Cancer Immunol Immunother DOI 10.1007/s00262-014-1525-z

Original Article

ImmTAC‑redirected tumour cell killing induces and potentiates antigen cross‑presentation by dendritic cells Giovanna Bossi · Sandrine Buisson · Joanne Oates · Bent K. Jakobsen · Namir J. Hassan 

Received: 19 April 2013 / Accepted: 30 January 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract Antigen cross-presentation by dendritic cells (DCs) is thought to play a critical role in driving a polyclonal and durable T cell response against cancer. It follows, therefore, that the capacity of emerging immunotherapeutic agents to orchestrate tumour eradication may depend on their ability to induce antigen cross-presentation. ImmTACs [immune-mobilising monoclonal TCRs (T cell receptors) against cancer] are a new class of soluble bispecific anti-cancer agents that combine pico-molar affinity TCR-based antigen recognition with T cell activation via a CD3-specific antibody fragment. ImmTACs specifically recognise human leucocyte antigen (HLA)-restricted tumour-associated antigens, presented by cancer cells, leading to T cell redirection and a potent anti-tumour response. Using an ImmTAC specific for a HLA-A*02-restricted peptide derived from the melanoma antigen gp100 (termed IMCgp100), we here observe that ImmTAC-driven melanoma-cell death leads to cross-presentation of melanoma antigens by DCs. These, in turn, can activate both melanoma-specific T cells and polyclonal T cells redirected by IMCgp100. Moreover, activation of melanoma-specific T cells by cross-presenting DCs is enhanced in the presence of IMCgp100; a feature that serves to increase the prospect of breaking tolerance in the tumour microenvironment. The mechanism of DC cross-presentation occurs via ‘crossdressing’ which involves the rapid and direct capture by Electronic supplementary material The online version of this article (doi:10.1007/s00262-014-1525-z) contains supplementary material, which is available to authorized users. G. Bossi · S. Buisson · J. Oates · B. K. Jakobsen · N. J. Hassan (*)  Immunocore Ltd, 57 Jubilee Avenue Milton Park, Abingdon, Oxon OX14 4RX, UK e-mail: [email protected]

DCs of membrane fragments from dying tumour cells. DC cross-presentation of gp100-peptide-HLA complexes was visualised and quantified using a fluorescently labelled soluble TCR. These data demonstrate how ImmTACs engage with the innate and adaptive components of the immune system enhancing the prospect of mediating an effective and durable anti-tumour response in patients. Keywords  ImmTAC · T cell receptor · Cross-presentation · Cross-dressing · Dendritic cell · Cancer immunotherapy

Introduction There is increasing evidence to suggest that an effective anti-tumour response in cancer patients requires the coordinated action of the innate and adaptive immune systems [1, 2]. In the adaptive immune response, studies in in vivo models and, more recently in cancer patients, have shown that CD8+ cytotoxic T cells (CTLs) are the key effectors involved in the killing of tumour cells [3, 4]. Within the innate immune system, dendritic cells (DCs) are pivotal to anti-tumour immunity. DCs can detect dying cancer cells, become activated, and display tumour antigens on their cell surface, leading to the potent activation of T cells. This process, referred to as antigen cross-presentation, can occur locally at the tumour site or in draining lymph nodes, resulting in the activation of naive T cells. There are several lines of evidence that support the role of DCs in anti-tumour immunity [5]. First, compared to other hematopoietic cells, DCs are particularly well endowed with co-stimulatory molecules to cross-present antigens to CD8+ T cells. Second, tumour-infiltrating DCs purified from tumour samples have the capacity to cross-present

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tumour antigens in vitro. Third, mice that are deficient in DCs fail to generate an anti-tumour T cell response [6–8]. It is therefore considered that the capacity for immunotherapeutic strategies to elicit effective anti-tumour responses in patients depends on the degree of engagement with both the adaptive and innate immune systems. The development of targeted immunotherapeutics based on CD8+ T cells, and in particular the T cell receptor (TCR), has been the focus of intense investigation in recent years (reviewed in [9]). Unlike antibodies, TCRs recognise antigens derived from intracellular proteins which first have been processed, then presented on the cell surface as peptide fragments in association with human leucocyte antigen (HLA) molecules. Since the vast majority of tumour-associated antigens is derived from intracellular proteins, TCR-based therapeutics offer immense scope as novel anti-cancer agents. However, there are major challenges associated with the use of naturally occurring TCRs. First, their inherent low affinity for antigen (typically in the low μM range), which in the context of TAAs is usually particularly low [10–12], and second, the poor stability of TCRs in a soluble form. The development of strategies to overcome these issues is now opening up the possibilities of TCR-based therapies. TCRs possessing enhanced affinity for antigen can be created by directed molecular evolution of the antigen binding regions [13–15]. For example, increases in TCR antigen affinity in excess of one millionfold have been achieved using phage display [15]. Producing TCR molecules which are both stable and soluble can be achieved by omitting the transmembrane domain and introducing a non-native disulphide bond, resulting in ‘monoclonal’ or ‘mTCRs’ [16]. These innovations have led us to develop a platform of novel bi-specific therapeutic agents, termed ImmTACs, which combine a high-affinity mTCR domain with a T cell activating anti-CD3 domain and are capable of generating a potent anti-tumour response in vitro and in vivo [17, 18]. The potential for ImmTACs to engage with and enhance the host’s immune response through antigen cross-presentation by dendritic cells is investigated here. In this study, we utilised an ImmTAC, known as IMCgp100, which specifically recognises a HLA-A*02-restricted peptide derived from the melanocyte differentiation antigen gp100 (gp100280–288) [19], and currently undergoing evaluation in a phase I clinical trial (NCT01211262). The data reported here show that IMCgp100-driven killing of melanoma cells induces cross-presentation of TAAs on DCs, leading to (1) activation of melanoma-specific T cells, which is potentiated in the presence of IMCgp100 and (2) activation of polyclonal T cells redirected by IMCgp100. In addition, using a fluorescently labelled mTCR specific for the gp100peptide-HLA complex, cross-presented gp100 is directly visualised on the DC cell surface, enabling a quantitative

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Cancer Immunol Immunother

assessment of cross-presentation. Finally, the data reveal that ImmTAC-driven cross-presentation may exploit a mechanism of antigen uptake termed ‘DC cross-dressing’.

Materials and Methods ImmTAC engineering IMCgp100, a gp100-specific ImmTAC, was prepared as previously described [17]. Briefly, a high-affinity TCR was generated from a wild-type gp100 TCR using directed molecular evolution and phage display selection [15]. The resulting high-affinity TCR beta chain was fused to a humanised CD3-specific scFv via a flexible linker and the alpha and beta chains of the resulting ImmTAC expressed in E. coli as inclusion bodies. ImmTACs were then refolded and purified as previously described [16]. Human T cell clones and cell lines MEL187.c5 and EBV176.c4.1 CD8+ T cell clones, specific for Melan A/MART-126–35 and EBV BRLF1259–267, respectively, were generated and maintained in-house as described previously [20]. Mel624 melanoma cells (HLAA*0201+; Melan A+ and gp100+) were obtained from Thymed; SK-Mel-24 (HLA-A*0201+; gp100− and Melan A−) and SK-Mel-28 (HLA-A*0201−; gp100+ and Melan A+) melanoma cell lines were obtained from the American Type Culture Collection (ATCC). The HLA-A*0201 status of the cell lines was determined by flow cytometry, following staining with mouse anti-human HLA-A*0201 PEconjugated antibody (Serotec) 7 days after transduction. Intracellular staining with mouse anti-gp100 (NK1/beteb; Hycult Biotech) and mouse anti-Mar1 (Ab-1; Calbiochem) antibodies was used to confirm the expression of gp100 and Melan A. Generation of human DCs, autologous CD8+ and CD4+ T cells Human peripheral blood mononuclear cells (PBMC) were prepared from healthy donors by Ficoll-Hypaque (Robbins Scientific) density gradient centrifugation. CD14+ monocyte cells isolated by positive selection with magnetic micro beads (Miltenyi) were differentiated for 7 days in culture in the presence of 800 U/ml GM-CSF and 1,000 U/ ml IL-4. Mature DCs (mDC) were generated by treatment with 100 ng/ml LPS for 24 h and 0.5 μg/ml soluble CD40L (Enzo Life Science) for a further 24 h. The purity and activation state of DCs were determined by staining with mouse anti-human CD80 PE-Cy7, CD83 APC, CD86 PE, HLA-DR PerCP-Cy5.5 and CD14 APC-Cy7 antibodies

Cancer Immunol Immunother

(BD BioSciences). Samples were acquired with FACSAriaII flow cytometer (BD BioSciences) and analysed with FlowJo software (TreeStar Inc.). Magnetic bead immunodepletion was used to enrich CD8+ and CD4+ T cells from freshly prepared peripheral blood lymphocytes (PBL) by negative selection according to the manufacturer’s instructions (Miltenyi). Preparation of apoptotic cells and DC phagocytosis

release)/(target maximum LDH release − target spontaneous LDH release) × 100. IFNγ ELISpot assays were carried out in triplicate according to the manufacturer’s instructions (BD BioSciences). Each well contained 2.5x104 DCs and 1x104 effector T cells (CD4+, CD8+) or 3 × 103 MEL187. c5 T cell clone cells. Plates were incubated overnight at 37 °C/5 % CO2 and quantified after development using an automated ELISpot reader (Immunospot Series 5 Analyzer, Cellular Technology Ltd.).

Apoptotic Mel624 cells were generated either by incubating the target cells with 0.1 nM IMCgp100 and a viral T cell clone (EBV176.c4.1) for 24 h or by treating the target cells with an apoptotic cocktail (5 μM staurosporine, 40  μg/ml anisomycin and 5 μM etoposide) for 5 h. The percentage of target killing or apoptotic rate was determined by pre-labelling targets with DiO (Life Technology) and determining the percentage of DiO+ cells that have become positive for annexin-V PE-conjugated and 7-AAD reagents (Guava Nexin Reagent; Millipore). In all cases, 100 % of target cells were determined to be apoptotic. When the apoptotic melanoma cells were generated by IMCgp100-redirected killing, EBV176.c4.1 T cells were eliminated by positive selection with anti-CD2 microbeads (Miltenyi). Immature DCs were incubated with apoptotic cells at 1:1 ratio for 24 h, washed with PBS and incubated for further 24 h in complete medium. The phagocytosis rate of apoptotic/necrotic cells by DCs was determine by labelling DCs with DiO dye and apoptotic cells with DiI dye (Life Technology) and detecting by FACS the percentage of DiO+ DC cells that became DiI+.

T cell proliferation was measured by CFSE labelling and the percentage of dividing cells determined. Briefly, autologous CD8+ T cells were resuspended at 1 × 106 cells/ml, in pre-warmed (37 °C) sterile PBS, supplemented with 0.1 % BSA. T cells were labelled with 2 μM CFSE (Life Technologies) for 10 min at 37 °C and quenched by addition of ice-cold RPMI with 10 % FCS for 5 min. 4 × 104 DCs HLA-A*0201+ and HLA-A*0201− that have been cocultured for 48 h with chemically induced apoptotic melanoma cells were plated in the presence of 2 × 105 autologous CD8+ T cells (5:1 effector to target ratio) in 200 μl R10. IMCgp100 was added at 1 nM final concentration. Cells were incubated at 37 °C/5 % CO2 for 7 days. 10,000 CFSE+ events were acquired on a FC500 flow cytometer (Beckman Coulter), and the files analysed with FlowJo version 7.6 (TreeStar Inc.).

Cellular assays

Cytokine analysis

CytoTox 96® non-radioactive cytotoxicity assay (Promega) was used to determine LDH release from dying target cells in the presence of increasing concentrations of IMCgp100. CD8+ T cells, obtained from a healthy donor, were used at 100,000 cells per well at an effector target ratio of 10:1. Experiments were performed as described previously [17]. Briefly, target and effector cells were plated in a 96-roundwell plate in triplicate, and IMCgp100 was added at the concentrations indicated in Fig. 1b. The plate was incubated for 24 h at 37 °C/5 % CO2, and the detection of LDH release from the target cells was measured in the supernatants according to the manufacturer’s instructions. To calculate the percentage lysis, the LDH signal derived from culture medium values alone was subtracted from all wells and volume correction control values were subtracted from target maximum release wells. Subsequently, the percentage lysis was calculated as (experimental LDH release − effector spontaneous LDH release − target spontaneous LDH

Polyclonal CD8+ T cells were incubated with DCs in the presence of 0.1 nM IMCgp100 for 3 days at 37 °C. 5 h before the end point, the co-culture was incubated with 1 μg/ml brefeldin A (GolgiPlug, BD BioSciences), 0.7 μl/ml monensin (GolgiStop, BD BioSciences) and mouse antihuman CD107 APC antibody (BD BioSciences) for 5 h. The cells were washed in PBS, stained with mouse antihuman CD8 APC-Cy7 antibody (BD BioScences) for 20 min on ice. After washing, cells were fixed and permeabilised using the Cytofix/Cytoperm kit (BD BioSciences) according to the manufacturer’s instructions. After permeabilisation, the cells were washed twice in the supplied buffer, and then stained with directly conjugated mouse anti-human IFNγ FITC, TNFα PE and IL-2 PE-Cy7 antibodies (BD BioSciences) and mouse anti-human MIP-1β PerCP (R&D Systems) for 1 h. The data were acquired on FACS AriaII flow cytometer (BD BioSciences) and analysed by FloJo version 7.6 (TreeStarInc.)

Data were analysed using Prism 5.0 software (GraphPad Software) T cell proliferation

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Cancer Immunol Immunother

Fig. 1  IMCgp100-driven killing of melanoma cell lines leads to antigen cross-presentation by dendritic cells. a Details of the cell lines used in this study. b IMCgp100 dose–response curve showing redirected T cell cytotoxicity against the cell lines detailed in (a). Cytotoxicity was determined by LDH release after 24 h. Polyclonal CD8+ T cells, obtained from a healthy donor, were used at 100,000 cells per well at an effector target ratio of 10:1. Data shown represent the average of three independent measurements ± SEM. c Flow diagram summarising the experimental approach used to monitor IMCgp100-driven cross-presentation. d Apoptotic Mel624 cells [denoted Mel624(ap)] were produced by IMCgp100-redirected

CD8+ T cell killing and subsequently incubated with immature DCs for 48 h to allow antigen uptake. IMCgp100 was used at a concentration of 0.1 nM. The resulting mature DCs were assessed for their ability to induce IFNγ release from Melan-A-specific CD8+ T cells. A further sample was prepared using LPS-matured DCs (mDC) only. Data shown represent the average of three independent measurements ± SEM. Additional control samples were prepared in a similar manner but in the absence of either DCs or Melan A T cells. These control measurements were performed in duplicate and are shown as average ± SEM

Microscopy

reagent (Open Biosystems). Supernatants collected at 24 h and 48 h were concentrated by centrifugation at 10,000×g for 16 h at 4 °C. Melanoma SK-Mel-28 cells were plated at 3 × 106 cells/well in a six well plate and transduced by addition of 1 ml crude lentiviral supernatant. HLAA*0201 transduction efficiency was determined by flow cytometry, following staining with mouse anti-human HLA-A*0201 PE-conjugated antibody (Serotec) 7 days after transduction.

Soluble high-affinity mTCRs were produced as previously described [15, 16]. Phase-contrast and PE-fluorescence images were acquired as previously described using a Zeiss 200 M/Universal Imaging system [21]. Z-stack fluorescent images were taken (21 individual planes, 0.7 μm apart) to cover the entire 3D surface of the cell. Counting the number of fluorescent spots on individual cells was undertaken manually using the 2D images. The number of cells counted for each sample is detailed in the legend to Fig. 4. Lentivirus manufacture and SK‑Mel‑28 cell transductions A T150 flask of semi-confluent HEK293T cells were transfected with 15 μg of lentivector encoding the human HLA-A*0201 antigen, along with a total of 43 μg of three packaging plasmids [22], using Express-In transfection

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Results IMCgp100‑driven tumour cell killing results in antigen cross‑presentation by dendritic cells The ability of IMCgp100 to redirect T cells to specifically target HLA-A*0201 cells expressing gp100 was examined by monitoring dose-dependent cytolysis of

Cancer Immunol Immunother

three melanoma cell lines (Fig. 1a). Cell lysis was determined by release of lactate dehydrogenase (LDH). In line with a previous study [17], IMCgp100 produced a potent redirected CD8+ T cell response against Mel624 cells (gp100+/HLA-A*0201+) (Fig. 1b). The specificity of the response was verified using cell lines which either do not express gp100 (SK-Mel-24) or express an alternative HLA type (SK-Mel-28). These data demonstrate that IMCgp100 retains specificity when used at concentrations of 10−9 M and below. Further experiments showed that insertion of the HLA-A*0201 gene into SK-Mel-28 cells led to IMCgp100-driven lysis of these cells. The difference in the level of cytotoxicity observed between Mel624 and SK-Mel-28 (with HLA-A*0201) may be explained by the higher levels of HLA displayed on Mel624 cells, the level of gp100 expression between the two cells lines is comparable (see supplementary Figure 1). It was then examined whether IMCgp100-driven killing of Mel624 cells could promote the cross-presentation of melanoma antigens on DCs. IMCgp100redirected viral-specific (i.e. irrelevant) CD8+ cytotoxic T cells were used to induce killing of Mel624 targets. Apoptosis was confirmed as described in the methods, and the viral T cells were removed by positive selection for CD2. The resulting apoptotic Mel624 cells were then incubated with an equivalent number of immature HLAA*0201 DCs for 48 h, allowing antigen uptake to occur. A functional assay was then used to determine whether the resulting DCs were cross-presenting the Melan-Amelanoma antigen. This was done by incubating them with CD8+ T cells specific for the HLA-A*02-restricted Melan A26–35 peptide (see Fig. 1c for an outline of the experimental approach). Activation of Melan A T cells, determined by IFNγ release, was observed with DCs exposed to apoptotic Mel624 cells (Fig. 1d), whereas with LPS-matured DCs (i.e. prepared in the absence of apoptotic Mel624), no activation was observed. To confirm that the response was mediated by DCs, rather than by apoptotic Mel624 cells directly, or as a result of contaminating IFNγ produced by IMCgp100-redirected viral T cells, additional samples were prepared in the same manner as described above, but in the absence of either immature DCs or Melan A T cells (Fig. 1d). Apoptotic Mel624 cells were not able to produce a T cell response in the absence of DCs, as expected, since they can only present antigen to CD8+ T cells in a 6–24 h window after induction of apoptosis, well outside the 48 h used here [23]. Furthermore, the lack of IFNγ in either of these control samples indicates that any residual IFNγ is effectively removed during the experiment. These data indicate that IMCgp100-driven tumour cell killing can induce cross-presentation of TAAs by dendritic cells.

Cross‑presentation in the presence of IMCgp100 enhances activation of TAA‑specific T cells To investigate the potential of IMCgp100 to respond to cross-presented gp100 a similar experimental strategy was followed to that used above, except that, for technical ease, apoptotic melanoma cells were prepared by chemically induced apoptosis (see Fig. 2a for an outline of the experimental approach). The data show that autologous polyclonal CD8+ T cells were activated in response to crosspresenting DCs in the presence of IMCgp100 (Fig. 2b). No activation was apparent in the absence of IMCgp100. We then sought to establish the effect of IMCgp100 on DCmediated activation of TAA-specific T cells. Comparable to the data shown in Fig. 1d, Melan A T cells were activated by cross-presenting DCs (Fig. 2c). However, in the presence of IMCgp100, activation of Melan A T cells increased significantly (p 

ImmTAC-redirected tumour cell killing induces and potentiates antigen cross-presentation by dendritic cells.

Antigen cross-presentation by dendritic cells (DCs) is thought to play a critical role in driving a polyclonal and durable T cell response against can...
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