Spontaneous antitumor T cell responses in cancer patients

Mutated proteins arising from somatic mutations in tumors are promising targets for cancer immunotherapy. They represent true tumor-specific antigens (TSAs) as they are exclusively expressed in tumors, reduce the risk of autoimmunity and are more likely to overcome tolerance compared to wild-type (wt) sequences. Hence, we designed a panel of long peptides (LPs, 28–35 aa) comprising driver gene mutations in TP35 and KRAS frequently found in gastrointestinal tumors to test their combined immunotherapeutic potential. We found increased numbers of T cells responsive against respective mutated and wt peptides in colorectal cancer patients that carry the tested mutations in their tumors than patients with other mutations. Further, active immunization of HLA(-A2/DR1)-humanized mice with mixes of the same mutated LPs yielded simultaneous, polyvalent CD8+/CD4+ T cell responses against the majority of peptides. Peptide-specific T cells possessed a multifunctional cytokine profile with CD4+ T cells showing a TH1-like phenotype. Two mutated peptides (Kras[G12V], p53[R248W]) induced significantly higher T cell responses than corresponding wt sequences and comprised HLA-A2/DR1-restricted mutated epitopes. However, vaccination with the same highly immunogenic LPs strongly increased systemic regulatory T cells (Treg) numbers in a syngeneic sarcoma model over-expressing these mutated protein variants and resulted in accelerated tumor outgrowth. In contrast, tumor outgrowth was delayed when vaccination was directed against tumor-intrinsic Kras/Tp53 mutations of lower immunogenicity. Conclusively, we show that LP vaccination targeting multiple mutated TSAs elicits polyvalent, multifunctional, and mutation-specific effector T cells capable of targeting tumors. However, the success of this therapeutic approach can be hampered by vaccination-induced, TSA-specific Tregs.

T‑cell costimulation is necessary to induce a response of naïve T cells. Whether T‑cell costimulation can also cause reactivation of unreactive, possibly anergized memory T cells (MTCs) from late‑stage cancer patients is unknown. To investigate this question, we developed a bispecific anti‑CD28 fusion protein (bsHN‑CD28) which can easily be attached to the vaccine ATV‑NDV. This virus‑modified autologous tumor cell vaccine has already shown effectivity in colon cancer patients following resection of liver metastases. In this phase Ⅰ clinical study, 14 colorectal carcinoma (CRC) patients with late‑stage disease which could not be operated anymore with curative intent were treated with the vaccine ATV‑NDV to which bsHN‑CD28 was attached. No severe adverse events were recorded. All patients showed an immunological response of tumor‑reactive T cells, at least once during the course of five vaccinations. Also, we demonstrate a dose‑response relationship with the costimulatory molecule added to the vaccine. A partial response of metastases was documented in four patients. The study suggests that the three‑component vaccine is safe and can reactivate possibly anergized T cells from a chronic disease like advanced‑stage cancer.

Human cytomegalovirus (HCMV) is a widespread and persistent beta-herpesvirus. The large DNA genome of HCMV encodes many proteins that are non-essential for viral replication including numerous proteins subverting host immunosurveillance. One of them is the barely characterized UL20, which is encoded adjacent to the well-defined immunoevasins UL16 and UL18. UL20 is a type I transmembrane glycoprotein with an immunoglobulin-like ectodomain that is highly polymorphic among HCMV strains. Here, we show that the homodimeric UL20, by virtue of its cytoplasmic domain, does not reach the cell surface but is targeted to endosomes and lysosomes. Accordingly, UL20 exhibits a short half-life because of rapid lysosomal degradation. Trafficking of UL20 to lysosomes is determined by several, independently functioning dileucine-based sorting motifs in the cytoplasmic domain of UL20 and involves the adaptor protein (AP) complex AP-1. Combined substitution of three dileucine motifs allowed strong cell surface expression of UL20 comparable to UL20 mutants lacking the cytoplasmic tail. Finally, we show that the intracellularly located UL20 also is subject to lysosomal degradation in the context of viral infection. Altogether, from these data, we hypothesize that UL20 is destined to efficiently sequester yet-to-be defined cellular proteins for degradation in lysosomes.

Vaccination with peptides comprising tumor-associated antigens (TAAs) is a promising approach in cancer immunotherapy. TAAs derived from mutated genes are exclusively expressed in the tumor and are not shared with normal tissue. This reduces the risk of autoimmunity compared to non-mutated proteins when employed for cancer peptide vaccination. Moreover, the effectiveness of the vaccine can be enhanced by combining epitopes to induce cytolytic CD8+ as well as T helper (CD4+) cell responses. We therefore use long peptides (28-35 amino acids) to facilitate a presentation of MHC I and II epitopes. More precisely, we designed a panel of long peptides, with sequences derived from the most frequent mutated variants of the tumor suppressor Trp53 and the oncoproteins Kras and Braf described for colorectal (CRC) and pancreatic carcinomas. To investigate the relevance of immune responses against the chosen mutations we screened blood and bone marrow from 26 CRC patients for T cell responses against the long peptides using IFNγ ELISpot analysis. We found a tendency towards stronger responses against the mutated peptides compared to those against corresponding wild-type (wt) peptides. Furthermore, we correlated the ELISpot results with the abundance of Trp53 and Kras mutations identified in the patients’ primary tumors and metastases. This revealed that patients carrying mutations were more likely to be responsive against wt and mutated peptides than patients with no detectable mutation. In order to analyze the potency of our long peptides for active vaccination, we utilized C57BL/6J mice as well as a MHC-Class I/II humanized mouse strain (β2m-deficient, HLA-A2/Db/hβ2m [HHD chimera] and HLA-DR1 double transgenic) in a multi-peptide vaccination setting. Immune responses were monitored with flow cytometry by measuring cytokine secretion after in vitro restimulation of T cells from immunized mice. Thereby we observed responses for the majority of the long peptides tested. Interestingly, some of the mutated peptides showed a significantly higher induction of cytokine levels than corresponding wt sequences suggesting mutation-specific responses. In addition we were able to monitor both CD4+ and CD8+ T cell responses, in which the presence of CD4+ T cells enhanced CD8+ T cell responsiveness. This observation was made for several peptides in different combinations suggesting the induction of a multi-epitope response. As a final goal we plan to investigate the tumor-protective capacity of our vaccination approach. Therefore, the MHC-Class I/II transgenic mice were treated with the carcinogen 3-methylcholanthrene to generate a syngeneic tumor cell line. We were able to establish a fibrosarcoma cell line growing in vivo, which is engineered to express the most-immunogenic mutations from our vaccination panel to be tested in tumor challenge experiments. Citation Format: Jasmin Quandt, Christoph Schlude, Michael Bartoschek, Angel Cid-Arregui, Philipp Beckhove, Frank Momburg. T cell responses against mutations in oncoproteins/tumor suppressor proteins and their induction by vaccination with long peptides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1261. doi:10.1158/1538-7445.AM2013-1261

Immunotherapy for patients suffering from multiple myeloma is a lively and emerging field in cancer research. Immunotherapeutic approaches offer unique treatment opportunities for this, to date, mostly incurable disease. Respective basic findings and recent clinical approaches are introduced and discussed. Although several obstacles still need to be overcome, it appears that clinically efficient immunotherapies will become available for multiple myeloma patients in the future.

The prognosis of colorectal carcinoma (CRC) patients is dependent on the establishment of distant metastasis, which have been shown to arise from a small population of long term tumor initiating cells (LT-TIC). Another major prognostic parameter is the T cell infiltrate in the primary tumor of CRC patients, suggesting that tumor specific T cell responses occur and that the T cell mediated immune surveillance might play an important role in controlling tumor relapse, metastasis and response to treatment. Based on these observations we hypothesize that T cell responses against antigens expressed on TIC might trigger more efficient immune surveillance. So far little is known about the target antigens of the spontaneous T cell responses in CRC patients. In order to identify those antigens in a systematic and unbiased manner, we established a two-dimensional protein separation technique (PF2D). This technique allowed to fractionate lysates from primary and metastatic tumor tissue and to select the fractions which were recognized by the patient's T cell repertoire, as assessed by IFN-γ ELISpot assays. The reactive fractions were further characterized with mass spectrometry (MS) and candidate proteins were verified by use of synthetic peptides in ELISpot. As a result, we have identified 21 novel CRC-associated target antigens of spontaneous T cell responses. We have shown in a cohort of 20 patients that the newly identified target antigens not only triggered responses more frequently than “canonical” tumor antigens that are commonly used for immunotherapy (up to 50% of the tested patients), but the frequency of the T cells specific for the novel tumor associated antigens (TAA) was significantly higher. Furthermore, as shown by gene expression analysis some of the newly identified TAAs were selectively overexpressed on LT-TIC of CRC, and by means of PF2D of LT-TIC enriched spheroid cultures and subsequent MS of the immunogenic fractions, we have demonstrated that they represent target antigens of patients' endogenous T cell responses in all of the tested LT-TIC cultures. In addition, we have identified 30 amino acids long sequences of the these TAA, which elicit CD8+ or CD4+ T cell responses and we are currently working on identification of T cell epitopes and generation of T cell clones specific for these antigens. These would be further used to test the hypothesis that LT-TIC specific T cell clones have the capability to reduce the metastatic potential and tumorigenicity of TIC enriched spheroids in vitro and in vivo. In conclusion we have shown that novel set of antigens expressed in a therapeutically relevant subset of CRC cells, can be highly immunogenic and might serve as better targets for immune monitoring and could be utilized for more efficient T cell based immunotherapy. Citation Format: Slava Stamova, Christoph Schlude, Saskia Rösch, Christel Herold-Mende, Taronish D. Dubash, Claudia R. Ball, Hanno Glimm, Martin A. Schneider, Philipp Beckhove. Identification of novel tumor associated antigens in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2896. doi:10.1158/1538-7445.AM2014-2896

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