Peptide-specific CD8 Research Articles

Abstract 6622: The identification of extranodal NK/T cell lymphoma-associated antigen for helper T cell-based immunotherapy

Abstract Extranodal natural killer/T-cell lymphoma, nasal type (NKTL) is a fatal destructive tumor that develops around the nasopharyngeal and nasal/paranasal areas. Although this disease was originally considered to be endemic to East Asia, Central and South America and was extremely rare in Europe and the United States, the incidence of NKTL has increased during the last several decades in countries where it was previously not epidemic. The combination of irradiation and chemotherapies such as SMILE and MPVIC-P are effective. However, these therapies cause severe and sometimes fatal adverse effects indicating that a novel strategy to treat NKTL is required. The positive results of clinical trials using immune checkpoint inhibitors in solid tumors proved that immune cells are capable of eliminating tumor. However, autoimmune diseases are inevitable adverse events in this type of therapy, which non-specifically activates T cells. Thus, the identification of epitope peptides in tumor has re-emerged as an attractive concept. I will describe our study to identify NKTL-derived T cell epitopes as a basis of cancer vaccine. The expression of c-Met and its ligand HGF plays a critical role in cell proliferation and is involved in numerous malignancies. Because c-Met expression and its role in NKTL remain unclear, we first studied the expression and function of c-Met in NKTL cells. As a result, we found that HGF and c-Met were expressed and contributed to the proliferation of cells in NK/T-cell lymphoma. Next, the helper epitopes were identified from c-Met protein using computer-based algorism. The identified epitope peptides could elicit peptide-specific helper CD4 T cells (HTLs), which showed antitumor responses in vitro. The addition of c-Met inhibitor suppressed TGF-β production from tumor followed by the augmentation of HTL responses. In summary, we identified novel c-Met HTL epitopes that can elicit effective antitumor responses against tumors expressing c-Met. Our results provide the rationale of combining c-Met targeting therapy and immunotherapy for NKTCLs and epithelial tumors. Citation Format: Takumi Kumai, Ryusuke Hayashi, Tatsuya Hayashi, Hiroya Kobayashi, Yasuaki Harabuchi. The identification of extranodal NK/T cell lymphoma-associated antigen for helper T cell-based immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6622.

Abstract 6699: CUE-102 Immuno-STATs for selective targeting and expansion of WT1-specific T cells for the treatment of HLA-A02+and/or HLA-A24+patients with WT1+ malignancies

Abstract WT1 has been ranked first amongst 75 tumor associated antigens in an effort by the National Cancer Institute to prioritize cancer antigens for therapeutic targeting. Development of novel modalities to target WT1 provide a significant opportunity to address high unmet medical need in WT1-positive hematological and solid tumor malignancies, including AML, ovarian, endometrial, breast, lung, colorectal and pancreatic cancer. We have developed two novel fusion proteins, termed Immuno-STATs (Selective Targeting and Activation of T cells), that are comprised of either HLA-A*02 or HLA-A*24 molecules (human leukocyte antigen) presenting peptide epitopes derived from WT1. Each CUE-102 Immuno-STAT molecule also contains four copies of affinity-attenuated human interleukin-2 (IL-2), and an effector attenuated human immunoglobulin G (IgG1) Fc domain. We present here the biochemical characterization of these molecules and their bioactivity across a variety of in vitro and in vivo studies, demonstrating their ability to selectively expand, from a naïve or pre-primed T cell repertoire, a polyfunctional population of WT1 peptide-specific CD8+ T cells capable of recognizing and responding to WT1 peptide-presenting target cells. Human peripheral blood mononuclear cells (PBMC) and WT1-specific CD8+ T cells were utilized to demonstrate cellular activity and specificity of CUE-102 Immuno-STATs. Repertoire analysis of WT1-specific T cells was done by single cell TCR sequencing. In vivo activity of CUE-102 molecules was assessed in HLA transgenic mice. CUE-102 Immuno-STATs presenting HLA-A*02 or A*24 specificities demonstrated selective binding and TCR triggering of WT1-specific CD8+ T cells. Signaling, cell-based assays and cytokine release studies confirmed significant functional attenuation of the IL-2 components of CUE-102. Primary stimulation of unprimed hPBMCs, or re-stimulation of hPBMCs after initial WT1 peptide stimulation, led to robust expansion of WT1-specific CD8+ T cells and demonstrated the ability of CUE-102 Immuno-STATs to stimulate and expand antigen-specific T cells from both a primed and pre-primed T cell repertoire. The expanded T cells exhibited a polyfunctional response upon challenge with WT1-presenting target cells, confirming that CUE-102 Immuno-STATs selectively expanded WT1-specific CD8+ T cells. The repertoire of the expanded cells, their polyfunctionality and ability to recognize and respond to WT1 peptide-presenting target cells suggest that CUE-102 Immuno-STATs have the potential to enhance anti-tumor immunity in patients with WT1-positive malignancies. Citation Format: Dharma Thapa, Alex Histed, Jonathan Soriano, Luke Witt, Zohra Merazga, Natasha Girgis, Miguel Moreta, Fulvio Diaz, Fan Zhao, Melissa Kemp, Paige Ruthardt, Anish Suri, Steve Quayle, John Ross, Saso Cemerski. CUE-102 Immuno-STATs for selective targeting and expansion of WT1-specific T cells for the treatment of HLA-A02+and/or HLA-A24+patients with WT1+ malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6699.

HLA DRB1*15:01-DQB1*06:02-Restricted Human CD4+ T Cells Are Selectively Activated With Amoxicillin-Peptide Adducts.

Amoxicillin-clavulanate is the most common cause of idiosyncratic drug-induced liver injury (DILI). Drug-specific CD4+ T cells have been detected in patients with DILI, suggestive of an immune etiology. Furthermore, genetic associations including the human leucocyte antigen (HLA) DRB1*15:01-DQB1*06:02 haplotype influence susceptibility. Amoxicillin forms protein adducts that are postulated to activate T cells, by conjugating with lysine residues. However, a role for such adducts has not been described. This study aimed to (1) investigate whether amoxicillin-modified HLA-DRB1*15:01-DQB1*06:02 binding peptides selectively activate DILI patient T cells and (2) define the nature of the T-cell response with respective to antigen structure. Peptides carrying lysine residues for amoxicillin binding in positions (KP) 2-6 and anchors for the HLA-DRB1*15:01-DQB1*06:02 haplotype were designed. The amoxicillin-modified peptides were characterized by mass spectrometry prior to culturing with patient peripheral blood mononuclear cell. T-cell clones were then tested for specificity with amoxicillin, unmodified- and amoxicillin-modified peptides, and structural variants. Amoxicillin-modified KP-2 and KP-3 peptide-specific CD4+ clones proliferated and secreted interferon gamma (IFN-γ), interleukin (IL)-10, perforin and/or IL-17/IL-22 in a dose-dependent manner and displayed no cross-reactivity with amoxicillin, unmodified peptide or with positional derivatives. The T cells response was HLA class II restricted and the amoxicillin-modified peptides bound selectively to HLA-DRB1*15:01 and/or DQB1*06:02. To conclude, we show that amoxicillin-modified peptides bind to both components of the risk haplotype to stimulate DILI patient T cells and describe the importance of the position of nucleophilic lysine residue in the HLA binding peptide sequence.

A peptide presentation by an MHC tetramer reduces the expansion of the peptide-specific T cells and the reduced expansion is reversible by PD-1 blockades

Abstract Background Peptide-MHC (pMHC) tetramers have been widely used in infectious disease and immune-oncology area to detect antigen-specific T cells. However, little is reported about their application in the context of pMHC presentation to stimulate T cells. Here we compared the expansion of antigen-specific T cells stimulated by an unlabeled pMHC tetramer and the peptide. We also analyzed the relationship among the programmed death-1 (PD-1), lymphocyte activation gene-3 (LAG-3), PD-L1, CD80 and CD38 expression on antigen-specific and non-specific T cells. Results Compared to the peptide stimulation, the pMHC tetramer induced lower expansion of the peptide-specific T cells. PD-1, PD-L1, CD80, and LAG3 expression was significantly higher on peptide-specific T cells than on non-specific T cells. Interestingly, peptide-specific T cells expanded by pMHC tetramer induced lower numbers of dysfunctional PD-1+CD38high CD8+ T cells. Furthermore, blockade of the PD-1 pathway by using an FDA approved antibody reversed such a restriction of expansion. Conclusion The pMHC tetramer stimulation resulted in the reduced expansion of peptide-specific CD8 T cells when compared to the peptide stimulation. Since the impaired expansion was reversed by the anti-PD1 therapeutic antibody, this artificial stimulation method could be used as a functional in vitro immune-oncology drug screening tool.

Immunological and Clinical Impact of Manipulated and Unmanipulated DLI after Allogeneic Stem Cell Transplantation of AML Patients.

Allogeneic stem cell transplantation (allo-SCT) is the preferred curative treatment for several hematological malignancies. The efficacy of allo-SCT depends on the graft-versus-leukemia (GvL) effect. However, the prognosis of patients with relapsed acute myeloid leukemia (AML) following allo-SCT is poor. Donor lymphocyte infusion (DLI) is utilized after allo-SCT in this setting to prevent relapse, to prolong progression free survival, to establish full donor chimerism and to restore the GvL effect in patients with hematological malignancies. Thus, there are different options for the administration of DLI in AML patients. DLI is currently used prophylactically and in the setting of an overt relapse. In addition, in the minimal residual disease (MRD) setting, DLI may be a possibility to improve overall survival. However, DLI might increase the risk of severe life-threatening complications such as graft-versus-host disease (GvHD) as well as severe infections. The transfusion of lymphocytes has been tested not only for the treatment of hematological malignancies but also chronic infections. In this context, manipulated DLI in a prophylactic or therapeutic approach are an option, e.g., virus-specific DLI using different selection methods or antigen-specific DLI such as peptide-specific CD8+ cytotoxic T lymphocytes (CTLs). In addition, T cells are also genetically engineered, using both chimeric antigen receptor (CAR) genetically modified T cells and T cell receptor (TCR) genetically modified T cells. T cell therapies in general have the potential to enhance antitumor immunity, augment vaccine efficacy, and limit graft-versus-host disease after allo-SCT. The focus of this review is to discuss the different strategies to use donor lymphocytes after allo-SCT. Our objective is to give an insight into the functional effects of DLI on immunogenic antigen recognition for a better understanding of the mechanisms of DLI. To ultimately increase the GvL potency without raising the risk of GvHD at the same time.

Modified Vaccinia Ankara-Vectored Vaccine Expressing Nucleoprotein and Matrix Protein 1 (M1) Activates Mucosal M1-Specific T-Cell Immunity and Tissue-Resident Memory T Cells in Human Nasopharynx-Associated Lymphoid Tissue.

BackgroundIncreasing evidence supports a critical role of CD8+ T-cell immunity against influenza. Activation of mucosal CD8+ T cells, particularly tissue-resident memory T (TRM) cells recognizing conserved epitopes would mediate rapid and broad protection. Matrix protein 1 (M1) is a well-conserved internal protein. MethodsWe studied the capacity of modified vaccinia Ankara (MVA)–vectored vaccine expressing nucleoprotein (NP) and M1 (MVA-NP+M1) to activate M1-specific CD8+ T-cell response, including TRM cells, in nasopharynx-associated lymphoid tissue from children and adults. ResultsAfter MVA-NP+M1 stimulation, M1 was abundantly expressed in adenotonsillar epithelial cells and B cells. MVA-NP+M1 activated a marked interferon γ–secreting T-cell response to M1 peptides. Using tetramer staining, we showed the vaccine activated a marked increase in M158–66 peptide-specific CD8+ T cells in tonsillar mononuclear cells of HLA-matched individuals. We also demonstrated MVA-NP+M1 activated a substantial increase in TRM cells exhibiting effector memory T-cell phenotype. On recall antigen recognition, M1-specific T cells rapidly undergo cytotoxic degranulation, release granzyme B and proinflammatory cytokines, leading to target cell killing. Conclusions MVA-NP+M1 elicits a substantial M1-specific T-cell response, including TRM cells, in nasopharynx-associated lymphoid tissue, demonstrating its strong capacity to expand memory T-cell pool exhibiting effector memory T-cell phenotype, therefore offering great potential for rapid and broad protection against influenza reinfection.

A new synthetic toll-like receptor 1/2 ligand is an efficient adjuvant for peptide vaccination in a human volunteer

BackgroundWe previously showed that the bacterial lipopeptide Pam3Cys-Ser-Ser, meanwhile established as a toll-like receptor (TLR) 1/2 ligand, acts as a strong adjuvant for the induction of virus specific CD8+ T cells in mice, when covalently coupled to a synthetic peptide.Case presentationWe now designed a new water-soluble synthetic Pam3Cys-derivative, named XS15 and characterized it in vitro by a TLR2 NF-κB luciferase reporter assay. Further, the capacity of XS15 to activate immune cells and stimulate peptide-specific CD8+ T and NK cells by 6-sulfo LacNAc+ monocytes was assessed by flow cytometry as well as cytokine induction using immunoassays. The induction of a functional immune response after vaccination of a volunteer with viral peptides was assessed by ELISpot assay and flow cytometry in peripheral blood cells and infiltrating cells at the vaccination site, as well as by immunohistochemistry and imaging.XS15 induced strong ex vivo CD8+ and TH1 CD4+ responses in a human volunteer upon a single injection of XS15 mixed to uncoupled peptides in a water-in-oil emulsion (Montanide™ ISA51 VG). A granuloma formed locally at the injection site containing highly activated functional CD4+ and CD8+ effector memory T cells. The total number of vaccine peptide-specific functional T cells was experimentally assessed and estimated to be 3.0 × 105 in the granuloma and 20.5 × 106 in peripheral blood.ConclusionThus, in one volunteer we show a granuloma forming by peptides combined with an efficient adjuvant in a water-in-oil-emulsion, inducing antigen specific T cells detectable in circulation and at the vaccination site, after one single vaccination only. The ex vivo T cell responses in peripheral blood were detectable for more than one year and could be strongly boosted by a second vaccination. Hence, XS15 is a promising adjuvant candidate for peptide vaccination, in particular for tumor peptide vaccines in a personalized setting.

Human leukocyte antigen (HLA) diversity and clinical applications in South Africa.

The major histocompatibility complex, known as the human leukocyte antigen (HLA) complex in humans, forms an integral component of adaptive T cell immunity by presenting self and non-self peptides to the T cell receptor, thereby allowing clonal expansion of responding peptide-specific CD4+ and CD8+ T cells. HLA likewise forms an integral part of the innate immune response through the binding of killer-cell immunoglobulin-like receptor (KIR) molecules, which regulate the response of natural killer (NK) cells. The HLA complex is found on the short arm of chromosome 6 and is the most polymorphic region in the human genome. Africans are genetically more diverse than other populations; however, information on HLA diversity among southern Africans, including South African populations, is limited. Paucity of African HLA data limits our understanding of disease associations, the ability to identify donor-recipient matches for transplantation and the development of disease-specific vaccines. This review discusses the importance of HLA in the clinical setting in South Africans and highlights how tools such as HLA imputation might augment standard HLA typing methods to increase our understanding of HLA diversity in our populations, which will better inform disease association studies, donor recruitment strategies into bone marrow registries and our understanding of human genetic diversity in South Africa.

Assessment of TCR signal strength of antigen-specific memory CD8+ T cells in human blood

AbstractAssessment of the quality and the breadth of antigen (Ag)-specific memory T cells in human samples is of paramount importance to elucidate the pathogenesis and to develop new treatments in various diseases. T-cell receptor (TCR) signal strength, primarily controlled by TCR affinity, affects many fundamental aspects of T-cell biology; however, no current assays for detection of Ag-specific CD8+ T cells can assess their TCR signal strength in human samples. Here, we provide evidence that interferon regulatory factor 4 (IRF4), a transcription factor rapidly upregulated in correlation with TCR signal strength, permits the assessment of the TCR signal strength of Ag-specific CD8+ T cells in human peripheral blood mononuclear cells (PBMCs). Coexpression of IRF4 and CD137 sensitively detected peptide-specific CD8+ T cells with extremely low background in PBMCs stimulated for 18 hours with MHC class I peptides. Our assay revealed that human memory CD8+ T cells with high-affinity TCRs have an intrinsic ability to highly express CD25. Furthermore, HIV-specific CD8+ T cells in chronic HIV+ subjects were found to display primarily low-affinity TCRs with low CD25 expression capacity. Impairment in the functions of HIV-specific CD8+ T cells might be associated with their suboptimal TCR signals, as well as impaired responsiveness to interleukin-2.

PF444 IDENTIFICATION OF T‐CELL RECEPTORS FROM THE ALLOGENEIC‐HLA REPERTOIRE SPECIFIC FOR MELANOMA‐ASSOCIATED ANTIGENS

Background:Melanoma‐associated antigens (MAGE) are highly expressed in multiple myelomas as well as solid tumors, but silent in normal cells except for male germ line cells. This tumor‐restricted expression makes it an excellent target for adoptive T cell therapy. However, due to thymic selection high‐affinity T cells against self‐antigens presented in the context of self‐HLA are deleted from the T‐cell repertoire of a patient. To overcome this problem, we identify T‐cell receptors (TCRs) from the allogeneic‐HLA repertoire instead of the autologous‐HLA repertoire.Aims:To identify high‐affinity T‐cell receptors from the allogeneic‐HLA repertoire specific for melanoma‐associated antigens.Methods:In this study we selected MAGE peptides by peptide elution experiments from HLA class I and subsequent mass spectrometric analysis of tumor cells. Only peptides binding in the most frequently occurring HLA‐alleles were selected. From the identified peptides, HLA‐tetramers were formed to isolate peptide specific CD8+ T cells from healthy allogeneic donors. Avidity of the selected T‐cell clones was determined by stimulation assays including a broad range of MAGE expressing tumor cell lines. To examine off‐target recognition, T‐cell clones were stimulated with MAGE negative cell lines and EBV‐transformed B lymphoblastic cells expressing all prevalent HLA molecules.Results:MAGE peptides were eluted from multiple myelomas, EBV‐transformed lymphoblastic cells, acute myeloid leukemia and ovarium carcinomas. For MAGE‐A1, T‐cell clones specific for four different epitopes presented in the context of HLA‐A∗01:01, HLA‐A∗03:01, HLA‐B∗07:02 and HLA‐C∗07:02 were selected. For MAGE‐A3, T‐cell clones specific for one epitope presented in the context of HLA‐A∗01:01 and HLA‐B∗35:01 were identified. The selected T‐cell clones demonstrated efficient recognition of MAGE‐A1 or MAGE‐A3 positive multiple myeloma and solid tumor cell lines without detectable cross‐reactivity.Summary/Conclusion:MAGE‐A1 and MAGE‐A3 specific T‐cell clones were isolated from the allogeneic‐HLA repertoire of healthy individuals. The identified clones were activated upon exposure to MAGE‐A1 or MAGE‐A3 positive tumor cell lines and showed limited off‐target recognition. These newly identified TCRs could potentially be used for TCR‐gene therapy strategies targeting MAGE‐A1 or MAGE‐A3 positive tumors.

Oligodeoxynucleotides ODN 2006 and M362 Exert Potent Adjuvant Effect through TLR-9/-6 Synergy to Exaggerate Mammaglobin-A Peptide Specific Cytotoxic CD8+T Lymphocyte Responses against Breast Cancer Cells.

Mammaglobin-A (MamA) is overexpressed in 40–80% of all human breast cancers. Recent phase I clinical trials of the MamA DNA vaccine showed encouraging safety outcomes. However, this vaccine elicited only a modest increase in MamA specific CD8+T lymphocyte (CTL) activation. As vaccine adjuvants play a critical role in enhancing the immunotherapeutic efficiency of vaccines, we tested the potential role of three synthetic CpG oligodeoxynucleotides (ODN2216—class A ODN, ODN2006—class B ODN, and ODN M362—class C ODN) to further enhance MamA specific CTL responses. Towards this, naïve CD8+T cells were obtained from healthy HLA-A2+ human donors. The HLA-A2 specific immunodominant epitope of MamA, MamA2.1 (LIYDSSLCDL), was utilized to activate naïve CD8+T cells. The THP-1 (HLA-A2+) cells were used as antigen presenting cells to stimulate naïve CD8+T cells along with (or without) co-treatment of various ODNs mentioned above. Activation of naïve CD8+T cells with the MamA2.1 peptide along with ODNs demonstrated enhanced MamA specific CTL mediated cytotoxicity on AU565 (HLA-A+/MamA+) breast cancer cells following co-treatment with ODN2006 and M362 compared to ODN2216 or MamA2.1 peptide alone. However, no significant cytotoxicity was noted upon treatment of MamA2.1 activated CTLs on MCF7 (HLA-A+/MamA−) cells, suggesting that the activation of CTLs is specific to the MamA antigen. Functional characterization studies demonstrated specific IL-12 mediated cross-talk between TLR-6 and -9 in THP-1 cells following stimulation with ODN2006 and M362, which was critical for the final cytotoxic activation of CD8+T lymphocytes. Based on these data, we conclude that ODN2006 and ODN M362 exerted a strong adjuvant effect through induction of the initial innate immune response through TLR9 upregulation followed by enhanced MamA specific CTL dependent adaptive immune responses. Our current data provide evidence for the application of Class-B/-C-CpG-ODNs as potential vaccine adjuvants towards enhancing the success of MamA based breast cancer vaccination.

Alum adjuvant is more effective than MF59 at prompting early germinal center formation in response to peptide-protein conjugates and enhancing efficacy of a vaccine against opioid use disorders

ABSTRACT Opioid use disorders (OUD) and fatal overdoses are a national emergency in the United States. Therapeutic vaccines offer a promising strategy to treat OUD and reduce the incidence of overdose. Immunization with opioid-based haptens conjugated to immunogenic carriers elicits opioid-specific antibodies that block opioid distribution to the brain and reduce opioid-induced behavior and toxicity in pre-clinical models. This study tested whether the efficacy of a lead oxycodone conjugate vaccine was improved by formulation in either aluminum hydroxide or the squalene-based oil-in-water emulsion MF59 adjuvant, which was recently FDA-approved for influenza vaccines in subjects 65+ years old. In adult BALB/c mice, alum formulation was more effective than MF59 at promoting the early expansion of hapten-specific B cells and the production of oxycodone-specific serum IgG antibodies, as well as blocking oxycodone distribution to the brain and oxycodone-induced motor activity. Alum was also more effective than MF59 at promoting early differentiation of peptide-specific MHCII-restricted CD4+ Tfh and GC-Tfh cells in adult C57Bl/6 mice immunized with a model peptide-protein conjugate. In contrast, alum and MF59 were equally effective in promoting hapten-specific B cells and peptide-specific MHCII-restricted CD4+ T cell differentiation in older C57Bl/6 mice. These data suggest that alum is a more effective adjuvant than MF59 for conjugate vaccines targeting synthetic small molecule haptens or peptide antigens in adult, but not aged, mice.

Abstract P2-09-04: Identification of a neoantigen targeted by tumor-infiltrating lymphocytes in a patient with Her2+ breast cancer

Abstract Background: Recent studies have demonstrated that the number of tumor infiltrating lymphocytes (TILs) positively correlates with outcome and response to chemotherapy in patients with HER2+ and Triple-Negative Breast Cancer (TNBC). Furthermore, first studies of immune-checkpoint inhibitors showed promising results in those patients. However, the targets of those TILs remain unknown. Neoantigens, which arise in the process of tumorigenesis, appear as potential targets. They can elicit high avidity, tumor-specific T-cell responses. Thus, it is the aim of our study to ascertainif these TILs are directed against tumor-specific mutations. Methods: TILs from breast cancer biopsies taken at the time point of diagnosis were expanded by unspecific stimulation. Additionally, we used the Gentle Macs Dissociator in combination with flow cytometry to investigate the number of TILs in the tumor tissue. Furthermore, we performed whole-genome sequencing of tumor tissue and as reference autologous blood cells to determine tumor-specific mutations. Mutations leading to a non-synonymous amino acid change were analyzed for RNA expression of the encoding gene as well as to determine potential neoantigens. Neoantigens were evaluated for their potential binding to the patient's specific HLA molecules. Peptides for potential neoantigens were synthesized, loaded onto autologous antigen presenting cells (APCs) and cocultured with TILs. All IFNγ producing T-cells were clonally expanded and retested for peptide specificity to identify neoantigen specific T-cell clones. Results: Our flow cytometric analysis of the tumor biopsy for more than 300 patients showed higher frequencies of TILs in TNBC as compared to other types of breast cancer or patients without malignancy. Screening for neoantigen specific T-cells in one patient led to identification of three peptide-specific CD4+ T-cell clones isolated from HER2+ breast cancer tissue taken at the time point of diagnosis. All T-cell clones specifically recognized the same tumor-specific mutation and not the wildtype counterpart. Furthermore, we demonstrated that these T-cell clones also recognized the endogenously expressed mutated antigen. This verified the ability of processing and presentation of the respective protein. Interestingly, we could also isolate a T-cell clone recognizing the same neoantigen in the resected tumor tissue after neoadjuvant therapy. Based on CDR3 sequencing we could prove that the four T-cell clones represented individual clones. This confirms the polyclonal nature of the immune response. Moreover, we showed that the same neoepitope was presented in two different HLA restriction molecules of the patient with three of the clones recognizing it in HLA-DPB1*0401 and one in HLA-DPB1*0201. These results further underline the immunogenicity of this neoantigen. Conclusion: In conclusion, our data demonstrate tumor-specificity of TILs in a patient with HER2+ breast cancer. Furthermore, we show the feasibility to identify individual cancer specific T-cell targets in breast cancer patients. These results may contribute to the development of targeted patient-specific immunotherapies in the future. Citation Format: Reimann H, Nguyen A, Hübner H, Erber R, Bausenwein J, Van der Meijden ED, Lux MP, Jud S, Griffioen M, Rauh C, Sanborn JZ, Benz SC, Rabizadeh S, Beckmann MW, Mackensen A, Rübner M, Fasching PA, Kremer AN. Identification of a neoantigen targeted by tumor-infiltrating lymphocytes in a patient with Her2+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-04.

Delayed IL-21 treatment preferentially expands peptide-specific CD8+ T cells by reducing bystander activation of T cells.

We previously reported a simple and practical procedure to generate peptide-specific CD8+ T cells using peptide and IL-2, which is applied to produce human telomerase reverse transcriptase (hTERT)-specific CD8+ T cells for clinical use. We have modified the procedure to enhance the amplification of peptide-specific CD8+ T cells adding IL-21. Using human leukocyte antigen (HLA)-A*0201-restricted cytomegalovirus/pp65-specific CD8+ T cells of healthy volunteers, we optimized the culture conditions by adjusting the dose and timing of IL-21 treatment. By adding IL-21, we accelerated the expansion rate of cytomegalovirus/pp65-specific CD8+ T cells by reducing bystander activation of T cells. We expect that the procedure including IL-21 would improve the production rate of hTERT- and Wilms tumor 1 (WT1)-specific CD8+ T cells for clinical trials.

Abstract B103: Altering the mevalonate pathway to enhance CD8+ T-cell responses

Abstract Inhibitors of the mevalonate pathway have been found to induce inflammatory reactions and delay the progression of cancer. Emerging evidence suggests these inhibitors sensitise immune cells to signalling from the myeloid differentiation primary response gene 88 (MyD88) and in turn enhance NF-κB dependent activation of antigen presenting cells (APCs), an essential element in inducing an immune response. To further explore the adjuvant effect of these inhibitors, we designed a vaccine comprising an inhibitor of the mevalonate pathway conjugated to a tumor-specific peptide. We hypothesized that immunisation with these vaccines would elicit functional peptide-specific CD8+ T-cells. Using an in vivo mouse model, we found the vaccine generates greater peptide-specific cytotoxicity against targeT-cells bearing a human papillomavirus antigen than its unconjugated components, a chemical control vaccine or peptide alone. Moreover, the vaccine delayed outgrowth of an antigen-expressing tumor. Using human PBMCs, we obtained preliminary evidence that a similar vaccine can promote greater in vitro activation of CD8+ T-cells specific for a cytomegalovirus antigen than peptide alone. These results suggest that inhibitors of the mevalonate pathway can act as adjuvants in eliciting cytotoxic T-cell responses, and may have utility for the treatment or prevention of viral infections or neoplasia. Citation Format: Olivia K. Burn, Astrid Authier-Hall, Collin Brooks, Regan Anderson, Andrew Marshall, Gavin Painter, Robert Weinkove, Ian Hermans. Altering the mevalonate pathway to enhance CD8+ T-cell responses [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B103.

Analysis of the Multiple Myeloma HLA Peptidome Identifies a Naturally Presented Bcma-Derived Peptide As an Immunogenic T-Cell Epitope for Immunotherapeutic Approaches

The B-cell maturation antigen (BCMA) is selectively expressed by cells of the B-lineage, including multiple myeloma (MM) cells, and constitutes a promising target for immunotherapeutic approaches. At present, BCMA is being evaluated as target for immunotherapeutic approaches, such as CAR T cells and bispecific antibodies, which have demonstrated promising results in phase I clinical trials. The utilization of cytotoxic T cells bearing T-cell receptors against BCMA constitutes an alternative promising approach to target MM cells. Therefore, the identification of BCMA-derived peptides that are naturally presented by human leukocyte antigens (HLA) and thus can serve as target structures for CD8+ T cells, is indispensable.In a previous study, we characterized the immunopeptidomic landscape of MM by mass spectrometry-based analysis of naturally presented HLA ligands from primary MM samples and MM cell lines (Walz et al., Blood, 2015). Comparative HLA peptidome profiling of the MM-derived HLA ligands versus the immunopeptidome of numerous benign samples from different tissues identified several strictly MM-associated antigens. Here, we evaluated this dataset for the presence of BCMA-derived MM-exclusive antigens and identified two HLA class I-restricted, BCMA-derived peptides in the immunopeptidome of our cohort comprising 15 primary MM samples and MM cell lines. Notably, one of these peptides showes strictly MM-associated presentation and was never detected on any benign tissues according to our extensive immunopeptidome database (135,354 HLA ligands originating from 16,626 source proteins detected in 337 samples from various benign tissues including blood, bone marrow, lung, kidney, liver, and spleen). This HLA-B*18-restricted ligand P(BCMA)B*18 is represented in 20% (3/15) of the analyzed MM immunopeptidomes.For immunological characterization of the P(BCMA)B*18 peptide, we performed in vitro artificial antigen-presenting cell-based priming experiments engaging naïve CD8+ T cells obtained from healthy volunteers (HV). Induction of tetramer-positive T-cell populations with frequencies ranging from 0.1-2.9% of viable CD8+ T cells was observed for all analyzed healthy whole blood donors, which demonstrates the immunogenicity of P(BCMA)B*18. Subsequently, we functionally characterized the induced P(BCMA)B*18-specific CD8+ T cells using intracellular cytokine staining. Upon stimulation with P(BCMA)B*18, we observed an increased IFNγ and TNF production specifically in the peptide-specific CD8+ T cells. In addition, the degranulation marker CD107a was found to be upregulated in the analyzed tetramer-positive T cells, confirming the activity of CD8+ T cells upon peptide-stimulation. Priming experiments using naïve CD8+ T cells obtained from MM patients as well as in vitro cytotoxicity assays with polyclonal peptide-specific effector T cells are presently ongoing in order to assess the capacity of P(BCMA)B*18-specific CD8+ T cells to induce antigen-specific cell lysis.Taken together, we identified a naturally presented and MM-associated, BCMA-derived peptide, which constitutes a promising target for tailored T cell-based immunotherapeutic approaches. DisclosuresSalih:Several patent applications: Patents & Royalties: e.g. EP3064507A1. Kowalewski:Immatics Biotechnologies GmbH: Employment. Weisel:Amgen, BMS, Celgene, Janssen, and Takeda: Honoraria; Amgen, BMS, Celgene, Janssen, Juno, Sanofi, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen, Celgene, Janssen, and Sanofi: Research Funding.

The HLA Ligandome Landscape of Chronic Myeloid Leukemia Delineates Novel T-Cell Epitopes for Immunotherapy

Chronic myeloid leukemia (CML) is characterized by the translocation t(9;22), which leads to the formation of the BCR-ABL fusion transcript. Several approved tyrosine kinase inhibitors (TKIs) target the resulting fusion protein, leading to an improved prognosis of CML patients. Currently, the main treatment goal is the achievement of a deep molecular response (MR), in which TKI therapy can be terminated. Several studies provide evidence that immunological control plays a major role for the course of CML and contributes to the achievement of deep MR in CML patients under TKI treatment (CMLTKI). This implies that reinforcing these immune responses might sustain long-term TKI-free survival or even cure for CML patients. Besides unspecific immunotherapy, such as interferon or immune checkpoint blocking antibodies, a more specific and minor side effect targeting of CML cells might be achieved by antigen-specific immunotherapy approaches. The prerequisite for such strategies is the identification of T-cell targets represented by tumor-associated human leukocyte antigen (HLA)-presented peptides on malignant cells.In this study, we used a mass spectrometry-based approach to identify naturally presented, CML-associated peptides in primary CML samples (HLA class I, n=21, 11,945 peptides, 5,478 source proteins; class II, n=20, 5,991 peptides, 1,302 proteins). Comparative HLA peptidome profiling using a comprehensive dataset of various benign tissues (e.g. blood, bone marrow, spleen, and lung) revealed frequently presented and strictly CML-associated antigens. In detail, the benign tissue dataset comprises hematological benign samples (class I, n=108, 51,233 peptides, 11,437 proteins; class II, n=88, 42,753 peptides, 4,877 proteins) and non-hematological benign tissues (28 tissues, n=166; class I, 128,590 peptides, 16,405 proteins; class II, 143,652 peptides, 13,410 proteins). We identified 50 CML-associated, HLA class I-restricted peptides with HLA allotype adjusted representation frequencies of ≥38% presented on HLA-A*02, -A*03, -A*11, and -B*07. HLA class II comparative profiling delineated 36 peptides exclusively and frequently presented in the HLA peptidome of ≥20% analyzed CML patients.For immunological characterization, we selected 8 HLA class I- and 6 class II-restricted highly CML-associated antigens. These peptides were analyzed in IFNγ ELISPOT assays using PBMCs from CMLTKI patients and healthy volunteers (HVs). Peptide-specific immune recognition was detected for 1/8 (13%) HLA class I peptides in 2/17 (12%) of CMLTKI patients. We hypothesized that this weak immune response might be due to an impaired CD8+ T cell function that reportedly is caused by TKI treatment. Thus, we compared T-cell responses against viral epitopes in IFNγ ELISPOT assays of CMLTKI patients with that of HVs and chronic lymphocytic leukemia (CLL) patients: in line with our hypothesis, we observed significantly reduced IFNγ release of T cells from CMLTKI patients compared to HVs and CLL patients (p<0.001), whereas CD8+ T-cell counts were not reduced. In contrast, no reduced IFNγ production was observed for HLA class II-restricted viral epitopes. These results were confirmed by memory T-cell responses detected for 6/6 (100%) HLA class II CML-associated peptides with frequencies up to 24% (4/17) of analyzed CMLTKI patients.To assess the immunogenicity of all HLA class I peptides, we performed in vitro artificial antigen presenting cell-based priming experiments using CD8+ T cells of HVs and CML patients. Effective priming of T cells was observed for 8/8 CML-associated peptides in ≥70% of analyzed HVs with frequencies of 0.1-33.9% (mean 2.2%) of CD8+ peptide-specific T cells. Notably, peptide-specific CD8+ T cells with frequencies of 0.1-2.2% (mean 0.4%) could also be induced in samples of CMLTKI patients that had not displayed preexisting immune responses. For 6/8 peptides, we observed multifunctionality of peptide-specific T cells by IFNγ and TNF production as well as upregulation of the degranulation marker CD107a. Cytotoxicity assays with polyclonal peptide-specific effector T cells confirmed the capacity to induce antigen-specific lysis for 3/4 analyzed peptides.Taken together, we here identified novel, naturally presented, CML-associated antigens and validated them as promising targets for tailored T cell-based immunotherapeutic approaches for CML patients. DisclosuresSalih:Several patent applications: Patents & Royalties: e.g. EP3064507A1. Kowalewski:Immatics Biotechnologies GmbH: Employment. Schuster:Immatics Biotechnologies GmbH: Employment. Brümmendorf:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy. Niederwieser:Miltenyi: Speakers Bureau; Novartis: Research Funding.

Phenotype of p53 wild-type epitope-specific T cells in the circulation of patients with head and neck cancer

CD8+ cytotoxic T-cell (CTL) specific for non-mutated, wild type (wt) sequence p53 peptides derived from wt or mutant p53 molecules expressed in head and neck squamous cell carcinomas (HNSCC) have been detected in the circulation of patients with this disease. The frequency and differentiation/maturation phenotypes of these anti-tumor specific CTL can reflect the host’s immunologic response. Therefore, we investigated the frequency and phenotypes of wt sequence p53 peptide-specific CTL in patients with HNSCC (n = 33) by flow cytometric analysis using HLA-A*0201 tetrameric peptides (tet) complexed with the wt sequence p53264–272 or p53149–157 peptide and co-staining with phenotypic markers. One main finding was that increasing frequencies of tet+ CD8+ T cells in patients’ circulation correlated with increased frequencies of inactive naïve tet+ cells, while those with effector memory and terminally differentiated phenotypes, which are associated with positive anti-tumor immune responses, decreased. We also found that the frequency of circulating tet+ CD8+ T cells negatively correlated with p53 expression in tumor tissues and tumor stage. Our findings support further clinical-based investigations to define the frequencies and phenotypes of wt sequence p53 peptide-specific CD8+ T cells to predict disease severity, enhance selection of patients for inclusion in vaccination trials and highlight prerequisites to enhance immune susceptibility by activation of inactive naïve tet+ T cells and/or enhancing circulating effector T cell activity by checkpoint blockage.

Mycobacterium tuberculosis peptide E7/HLA-DRB1 tetramers with different HLA-DR alleles bound CD4+ T cells might share identical CDR3 region

Human CD4+ T cells play an important role in the immune response to Mycobacterium tuberculosis (MTB). However, little is known about the spectratyping characteristics of the CD4+ T-cell receptor (TCR) α- and β-chains CDR3 region in tuberculosis (TB) patients. We sorted MTB peptide E7-bound CD4+ T cells by using E7/HLA-DR tetramers constructed with different HLA-DRB1 alleles and extracted the CDR3 amino-acid sequences of TCR α- and β-chains. The results showed that the CDR3 sequences of E7-bound CD4+ T cells were completely or partially identical in a single patient. The sequences of MTB peptide C5-bound CD4+ T cells shared another, and non-peptide bound CD4+ T cells, as well as unbound CD4+ T cells with tetramers were different from each other. Specifically, diverse CDR3 sequences of E7-bound CD4+ T cells displayed similar protein tertiary structure in one TB patient. In summary, the TCR α- and β-chains of CDR3 lineage of CD4+ T cells in TB patients apparently drifted, and the predominant CDR3 sequences of TCR α- and β-chains that recognized the MTB antigen exhibited peptide specificity, and certain HLA-DR restriction was also established. This study elucidates the possible causes and mechanisms of peptide-specific CD4+ T-cell-related presentation against MTB.

Vaccination with ApoB100 Derived HLA-A2 Restricted CD8 T Cell Epitopes Did Not Reduce Atherosclerosis in Male LDLrKO hApoB100tg HLA-A2tg Mice

CD8 T cells make up a large amount of lymphocytes in atherosclerotic plaques , however their role in atherosclerosis has not been thoroughly studied. Since ApoB100 is a key antigen in atherosclerosis and a plausible vaccination target, we were interested in the role of ApoB100 specific CD8 T cells in atherosclerosis. To be directly relevant for human vaccination HLA-A2 transgenic (HLA-A2) and human ApoB100 transgenic (hApoB100tg) mice were used. HLA-A2 restricted CD8 T cell epitopes derived from human ApoB100 were in silico predicted using The “Proteasomal cleavage/TAP transport/MHC class I combined predictor”of the immune epitope database. Best 6 epitopes were synthesized and binding to HLA-A2 assesed with T2 cell binding assays . To assess induction of peptide specific CD8 T cells, HLA-A2 transgenic mice were vaccinated with peptide pulsed DCs. Peptide recall responses were measured with flow cytometry . To study whether vaccination with ApoB100 derived CD8 T cell epitopes could influence atherosclerosis , male hApoB100tg HLA-A2tg LDLrKO mice (n=10-11) were vaccinated with peptide pulsed bone marrow derived DCs and boosted a week later with with αCD40 and Poly(I:C) and peptides. Animals were fed western type diet for 11 weeks following DC vaccination, after which they were sacrificed to assess aortic root lesion development. All predicted HLA-A2 restricted CD8 T cell epitopes stabilized HLA-A2 in T2 cell binding assays , indicating binding of all peptides to HLA-A2. For 5 of 6 peptides peptide specific recall response were measured a week after vaccination with peptide pulsed DCs. Vaccination with these CD8 T cell epitopes did not lead to reduced atherosclerosis . 10 weeks after the booster vaccination still peptide specific CD8 T cell responses could be detected, indicating that the vaccination was succesful. We have identified 5 HLA-A2 restricted ApoB100 derived CD8 T cell epitopes. Vaccination with these peptides did induce long lasting ApoB100 specific CD8 T cells, however did not affect atherosclerosis in male hApoB100tg HLA-A2tg LDLrKO mice