13 results on '"Sylvie Pollmann"'
Search Results
2. Targeting KRAS mutations with HLA class II-restricted TCRs for the treatment of solid tumors
- Author
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Pierre Dillard, Nicholas Casey, Sylvie Pollmann, Patrik Vernhoff, Gustav Gaudernack, Gunnar Kvalheim, Sébastien Wälchli, and Else Marit Inderberg
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immunotherapy ,adoptive cell therapy ,tcr ,kras ,Immunologic diseases. Allergy ,RC581-607 ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
T-cell receptor (TCR) redirected T cells are considered as the next generation of care for the treatment of numerous solid tumors. KRAS mutations are driver neoantigens that are expressed in over 25% of all cancers and are thus regarded as ideal targets for Adoptive Cell Therapy (ACT). We have isolated four KRAS-specific TCRs from a long-term surviving pancreatic cancer patient vaccinated with a mix of mutated KRAS peptides. The sequence of these TCRs could be identified and expressed in primary cells. We demonstrated stable expression of all TCRs as well as target-specific functionality when expressing T cells were co-incubated with target cells presenting KRAS peptides. In addition, these TCRs were all partially co-receptor independent since they were functional in both CD4 and CD8 T cells, thus indicating high affinity. Interestingly, we observed that certain TCRs were able to recognize several KRAS mutations in complex with their cognate Human leukocyte antigen (HLA), suggesting that, here, the point mutations were less important for the HLA binding and TCR recognition, whereas others were single-mutation restricted. Finally, we demonstrated that these peptides were indeed processed and presented, since HLA-matched antigen presenting cells exogenously loaded with KRAS proteins were recognized by TCR-transduced T cells. Taken together, our data demonstrate that KRAS mutations are immunogenic for CD4 T cells and are interesting targets for TCR-based cancer immunotherapy.
- Published
- 2021
- Full Text
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3. Targeting Telomerase with an HLA Class II-Restricted TCR for Cancer Immunotherapy
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Anna K. Winge-Main, Gunnar Kvalheim, Gunhild Mari Mælandsmo, Solrun Melkorka Maggadottir, Sébastien Wälchli, Marit Renée Myhre, Gustav Gaudernack, Pierre Dillard, Vivi Ann Flørenes, Sylvie Pollmann, Mathilde Menard, Else Marit Inderberg, and Hakan Köksal
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Telomerase ,medicine.medical_treatment ,Population ,Receptors, Antigen, T-Cell ,Apoptosis ,Mice, SCID ,CD8-Positive T-Lymphocytes ,Biology ,Cell therapy ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Cancer immunotherapy ,Mice, Inbred NOD ,Drug Discovery ,Tumor Cells, Cultured ,Genetics ,medicine ,Animals ,Humans ,Telomerase reverse transcriptase ,education ,Melanoma ,Molecular Biology ,Cell Proliferation ,030304 developmental biology ,Pharmacology ,0303 health sciences ,education.field_of_study ,T-cell receptor ,Histocompatibility Antigens Class II ,Immunotherapy ,Xenograft Model Antitumor Assays ,030220 oncology & carcinogenesis ,Cancer research ,Molecular Medicine ,CD8 ,T-Lymphocytes, Cytotoxic - Abstract
T cell receptor (TCR)-engineered T cell therapy is a promising cancer treatment approach. Human telomerase reverse transcriptase (hTERT) is overexpressed in the majority of tumors and a potential target for adoptive cell therapy. We isolated a novel hTERT-specific TCR sequence, named Radium-4, from a clinically responding pancreatic cancer patient vaccinated with a long hTERT peptide. Radium-4 TCR-redirected primary CD4+ and CD8+ T cells demonstrated in vitro efficacy, producing inflammatory cytokines and killing hTERT+ melanoma cells in both 2D and 3D settings, as well as malignant, patient-derived ascites cells. Importantly, T cells expressing Radium-4 TCR displayed no toxicity against bone marrow stem cells or mature hematopoietic cells. Notably, Radium-4 TCR+ T cells also significantly reduced tumor growth and improved survival in a xenograft mouse model. Since hTERT is a universal cancer antigen, and the very frequently expressed HLA class II molecules presenting the hTERT peptide to this TCR provide a very high (>75%) population coverage, this TCR represents an attractive candidate for immunotherapy of solid tumors.
- Published
- 2021
4. Targeting KRAS mutations with HLA class II-restricted TCRs for the treatment of solid tumors
- Author
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Else Marit Inderberg, Pierre Dillard, Nicholas Casey, Patrik Vernhoff, Sébastien Wälchli, Gunnar Kvalheim, Sylvie Pollmann, and Gustav Gaudernack
- Subjects
0301 basic medicine ,medicine.medical_treatment ,Immunology ,Receptors, Antigen, T-Cell ,chemical and pharmacologic phenomena ,Human leukocyte antigen ,Biology ,medicine.disease_cause ,Cell therapy ,Proto-Oncogene Proteins p21(ras) ,03 medical and health sciences ,0302 clinical medicine ,Cancer immunotherapy ,Antigens, Neoplasm ,HLA Antigens ,medicine ,KRAS ,Immunology and Allergy ,Cytotoxic T cell ,Humans ,Antigen-presenting cell ,RC254-282 ,Original Research ,Adoptive Cell Therapy ,T-cell receptor ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,hemic and immune systems ,Immunotherapy ,RC581-607 ,Pancreatic Neoplasms ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,Mutation ,Cancer research ,Immunologic diseases. Allergy ,TCR ,Research Article - Abstract
T-cell receptor (TCR) redirected T cells are considered as the next generation of care for the treatment of numerous solid tumors. KRAS mutations are driver neoantigens that are expressed in over 25% of all cancers and are thus regarded as ideal targets for Adoptive Cell Therapy (ACT). We have isolated four KRAS-specific TCRs from a long-term surviving pancreatic cancer patient vaccinated with a mix of mutated KRAS peptides. The sequence of these TCRs could be identified and expressed in primary cells. We demonstrated stable expression of all TCRs as well as target-specific functionality when expressing T cells were co-incubated with target cells presenting KRAS peptides. In addition, these TCRs were all partially co-receptor independent since they were functional in both CD4 and CD8 T cells, thus indicating high affinity. Interestingly, we observed that certain TCRs were able to recognize several KRAS mutations in complex with their cognate Human leukocyte antigen (HLA), suggesting that, here, the point mutations were less important for the HLA binding and TCR recognition, whereas others were single-mutation restricted. Finally, we demonstrated that these peptides were indeed processed and presented, since HLA-matched antigen presenting cells exogenously loaded with KRAS proteins were recognized by TCR-transduced T cells. Taken together, our data demonstrate that KRAS mutations are immunogenic for CD4 T cells and are interesting targets for TCR-based cancer immunotherapy.
- Published
- 2021
5. Correction to: Preclinical assessment of transiently TCR redirected T cells for solid tumour immunotherapy
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Gunnar Kvalheim, Sylvie Pollmann, Else Marit Inderberg, Gustav Gaudernack, Sébastien Wälchli, Nadia Mensali, Pierre Dillard, and Marit Renée Myhre
- Subjects
Cytotoxicity, Immunologic ,Oncology ,Cancer Research ,medicine.medical_specialty ,T-Lymphocytes ,medicine.medical_treatment ,Immunology ,T-Cell Antigen Receptor Specificity ,Mistake ,Mice, SCID ,Cross Reactions ,Cancer Vaccines ,Immunotherapy, Adoptive ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Text mining ,Internal medicine ,medicine ,Animals ,Humans ,Immunology and Allergy ,RNA, Messenger ,Solid tumour ,Receptors, Chimeric Antigen ,business.industry ,T-cell receptor ,Correction ,Neoplasms, Experimental ,Immunotherapy ,HCT116 Cells ,Xenograft Model Antitumor Assays ,Electroporation ,Colorectal Neoplasms ,business ,030215 immunology - Abstract
Off-target toxicity due to the expression of target antigens in normal tissue or TCR cross-reactivity represents a major risk when using T cell receptor (TCR)-engineered T cells for treatment of solid tumours. Due to the inherent cross-reactivity of TCRs it is difficult to accurately predict their target recognition pre-clinically. It has become evident that direct testing in a human being represents the best evaluation of the risks. There is, therefore, a clear unmet need for assessing the safety of a therapeutic TCR in a more controllable manner than by the injection of permanently modified cellular products. Using transiently modified T cells combined with dose escalation has already been shown feasible for chimeric antigen receptor (CAR)-engineered T cells, but nothing is yet reported for TCR. We performed a preclinical evaluation of a therapeutic TCR transiently expressed in T cells by mRNA electroporation. We analyzed if the construct was active in vitro, how long it was detectable for and if this expression format was adapted to in vivo efficacy assessment. Our data demonstrate the potential of mRNA engineered T cells, although less powerful than permanent redirection, to induce a significant response. Thus, these findings support the development of mRNA based TCR-therapy strategies as a feasible and efficacious method for evaluating TCR safety and efficacy in first-in-man testing.
- Published
- 2019
6. Preclinical assessment of transiently TCR redirected T cells for solid tumour immunotherapy
- Author
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Marit Renée Myhre, Gustav Gaudernack, Pierre Dillard, Else Marit Inderberg, Sébastien Wälchli, Nadia Mensali, Sylvie Pollmann, and Gunnar Kvalheim
- Subjects
Cancer Research ,medicine.medical_treatment ,mRNA ,Immunology ,Solid tumour ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,In vivo ,Immunology and Allergy ,Medicine ,In vivo model ,030304 developmental biology ,0303 health sciences ,Messenger RNA ,business.industry ,Electroporation ,T-cell receptor ,Immunotherapy ,Chimeric antigen receptor ,In vitro ,Oncology ,030220 oncology & carcinogenesis ,Cancer research ,Original Article ,T cell receptor ,business - Abstract
Off-target toxicity due to the expression of target antigens in normal tissue or TCR cross-reactivity represents a major risk when using T cell receptor (TCR)-engineered T cells for treatment of solid tumours. Due to the inherent cross-reactivity of TCRs it is difficult to accurately predict their target recognition pre-clinically. It has become evident that direct testing in a human being represents the best evaluation of the risks. There is, therefore, a clear unmet need for assessing the safety of a therapeutic TCR in a more controllable manner than by the injection of permanently modified cellular products. Using transiently modified T cells combined with dose escalation has already been shown feasible for chimeric antigen receptor (CAR)-engineered T cells, but nothing is yet reported for TCR. We performed a preclinical evaluation of a therapeutic TCR transiently expressed in T cells by mRNA electroporation. We analyzed if the construct was active in vitro, how long it was detectable for and if this expression format was adapted to in vivo efficacy assessment. Our data demonstrate the potential of mRNA engineered T cells, although less powerful than permanent redirection, to induce a significant response. Thus, these findings support the development of mRNA based TCR-therapy strategies as a feasible and efficacious method for evaluating TCR safety and efficacy in first-in-man testing. Electronic supplementary material The online version of this article (10.1007/s00262-019-02356-2) contains supplementary material, which is available to authorized users.
- Published
- 2019
7. Preclinical development of CD37CAR T-cell therapy for treatment of B-cell lymphoma
- Author
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June Helen Myklebust, Sébastien Wälchli, Are Kolstad, Yngvild Nuvin Blaker, Solrun Melkorka Maggadottir, Sarah Elisabet Josefsson, Kanutte Huse, Pierre Dillard, Gunnar Kvalheim, Hakan Köksal, Else Marit Inderberg, Anne Fåne, Harald Holte, Sylvie Pollmann, Erlend B. Smeland, and Klaus Beiske
- Subjects
0301 basic medicine ,Adoptive cell transfer ,Lymphoma, B-Cell ,Immunobiology and Immunotherapy ,Tetraspanins ,T cell ,Antigens, CD19 ,Mice, SCID ,Jurkat cells ,CD19 ,Jurkat Cells ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,Antigens, Neoplasm ,Mice, Inbred NOD ,immune system diseases ,Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,B-cell lymphoma ,Receptors, Chimeric Antigen ,biology ,business.industry ,Hematology ,medicine.disease ,Adoptive Transfer ,Xenograft Model Antitumor Assays ,Chimeric antigen receptor ,Lymphoma ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,biology.protein ,Cancer research ,K562 Cells ,business - Abstract
T cells modified to express chimeric antigen receptor (CAR) targeting CD19 (CD19CAR) have produced remarkable clinical responses in patients with relapsed/refractory B-cell acute lymphoblastic leukemia. CD19CAR T-cell therapy has also demonstrated prominent effects in B-cell non-Hodgkin lymphoma (B-NHL) patients. However, a subset of patients who relapse after CD19CAR T-cell therapy have outgrowth of CD19− tumor cells. Hence, development of alternative CARs targeting other B-cell markers represents an unmet medical need for B-cell acute lymphoblastic leukemia and B-NHL. Here, we confirmed previous data by showing that, overall, B-NHL has high expression of CD37. A second-generation CD37CAR was designed, and its efficacy in T cells was compared with that of CD19CAR. In vitro assessment of cytotoxicity and T-cell function upon coculture of the CAR T cells with different target B-cell lymphoma cell lines demonstrated comparable efficacy between the 2 CARs. In an aggressive B-cell lymphoma xenograft model, CD37CAR T cells were as potent as CD19CAR T cells in controlling tumor growth. In a second xenograft model, using U2932 lymphoma cells containing a CD19− subpopulation, CD37CAR T cells efficiently controlled tumor growth and prolonged survival, whereas CD19CAR T cells had limited effect. We further show that, unlike CD19CAR, CD37CAR was not sensitive to antigen masking. Finally, CD37CAR reactivity was restricted to B-lineage cells. Collectively, our results demonstrated that CD37CAR T cells also can effectively eradicate B-cell lymphoma tumors when CD19 antigen expression is lost and support further clinical testing for patients with relapsed/refractory B-NHL.
- Published
- 2019
8. Abstract 1422: Preclinical development of CD37CAR T-cell therapy for treatment of B-cell lymphoma
- Author
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Erlend B. Smeland, Harald Holte, Anne Fåne, Yngvild Nuvin Blaker, Sylvie Pollmann, June Helen Myklebust, Klaus Beiske, Sébastien Wälchli, Solrun Melkorka Maggadottir, Else Marit Inderberg, Ane Kolstad, Pierre Dillard, Kanutte Huse, Hakan Köksal, Gunnar Kvalheim, and Sarah Josefsson
- Subjects
Cancer Research ,biology ,business.industry ,T cell ,Cancer ,medicine.disease ,Chimeric antigen receptor ,CD19 ,Lymphoma ,medicine.anatomical_structure ,Oncology ,Antigen ,Cell culture ,hemic and lymphatic diseases ,medicine ,Cancer research ,biology.protein ,business ,B-cell lymphoma - Abstract
T cells modified to express chimeric antigen receptor (CAR) targeting CD19 have produced remarkable clinical responses in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). CD19CAR T-cell therapy has also demonstrated prominent effects in B-cell non-Hodgkin lymphoma (B-NHL) patients. However, a subset of patients who relapse after CD19CAR T-cell therapy have outgrowth of CD19-negative tumor cells. Hence, development of alternative CARs targeting other B-cell markers represents an unmet medical need for B-ALL and B-NHL. Here, we confirmed previous data by showing that B-NHL overall have high expression of CD37. A second generation CD37CAR was designed and its efficacy in T cells was compared to that of CD19CAR. In vitro assessment of cytotoxicity and T-cell function upon co-culture of the CAR T cells with different target B-cell lymphoma cell lines demonstrated comparable efficacy between the two CARs. In an aggressive B-cell lymphoma xenograft model, CD37CAR T cells were as potent as CD19CAR T cells in controlling tumor growth. In a second xenograft model, using U2932 lymphoma cells containing a CD19-negative subpopulation, CD37CAR T cells efficiently controlled tumors and cured the mice while CD19CAR T cells had limited effect. We further show that, unlike CD19CAR, CD37CAR was not sensitive to antigen masking. Finally, CD37CAR reactivity was restricted to B-lineage cells. Collectively, our results demonstrated that CD37CAR T cells effectively can eradicate B-cell lymphoma tumors also when CD19 antigen expression is lost, and support further clinical testing for patients with relapsed/refractory B-NHL. Citation Format: Pierre Dillard, Hakan Köksal, Sarah Josefsson, Solrun Melkorka Maggadottir, Sylvie Pollmann, Anne Fåne, Yngvild Nuvin Blaker, Klaus Beiske, Kanutte Huse, Ane Kolstad, Harald Holte, Gunnar Kvalheim, Erlend Bremertun Smeland, June Myklebust, Else Marit Inderberg, Sebastien Wälchli. Preclinical development of CD37CAR T-cell therapy for treatment of B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1422.
- Published
- 2019
9. Anti-PAD4 autoantibodies in rheumatoid arthritis: levels in serum over time and impact on PAD4 activity as measured with a small synthetic substrate
- Author
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Tore K Kvien, Ludvig M. Sollid, Burkhard Fleckenstein, Eirik Hornes Halvorsen, Sylvie Pollmann, Øyvind Molberg, and Maria Stensland
- Subjects
Hydrolases ,Immunology ,Arthritis ,Arginine ,Severity of Illness Index ,Catalysis ,Immunoglobulin G ,Substrate Specificity ,Arthritis, Rheumatoid ,chemistry.chemical_compound ,Protein-Arginine Deiminase Type 4 ,Rheumatology ,Antigen ,Medicine & Public Health ,medicine ,Citrulline ,Humans ,Immunology and Allergy ,Rheumatoid arthritis ,Autoantibodies ,Immunoassay ,Peptidylarginine deiminase 4 ,medicine.diagnostic_test ,biology ,Norway ,business.industry ,Autoantibody ,Citrullination ,medicine.disease ,Kinetics ,chemistry ,Case-Control Studies ,Disease Progression ,Protein-Arginine Deiminases ,biology.protein ,Original Article ,Antibody ,business ,Biomarkers - Abstract
Isoform 4 of the human peptidylarginine deiminase (hPAD4) enzyme may be responsible for the citrullination of antigens in rheumatoid arthritis (RA) and has been shown to be itself the target of disease-specific autoantibodies. Here, we have tested whether the level of serum anti-hPAD4 antibodies in RA patients is stable over a period of 10 years and whether the antibodies influence hPAD4-mediated deimination of the small substrate N-α-benzoyl-L-arginine ethyl ester. RA sera (n = 128) obtained at baseline and after 10 years were assessed for anti-hPAD4 antibodies by a specific immunoassay. For 118 RA patients, serum anti-hPAD4 IgG levels were stable over 10 years. Seven patients who were negative for anti-PAD4 IgG at baseline had become positive after 10 years. Further, total IgG from selected RA patients and controls were purified, and a fraction was depleted for anti-hPAD4 antibodies. Kinetic deimination assays were performed with total IgG and depleted fractions. The k ( cat ) and K ( m ) values of hPAD4-mediated deimination of N-α-benzoyl-L-arginine ethyl ester were not affected by the depletion of the anti-hPAD4 antibodies from the total IgG pool. In conclusion, RA patients remain positive for anti-hPAD4 antibodies over time and some patients who are initially anti-hPAD4 negative become positive later in the disease course. The anti-hPAD4 antibodies did not affect the enzymatic activity of hPAD4 when the small substrate N-α-benzoyl-L-arginine ethyl ester was used. However, this finding may not exclude an effect of these autoantibodies on citrullination of protein substrates in RA.
- Published
- 2011
10. Serum IgG antibodies to peptidylarginine deiminase 4 in rheumatoid arthritis and associations with disease severity
- Author
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D. van der Heijde, Øyvind Molberg, Sigrid Ødegård, Robert Landewé, Sylvie Pollmann, Inge-Margrethe Gilboe, T.K. Kvien, E. H. Halvorsen, and Other departments
- Subjects
Adult ,Male ,Adolescent ,Hydrolases ,Immunology ,Arthritis ,Peptides, Cyclic ,Severity of Illness Index ,General Biochemistry, Genetics and Molecular Biology ,Immunoglobulin G ,Arthritis, Rheumatoid ,Cohort Studies ,Protein-Arginine Deiminase Type 4 ,Rheumatology ,Rheumatoid Factor ,medicine ,Humans ,Lupus Erythematosus, Systemic ,Immunology and Allergy ,Rheumatoid factor ,Fluorometry ,Aged ,Autoantibodies ,Aged, 80 and over ,Lupus erythematosus ,biology ,business.industry ,Autoantibody ,Citrullination ,Middle Aged ,medicine.disease ,Connective tissue disease ,Recombinant Proteins ,Radiography ,Rheumatoid arthritis ,Protein-Arginine Deiminases ,biology.protein ,Female ,business - Abstract
Background: Antibodies targeting citrullinated antigens are specific for rheumatoid arthritis (RA). Citrullination is catalysed by the peptidylarginine deiminase (PAD) enzyme family. Critical enzymes are often targeted by disease-specific antibodies in complex immune-mediated diseases. Here, we have tested for autoantibodies against human recombinant PAD4 (hPAD4) in Caucasian RA patients. Methods: A time-resolved fluorometric immunoassay based on hPAD4 was developed to analyse sera from two RA cohorts (n = 237 and n = 177), one systemic lupus erythaematosus (SLE) cohort (n = 84) and 148 healthy controls. Simple and multiple analyses were performed to examine possible associations between anti-hPAD4 and disease variables. Results: Raised levels of anti-hPAD4 IgG were found in both RA cohorts compared to the controls, and 23% of the RA patients were anti-hPAD4 IgG positive. Anti-hPAD4 was associated with anti-cyclic citrullinated peptide (CCP) and rheumatoid factor (RF), as well as increased physical disability. Anti-hPAD4 was also associated with higher longitudinal radiographic damage scores and increased clinical joint pathology, but weaker than anti-CCP. No associations were found between anti-hPAD4 and selected Human leukocyte antigen (HLA)-DRB1 variants. Conclusions: Approximately 23% of Caucasian RA patients have serum IgG antibodies against hPAD4.The presence of serum anti-hPAD4 IgG was in simple analyses associated with a more severe disease phenotype, and the association with physical disability was maintained in multiple analyses.
- Published
- 2008
11. Primary sequence, together with other factors, influence peptide deimination by peptidylarginine deiminase-4
- Author
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Ludvig M. Sollid, Sylvie Pollmann, Burkhard Fleckenstein, Øyvind Molberg, and Maria Stensland
- Subjects
Arginine ,Hydrolases ,Clinical Biochemistry ,Molecular Sequence Data ,Peptide ,Biochemistry ,chemistry.chemical_compound ,Protein-Arginine Deiminase Type 4 ,Citrulline ,Humans ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,Citrullination ,Amino acid ,Kinetics ,Enzyme ,chemistry ,Cyclization ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Protein-Arginine Deiminases ,Peptides ,Cysteine - Abstract
Enzymes of the peptidylarginine deiminase (PAD) family catalyze the posttranslational deimination of polypeptide-bound arginine residues. Here, we report the selection of peptide substrates by PAD-4, an isoform thought to be involved in the pathogenesis of rheumatoid arthritis. First, we investigated whether PAD-4-mediated deimination is influenced by the nature of amino acid residues flanking the targeted arginine. Using two peptide substrates, residues in positions -2, -1, +1, and +2 relative to the central arginine targeted by PAD-4 were systematically replaced by all natural l-amino acids except cysteine. Each peptide was treated with recombinant human PAD-4 and deimination was analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In all four flanking positions, amino acids which positively or negatively influenced deimination were identified. We next designed peptides with expected high or low deimination rates and determined their K m and k cat values. These peptides showed PAD-4 substrate behavior as predicted, demonstrating that residues flanking the targeted arginine are important for deimination. Further truncation of peptide substrates suggested additional effects on deimination by residues outside the -2 to +2 region. Finally, we observed that a methylated lysine residue flanking the targeted arginine influences PAD-4-mediated deimination, also suggesting that posttranslational modifications can affect substrate efficiency.
- Published
- 2008
12. The N-terminal flanking region of the TRP2360-368 melanoma antigen determines proteasome activator PA28 requirement for epitope liberation
- Author
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Theresa Bergann, Alice J. A. M. Sijts, Ilse Drung, Peter Henklein, Hardy Weisshoff, Kathrin Textoris-Taube, Sylvie Pollmann, Clemens Mügge, Peter-Michael Kloetzel, Ulrike Seifert, Ulrike Kuckelkorn, Strategic Infection Biology, and Dep Infectieziekten Immunologie
- Subjects
Proteasome Endopeptidase Complex ,Muscle Proteins ,Biology ,Major histocompatibility complex ,Biochemistry ,Epitope ,Protein Structure, Secondary ,Epitopes ,Antigen ,Antigens, Neoplasm ,HLA-A2 Antigen ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Melanoma ,Nuclear Magnetic Resonance, Biomolecular ,Linear epitope ,HLA-A Antigens ,Activator (genetics) ,Cell Biology ,Molecular biology ,Proteasome ,biology.protein ,Peptides - Abstract
Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens. The interferon-gamma-inducible proteasome activator PA28 plays an important role in the generation of MHC ligands by proteasomes. Generation of the HLA-A(*)0201 restricted melanoma antigen TRP2(360-368) by the proteasome has been shown to be dependent on the function of PA28 in vitro and in vivo (Sun, Y., Sijts, A. J., Song, M., Janek, K., Nussbaum, A. K., Kral, S., Schirle, M., Stevanovic, S., Paschen, A., Schild, H., Kloetzel, P. M., and Schadendorf, D. (2002) Cancer Res. 62, 2875-2882). Here we analyzed the role of the epitope sequence environment in determining this PA28 dependence. Experiments using the melanoma TRP2(288-296) epitope and the murine cytomegalovirus-derived pp89 epitope precursor peptide for epitope replacement revealed that the TRP2(360-368) flanking sequences can transfer PA28 dependence onto otherwise PA28 independent epitopes. Moreover, the N-terminal flanking sequence is sufficient to establish PA28 dependence of an epitope by allowing PA28-induced coordinated dual cleavages. These results show that N-terminal flanking sequences strongly influence epitope generation efficiency and that PA28 function is particularly relevant for the generation of normally poorly excised peptide products.
- Published
- 2007
13. DeltaPhage—a novel helper phage for high-valence pIX phagemid display
- Author
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Nicolay Rustad Nilssen, Terje Frigstad, Bjarne Bogen, Inger Sandlie, Norbert Roos, Sylvie Pollmann, and Geir Åge Løset
- Subjects
Genetics ,Phage display ,Phagemid ,Genetic Vectors ,Receptors, Antigen, T-Cell ,Enzyme-Linked Immunosorbent Assay ,Biology ,Key features ,Cell Line ,Complementation ,Mice ,Start codon ,Peptide Library ,Helper virus ,Methods Online ,Animals ,Bacteriophages ,Antigens ,Peptide library ,Helper Viruses - Abstract
Phage display has been instrumental in discovery of novel binding peptides and folded domains for the past two decades. We recently reported a novel pIX phagemid display system that is characterized by a strong preference for phagemid packaging combined with low display levels, two key features that support highly efficient affinity selection. However, high diversity in selected repertoires are intimately coupled to high display levels during initial selection rounds. To incorporate this additional feature into the pIX display system, we have developed a novel helper phage termed DeltaPhage that allows for high-valence display on pIX. This was obtained by inserting two amber mutations close to the pIX start codon, but after the pVII translational stop, conditionally inactivating the helper phage encoded pIX. Until now, the general notion has been that display on pIX is dependent on wild-type complementation, making high-valence display unachievable. However, we found that DeltaPhage does facilitate high-valence pIX display when used with a non-suppressor host. Here, we report a side-by-side comparison with pIII display, and we find that this novel helper phage complements existing pIX phagemid display systems to allow both low and high-valence display, making pIX display a complete and efficient alternative to existing pIII phagemid display systems.
- Published
- 2012
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