Else Marit Inderberg

T cell therapy targeting a public neoantigen in microsatellite instable colon cancer reduces in vivo tumor growth

ABSTRACT T-cell receptor (TCR) transfer is an attractive strategy to increase the number of cancer-specific T cells in adoptive cell therapy. However, recent clinical and pre-clinical findings indicate that careful consideration of the target antigen is required to limit the risk of off-target toxicity. Directing T cells against mutated proteins such as frequently occurring frameshift mutations may thus be a safer alternative to tumor-associated self-antigens. Furthermore, such frameshift mutations result in novel polypeptides allowing selection of TCRs from the non-tolerant T-cell repertoire circumventing the problem of low affinity TCRs due to central tolerance. The transforming growth factor β Receptor II frameshift mutation (TGFβRIImut) is found in Lynch syndrome cancer patients and in approximately 15% of sporadic colorectal and gastric cancers displaying microsatellite instability (MSI). The -1A mutation within a stretch of 10 adenine bases (nucleotides 709–718) of the TGFβRII gene gives rise to immunogenic peptides previously used for vaccination of MSI+ colorectal cancer patients in a Phase I clinical trial. From a clinically responding patient, we isolated a cytotoxic T lymphocyte (CTL) clone showing a restriction for HLA-A2 in complex with TGFβRIImut peptide. Its TCR was identified and shown to redirect T cells against colon carcinoma cell lines harboring the frameshift mutation. Finally, T cells transduced with the HLA-A2-restricted TGFβRIImut-specific TCR were demonstrated to significantly reduce the growth of colorectal cancer and enhance survival in a NOD/SCID xenograft mouse model.

Immune response and long-term clinical outcome in advanced melanoma patients vaccinated with tumor-mRNA-transfected dendritic cells

ABSTRACT The most effective anticancer immune responses are probably directed against patient-specific neoantigens. We have developed a melanoma vaccine targeting this individual mutanome based on dendritic cells (DCs) loaded with autologous tumor-mRNA. Here, we report a phase I/II trial evaluating toxicity, immune response and clinical outcome in 31 metastatic melanoma patients. The first cohort (n = 22) received the vaccine without any adjuvant; the next cohort (n = 9) received adjuvant IL2. Each subject received four weekly intranodal or intradermal injections, followed by optional monthly vaccines. Immune response was evaluated by delayed-type hypersensitivity (DTH), T cell proliferation and cytokine assays. Data were collected for 10 y after inclusion of the last patient. No serious adverse events were detected. In the intention-to-treat-cohort, we demonstrated significantly superior survival compared to matched controls from a benchmark meta-analysis (1 y survival 43% vs. 24%, 2 y 23% vs. 6.6%). A tumor-specific immune response was demonstrated in 16/31 patients. The response rate was higher after intradermal than intranodal vaccination (80% vs. 38%). Immune responders had improved survival compared to non-responders (median 14 mo vs. 6 mo; p = 0.030), and all eight patients surviving >20 mo were immune responders. In addition to the tumor-specific response, most patients developed a response against autologous DC antigens. The cytokine profile was polyfunctional and did not follow a Th1/Th2 dichotomy. We conclude that the favorable safety profile and evidence of a possible survival benefit warrant further studies of the RNA/DC vaccine. The vaccine appears insufficient as monotherapy, but there is a strong rationale for combination with checkpoint modulators.

A TCR-based Chimeric Antigen Receptor

Effector T cells equipped with engineered antigen receptors specific for cancer targets have proven to be very efficient. Two methods have emerged: the Chimeric Antigen Receptors (CARs) and T-cell Receptor (TCR) redirection. Although very potent, CAR recognition is limited to membrane antigens which represent around 1% of the total proteins expressed, whereas TCRs have the advantage of targeting any peptide resulting from cellular protein degradation. However, TCRs depend on heavy signalling machinery only present in T cells which restricts the type of eligible therapeutic cells. Hence, an introduced therapeutic TCR will compete with the endogenous TCR for the signalling proteins and carries the potential risk of mixed dimer formation giving rise to a new TCR with unpredictable specificity. We have fused a soluble TCR construct to a CAR-signalling tail and named the final product TCR-CAR. We here show that, if expressed, the TCR-CAR conserved the specificity and the functionality of the original TCR. In addition, we demonstrate that TCR-CAR redirection was not restricted to T cells. Indeed, after transduction, the NK cell line NK-92 became TCR positive and reacted against pMHC target. This opens therapeutic avenues combing the killing efficiency of NK cells with the diversified target recognition of TCRs.

Artesunate shows potent anti-tumor activity in B-cell lymphoma

BackgroundAlthough chemo-immunotherapy has led to an improved overall survival for most B-cell lymphoma types, relapsed and refractory disease remains a challenge. The malaria drug artesunate has previously been identified as a growth suppressor in some cancer types and was tested as a new treatment option in B-cell lymphoma.MethodsWe included artesunate in a cancer sensitivity drug screen in B lymphoma cell lines. The preclinical properties of artesunate was tested as single agent in vitro in 18 B-cell lymphoma cell lines representing different histologies and in vivo in an aggressive B-cell lymphoma xenograft model, using NSG mice. Artesunate-treated B lymphoma cell lines were analyzed by functional assays, gene expression profiling, and protein expression to identify the mechanism of action.ResultsDrug screening identified artesunate as a highly potent anti-lymphoma drug. Artesunate induced potent growth suppression in most B lymphoma cells with an IC50 comparable to concentrations measured in serum from artesunate-treated malaria patients, while leaving normal B-cells unaffected. Artesunate markedly inhibited highly aggressive tumor growth in a xenograft model. Gene expression analysis identified endoplasmic reticulum (ER) stress and the unfolded protein response as the most affected pathways and artesunate-induced expression of the ER stress markers ATF-4 and DDIT3 was specifically upregulated in malignant B-cells, but not in normal B-cells. In addition, artesunate significantly suppressed the overall cell metabolism, affecting both respiration and glycolysis.ConclusionsArtesunate demonstrated potent apoptosis-inducing effects across a broad range of B-cell lymphoma cell lines in vitro, and a prominent anti-lymphoma activity in vivo, suggesting it to be a relevant drug for treatment of B-cell lymphoma.

Abstract 2516: A new generation of dendritic cells to improve cancer therapy shows prolonged progression-free survival in patients with solid tumors

We have previously used different protocols for GMP production of monocyte derived dendritic cells (DCs), loaded them with mRNA and conducted several clinical trials to investigate their therapeutic impact in treatment of different solid tumors like prostate carcinoma, glioblastoma or melanoma. These studies showed that only a group of patients mounted an immune response after DC vaccination within a timeframe of 3 to 6 months. A new generation of IL-12p70 producing DCs gave superior immunological responses in pre-clinical data when compared with DCs generated according to the standard protocol used for DC production so far. Aim one of our study is to show the feasibility of generating IL-12p70 producing monocyte derived DCs with starting material from patients with different types of malignancies in a large scale GMP setting and aim two is to investigate the clinical response of these new generation DCs in patients suffering from different types of cancer and acute myeloid leukemia (AML). Autologous mature DCs were loaded with mRNA (messenger Ribonucleic Acid) either encoding the tumor specific antigens hTERT and survivin, plus where available, autologous tumor mRNA, or the AML specific antigens WT-1 or PRAME. Patients received 4 initial vaccinations in weekly intervals, a delayed type hypersensitivity challenge in week 6 and further boost vaccinations every month. With each vaccination either 2,5 x10^6 or 5×10^6 DCs were injected intradermal. So far we have started treatment of one lung cancer patient, one prostate cancer patient, 4 glioblastoma patients and 3 AML-patients. The lung cancer patient suffering from stage IV disease with lung and brain metastases has now been vaccinated since 12/2011 and remained in stable disease. The glioblastoma patients, also with stage IV diseases, have been vaccinated for 16, 12, 12 and 11 months. All of them are in complete remission. One patient developed a pseudo relapse after 7 and again after 9 months of non-malignant origin. The Prostate cancer patient progressed before an immune response could be expected and immediately dropped out of the vaccination program after start of DC treatment. The first AML patient has started DC treatment in May 2014, the other two just recently and therefore are still too early to evaluate. Our investigations show that mRNA loaded new generation DCs can successfully be produced from patients with different types of cancer. All patients tolerated DC vaccination well. Some patients developed flu-like symptoms on the day of vaccination. Strong DTH responses at the injection sites appeared 2 to 4 days after DC injection from the second vaccination time point on indicating the presence of specific immune responses. Solid tumor patients suffering from advanced disease treated with our DC vaccines have a prolonged progression free survival, showing that this immunotherapeutic approach will be a promising alternative to current standard therapies. Citation Format: Iris Bigalke, Kirsti Honnashagen, Marianne Lundby, Guri Solum, Lisbeth Skoge, Else M. Suso Inderberg, Julitta Kasten, Stein Saboe-Larssen, Dolores J. Schendel, Gunnar Kvalheim. A new generation of dendritic cells to improve cancer therapy shows prolonged progression-free survival in patients with solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2516. doi:10.1158/1538-7445.AM2015-2516

Human c-SRC kinase (CSK) overexpression makes T cells dummy

Adoptive cell therapy with T-cell receptor (TCR)-engineered T cells represents a powerful method to redirect the immune system against tumours. However, although TCR recognition is restricted to a specific peptide–MHC (pMHC) complex, increasing numbers of reports have shown cross-reactivity and off-target effects with severe consequences for the patients. This demands further development of strategies to validate TCR safety prior to clinical use. We reasoned that the desired TCR signalling depends on correct pMHC recognition on the outside and a restricted clustering on the inside of the cell. Since the majority of the adverse events are due to TCR recognition of the wrong target, we tested if blocking the signalling would affect the binding. By over-expressing the c-SRC kinase (CSK), a negative regulator of LCK, in redirected T cells, we showed that peripheral blood T cells inhibited anti-CD3/anti-CD28-induced phosphorylation of ERK, whereas TCR proximal signalling was not affected. Similarly, overexpression of CSK together with a therapeutic TCR prevented pMHC-induced ERK phosphorylation. Downstream effector functions were also almost completely blocked, including pMHC-induced IL-2 release, degranulation and, most importantly, target cell killing. The lack of effector functions contrasted with the unaffected TCR expression, pMHC recognition, and membrane exchange activity (trogocytosis). Therefore, co-expression of CSK with a therapeutic TCR did not compromise target recognition and binding, but rendered T cells incapable of executing their effector functions. Consequently, we named these redirected T cells “dummy T cells” and propose to use them for safety validation of new TCRs prior to therapy.

T cells expressing checkpoint receptor TIGIT are enriched in follicular lymphoma tumors and characterized by reversible suppression of T-cell receptor signaling

Purpose: T cells infiltrating follicular lymphoma (FL) tumors are considered dysfunctional, yet the optimal target for immune checkpoint blockade is unknown. Characterizing coinhibitory receptor expression patterns and signaling responses in FL T-cell subsets might reveal new therapeutic targets. Experimental Design: Surface expression of 9 coinhibitory receptors governing T-cell function was characterized in T-cell subsets from FL lymph node tumors and from healthy donor tonsils and peripheral blood samples, using high-dimensional flow cytometry. The results were integrated with T-cell receptor (TCR)-induced signaling and cytokine production. Expression of T-cell immunoglobulin and ITIM domain (TIGIT) ligands was detected by immunohistochemistry. Results: TIGIT was a frequently expressed coinhibitory receptor in FL, expressed by the majority of CD8 T effector memory cells, which commonly coexpressed exhaustion markers such as PD-1 and CD244. CD8 FL T cells demonstrated highly reduced TCR-induced phosphorylation (p) of ERK and reduced production of IFNγ, while TCR proximal signaling (p-CD3ζ, p-SLP76) was not affected. The TIGIT ligands CD112 and CD155 were expressed by follicular dendritic cells in the tumor microenvironment. Dysfunctional TCR signaling correlated with TIGIT expression in FL CD8 T cells and could be fully restored upon in vitro culture. The costimulatory receptor CD226 was downregulated in TIGIT+ compared with TIGIT− CD8 FL T cells, further skewing the balance toward immunosuppression. Conclusions: TIGIT blockade is a relevant strategy for improved immunotherapy in FL. A deeper understanding of the interplay between coinhibitory receptors and key T-cell signaling events can further assist in engineering immunotherapeutic regimens to improve clinical outcomes of cancer patients. Clin Cancer Res; 24(4); 870–81. ©2017 AACR.

Abstract 3773: Tapping CD4 T cells for cancer immunotherapy

T-cell based immunotherapy represents an attractive strategy for the treatment of cancer. Whereas cellular anti-tumor immune responses have typically been attributed to CD8 T cells, CD4 T cells play a critical role in tumor elimination and the priming and maintenance of CD8 T-cell responses. Recent findings have highlighted new opportunities for CD4 T cells in cancer immunotherapy. We have isolated CD4+ T cells reactive against tumor antigens from patients who experienced clinical benefit from treatment with cancer vaccines targeting antigens such as hTERT, survivin and frequent neoantigens such as frameshift mutated TGFβRII. Strong T-cell responses against the vaccine or unrelated cancer antigens suggesting epitope spreading correlated with enhanced survival and tumor regression in late stage cancer patients. These HLA class II restricted T-cell clones recognised target cells loaded with long peptides or protein and for some CD4+ T cell clones we could also show direct tumor recognition. TCRs were expressed in expanded third-party T cells by mRNA electroporation or retroviral transduction and tested for functionality. Both CD8+ and CD4+ T cells expressing the TCRs produced TNF-α, IFN-γ and redirected T cells had the capacity to kill following co-incubation with their targets. Selecting highly functional CD4+ T-cell clones reactive against tumor-associated or -specific antigens from patients with clinical responses after treatment with immunotherapy is a successful method for identifying highly functional HLA class II restricted TCRs for adoptive immunotherapy. These HLA class II-restricted TCRs may be of therapeutic value both in haematopoietic malignancies and in melanoma where tumor cells frequently express HLA class II. Furthermore, combining HLA class I- and class II-restricted TCRs for T-cell redirection may provide a more potent therapeutic effect in adoptive T cell therapy. Citation Format: Sebastien Walchli, Marit R. Myhre, Nadia Mensali, Anne Fane, Kari Lislerud, Gunnar Kvalheim, Gustav Gaudernack, Else M. Inderberg. Tapping CD4 T cells for cancer immunotherapy [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 3773. doi:10.1158/1538-7445.AM2017-3773

T-helper cell receptors from long-term survivors after telomerase cancer vaccination for use in adoptive cell therapy

ABSTRACT We herein report retargeting of T-helper (Th) cells against the universal cancer antigen telomerase for use in adoptive cell therapy. The redirected Th cells may counter tumor tolerance, transform the inflammatory milieu, and induce epitope spreading and cancer senescence. We have previously conducted a series of trials evaluating vaccination with telomerase peptides. From long-term survivors, we isolated >100 CD4+ Th-cell clones recognizing telomerase epitopes. The clones were characterized with regard to HLA restriction, functional avidity, fine specificity, proliferative capacity, cytokine profile, and recognition of naturally processed epitopes. DP4 is the most prevalent HLA molecule worldwide. Two DP4-restricted T-cell clones with different functional avidity, C13 and D71, were selected for molecular T-cell receptor (TCR) cloning. Both clones showed a high proliferative capacity, recognition of naturally processed telomerase epitopes, and a polyfunctional and Th1-weighted cytokine profile. TCR C13 and D71 were cloned into the retroviral vector MP71 together with the compact and GMP-applicable marker/suicide gene RQR8. Both TCRs were expressed well in recipient T cells after PBMC transduction. The transduced T cells co-expressed RQR8 and acquired the desired telomerase specificity, with a polyfunctional response including production of TNFa, IFNγ, and CD107a. Interestingly, the DP4-restricted TCRs were expressed and functional both in CD4+ and CD8+ T cells. The findings demonstrate that the cloned TCRs confer recipient T cells with the desired hTERT-specificity and functionality. We hypothesize that adoptive therapy with Th cells may offer a powerful novel approach for overcoming tumor tolerance and synergize with other forms of immunotherapy.

Abstract 2306: Treatment with hTERT/survivin mRNA-loaded dendritic cells combined with autologous ex vivo expanded T cells improves progression free survival in stage IV melanoma patients when compared to dendritic cell vaccines alone

In previous studies with tumor specific antigen loaded dendritic cells (DCs) we have shown that 50% of patients with metastatic malignant melanoma mount specific immune responses and clinical effects. However responses were only transient. Here we investigated, in a phase I/II study, if the combination of autologous DCs loaded with mRNA hTERT and survivin followed by transfusion of autologous ex-vivo expanded polyclonal T cells can strengthen the immune response and improve survival. Sixteen melanoma patients with non resectable metastases were included. Monocyte derived DCs were generated according to standard protocol using Jonuleit maturation cocktail. Patients received 4 vaccines in weekly intervals, a DTH-challenge in week 6, followed by monthly boosts with either 5 or 10 x10E+6 DCs. Blood samples were taken before and during DC vaccination and used for testing of specific immune responses against hTERT and survivin. Patients with immune responses were offered additional treatment with T cells. Fourteen patients could be evaluated, seven showing specific immune responses against hTERT and/or survivin. Three of them received a dose of 3×1010 expanded T cells after pre-treatment with Fludarabin and Cyclophosaphamide. DC vaccination was continued the day after T cell transfer. Patient (Pt) 1 and 2 showed an increase of hTERT specific T cells in peripheral blood after T cell transfer. Pt 1 lost the hTERT specific response 20 months after beginning of DC-vaccination correlating with progression of the disease. Pt 2 lost the hTERT response 29 months after start of DC-vaccination and proceeded with a response against surviving. Thereafter progression of disease occurred. Pt 3 had a response against survivin prior to T cell collection and at the same time progression occurred. Following administration of expanded T-cells a strong increase of survivin specific T cells in peripheral blood was detected. In spite of this the patient only had a progression free survival of 11 months. Patients were given the checkpoint inhibitor Ipilimumab when progressing and Pt 1 and Pt 2 are still in stable disease. Patients who did not mount any immune response during DC therapy had a mean PFS of 4,7 (2-7) months while patients with specific immune responses show a mean PFS of 9 (6-14) months. Retrospectively we performed an extended analysis of the DCs and their impact on immune responses. Flow-analysis showed a patient individual expression of maturation markers and co-stimulatory molecules. Mimicking T cell encounter by CD40L stimulation showed individual differences of IL-10 release but no IL-12p70. Differences in quality of the DCs did not have an impact on immune responses. Our data show that patients receiving expanded T cells after mounting a specific immune response have a longer PFS when compared to patients with DC vaccination alone. Citation Format: Iris Bigalke, Elin Aamdal, Siri Torhaug, Marianne Lundby, Else M. Inderberg, Gustav Gaudernack, Anne M. Rasmussen, Steinar Aamdal, Gunnar Kvalheim. Treatment with hTERT/survivin mRNA-loaded dendritic cells combined with autologous ex vivo expanded T cells improves progression free survival in stage IV melanoma patients when compared to dendritic cell vaccines alone. [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 2306.