. Simultaneous inoculation of tumour-pulsed donor DC and DLI synergise in stimulation of potent GVT reactions to the extent of eradication of established NB tumours.

CD40 ligand gene therapy directly induces apoptosis in carcinomas

14082 Background: CD40 is a member of the TNFReceptor superfamily expressed on a variety of immune cells. Interaction of CD40 with its ligand (CD40L) plays a key role in orchestrating immune responses. We have shown that CD40 is also expressed on a variety of carcinomas. CD40 ligation in this context can induce cancer cell death. Thus CD40 represents a rational target with direct cytotoxicity and induction of anti-tumour immunity. Methods: We generated an adenovirus encoding CD40L (AdCD40L) and investigated its effect in CD40-expressing carcinoma cells (bladder, ovary, hepatocellular) as follows:(i)comparison of Ad-delivered CD40L with recombinant soluble ligand (rsCD40L);(ii)inhibition of cleavage of CD40L from the cell membrane using metalloproteinase inhibitors (MMPI);(iii)generation of a mutant CD40L resistant to MMP cleavage. Results: (i)CD40 ligation in carcinomas stimulates survival and apoptotic signaling. rsCD40L is only cytotoxic in the presence of protein synthesis inhibition, which blocks survival pathways and allows apoptosis. In contrast, AdCD40L is cytotoxic even in the absence of protein synthesis inhibitor;(ii)AdCD40L results in expression at the cell membrane but this is cleaved from the membrane and detected as soluble ligand in supernatant of infected cells. Cleavage is inhibited by MMPI and this increases apoptosis, suggesting that membrane-bound CD40L is a more potent apoptotic stimulus than soluble ligand;(iii)we identified the cleavage site of CD40L and generated a mutant by deletion of this region and cloned this mCD40L into an adenovirus. AdmCD40L results in membrane expression of CD40L, with no detectable soluble CD40L, confirming its resistance to cleavage. AdmCD40L is significantly more cytotoxic than wild-type AdCD40L. Conclusions: The effect of CD40 ligation depends on the cellular context and the method of CD40L delivery: adenovirus delivery results in membrane-bound expression and this provides a stronger apoptotic stimulus than rsCD40L. Apoptosis is further enhanced by a mutant CD40L that is resistant to cleavage from the cell membrane. Thus AdmCD40L is a novel therapeutic that is a potent inducer of apoptosis. Studies are underway to assess these effects in vivo and to elucidate the contribution of immunostimulatory effects in syngeneic models. No significant financial relationships to disclose.

Potential prognostic and therapeutic roles for cytokines in breast cancer (Review)

Cytokines are known to have both stimulatory and inhibitory effects on breast cancer growth depending on their relative concentrations and the presence of other modulating factors in the tumour microenvironment. Certain cytokines appear to prevent an effective immune response being mounted, permitting cancer growth, whereas others promote the immune system's anti-tumour capability. Furthermore, the systemic levels of certain cytokines, e.g. IL-6 and IL-18, independently show promising correlations with disease stage and progression. With advances in methods for delivery of cytokines to a tumour site, the enhanced induction of anti-tumour immunity by targeted cytokine release is becoming a realistic option. Here, we review the role of cytokines in the immune response against breast cancer and assess their potential as prognostic indicators and/or use in immune therapy. A literature search was conducted using Medline, restricted to articles published in the English language, using combinations of the following MeSH terms: cytokines, breast cancer, immunology, immunotherapy and interleukins. Focused searches using keywords relevant to the role of cytokines in breast cancer immunology yielded >200 references.

Induction of antitumour immunity using survivin peptide-pulsed dendritic cells in a murine lymphoma model.

Survivin is overexpressed in several types of haematological malignancies making it an attractive target for therapeutic cytotoxic T-lymphocyte responses. Here, we identify two peptide epitopes derived from the murine survivin protein and demonstrate that Balb/c mice treated with syngeneic dendritic cells pulsed with the survivin epitopes were able to reject an otherwise lethal tumour inoculation of the A20 lymphoma. For the first time, these data provide evidence for the use of survivin peptide epitopes in T cell-based immunotherapeutic concepts against a B-cell lymphoma in vivo.

Enhanced inhibition of tumour growth and metastasis, and induction of antitumour immunity by IL-2-IgG2b fusion protein.

Cytokine-immunoglobulin (Ig)-fusion proteins have attracted increasing interest as antitumour agents. Here, we have investigated the antimetastatic and antitumour responses elicited in vivo by mammary adenocarcinoma cells (TS/A) engineered to secrete interleukin (IL)-2-IgG fusion proteins. TS/A cells were transfected with DNA coding for IL-2-IgG2b, IgG2b or IL-2, and injected subcutaneously into syngeneic mice. Animals injected with TS/A-IL-2 or TS/A-IL-2-IgG2b both efficiently rejected tumours, whereas treatment with parental cells or TS/A-IgG2b was lethal. Interestingly, only mice vaccinated with IL-2-IgG2b fusion protein-secreting cells showed a long-lasting protective immunity against a later challenge with parental tumour cells. Moreover, the metastatic potential of TS/A-IL-2-IgG2b-transfected cells was dramatically decreased compared with TS/A-IL-2-cells, with a virtual absence of lung metastases after intravenous injection. Adenocarcinomas secreting IL-2-IgG2b exhibited a more prominent, early and persistent infiltration of CD4+, CD8+ and natural killer (NK) cells than TS/A-IL-2 cells. Therefore, upon transfection into adenocarcinoma cells, the IgG2b part of IL-2 fusion protein exerts intriguing added antitumour properties over IL-2 alone, thus contributing to a long-lasting tumour immunity, probably by the recruitment of specific immune effector cells. These findings suggest a promising new oncotherapeutic strategy for poorly immunogenic tumours: vaccination with tumour cells engineered to secrete IL-2-IgG2b fusion protein.

Induction of anti-tumour immunity by suicide-gene-modified HPV-16-transformed hamster cells.

From K3/II, which is a highly oncogenic HPV16-transformed Syrian hamster cell line, thymidine-kinase(TK)-less cells, denoted B 49, were derived. B49 cells were transfected by a plasmid containing the herpes-simplex-virus TK gene (HSV TK) and several sub-lines expressing this gene were isolated from the transfected cultures. The HSV TK+ cells were highly sensitive to ganciclovir (GCV) and other anti-viral substances whose inhibitory effect is based on their phosphorylation by HSV TK. One of the cell lines, denoted KL1/6, exhibited relatively high stability of the HSV TK+ phenotype and was used in subsequent experiments. When KL1/6 cells were co-cultivated in the presence of GCV with various other cell lines of hamster, mouse or monkey origin, the by-stander effect (BE) was observed. GCV treatment of hamsters prevented development of tumours after the administration of KL1/6 cells but not K3/II cells. The treatment of animals with already established KL1/6-induced tumours resulted in tumour regression in all instances, but complete regression was observed only in animals carrying small tumours. The BE of KL1/6 cells on K3/II cells was also seen in vivo. In addition, concomitant immunity was observed in animals simultaneously inoculated with KL1/6 cells and K3/11 cells at 2 separate sites of the body. This effect was evident not only in animals in which KL1/6 tumours developed, but also in those in which tumour outgrowth was prevented by GCV treatment. In other experiments it was demonstrated that one KL1/6 + GCV treatment resulted in partial resistance, 2 such treatments in complete resistance to the challenge with K3/II cells.

Induction of anti-tumour immunity in syngeneic mice by a leukaemic cell line.

Induction of anti-tumour immunity in syngeneic mice by LBC cell line derived from a non-immunogenic T cell leukaemia was studied. The immunization of BALB/c mice with LBC irradiated cells induced in them anti-tumour spleen cells, cytotoxic T lymphocytes and anti-LBC antibodies. The anti-LBC antibodies reacted with components of 14, 16 and 27 kDa present on LB tumour cells, LBC cell line and normal thymocytes, but not with normal lymph node cells. Furthermore, immunization of the autologous hosts with LBC cells partially protected them against subsequent challenge with the original LB leukaemic cells. These findings demonstrate that culture conditions induced modifications in the antigenic properties of the leukaemic cells, allowing LBC cells to stimulate an immune response directed against components expressed at early stages during T cell maturation. These results also suggest that the immune response is responsible for the prolongation of the survival time of the mice inoculated with the parental leukaemic cells.

Effect of a synthetic adjuvant for inducing anti-tumour immunity

An acylated derivative of muramyl dipeptide (MDP), 6-O-(2-tetradecyl-hexadecanoyl)-muramyl-dipeptide (B30-MDP) is a strong adjuvant effective in inducing cell-mediated immunity. We used B30-MDP as an adjuvant for induction of anti-tumour immunity. Guinea-pigs which were injected repeatedly with a mixture of X-ray-treated leukaemic cells and B30-MDP dissolved in phosphate buffered saline resisted a challenge of leukaemia cells and showed no sign of leukocytosis. The immunity induced was tumour-specific and retained for more than 100 days. These results suggest that B30-MDP is useful as a simple but potent immunotherapeutic tool.