Cornelis Melief - Academia.edu (original) (raw)

Papers by Cornelis Melief

Research paper thumbnail of A placebo-controlled randomized HPV16 synthetic long-peptide vaccination study in women with high-grade cervical squamous intraepithelial lesions

Cancer Immunology, Immunotherapy, 2012

The aim of this study was to investigate the capacity of an HPV16 E6/E7 synthetic overlapping lon... more The aim of this study was to investigate the capacity of an HPV16 E6/E7 synthetic overlapping longpeptide vaccine to stimulate the HPV16-specific T-cell response, to enhance the infiltration of HPV16-specific type 1 T cells into the lesions of patients with HPV16? high-grade cervical squamous intraepithelial lesion (HSIL) and HPV clearance. This was a placebo-controlled randomized phase II study in patients with HPV16-positive HSIL. HPV16-specific T-cell responses were determined pre-and post-vaccination by ELISPOT, proliferation assay and cytokine assays in PBMC and HSIL-infiltrating lymphocytes, and delayed-type hypersensitivity skin tests. Motivational problems of this patient group to postpone treatment of their premalignant lesions affected the inclusion rates and caused the study to stop prematurely. Of the accrued patients, 4 received a placebo and 5 received 1-2 vaccinations. Side effects mainly were flu-like symptoms and injection site reactions. A strong HPV-specific IFNcassociated T-cell response was detected by ELISPOT in all vaccinated patients. The outcome of the skin tests correlated well with the ELISPOT analysis. The cytokine profile associated with HPV16-specific proliferation varied from robust type 1 to dominant type 2 responses. No conclusions could be drawn on vaccine-enhanced T-cell infiltration of the lesion, and there was no HPV clearance at the time of LEEP excision. Thus, vaccination of HSIL patients results in increased HPV16-specific T-cell immunity. Further development of this type of treatment relies on the ability to motivate patients and in the reduction in the side effects.

Research paper thumbnail of Synergy of therapeutic vaccination against HPV16 oncogenic proteins and standard chemotherapeutics

Journal for ImmunoTherapy of Cancer, 2014

Research paper thumbnail of DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells

Research paper thumbnail of The Rat cim Effect: TAP Allele-Dependent Changes in a Class I MHC Anchor Motif and Evidence Against C-Terminal Trimming of Peptides in the ER

Immunity, 1996

In the rat, TAP2 exists as two main allotypes, TAP2A Louise Richardson, 1 Remco P. Brandt, 4 and ... more In the rat, TAP2 exists as two main allotypes, TAP2A Louise Richardson, 1 Remco P. Brandt, 4 and TAP2B, differing by 25 aa (Powis et al., 1992; Joly Cornelis J. Melief, 4 Jonathan C. Howard, 1, 5 et al., 1994). Recently, TAP-dependent peptide transport and Geoffrey W. Butcher 1 assays in vitro have demonstrated that rat TAP com-1 Laboratory of Immunogenetics plexes containing TAP2A or TAP2B display differing Department of Immunology peptide selectivities (Heemels et al., 1993; Momburg et 2 Microchemical Facility al., 1994). While TAP2A allowed the transport of syn-The Babraham Institute thetic peptides with most amino acids (except proline) Cambridge, CB2 4AT at the C terminus, TAP2B was more restricted to trans-United Kingdom porting peptides with hydrophobic residues at the C 3 Department of Biochemistry terminus. It has also been determined that TAP2B can University of Dundee permit the transport of longer peptides more efficiently Dundee than TAP2A (Heemels and Ploegh, 1994). This functional United Kingdom polymorphism affects the behavior of the rat class I 4 Department of Immunohematology MHC molecule, RT1.A a , in a set of phenomena named and Bloodbank class I modification (cim). In cim, intracellular assembly University Hospital of RT1.A a has been shown to be much slower in cim b Leiden cells (homozygous for TAP2B) than in cim a (TAP2A) cells The Netherlands (Powis et al., 1991b). Furthermore, alloreactive cytotoxic 5 Institute for Genetics T lymphocyte responses can be generated that discrimi-University of Cologne nate between RT1.A a expressed by cim a and cim b cells Cologne (Livingstone et al., 1989). In conjunction with initial stud-Federal Republic of Germany ies of eluted radiolabeled peptides, this implies the presentation of different peptide subsets (Powis et al., 1992).

Research paper thumbnail of Chemically synthesized protein as tumour-specific vaccine: immunogenicity and efficacy of synthetic HPV16 E7 in the TC-1 mouse tumour model

Vaccine, 2004

Many successful candidate vaccines capable of combating tumours in animal models come to an untim... more Many successful candidate vaccines capable of combating tumours in animal models come to an untimely end because of the costs associated with the approval and production of the GMP-grade materials, which are usually of biological origin, for use in humans. We have used a GMP-compatible method to chemically synthesize a pure synthetic E7 protein of the human papillomavirus type 16 (HPV16-E7). This oncogen-derived protein is constitutively expressed in cervical cancer and its precursors and is thus considered as an excellent target for tumour-specific immunity. Injection of a mixture of the synthetic HPV16-E7 protein and the synthetic adjuvant CpG in mice resulted in strong functional HPV16-specific cytotoxic T-lymphocyte responses as measured by CD8+ MHC class I-tetramer staining, the detection of antigen-specific intracellular IFNgamma production and the ability to protect mice against a challenge with HPV16-E7+ TC-1 tumour cells in both prophylactic and therapeutic vaccination regimens. Our results demonstrate the potential use of pure synthetic vaccines that can be efficiently produced under GMP at low cost, which will stimulate the translation of new vaccination strategies into phase I/II clinical trials.

Research paper thumbnail of Potential immunogenicity of oncogene and tumor suppressor gene products

Current Opinion in Immunology, 1993

The immunogenicity of viral oncoproteins has been established beyond doubt. Cytotoxic T lymphocyt... more The immunogenicity of viral oncoproteins has been established beyond doubt. Cytotoxic T lymphocytes directed against viral oncogene products can eradicate large established tumor masses. This stage has not yet been reached for cellular oncogene and tumor suppressor gene products, but T cells have been raised against MHC-binding peptides encoded by both mutant and wild-type alleles of the ras oncogene and the p53 tumor suppressor gene. In addition, T cells specific for joining region peptides of abnormal fusion proteins resulting from chromosome translocation in tumor cells have been generated. Some of these peptides are processed in cells infected with, for example, vaccinia-ras, but direct anti-tumor effects of peptide specific T lymphocytes remain to be demonstrated.

Research paper thumbnail of The Current Status of Therapeutic HPV Vaccine

Annals of Medicine, 1996

Cervical carcinoma is the second most common cause of cancer-related deaths in women worldwide. R... more Cervical carcinoma is the second most common cause of cancer-related deaths in women worldwide. Recurrences occur in 15% of the patients after optimal treatment of low-risk early-stage disease. Treatment results of recurrent disease are relatively poor and for this reason new therapeutic strategies are warranted. Viral infection with human papillomavirus seems to have an essential part in the aetiology of cervical carcinoma. Evidence for the assumption that cervical carcinoma, among other malignancies such as melanomas, renal malignancies and Kaposi sarcoma, are immunogenic is provided by the fact that these malignancies grow more rapidly in the presence of systemic immunosuppression. Spontaneous regression for these tumour types is also described and immunohistochemical studies show extensive infiltrates in the tumour, consisting of immunocompetent cells. It is thus postulated that cellular immunity, and mainly the T-cell system plays an important role in the antitumour defence in cervical carcinoma. This review describes the rationale for the use of immunotherapy as treatment for cervical carcinoma as well as the results of recent developments in tumour immunology and its implications for the clinical use of immunotherapeutical approaches.

Research paper thumbnail of Immature Dendritic Cells Acquire CD8+ Cytotoxic T Lymphocyte Priming Capacity upon Activation by T Helper Cell-independent or -dependent Stimuli

Journal of Experimental Medicine, 2000

The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC ma... more The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC maturation in CTL induction in vitro and in vivo. Maturation of D1 cells, characterized by markedly increased expression of MHC and costimulatory molecules, was induced by incubation with lipopolysaccharide, agonistic CD40 antibody, or specific CD4 ϩ T helper (Th) cells. Activated, but not immature, D1 cells efficiently primed alloreactive T cell responses in vitro. Similarly, priming of CTL immunity in vivo in CD4-depleted mice was only observed if these mice were immunized with activated D1 cells. This study provides formal evidence that activation of DCs, induced by Th-independent as well as Th-dependent stimuli, is essential for efficient induction of CTL responses.

Research paper thumbnail of Detection of T Helper Responses, But Not of Human Papillomavirus-Specific Cytotoxic T Lymphocyte Responses, After Peptide Vaccination of Patients With Cervical Carcinoma

Journal of Immunotherapy, 2000

Human papillomavirus type 16 (HPV16)-encoded E7 oncoprotein is constitutively expressed in cervic... more Human papillomavirus type 16 (HPV16)-encoded E7 oncoprotein is constitutively expressed in cervical carcinoma cells and is required for cellular transformation to be maintained. The E7 protein, therefore, forms an attractive target for T-cell-mediated immune intervention to prevent or treat HPV16+ tumors. The authors performed a peptide-based phase I/II vaccination trial to induce anti-tumor immune responses in patients with recurrent or residual cervical carcinoma. Fifteen HLA-A*0201+ patients with HPV16+ cervical carcinoma received vaccinations with synthetic peptides representing 2 HPV16 E7-encoded, HLA-A*0201-restricted cytotoxic T lymphocyte epitopes and a pan-HLA-DR-binding T-helper epitope, PADRE, in adjuvant. No signs of toxicity were observed. Two patients had stable disease for more than 1 year after vaccination, 3 patients died of the disease during or shortly after the vaccination period, and 10 patients maintained progressive cervical carcinoma. Specific immune responses directed against the vaccine components were analyzed in peripheral blood samples. No cytotoxic T lymphocyte responses against the HPV16 E7 peptides were detectable. After vaccination, strong PADRE helper peptide-specific proliferation was detected in 4 of 12 patients. In conclusion, peptide vaccination with 2 HPV16 E7 cytotoxic T lymphocyte epitopes and a universal T helper epitope is well tolerated by patients with advanced cervical carcinoma. Despite a reduction of in vitro cytolytic or proliferative recall responses to some, but not all, conventional antigens in this patient group, peptide-specific proliferative responses were induced in 4 patients. Based on the current study, it is now feasible to perform peptide vaccination in earlier stages of HPV16-induced cervical disease.

Research paper thumbnail of The HPV16 E7 peptide based vaccine trial in end stage cervical carcinoma: An interim report

Immunol Lett, 1997

melanoma cells may kill CDQ5-sensitive activated T cells. However, our data does not exclude that... more melanoma cells may kill CDQ5-sensitive activated T cells. However, our data does not exclude that other CD95 expressing cells, i.e. granulocytes or NK cells, contribute to the obsenred pool of apoptotfc cells within the tumor. In summary, CDQBL expression seem to be acquired during the progression of melanoma and is associated with substantial apoptosis in tumor infiltrating leukocytes, thereby providing another immune escape mechanism. 1 P.5.10.15 ( Plasma cell expression of CD28 and CD40 correlates wlth perlpheral T-cell defects In multiple myeloma

Research paper thumbnail of Natural T-helper immunity against human papillomavirus type 16 (hpv16) e7-derived peptide epitopes in patients with hpv16-positive cervical lesions: Identification of 3 human leukocyte antigen class ii-restricted epitopes

International Journal of Cancer, 2001

Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine... more Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine-induced immune defense against tumors. Since the majority of cervical cancers express human papillomavirus type 16 (HPV16) E7 oncoprotein, it is important to investigate the Th response against this target antigen in detail. By means of PBMC cultures from HLA-typed healthy donors, we identified the central part of HPV16 E7 (E7 41-72 ) as the major immunogenic region within this antigen. Furthermore, we mapped 3 distinct Th epitopes within this region (DR15/E7 50 -62 , DR3/ E7 43-77 , DQ2/E7 35-50 ). In a parallel approach, employing IFN-␥ ELISPOT analysis, we detected Th immunity against HPV16 E7 in subjects with HPV16 ؉ lesions. Several of these responses matched with the 3 Th epitopes defined in our study. A number of other HPV16 ؉ subjects did not display any E7-specific type 1 cytokine-producing T-cell immunity, indicating failure of the immune response. Our combined data argue for more extensive as well as longitudinal analysis of HPV16-specific T-cell immunity using the ELISPOT assay described, as well as for HPV-specific vaccination of individuals with HPV ؉ lesions.

Research paper thumbnail of Immunotherapy of cancer by peptide-based vaccines for the induction of tumor-specific T cell immunity

Immunotechnology, 1996

Recent progress in defining the molecular nature of antigens and in finding ways to manipulate T ... more Recent progress in defining the molecular nature of antigens and in finding ways to manipulate T cell-mediated immune responses may provide new modalities for cancer treatment. In this report, we review preclinical studies as well as the first clinical trials with vaccination strategies aiming at the induction of anti-tumor immunity. In particular, we focus on the development of a vaccine against human papillomavirus-induced cervical carcinoma.

Research paper thumbnail of Natural T-helper immunity against human papillomavirus type 16 (HPV16) E7-derived peptide epitopes in patients with HPV16-positive cervical lesions: identification of 3 human leukocyte antigen class II-restricted epitopes

International Journal of Cancer, Mar 1, 2001

Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine... more Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine-induced immune defense against tumors. Since the majority of cervical cancers express human papillomavirus type 16 (HPV16) E7 oncoprotein, it is important to investigate the Th response against this target antigen in detail. By means of PBMC cultures from HLA-typed healthy donors, we identified the central part of HPV16 E7 (E7 41-72 ) as the major immunogenic region within this antigen. Furthermore, we mapped 3 distinct Th epitopes within this region (DR15/E7 50 -62 , DR3/ E7 43-77 , DQ2/E7 35-50 ). In a parallel approach, employing IFN-␥ ELISPOT analysis, we detected Th immunity against HPV16 E7 in subjects with HPV16 ؉ lesions. Several of these responses matched with the 3 Th epitopes defined in our study. A number of other HPV16 ؉ subjects did not display any E7-specific type 1 cytokine-producing T-cell immunity, indicating failure of the immune response. Our combined data argue for more extensive as well as longitudinal analysis of HPV16-specific T-cell immunity using the ELISPOT assay described, as well as for HPV-specific vaccination of individuals with HPV ؉ lesions.

Research paper thumbnail of Skin reactions to human papillomavirus (HPV) 16 specific antigens intradermally injected in healthy subjects and patients with cervical neoplasia

International Journal of Cancer, 2008

We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin t... more We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin test to detect cellular immune responses to HPV16 E2, E6 and E7 in vivo. Women with cervical neoplasia (n 5 11) and healthy individuals (n 5 19) were intradermally challenged with 8 different pools of HPV16 E2, E6 and E7 peptides. The skin test was safe as the injections were perceived as mildly painful and no adverse events were observed. The majority of skin reactions appeared significantly earlier in HPV161 patients (<8 days) than in healthy subjects (8-25 days). The development of late skin reactions in healthy subjects was associated with the appearance of circulating HPV16-specific T cells and the infiltration of both HPV16-specific CD41 Th1/Th2 and CD81 T cells into the skin. These data show that the intradermal injection of pools of HPV16 synthetic long peptides is safe and results in the migration of HPV16-specific T cells into the skin as well as in an increase in the number of circulating HPV16-specific T cells. The use of this test to measure HPV16-specific immunity is currently tested in a low resource setting for the measurement of spontaneously induced T-cell responses as well as in our HPV16 vaccination trials for the detection of vaccine-induced immunity. '

Research paper thumbnail of Immune Escape of Tumors In Vivo by Expression of Cellular FLICE-inhibitory Protein

Summary The antiapoptotic protein cellular FLICE (Fas-associated death domain-like IL-1 b -conver... more Summary The antiapoptotic protein cellular FLICE (Fas-associated death domain-like IL-1 b -converting enzyme) inhibitory protein (cFLIP) protects cells from CD95(APO-1/Fas)-induced apoptosis in vitro and was found to be overexpressed in human melanomas. However, cytotoxic T cell- induced apoptosis, which is critically involved in tumor control in vivo, is not inhibited by cFLIP in vitro, as only CD95- and not perforin-dependent

Research paper thumbnail of Natural T cell response against HPV16 and development of optimal peptide based vaccine

European Journal of Cancer, 2001

Research paper thumbnail of Natural T-helper immunity against human papillomavirus type 16 (hpv16) e7-derived peptide epitopes in patients with hpv16-positive cervical lesions: Identification of 3 human leukocyte antigen class ii-restricted epitopes

International Journal of Cancer, 2001

Tumor-specificT-helper(Th)immunitywasfoundtoplay apivotalroleinthenaturalandvaccine-inducedimmune... more Tumor-specificT-helper(Th)immunitywasfoundtoplay apivotalroleinthenaturalandvaccine-inducedimmune defenseagainsttumors.Sincethemajorityofcervicalcan-cersexpresshumanpapillomavirustype16(HPV16)E7oncoprotein,itisimportanttoinvestigatetheThresponse againstthistargetantigenindetail.BymeansofPBMC culturesfromHLA-typedhealthydonors,weidentifiedthe centralpartofHPV16E7(E7 41-72 )asthemajorimmunogenicregionwithinthisantigen.Furthermore,wemapped3 distinctThepitopeswithinthisregion(DR15/E7 50-62 ,DR3/ E7 43-77 ,DQ2/E7 35-50 ).Inaparallelapproach,employing IFN-␥ELISPOTanalysis,wedetectedThimmunityagainst HPV16E7insubjectswithHPV16 ؉ lesions.Severalofthese responsesmatchedwiththe3Thepitopesdefinedinour study.AnumberofotherHPV16 ؉ subjectsdidnotdisplay anyE7-specifictype1cytokine-producingT-cellimmunity, indicatingfailureoftheimmuneresponse.Ourcombined dataargueformoreextensiveaswellaslongitudinalanalysis ofHPV16-specificT-cellimmunityusingtheELISPOTassay described,aswellasforHPV-specificvaccinationofindivid-ualswithHPV ؉ lesions.

Research paper thumbnail of Tumor-Expressed B7-H1 and B7-DC in Relation to PD-1+ T-Cell Infiltration and Survival of Patients with Cervical Carcinoma

Clinical Cancer Research, 2009

The interaction between programmed cell death 1 (PD-1), expressed by activated effector or regula... more The interaction between programmed cell death 1 (PD-1), expressed by activated effector or regulatory T cells, and B7-H1 (PD-L1) and B7-DC (PD-L2) results in the inhibition of T-cell function. The aim of this study was to determine B7-H1, B7-DC, and PD-1 expression in cervical carcinoma. A tissue microarray of a well-defined group of 115 patients was stained with antibodies against B7-H1 and B7-DC. Three-color fluorescent immunohistochemistry was used to study the number and phenotype of tumor-infiltrating T cells expressing PD-1. Additional analyses consisted of in vitro T-cell suppression assays. B7-H1 was expressed in 19%, and B7-DC was expressed by 29% of the 115 tumors. PD-1 was expressed by more than half of both the infiltrating CD8+ T cells and CD4+Foxp3+ T cells, irrespective of B7-H1 or B7-DC expression by tumors. The expression of B7-H1 did not show a direct impact on patient survival. However, subgroup analysis revealed that patients with a relative excess of infiltrating regulatory T cells displayed a better survival when the tumor was B7-H1 positive (P = 0.033). Additional studies showed that the presence of B7-H1 during the activation of CD4+Foxp3+ regulatory T cells impaired their suppressive function in a functional in vitro assay. B7-H1 is expressed on only a minority of cervical cancers and does not influence the survival of patients with cervical cancer. PD-1 is expressed by a vast number of infiltrating CD8 T cells, suggesting that blocking of PD-1 could have therapeutic potential in cervical cancer patients.

Research paper thumbnail of Human Papillomavirus Type 16-Positive Cervical Cancer Is Associated with Impaired CD4+ T-Cell Immunity against Early Antigens E2 and E6

Cancer Research, 2004

Cervical cancer is the possible outcome of genital infection with highrisk human papillomavirus (... more Cervical cancer is the possible outcome of genital infection with highrisk human papillomavirus (HPV) and is preceded by a phase of persistent HPV infection during which the host immune system fails to eliminate the virus. Fortunately, the majority of genital HPV infections are cleared before the development of (pre)malignant lesions. Analysis of CD4؉ T-helper (Th) immunity against the E2, E6, and E7 antigens of HPV16 in healthy women revealed strong proliferative E2-and E6-specific responses associated with prominent IFN-␥ and interleukin 5 secretion. This indicates that the naturally arising virus-induced immune response displays a mixed Th1/Th2 cytokine profile. Of all HPV16؉ cervical cancer patients, approximately half failed to mount a detectable immune response against the HPV16-derived peptides.

Research paper thumbnail of Phase I Immunotherapeutic Trial with Long Peptides Spanning the E6 and E7 Sequences of High-Risk Human Papillomavirus 16 in End-Stage Cervical Cancer Patients Shows Low Toxicity and Robust Immunogenicity

Clinical Cancer Research, 2008

Purpose: To determine the toxicity, safety, and immunogenicity of a human papillomavirus 16 (HPV1... more Purpose: To determine the toxicity, safety, and immunogenicity of a human papillomavirus 16 (HPV16) E6 and E7 long peptide vaccine administered to end-stage cervical cancer patients. Experimental Design:Three groups of end-stage cervical cancer patients (in total n =35)were s.c. vaccinated with HPV16 E6 combined with or separated from HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant, four times at 3-week intervals. Group 1 received 300 Ag/ peptide at a single site and group 2 received 100 Ag/peptide of the E6 peptides in one limb and 300 Ag/peptide of the E7 peptides in a second limb. Group 3 received separate injections of E6 and E7 peptides, each at a dose of 50 Ag/peptide. The primary end point was to determine safety and toxicity of the HPV16 long peptides vaccine. In addition, the vaccine-induced T-cell response was assessed by IFNg enzyme-linked immunospot. Results: No toxicity beyond grade 2 was observed during and after four vaccinations. In a few patients, transient flu-like symptoms were observed. Enzyme-linked immunospot analysis of the vaccine-induced immune response revealed that coinjection of the E6 and E7 peptides resulted in a strong and broad T-cell response dominated by immunity against E6. Injection of the E6 and E7 peptides at two different sites increased the E7 response but did not affect the magnitude of the E6-induced immune response. Conclusions: The HPV16 E6 and E7 long peptide-based vaccine is well tolerated and capable of inducing a broad IFNg-associated T-cell response even in end-stage cervical cancer patients.

Research paper thumbnail of A placebo-controlled randomized HPV16 synthetic long-peptide vaccination study in women with high-grade cervical squamous intraepithelial lesions

Cancer Immunology, Immunotherapy, 2012

The aim of this study was to investigate the capacity of an HPV16 E6/E7 synthetic overlapping lon... more The aim of this study was to investigate the capacity of an HPV16 E6/E7 synthetic overlapping longpeptide vaccine to stimulate the HPV16-specific T-cell response, to enhance the infiltration of HPV16-specific type 1 T cells into the lesions of patients with HPV16? high-grade cervical squamous intraepithelial lesion (HSIL) and HPV clearance. This was a placebo-controlled randomized phase II study in patients with HPV16-positive HSIL. HPV16-specific T-cell responses were determined pre-and post-vaccination by ELISPOT, proliferation assay and cytokine assays in PBMC and HSIL-infiltrating lymphocytes, and delayed-type hypersensitivity skin tests. Motivational problems of this patient group to postpone treatment of their premalignant lesions affected the inclusion rates and caused the study to stop prematurely. Of the accrued patients, 4 received a placebo and 5 received 1-2 vaccinations. Side effects mainly were flu-like symptoms and injection site reactions. A strong HPV-specific IFNcassociated T-cell response was detected by ELISPOT in all vaccinated patients. The outcome of the skin tests correlated well with the ELISPOT analysis. The cytokine profile associated with HPV16-specific proliferation varied from robust type 1 to dominant type 2 responses. No conclusions could be drawn on vaccine-enhanced T-cell infiltration of the lesion, and there was no HPV clearance at the time of LEEP excision. Thus, vaccination of HSIL patients results in increased HPV16-specific T-cell immunity. Further development of this type of treatment relies on the ability to motivate patients and in the reduction in the side effects.

Research paper thumbnail of Synergy of therapeutic vaccination against HPV16 oncogenic proteins and standard chemotherapeutics

Journal for ImmunoTherapy of Cancer, 2014

Research paper thumbnail of DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells

Research paper thumbnail of The Rat cim Effect: TAP Allele-Dependent Changes in a Class I MHC Anchor Motif and Evidence Against C-Terminal Trimming of Peptides in the ER

Immunity, 1996

In the rat, TAP2 exists as two main allotypes, TAP2A Louise Richardson, 1 Remco P. Brandt, 4 and ... more In the rat, TAP2 exists as two main allotypes, TAP2A Louise Richardson, 1 Remco P. Brandt, 4 and TAP2B, differing by 25 aa (Powis et al., 1992; Joly Cornelis J. Melief, 4 Jonathan C. Howard, 1, 5 et al., 1994). Recently, TAP-dependent peptide transport and Geoffrey W. Butcher 1 assays in vitro have demonstrated that rat TAP com-1 Laboratory of Immunogenetics plexes containing TAP2A or TAP2B display differing Department of Immunology peptide selectivities (Heemels et al., 1993; Momburg et 2 Microchemical Facility al., 1994). While TAP2A allowed the transport of syn-The Babraham Institute thetic peptides with most amino acids (except proline) Cambridge, CB2 4AT at the C terminus, TAP2B was more restricted to trans-United Kingdom porting peptides with hydrophobic residues at the C 3 Department of Biochemistry terminus. It has also been determined that TAP2B can University of Dundee permit the transport of longer peptides more efficiently Dundee than TAP2A (Heemels and Ploegh, 1994). This functional United Kingdom polymorphism affects the behavior of the rat class I 4 Department of Immunohematology MHC molecule, RT1.A a , in a set of phenomena named and Bloodbank class I modification (cim). In cim, intracellular assembly University Hospital of RT1.A a has been shown to be much slower in cim b Leiden cells (homozygous for TAP2B) than in cim a (TAP2A) cells The Netherlands (Powis et al., 1991b). Furthermore, alloreactive cytotoxic 5 Institute for Genetics T lymphocyte responses can be generated that discrimi-University of Cologne nate between RT1.A a expressed by cim a and cim b cells Cologne (Livingstone et al., 1989). In conjunction with initial stud-Federal Republic of Germany ies of eluted radiolabeled peptides, this implies the presentation of different peptide subsets (Powis et al., 1992).

Research paper thumbnail of Chemically synthesized protein as tumour-specific vaccine: immunogenicity and efficacy of synthetic HPV16 E7 in the TC-1 mouse tumour model

Vaccine, 2004

Many successful candidate vaccines capable of combating tumours in animal models come to an untim... more Many successful candidate vaccines capable of combating tumours in animal models come to an untimely end because of the costs associated with the approval and production of the GMP-grade materials, which are usually of biological origin, for use in humans. We have used a GMP-compatible method to chemically synthesize a pure synthetic E7 protein of the human papillomavirus type 16 (HPV16-E7). This oncogen-derived protein is constitutively expressed in cervical cancer and its precursors and is thus considered as an excellent target for tumour-specific immunity. Injection of a mixture of the synthetic HPV16-E7 protein and the synthetic adjuvant CpG in mice resulted in strong functional HPV16-specific cytotoxic T-lymphocyte responses as measured by CD8+ MHC class I-tetramer staining, the detection of antigen-specific intracellular IFNgamma production and the ability to protect mice against a challenge with HPV16-E7+ TC-1 tumour cells in both prophylactic and therapeutic vaccination regimens. Our results demonstrate the potential use of pure synthetic vaccines that can be efficiently produced under GMP at low cost, which will stimulate the translation of new vaccination strategies into phase I/II clinical trials.

Research paper thumbnail of Potential immunogenicity of oncogene and tumor suppressor gene products

Current Opinion in Immunology, 1993

The immunogenicity of viral oncoproteins has been established beyond doubt. Cytotoxic T lymphocyt... more The immunogenicity of viral oncoproteins has been established beyond doubt. Cytotoxic T lymphocytes directed against viral oncogene products can eradicate large established tumor masses. This stage has not yet been reached for cellular oncogene and tumor suppressor gene products, but T cells have been raised against MHC-binding peptides encoded by both mutant and wild-type alleles of the ras oncogene and the p53 tumor suppressor gene. In addition, T cells specific for joining region peptides of abnormal fusion proteins resulting from chromosome translocation in tumor cells have been generated. Some of these peptides are processed in cells infected with, for example, vaccinia-ras, but direct anti-tumor effects of peptide specific T lymphocytes remain to be demonstrated.

Research paper thumbnail of The Current Status of Therapeutic HPV Vaccine

Annals of Medicine, 1996

Cervical carcinoma is the second most common cause of cancer-related deaths in women worldwide. R... more Cervical carcinoma is the second most common cause of cancer-related deaths in women worldwide. Recurrences occur in 15% of the patients after optimal treatment of low-risk early-stage disease. Treatment results of recurrent disease are relatively poor and for this reason new therapeutic strategies are warranted. Viral infection with human papillomavirus seems to have an essential part in the aetiology of cervical carcinoma. Evidence for the assumption that cervical carcinoma, among other malignancies such as melanomas, renal malignancies and Kaposi sarcoma, are immunogenic is provided by the fact that these malignancies grow more rapidly in the presence of systemic immunosuppression. Spontaneous regression for these tumour types is also described and immunohistochemical studies show extensive infiltrates in the tumour, consisting of immunocompetent cells. It is thus postulated that cellular immunity, and mainly the T-cell system plays an important role in the antitumour defence in cervical carcinoma. This review describes the rationale for the use of immunotherapy as treatment for cervical carcinoma as well as the results of recent developments in tumour immunology and its implications for the clinical use of immunotherapeutical approaches.

Research paper thumbnail of Immature Dendritic Cells Acquire CD8+ Cytotoxic T Lymphocyte Priming Capacity upon Activation by T Helper Cell-independent or -dependent Stimuli

Journal of Experimental Medicine, 2000

The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC ma... more The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC maturation in CTL induction in vitro and in vivo. Maturation of D1 cells, characterized by markedly increased expression of MHC and costimulatory molecules, was induced by incubation with lipopolysaccharide, agonistic CD40 antibody, or specific CD4 ϩ T helper (Th) cells. Activated, but not immature, D1 cells efficiently primed alloreactive T cell responses in vitro. Similarly, priming of CTL immunity in vivo in CD4-depleted mice was only observed if these mice were immunized with activated D1 cells. This study provides formal evidence that activation of DCs, induced by Th-independent as well as Th-dependent stimuli, is essential for efficient induction of CTL responses.

Research paper thumbnail of Detection of T Helper Responses, But Not of Human Papillomavirus-Specific Cytotoxic T Lymphocyte Responses, After Peptide Vaccination of Patients With Cervical Carcinoma

Journal of Immunotherapy, 2000

Human papillomavirus type 16 (HPV16)-encoded E7 oncoprotein is constitutively expressed in cervic... more Human papillomavirus type 16 (HPV16)-encoded E7 oncoprotein is constitutively expressed in cervical carcinoma cells and is required for cellular transformation to be maintained. The E7 protein, therefore, forms an attractive target for T-cell-mediated immune intervention to prevent or treat HPV16+ tumors. The authors performed a peptide-based phase I/II vaccination trial to induce anti-tumor immune responses in patients with recurrent or residual cervical carcinoma. Fifteen HLA-A*0201+ patients with HPV16+ cervical carcinoma received vaccinations with synthetic peptides representing 2 HPV16 E7-encoded, HLA-A*0201-restricted cytotoxic T lymphocyte epitopes and a pan-HLA-DR-binding T-helper epitope, PADRE, in adjuvant. No signs of toxicity were observed. Two patients had stable disease for more than 1 year after vaccination, 3 patients died of the disease during or shortly after the vaccination period, and 10 patients maintained progressive cervical carcinoma. Specific immune responses directed against the vaccine components were analyzed in peripheral blood samples. No cytotoxic T lymphocyte responses against the HPV16 E7 peptides were detectable. After vaccination, strong PADRE helper peptide-specific proliferation was detected in 4 of 12 patients. In conclusion, peptide vaccination with 2 HPV16 E7 cytotoxic T lymphocyte epitopes and a universal T helper epitope is well tolerated by patients with advanced cervical carcinoma. Despite a reduction of in vitro cytolytic or proliferative recall responses to some, but not all, conventional antigens in this patient group, peptide-specific proliferative responses were induced in 4 patients. Based on the current study, it is now feasible to perform peptide vaccination in earlier stages of HPV16-induced cervical disease.

Research paper thumbnail of The HPV16 E7 peptide based vaccine trial in end stage cervical carcinoma: An interim report

Immunol Lett, 1997

melanoma cells may kill CDQ5-sensitive activated T cells. However, our data does not exclude that... more melanoma cells may kill CDQ5-sensitive activated T cells. However, our data does not exclude that other CD95 expressing cells, i.e. granulocytes or NK cells, contribute to the obsenred pool of apoptotfc cells within the tumor. In summary, CDQBL expression seem to be acquired during the progression of melanoma and is associated with substantial apoptosis in tumor infiltrating leukocytes, thereby providing another immune escape mechanism. 1 P.5.10.15 ( Plasma cell expression of CD28 and CD40 correlates wlth perlpheral T-cell defects In multiple myeloma

Research paper thumbnail of Natural T-helper immunity against human papillomavirus type 16 (hpv16) e7-derived peptide epitopes in patients with hpv16-positive cervical lesions: Identification of 3 human leukocyte antigen class ii-restricted epitopes

International Journal of Cancer, 2001

Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine... more Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine-induced immune defense against tumors. Since the majority of cervical cancers express human papillomavirus type 16 (HPV16) E7 oncoprotein, it is important to investigate the Th response against this target antigen in detail. By means of PBMC cultures from HLA-typed healthy donors, we identified the central part of HPV16 E7 (E7 41-72 ) as the major immunogenic region within this antigen. Furthermore, we mapped 3 distinct Th epitopes within this region (DR15/E7 50 -62 , DR3/ E7 43-77 , DQ2/E7 35-50 ). In a parallel approach, employing IFN-␥ ELISPOT analysis, we detected Th immunity against HPV16 E7 in subjects with HPV16 ؉ lesions. Several of these responses matched with the 3 Th epitopes defined in our study. A number of other HPV16 ؉ subjects did not display any E7-specific type 1 cytokine-producing T-cell immunity, indicating failure of the immune response. Our combined data argue for more extensive as well as longitudinal analysis of HPV16-specific T-cell immunity using the ELISPOT assay described, as well as for HPV-specific vaccination of individuals with HPV ؉ lesions.

Research paper thumbnail of Immunotherapy of cancer by peptide-based vaccines for the induction of tumor-specific T cell immunity

Immunotechnology, 1996

Recent progress in defining the molecular nature of antigens and in finding ways to manipulate T ... more Recent progress in defining the molecular nature of antigens and in finding ways to manipulate T cell-mediated immune responses may provide new modalities for cancer treatment. In this report, we review preclinical studies as well as the first clinical trials with vaccination strategies aiming at the induction of anti-tumor immunity. In particular, we focus on the development of a vaccine against human papillomavirus-induced cervical carcinoma.

Research paper thumbnail of Natural T-helper immunity against human papillomavirus type 16 (HPV16) E7-derived peptide epitopes in patients with HPV16-positive cervical lesions: identification of 3 human leukocyte antigen class II-restricted epitopes

International Journal of Cancer, Mar 1, 2001

Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine... more Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine-induced immune defense against tumors. Since the majority of cervical cancers express human papillomavirus type 16 (HPV16) E7 oncoprotein, it is important to investigate the Th response against this target antigen in detail. By means of PBMC cultures from HLA-typed healthy donors, we identified the central part of HPV16 E7 (E7 41-72 ) as the major immunogenic region within this antigen. Furthermore, we mapped 3 distinct Th epitopes within this region (DR15/E7 50 -62 , DR3/ E7 43-77 , DQ2/E7 35-50 ). In a parallel approach, employing IFN-␥ ELISPOT analysis, we detected Th immunity against HPV16 E7 in subjects with HPV16 ؉ lesions. Several of these responses matched with the 3 Th epitopes defined in our study. A number of other HPV16 ؉ subjects did not display any E7-specific type 1 cytokine-producing T-cell immunity, indicating failure of the immune response. Our combined data argue for more extensive as well as longitudinal analysis of HPV16-specific T-cell immunity using the ELISPOT assay described, as well as for HPV-specific vaccination of individuals with HPV ؉ lesions.

Research paper thumbnail of Skin reactions to human papillomavirus (HPV) 16 specific antigens intradermally injected in healthy subjects and patients with cervical neoplasia

International Journal of Cancer, 2008

We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin t... more We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin test to detect cellular immune responses to HPV16 E2, E6 and E7 in vivo. Women with cervical neoplasia (n 5 11) and healthy individuals (n 5 19) were intradermally challenged with 8 different pools of HPV16 E2, E6 and E7 peptides. The skin test was safe as the injections were perceived as mildly painful and no adverse events were observed. The majority of skin reactions appeared significantly earlier in HPV161 patients (<8 days) than in healthy subjects (8-25 days). The development of late skin reactions in healthy subjects was associated with the appearance of circulating HPV16-specific T cells and the infiltration of both HPV16-specific CD41 Th1/Th2 and CD81 T cells into the skin. These data show that the intradermal injection of pools of HPV16 synthetic long peptides is safe and results in the migration of HPV16-specific T cells into the skin as well as in an increase in the number of circulating HPV16-specific T cells. The use of this test to measure HPV16-specific immunity is currently tested in a low resource setting for the measurement of spontaneously induced T-cell responses as well as in our HPV16 vaccination trials for the detection of vaccine-induced immunity. '

Research paper thumbnail of Immune Escape of Tumors In Vivo by Expression of Cellular FLICE-inhibitory Protein

Summary The antiapoptotic protein cellular FLICE (Fas-associated death domain-like IL-1 b -conver... more Summary The antiapoptotic protein cellular FLICE (Fas-associated death domain-like IL-1 b -converting enzyme) inhibitory protein (cFLIP) protects cells from CD95(APO-1/Fas)-induced apoptosis in vitro and was found to be overexpressed in human melanomas. However, cytotoxic T cell- induced apoptosis, which is critically involved in tumor control in vivo, is not inhibited by cFLIP in vitro, as only CD95- and not perforin-dependent

Research paper thumbnail of Natural T cell response against HPV16 and development of optimal peptide based vaccine

European Journal of Cancer, 2001

Research paper thumbnail of Natural T-helper immunity against human papillomavirus type 16 (hpv16) e7-derived peptide epitopes in patients with hpv16-positive cervical lesions: Identification of 3 human leukocyte antigen class ii-restricted epitopes

International Journal of Cancer, 2001

Tumor-specificT-helper(Th)immunitywasfoundtoplay apivotalroleinthenaturalandvaccine-inducedimmune... more Tumor-specificT-helper(Th)immunitywasfoundtoplay apivotalroleinthenaturalandvaccine-inducedimmune defenseagainsttumors.Sincethemajorityofcervicalcan-cersexpresshumanpapillomavirustype16(HPV16)E7oncoprotein,itisimportanttoinvestigatetheThresponse againstthistargetantigenindetail.BymeansofPBMC culturesfromHLA-typedhealthydonors,weidentifiedthe centralpartofHPV16E7(E7 41-72 )asthemajorimmunogenicregionwithinthisantigen.Furthermore,wemapped3 distinctThepitopeswithinthisregion(DR15/E7 50-62 ,DR3/ E7 43-77 ,DQ2/E7 35-50 ).Inaparallelapproach,employing IFN-␥ELISPOTanalysis,wedetectedThimmunityagainst HPV16E7insubjectswithHPV16 ؉ lesions.Severalofthese responsesmatchedwiththe3Thepitopesdefinedinour study.AnumberofotherHPV16 ؉ subjectsdidnotdisplay anyE7-specifictype1cytokine-producingT-cellimmunity, indicatingfailureoftheimmuneresponse.Ourcombined dataargueformoreextensiveaswellaslongitudinalanalysis ofHPV16-specificT-cellimmunityusingtheELISPOTassay described,aswellasforHPV-specificvaccinationofindivid-ualswithHPV ؉ lesions.

Research paper thumbnail of Tumor-Expressed B7-H1 and B7-DC in Relation to PD-1+ T-Cell Infiltration and Survival of Patients with Cervical Carcinoma

Clinical Cancer Research, 2009

The interaction between programmed cell death 1 (PD-1), expressed by activated effector or regula... more The interaction between programmed cell death 1 (PD-1), expressed by activated effector or regulatory T cells, and B7-H1 (PD-L1) and B7-DC (PD-L2) results in the inhibition of T-cell function. The aim of this study was to determine B7-H1, B7-DC, and PD-1 expression in cervical carcinoma. A tissue microarray of a well-defined group of 115 patients was stained with antibodies against B7-H1 and B7-DC. Three-color fluorescent immunohistochemistry was used to study the number and phenotype of tumor-infiltrating T cells expressing PD-1. Additional analyses consisted of in vitro T-cell suppression assays. B7-H1 was expressed in 19%, and B7-DC was expressed by 29% of the 115 tumors. PD-1 was expressed by more than half of both the infiltrating CD8+ T cells and CD4+Foxp3+ T cells, irrespective of B7-H1 or B7-DC expression by tumors. The expression of B7-H1 did not show a direct impact on patient survival. However, subgroup analysis revealed that patients with a relative excess of infiltrating regulatory T cells displayed a better survival when the tumor was B7-H1 positive (P = 0.033). Additional studies showed that the presence of B7-H1 during the activation of CD4+Foxp3+ regulatory T cells impaired their suppressive function in a functional in vitro assay. B7-H1 is expressed on only a minority of cervical cancers and does not influence the survival of patients with cervical cancer. PD-1 is expressed by a vast number of infiltrating CD8 T cells, suggesting that blocking of PD-1 could have therapeutic potential in cervical cancer patients.

Research paper thumbnail of Human Papillomavirus Type 16-Positive Cervical Cancer Is Associated with Impaired CD4+ T-Cell Immunity against Early Antigens E2 and E6

Cancer Research, 2004

Cervical cancer is the possible outcome of genital infection with highrisk human papillomavirus (... more Cervical cancer is the possible outcome of genital infection with highrisk human papillomavirus (HPV) and is preceded by a phase of persistent HPV infection during which the host immune system fails to eliminate the virus. Fortunately, the majority of genital HPV infections are cleared before the development of (pre)malignant lesions. Analysis of CD4؉ T-helper (Th) immunity against the E2, E6, and E7 antigens of HPV16 in healthy women revealed strong proliferative E2-and E6-specific responses associated with prominent IFN-␥ and interleukin 5 secretion. This indicates that the naturally arising virus-induced immune response displays a mixed Th1/Th2 cytokine profile. Of all HPV16؉ cervical cancer patients, approximately half failed to mount a detectable immune response against the HPV16-derived peptides.

Research paper thumbnail of Phase I Immunotherapeutic Trial with Long Peptides Spanning the E6 and E7 Sequences of High-Risk Human Papillomavirus 16 in End-Stage Cervical Cancer Patients Shows Low Toxicity and Robust Immunogenicity

Clinical Cancer Research, 2008

Purpose: To determine the toxicity, safety, and immunogenicity of a human papillomavirus 16 (HPV1... more Purpose: To determine the toxicity, safety, and immunogenicity of a human papillomavirus 16 (HPV16) E6 and E7 long peptide vaccine administered to end-stage cervical cancer patients. Experimental Design:Three groups of end-stage cervical cancer patients (in total n =35)were s.c. vaccinated with HPV16 E6 combined with or separated from HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant, four times at 3-week intervals. Group 1 received 300 Ag/ peptide at a single site and group 2 received 100 Ag/peptide of the E6 peptides in one limb and 300 Ag/peptide of the E7 peptides in a second limb. Group 3 received separate injections of E6 and E7 peptides, each at a dose of 50 Ag/peptide. The primary end point was to determine safety and toxicity of the HPV16 long peptides vaccine. In addition, the vaccine-induced T-cell response was assessed by IFNg enzyme-linked immunospot. Results: No toxicity beyond grade 2 was observed during and after four vaccinations. In a few patients, transient flu-like symptoms were observed. Enzyme-linked immunospot analysis of the vaccine-induced immune response revealed that coinjection of the E6 and E7 peptides resulted in a strong and broad T-cell response dominated by immunity against E6. Injection of the E6 and E7 peptides at two different sites increased the E7 response but did not affect the magnitude of the E6-induced immune response. Conclusions: The HPV16 E6 and E7 long peptide-based vaccine is well tolerated and capable of inducing a broad IFNg-associated T-cell response even in end-stage cervical cancer patients.