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Supplementary Table 3. Expanded T-cell lines after first MHC-multimer pull down. Expanded T-cell lines after second MHC-multimer pull down.

Supplementary Table 2. MiHA validation scores

Supplementary Figure 2. Gene expression profiles of CLYBL (A) and TEP1 (B) were analyzed.

Purpose: T-cell recognition of minor histocompatibility antigens (MiHA) not only plays an important role in the beneficial graft-versus-leukemia (GVL) effect of allogeneic stem cell transplantation (allo-SCT) but also mediates serious GVH complications associated with allo-SCT. Using a reverse immunology approach, we aim to develop a method enabling the identification of T-cell responses directed against predefined antigens, with the goal to select those MiHAs that can be used clinically in combination with allo-SCT.Experimental Design: In this study, we used a recently developed MiHA selection algorithm to select candidate MiHAs within the HLA-presented ligandome of transformed B cells. From the HLA-presented ligandome that predominantly consisted of monomorphic peptides, 25 polymorphic peptides with a clinically relevant allele frequency were selected. By high-throughput screening, the availability of high-avidity T cells specific for these MiHA candidates in different healthy donors was analyzed.Results: With the use of MHC multimer enrichment, analyses of expanded T cells by combinatorial coding MHC multimer flow cytometry, and subsequent single-cell cloning, positive T-cell clones directed to two new MiHA: LB-CLYBL-1Y and LB-TEP1-1S could be demonstrated, indicating the immunogenicity of these two MiHAs.Conclusions: The biologic relevance of MiHA LB-CLYBL-1Y was demonstrated by the detection of LB-CLYBL-1Y–specific T cells in a patient suffering from acute myeloid leukemia (AML) that experienced an anti-leukemic response after treatment with allo-SCT. Clin Cancer Res; 21(9); 2177–86. ©2015 AACR.

Supplementary Table 1. Eluted published MiHA epitopes

Supplementary Figure 1. The HLA-binding affinity of 25 eluted highly potential MiHA candidates was analyzed by a binding assay that is based on HLA-recovery after UV-exchange monomer technology.

Eluforsen (previously known as QR-010) is a 33-mer 2′-O-methyl modified phosphorothioate antisense oligonucleotide targeting the F508del mutation in the gene encoding CFTR protein of cystic fibrosis patients. In this study, eluforsen was incubated with endo- and exonucleases and mouse liver homogenates to elucidate its in vitro metabolism. Mice and monkeys were used to determine in vivo liver and lung metabolism of eluforsen following inhalation. We developed a liquid chromatography-mass spectrometry method for the identification and semi-quantitation of the metabolites of eluforsen and then applied the method for in vitro and in vivo metabolism studies. Solid-phase extraction was used following proteinase K digestion for sample preparation. Chain-shortened metabolites of eluforsen by 3′ exonuclease were observed in mouse liver in an in vitro incubation system and by either 3′ exonuclease or 5′ exonuclease in liver and lung samples from an in vivo mouse and monkey study. This study provides approaches for further metabolite characterization of 2′-ribose-modified phosphorothioate oligonucleotides in in vitro and in vivo studies to support the development of oligonucleotide therapeutics. Keywords: eluforsen, antisense oligonucleotide, metabolism, cystic fibrosis, ion-pair, liquid chromatography, mass spectrometry

Immunotherapy of B-cell malignancies using CD19-targeted chimeric antigen receptor-transduced T cells or CD20-targeted therapeutic monoclonal antibodies has shown clinical efficacy. However, refractory disease and the emergence of antigen-loss tumor escape variants after treatment demonstrate the need to target additional antigens. Here we aimed to target the B-cell receptor-associated protein CD79b by a T-cell receptor (TCR)-based approach. Because thymic selection depletes high-avidity T cells recognizing CD79b-derived peptides presented in self-HLA molecules, we aimed to isolate T cells recognizing these peptides presented in allogeneic HLA. Peptide-HLA tetramers composed of CD79b peptides bound to either HLA-A2 or HLA-B7 were used to isolate T-cell clones from HLA-A*0201 and B*0702-negative individuals. For 3 distinct T-cell clones, CD79b specificity was confirmed through CD79b gene transduction and CD79b-specific shRNA knockdown. The CD79b-specific T-cell clones were highly reactive against CD79b-expressing primary B-cell malignancies, whereas no recognition of nonhematopoietic cells was observed. Although lacking CD79b-cell surface expression, intermediate reactivity toward monocytes, hematopoietic progenitor cells, and T-cells was observed. Quantitative reverse transcriptase polymerase chain reaction revealed low CD79b gene expression in these cell types. Therefore, aberrant gene expression must be taken into consideration when selecting common, apparently lineage-specific self-antigens as targets for TCR-based immunotherapies.

Multiple fractionation or separation methods are often combined in proteomics to improve signal-to-noise and proteome coverage and to reduce interference between peptides in quantitative proteomics. Furthermore, a given fractionation method provides additional information on the analytes, such as molecular weight, hydrophobicity or isoelectric point that can be used to improve identification, and to discover protein splice variants or large post-translational modifications. Here we describe a Taverna scientific workflow for analysis and comparison between strong cation exchange (SCX) chromatography, peptide isoelectric focusing (pIEF) and SDS-PAGE performed using robust capillary LC and ion trap tandem mass spectrometry.

Development of the brain involves the formation and maturation of numerous synapses. This process requires prominent changes of the synaptic proteome and potentially involves thousands of different proteins at every synapse. To date the proteome analysis of synapse development has been studied sparsely. Here, we analyzed the cortical synaptic membrane proteome of juvenile postnatal days 9 (P9), P15, P21, P27, adolescent (P35) and different adult ages P70, P140 and P280 of C57Bl6/J mice. Using a quantitative proteomics workflow we quantified 1560 proteins of which 696 showed statistically significant differences over time. Synaptic proteins generally showed increased levels during maturation, whereas proteins involved in protein synthesis generally decreased in abundance. In several cases, proteins from a single functional molecular entity, e.g., subunits of the NMDA receptor, showed differences in their temporal regulation, which may reflect specific synaptic development features of connectivity, strength and plasticity. SNARE proteins, Snap 29/47 and Stx 7/8/12, showed higher expression in immature animals. Finally, we evaluated the function of Cxadr that showed high expression levels at P9 and a fast decline in expression during neuronal development. Knock down of the expression of Cxadr in cultured primary mouse neurons revealed a significant decrease in synapse density.

Purpose: T-cell recognition of minor histocompatibility antigens (MiHA) not only plays an important role in the beneficial graft-versus-leukemia (GVL) effect of allogeneic stem cell transplantation (allo-SCT) but also mediates serious GVH complications associated with allo-SCT. Using a reverse immunology approach, we aim to develop a method enabling the identification of T-cell responses directed against predefined antigens, with the goal to select those MiHAs that can be used clinically in combination with allo-SCT. Experimental Design: In this study, we used a recently developed MiHA selection algorithm to select candidate MiHAs within the HLA-presented ligandome of transformed B cells. From the HLA-presented ligandome that predominantly consisted of monomorphic peptides, 25 polymorphic peptides with a clinically relevant allele frequency were selected. By high-throughput screening, the availability of high-avidity T cells specific for these MiHA candidates in different healthy donors was analyzed. Results: With the use of MHC multimer enrichment, analyses of expanded T cells by combinatorial coding MHC multimer flow cytometry, and subsequent single-cell cloning, positive T-cell clones directed to two new MiHA: LB-CLYBL-1Y and LB-TEP1-1S could be demonstrated, indicating the immunogenicity of these two MiHAs. Conclusions: The biologic relevance of MiHA LB-CLYBL-1Y was demonstrated by the detection of LB-CLYBL-1Y–specific T cells in a patient suffering from acute myeloid leukemia (AML) that experienced an anti-leukemic response after treatment with allo-SCT. Clin Cancer Res; 21(9); 2177–86. ©2015 AACR.

Human leukocyte antigen (HLA) class I molecules generally present peptides (p) of 8 to 11 amino acids (aa) in length. Although an increasing number of examples with lengthy (>11 aa) peptides, presented mostly by HLA-B alleles, have been reported. Here we characterize HLA-A*02:01 restricted, in addition to the HLA-B*0702 and HLA-B*4402 restricted, lengthy peptides (>11 aa) arising from the B-cell ligandome. We analyzed a number of 15-mer peptides presented by HLA-A*02:01, and confirmed pHLA-I formation by HLA folding and thermal stability assays. Surprisingly the binding affinity and stability of the 15-mer epitopes in complex with HLA-A*02:01 were comparable with the values observed for canonical length (8 to 11 aa) HLA-A*02:01-restricted peptides. We solved the structures of two 15-mer epitopes in complex with HLA-A*02:01, within which the peptides adopted distinct super-bulged conformations. Moreover, we demonstrate that T-cells can recognize the 15-mer peptides in the context of HLA-A*02:01, indicating that these 15-mer peptides represent immunogenic ligands. Collectively, our data expand our understanding of longer epitopes in the context of HLA-I, highlighting that they are not limited to the HLA-B family, but can bind the ubiquitous HLA-A*02:01 molecule, and play an important role in T-cell immunity.

Indian and Chinese rhesus macaques are often used in biomedical research. Genetic analyses of the major histocompatibility class I region have revealed that these macaques display a substantial level of polymorphism at Mamu-A and Mamu-B loci, which have been subject to duplication. Only a few Mamu class I allotypes are characterised for their peptide-binding motifs, although more information of this nature would contribute to a better interpretation of T cell-mediated immune responses. Here, we present the results of the characterisation of the functional properties of Mamu-B*037:01, an allotype commonly encountered in rhesus macaques of Indian and Chinese origin. Mamu-B*037:01 is seen to have a strong preference for acidic amino acids at the third residue, and for arginine, lysine, and tyrosine at the carboxyl terminus. This peptide-binding motif is not described in the human population.

T cell recognition of minor histocompatibility Ags (MiHA) plays an important role in the graft-versus-tumor effect of allogeneic stem cell transplantation. Selective infusion of T cells reactive for hematopoiesis-restricted MiHA presented in the context of HLA class I or II molecules may help to separate the graft-versus-tumor effects from graft-versus-host disease effects after allogeneic stem cell transplantation. Over the years, increasing numbers of MiHA have been identified by forward immunology approaches, and the relevance of these MiHA has been illustrated by correlation with clinical outcome. As the tissue distribution of MiHA affects the clinical outcome of T cell responses against these Ags, it would be beneficial to identify additional predefined MiHA that are exclusively expressed on hematopoietic cells. Therefore, several reverse immunology approaches have been explored for the prediction of MiHA. Thus far, these approaches frequently resulted in the identification of T cells directed against epitopes that are not naturally processed and presented. In this study we established a method for the identification of biologically relevant MiHA, implementing mass spectrometry–based HLA-peptidomics into a reverse immunology approach. For this purpose, HLA class I binding peptides were eluted from transformed B cells, analyzed by mass spectrometry, and matched with a database dedicated to identifying polymorphic peptides. This process resulted in a set of 40 MiHA candidates that were evaluated in multiple selection steps. The identification of LB-NISCH-1A demonstrated the technical feasibility of our approach. On the basis of these results, we present an approach that can be of value for the efficient identification of MiHA or other T cell epitopes.

Peptides presented by human leukocyte antigen (HLA) molecules on the cell surface play a crucial role in adaptive immunology, mediating the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies on T cell action, and in cellular immunotherapy and transplantation. In this study we present the in-depth identification and relative quantification of 14,500 peptide ligands constituting the HLA-ligandome of B-cells. This large number of identified ligands provides a general insight in the presented peptide repertoire and antigen presentation. Our uniquely large set of HLA-ligands allowed us to characterize in detail the peptides constituting the ligandome in terms of relative abundance, peptide length distribution, physicochemical properties, binding affinity to the HLA molecule and presence of post-translational modifications. The presented B-lymphocyte ligandome is shown to be a rich source of information by the presence of minor histocompatibility antigens, virus-derived epitopes and post-translationally modified HLA ligands and can be a good starting point to solve a wealth of specific immunological questions. These HLA ligands can form the basis for reversed immunology approaches to identify T cell epitopes, not based on in silico predictions, but based on the bona fide eluted HLA-ligandome.

The non-classical HLA-E is a conserved class I molecule that mainly presents monomorphic leader peptides derived from other HLA class I molecules. These leader peptides comprise an optimized sequence for tight and deep binding into the HLA-E groove. In a TAP-deficient environment, as it can be generated during viral infection or in tumor tissue, loading of the classical leader peptide sequences is hampered leading to an alternative HLA-E peptide repertoire. In this study, we characterized this alternative peptide repertoire using cells in which TAP activity is inhibited. We identified more than 500 unique peptide sequences carried by HLA-E and found that their binding motif is different from the dominant leader peptides. Hydrophobic amino acids were only found at positions 2 and 9, in close resemblance to the peptide binding motif of HLA-A*0201. HLA-E-eluted peptides were indeed able to bind this classical HLA class I molecule. Our findings suggest that the dominant leader peptides uniquely conform to HLA-E, but that in their absence a peptide pool is presented like that of HLA-A*0201.

Therapeutic reactivity of CD20-specific monoclonal antibodies (mAb) or CD19-specific chimeric antigen receptor (CAR)-transduced T cells is exerted by targeting extracellular antigens. In contrast to mAbs and CARs, T cell receptors (TCRs) recognize antigen-derived peptides that are bound to human leukocyte antigen (HLA) molecules on the cell surface. Since HLA molecules constantly sample the entire endogenous proteome of a cell, extracellular and intracellular antigens are presented and can thus be recognized by a TCR. Here, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for immunotherapy. Bob1 is highly expressed in CD19+ B cells, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM) and is absent in the non-B lineages including CD34+ hematopoietic progenitor cells (HPCs), T cells, fibroblasts, keratinocytes and gastrointestinal tract. Bob1 is localized intracellularly but HLA-presented Bob1-derived peptides are accessible on the cell surface to TCRs and can thus be recognized by T cells. From the HLA-presented ligandome (Mol Cell Proteomics, 2013;12:1829) we identified naturally processed Bob1-derived peptides displayed in HLA-A*0201 (HLA-A2) and in HLA-B*0702 (HLA-B7). Since auto-reactivity towards self-antigens such as Bob1 is prevented by depleting high-avidity T cells recognizing self-antigens in self-HLA, we exploited the immunogenicity of these peptides presented in allogeneic HLA. From a HLA-A2/B7-negative healthy individual we isolated T cell clone 4G11 demonstrating high sensitivity and specificity for Bob1-derived peptide Bob144 presented in HLA-B7. Bob1-dependent recognition was demonstrated by transduction of Bob1 into cell lines that otherwise lack Bob1 expression. No harmful toxicities of clone 4G11 were observed against a wide panel of Bob1-negative stimulator cells including HLA-B7-positive CD34+ HPCs, T cells, monocytes, immature and mature dendritic cells, and fibroblasts even under simulated inflamed conditions. Furthermore, stringent HLA-B7-restricted recognition was observed for clone 4G11 when tested against a stimulator panel expressing a wide range of common and rare HLA class I and II molecules. Clone 4G11 demonstrated clinical applicability by efficiently recognizing HLA-B7+ primary ALL, CLL and MCL. Furthermore, reproducible strong recognition of purified primary HLA-B7+ MM could be demonstrated. Therefore, the TCR of clone 4G11 may be used for immunotherapy by administering TCR-transduced T cells to patients suffering from B cell malignancies including multiple myeloma. Retroviral gene transfer of TCR 4G11 led to efficient cell surface expression demonstrated by binding of TCR-transduced CD8+ T cells to pMHC-tetramer composed of peptide Bob144 bound to HLA-B7. TCR-modified CD8+ T cells strongly recognized Bob1-expressing HLA-B7+ multiple myeloma cell lines U266 and UM9, and ALL cell lines. TCR-modified T cells efficiently lysed HLA-B7+ primary ALL, CLL and MCL at very low effector-to-target ratios. In addition, highly purified primary multiple myeloma samples were also readily lysed. Furthermore, TCR-transduced T cells strongly proliferated in an antigen-specific manner when stimulated with primary malignant cell samples including ALL, CLL, and MCL or MM cell lines. As expected, TCR-transduced T cells also lysed autologous primary and CD40L-stimulated B cells since these targets cells also express Bob1. In contrast, no lysis of Bob1-negative autologous primary and activated T cells, or monocytes was observed when co-cultured with TCR-transduced T cells. In summary, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for TCR-based immunotherapies of B cell malignancies. Bob1-specific T cell clone 4G11 efficiently recognized primary B cell leukemia and multiple myeloma. Gene transfer of TCR of clone 4G11 installed Bob1-reactivity and specificity onto recipient T cells shown here by cytolytic capacity and proliferation upon antigen encounter. TCR gene transfer approaches using this Bob1-specific TCR can bring novel treatment modalities and possibly curative therapy to patients with B cell malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

T cell epitopes derived from polymorphic proteins or from proteins encoded by alternative reading frames (ARFs) play an important role in (tumor) immunology. Identification of these peptides is successfully performed with mass spectrometry. In a mass spectrometry-based approach, the recorded tandem mass spectra are matched against hypothetical spectra generated from known protein sequence databases. Commonly used protein databases contain a minimal level of redundancy, and thus, are not suitable data sources for searching polymorphic T cell epitopes, either in normal or ARFs. At the same time, however, these databases contain much non-polymorphic sequence information, thereby complicating the matching of recorded and theoretical spectra, and increasing the potential for finding false positives. Therefore, we created a database with peptides from ARFs and peptide variation arising from single nucleotide polymorphisms (SNPs). It is based on the human mRNA sequences from the well-annotated reference sequence (RefSeq) database and associated variation information derived from the Single Nucleotide Polymorphism Database (dbSNP). In this process, we removed all non-polymorphic information. Investigation of the frequency of SNPs in the dbSNP revealed that many SNPs are non-polymorphic “SNPs”. Therefore, we removed those from our dedicated database, and this resulted in a comprehensive high quality database, which we coined the Human Short Peptide Variation Database (HSPVdb). The value of our HSPVdb is shown by identification of the majority of published polymorphic SNP- and/or ARF-derived epitopes from a mass spectrometry-based proteomics workflow, and by a large variety of polymorphic peptides identified as potential T cell epitopes in the HLA-ligandome presented by the Epstein–Barr virus cells. Electronic supplementary material The online version of this article (doi:10.1007/s00251-010-0497-1) contains supplementary material, which is available to authorized users.

Therapeutic monoclonal antibodies (mAb) such as Rituximab and Ofatumumab have demonstrated the clinical efficacy of targeting the B-cell restricted antigen CD20 for the treatment of B-cell lymphomas and leukemia. Although CD20 is also expressed on healthy B-cell cells which are depleted in the course of therapy, long-term B-cell aplasia is well manageable. However, non-responsive or refractory disease to CD20-targeted mAb treatment has been reported with various mechanisms of resistance: downregulation of CD20 expression, internalization of CD20:mAb complex, inhibition of complement-dependent cytotoxicity and absence of an effector cell repertoire in patients treated with chemotherapy prior to mAb infusion. Therefore, additional therapeutic strategies are required. T-cell receptor (TCR) gene transfer is an attractive strategy to equip T-cells with TCRs of defined antigen-specificity. Due to their high sensitivity for cognate antigen presented in HLA, TCRs can induce T-cell activation even when antigen expression is very low. However, the broad application of TCR-based adoptive immunotherapy directed against self-antigens such as CD20 is hampered by lack of an effective immune response against self-antigens. T-cells carrying high-affinity TCRs reactive to such self-antigens are deleted by negative selection during thymic development to prevent auto-reactivity. An attractive strategy to target self-antigens is to exploiting the immunogenicity of such antigens presented in the context of allogeneic HLA (alloHLA). Here, we used the CD20-derived peptide SLFLGILSV (CD20SLF) binding in HLA-A2 to isolate CD20-reactive T-cells carrying high-affinity TCRs. From peripheral blood mononuclear cells of HLA-A*0201 (HLA-A2)-negative healthy individuals CD8+ T-cells binding to peptide-HLA tetramers composed of CD20SLF bound to HLA-A2 were isolated and clonally expanded. Two high-avidity T-cell clones were identified specific for HLA-A2-bound CD20SLF. CD20-dependent recognition was demonstrated for both clones by transducing the CD20 gene in HLA-A2-positive cell lines which otherwise lack CD20 expression. Both CD20-specific T-cell clones efficiently recognized CD20-expressing HLA-A2-positive primary B-cell malignancies including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In addition, the CD20-specific T-cell clones were able to more efficiently recognize ALL cell-lines than CD20-specific mAbs. We demonstrated that on target cells with only very low CD20 surface expression, the CD20-specific T-cell clones could still efficiently recognize endogenously processed CD20-derived peptide in the context of HLA-A2. Furthermore, no recognition of HLA-A2-positive but CD20-negative cell subsets including CD34+hematopoietic progenitor cells, T-cells, immature and mature dendritic cells could be demonstrated. Additionally, recognition of HLA-A2-positive non-hematopoietic cells such as fibroblasts even under simulated inflamed conditions was absent. Transduction of the identified TCRs resulted in efficient expression of the introduced CD20-specific TCRs and conferred CD20-specificity onto recipient cells. In summary, we exploited the immunogenicity of alloHLA to raise high-avidity T-cells against self-antigens such as CD20. The identified CD20-specific T-cell clones efficiently recognized CD20-expressing primary ALL, CLL and MCL. These T-cells clones more efficiently recognized B-cell malignancies than CD20-targeted mAbs while no recognition of CD20-negative hematopoietic and non-hematopoietic cells was observed. Transduction of these CD20-specific TCRs conferred CD20-specificity onto recipient cells. These CD20-specific TCRs can be useful to treat patients with CD20low B-cell malignancies by administering TCR-engineered T cells with potent effector function. Disclosures No relevant conflicts of interest to declare.

Therapeutic reactivity of CD20-specific monoclonal antibodies (mAb) or CD19-specific chimeric antigen receptor (CAR)-transduced T cells is exerted by targeting extracellular antigens. However, loss of CD20 and CD19 expression or absence of these molecules on other malignancies such as multiple myeloma restricts their application. Here, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for immunotherapy. Bob1 is highly expressed in CD19+ B cells, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM) and is absent in the non-B lineages including CD34+ hematopoietic progenitor cells (HPCs), T cells, fibroblasts, keratinocytes and gastrointestinal tract. Bob1 is localized intracellularly but HLA-presented Bob1-derived peptides are accessible on the cell surface to T cell receptors (TCRs) and can thus be recognized by T cells. From the HLA-presented ligandome (Mol Cell Proteomics, 2013;12:1829) we identified naturally processed Bob1-derived peptides displayed in HLA-A*0201 (HLA-A2) and in HLA-B*0702 (HLA-B7). Since auto-reactivity towards self-antigens such as Bob1 is prevented by depleting high-avidity T cells recognizing self-antigens in self-HLA, we exploited the immunogenicity of these peptides presented in allogeneic HLA. From a total of 3 x 109 peripheral blood mononuclear cells from 6 different HLA-A2/B7-negative healthy donors, we isolated and clonally expanded more than 1000 CD8+ T cells binding to peptide-MHC-tetramers composed of the Bob1-derived peptides bound to HLA-A2 or HLA-B7. The T cell clones were tested for stringent peptide-specificity by stimulation with Bob1-negative K562 cells expressing either HLA-A2 or B7 unloaded or pulsed with Bob1-derived peptides. This resulted in the selection of 15 T cell clones highly specific for Bob1. To identify the T cell clones of highest avidity, T cell clones were compared for peptide-sensitivity by testing the recognition of stimulator cells loaded with titrated amounts of Bob1-derived peptides and of Bob1-expressing HLA-A2/B7-positive EBV-transformed B cells. T cell clone 4G11 was selected because of high sensitivity and specificity for Bob1-derived peptide Bob144 presented in HLA-B7 and T cell clone 3C10 specifically recognized peptide Bob1245 bound to HLA-A2. Bob1-dependent recognition was demonstrated by transduction of Bob1 into cell lines that otherwise lack Bob1 expression. To investigate whether harmful toxicities could be caused by these T cell clones, we tested their reactivity against a wide panel of Bob1-negative stimulator cells demonstrating absence of recognition of HLA-B7-positive CD34+ HPCs, T cells, monocytes, immature and mature dendritic cells, and fibroblasts even under simulated inflamed conditions. Stringent HLA-B7-restricted recognition was observed for clone 4G11 when tested against a stimulator panel expressing a wide range of common and rare HLA class I and II molecules. These data illustrate a safe reactivity profile with little chance of off-target toxicity. To test their clinical applicability, clone 4G11 and 3C10 were tested for recognition of various primary B cell malignancies. Clone 4G11 efficiently recognized HLA-B7-positive primary ALL, CLL and mantle cell lymphoma while clone 3C10 recognized HLA-A2-positive primary B cell malignancies albeit to a lesser degree. Furthermore, reproducible strong recognition of purified primary HLA-B7-positive multiple myeloma could be demonstrated for clone 4G11. Therefore, T cell clone 4G11’s TCR may be used for immunotherapy by administering TCR-transduced T cells to multiple myeloma patients. To test whether introduction of 4G11’s TCR confers Bob1-reactivity onto recipient cells, the TCR was cloned into a retroviral vector. Highly specific reactivity against HLA-B7-positive Bob1-expressing target cells could be installed to TCR-transduced recipient T cells. In summary, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for TCR-based immunotherapies of B cell malignancies and multiple myeloma. Bob1-specific T cell clone 4G11 efficiently recognized primary B cell leukemia and multiple myeloma. TCR gene transfer approaches using Bob1-specific TCRs can bring novel treatment modalities for patients with B cell malignancies or multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Meeting abstracts Studies using T-cell receptor (TCR) or chimeric antigen receptor (CAR) transduced T-cells have shown the effectiveness of adoptive immunotherapy to treat different malignancies. The efficacy and safety of such interventions greatly depends on good target selection to prevent on

Abstract 4038 T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, MiHA recognition is also associated with graft-versus-host disease (GVHD). It is assumed that the selective infusion of T-cells reactive with hematopoiesis-restricted MiHA may help to separate the GVT and GVHD effects of allo-SCT. However, the number of attractive MiHA identified to date remains limited. In this study we aimed to determine whether it is feasible to identify MiHA using HLA-peptidomics in a reverse-immunology approach, based on bona fide eluted MiHA epitopes. Successful development of such a technology could allow the rapid identification of new MiHA, required to make antigen-selective adoptive T-cell therapy a realistic option. In addition, when compared to classical forward approaches, this strategy may provide tools to efficiently identify favorable GVT-involved MiHA, rather than random identifying targets of activated T-cells isolated during a GVT-response. To identify biological relevant MiHA candidates, HLA class I peptides were isolated from lysed EBV-transformed B-cells (EBV-LCL), analyzed by mass spectrometry (MS) and matched with a human protein database (IPI). This effort resulted in a set of fifteen thousand peptides, encoded in the normal reading frame with high probability MS scores. To identify potential MiHA candidates, the total set was matched with our newly developed public available Human Short Peptide Variation Database (http://srs.bioinformatics.nl/hspv), dedicated to polymorphic peptides. The quality of this peptide set was demonstrated by a detection efficiency of fifty percent of known MiHA including various length variants and eluted MiHA counterparts. Subsequently the combined use of gene expression databases, validated single nucleotide polymorphism (SNP) arrays and HLA-peptide binding assays resulted in a further selection of 27 high potential HLA-A*0201 and B*0701 MiHA candidates. This set was used for the generation of pMHC tetramers by UV-mediated exchange technology. Next, pMHC tetramer positive specific T-cell lines were generated from eighteen healthy SNP-typed PBMC donors following MACS isolation. To decrease the incidence of isolating low affinity T-cells, due to self-tolerance induction, pMHC tetramer isolations were only performed using donors homozygous negative for the specific SNP. After repeated pMHC tetramer pull down, in vitro expanded cell samples were analyzed on a multi-color FACS LSRII flow cytometer and clonally expanded following FACS cell sorting. Using this approach we were able to detect 16 unique pMHC tetramer positive T-cell populations corresponding with 70% of eluted MiHA candidates. Most of these pMHC tetramer positive T-cell populations were detected in multiple individuals, and appeared to be oligoclonal. Although most T-cell clones produced IFN-γ when co-cultured with peptide-pulsed target cells, there appeared to be a wide variety of peptide affinity among the pMHC tetramer positive T-cell clones. High throughput screening of all clones for MiHA specific recognition patterns of SNP-typed EBV-LCL panels revealed a clear correlation between the peptide-affinity of the T-cell clone and its capacity to recognize endogenously processed and presented peptide. Collectively these efforts resulted in the validation of two previously described MiHA and the identification of three new biological relevant MiHA. In summary, this study resulted in the establishment of an algorithm for the high-throughput identification of MiHA based on the combined use of HLA-peptidomics and reverse-immunology by pMHC tetramers. Our data indicate that the technology developed within this project can be of great value to the efficient identification of novel MiHA with potential clinical value especially when epitope selection criteria are supplemented with gene expression data, allowing pre-selection for those MiHA candidates with a hematopoiesis restricted gene expression patterns that may direct reactivity towards GVT. Disclosures: No relevant conflicts of interest to declare.