ABSTRACT
Vaccination of patients against neoantigens expressed in concurrent tumors, recurrent tumors, or tumors developing in individuals at risk of cancer is posing major challenges in terms of which antigens to target and is limited to patients expressing neoantigens in their tumors. Here, we describe a vaccination strategy against antigens that were induced in tumor cells by downregulation of the peptide transporter associated with antigen processing (TAP). Vaccination against TAP downregulation-induced antigens was more effective than vaccination against mutation-derived neoantigens, was devoid of measurable toxicity, and inhibited the growth of concurrent and future tumors in models of recurrence and premalignant disease. Human CD8+ T cells stimulated with TAPlow dendritic cells elicited a polyclonal T-cell response that recognized tumor cells with experimentally reduced TAP expression. Vaccination against TAP downregulation-induced antigens overcomes the main limitations of vaccinating against mostly unique tumor-resident neoantigens and could represent a simpler vaccination strategy that will be applicable to most patients with cancer.
Subject(s)
Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/administration & dosage , Neoplasm Recurrence, Local/therapy , Neoplasms/therapy , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/immunology , Animals , Antigen Presentation/immunology , Cancer Vaccines/immunology , Cell Line, Tumor , Disease Models, Animal , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasm Recurrence, Local/immunology , Neoplasms/immunology , RNA, Small Interfering/geneticsABSTRACT
Neoantigen burden is a major determinant of tumor immunogenicity, underscored by recent clinical experience with checkpoint blockade therapy. Yet the majority of patients do not express, or express too few, neoantigens, and hence are less responsive to immune therapy. Here we describe an approach whereby a common set of new antigens are induced in tumor cells in situ by transient downregulation of the transporter associated with antigen processing (TAP). Administration of TAP siRNA conjugated to a broad-range tumor-targeting nucleolin aptamer inhibited tumor growth in multiple tumor models without measurable toxicity, was comparatively effective to vaccination against prototypic mutation-generated neoantigens, potentiated the antitumor effect of PD-1 antibody or Flt3 ligand, and induced the presentation of a TAP-independent peptide in human tumor cells. Treatment with the chemically-synthesized nucleolin aptamer-TAP siRNA conjugate represents a broadly-applicable approach to increase the antigenicity of tumor lesions and thereby enhance the effectiveness of immune potentiating therapies.
Subject(s)
ATP-Binding Cassette Transporters/immunology , Antigens, Neoplasm/immunology , Immunotherapy , Neoplasms/immunology , Neoplasms/prevention & control , ATP-Binding Cassette Transporters/genetics , Animals , Antigens, Neoplasm/genetics , Aptamers, Nucleotide , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Cancer Vaccines , Cell Line, Tumor , Down-Regulation , Epitopes/immunology , Female , Humans , Immunization , Immunogenicity, Vaccine , Male , Membrane Proteins/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Targeted Therapy , NIH 3T3 Cells , Neoplasms, Experimental , Oligodeoxyribonucleotides , Phosphoproteins , Programmed Cell Death 1 Receptor/immunology , RNA, Small Interfering/administration & dosage , RNA-Binding Proteins , Spleen/immunology , Spleen/pathology , Vaccination , NucleolinABSTRACT
Defining how epidermal growth factor receptor (EGFR)-targeting therapies influence the immune response is essential to increase their clinical efficacy. A growing emphasis is being placed on immune regulator genes that govern tumor - T cell interactions. Previous studies showed an increase in HLA class I cell surface expression in tumor cell lines treated with anti-EGFR agents. In particular, earlier studies of the anti-EGFR blocking antibody cetuximab, have suggested that increased tumor expression of HLA class I is associated with positive clinical response. We investigated the effect of another commercially available anti-EGFR antibody nimotuzumab on HLA class I expression in tumor cell lines. We observed, for the first time, that nimotuzumab increases HLA class I expression and its effect is associated with a coordinated increase in mRNA levels of the principal antigen processing and presentation components. Moreover, using 7A7 (a specific surrogate antibody against murine EGFR), we obtained results suggesting the importance of the increased MHC-I expression induced by EGFR-targeted therapies display higher in antitumor immune response. 7A7 therapy induced upregulation of tumor MHC-I expression in vivo and tumors treated with this antibody display higher susceptibility to CD8+ T cells-mediated lysis. Our results represent the first evidence suggesting the importance of the adaptive immunity in nimotuzumab-mediated antitumor activity. More experiments should be conducted in order to elucidate the relevance of this mechanism in cancer patients. This novel immune-related antitumor mechanism mediated by nimotuzumab opens new perspectives for its combination with various immunotherapeutic agents and cancer vaccines.
ABSTRACT
Searching for biomarkers that associated with the acquired resistance of malignant cells to epidermal growth factor receptor (EGFR)-targeting monoclonal antibodies is crucial to improve the clinical benefits of these therapeutic agents. We have recently demonstrated that molecular alterations in both oncogenic and immunological pathways may be responsible for such an insensitivity. Our findings suggest that a combination of targeted anticancer agents and immunomodulatory drugs may be useful for overcoming the acquired resistance of cancer cells to EGFR-specific monoclonal antibodies.
ABSTRACT
Despite promising results in the use of anti-epidermal growth factor receptor (EGFR) Abs for cancer therapy, several issues remain to be addressed. An increasing emphasis is being placed on immune effector mechanisms. It has become clear for other Abs directed to tumor targets that their effects involve the adaptive immunity, mainly by the contribution of Fc region-mediated mechanisms. Given the relevance of EGFR signaling for tumor biology, we wonder whether the oncogene inhibition could contribute to Ab-induced vaccine effect. In a mouse model in which 7A7 (an anti-murine EGFR Ab) and AG1478 (an EGFR-tyrosine kinase inhibitor) displayed potent antimetastatic activities, depletion experiments revealed that only in the case of the Ab, the effect was dependent on CD4(+) and CD8(+) T cells. Correspondingly, 7A7 administration elicited a remarkable tumor-specific CTL response in hosts. Importantly, experiments using 7A7 F(ab')(2) suggested that in vivo Ab-mediated EGFR blockade may play an important role in the linkage with adaptive immunity. Addressing the possible mechanism involved in this effect, we found quantitative and qualitative differences between 7A7 and AG1478-induced apoptosis. EGFR blocking by 7A7 not only prompted a higher proapoptotic effect on tumor metastases compared with AG1478, but also was able to induce apoptosis with immunogenic potential in an Fc-independent manner. As expected, 7A7 but not AG1478 stimulated exposure of danger signals on tumor cells. Subcutaneous injection of 7A7-treated tumor cells induced an antitumor immune response. This is the first report, to our knowledge, of a tumor-specific CTL response generated by Ab-mediated EGFR inhibition, suggesting an important contribution of immunogenic apoptosis to this effect.
Subject(s)
Antibodies, Monoclonal/physiology , Antibody Specificity/physiology , Apoptosis/immunology , Carcinoma, Lewis Lung/immunology , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/immunology , Animals , Antibodies, Blocking/physiology , Carcinoma, Lewis Lung/pathology , Carcinoma, Lewis Lung/secondary , Cell Line, Tumor , Cells, Cultured , ErbB Receptors/metabolism , Female , Immunoglobulin Fc Fragments/physiology , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Melanoma, Experimental/prevention & control , Mice , Mice, Inbred C57BL , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , T-Lymphocytes, Cytotoxic/pathologyABSTRACT
Nimotuzumab is an EGFR-targeting antibody that has demonstrated encouraging clinical results in the absence of severe side-effects observed with other approved anti-EGFR antibodies. We investigated whether different clinical behavior of nimotuzumab is related to its bivalent/monovalent binding profile. Binding properties of nimotuzumab and cetuximab, the most development of anti-EGFR antibodies, were studied in vitro using chip surfaces and cells with varying EGFR expression levels. Experimental observations demonstrated that in contrast to cetuximab, the intrinsic properties of nimotuzumab required bivalent binding for stable attachment to the cellular surface, leading to nimotuzumab selectively binding to cells that express moderate to high EGFR expression levels. At these conditions, both antibodies bound bivalently, and accumulated to similar degrees. When EGFR density is low, nimotuzumab monovalent interaction was transient, whereas cetuximab continued to interact strongly with the receptors. We compared the in vitro anti-tumor efficacy of nimotuzumab and cetuximab. Cetuximab decreased the cell viability and induced apoptosis for all the tested cell lines, effects which did not depend on EGFR expression level. In contrast, nimotuzumab also provoked significant anti-cellular effects, but its anti-tumor capacity decreased together with EGFR expression level. Cetuximab Fab fragment was able to impact tumor cell survival, whereas nimotuzumab fragment totally lost this effect. Tumor-xenograft experiments using cells with a high EGFR expression revealed similar tumor growth inhibiting effects for both antibodies. This study suggests an explanation for nimotuzumab clinical profile, whereby anti-tumor activity is obtained in absence of severe toxicities due to its properties of bivalent binding to EGFR.
Subject(s)
Antibodies, Monoclonal , Antibody Affinity/immunology , Neoplasms/drug therapy , Neoplasms/genetics , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Cetuximab , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , ErbB Receptors/metabolism , Gene Expression Regulation, Neoplastic/genetics , Humans , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin Fab Fragments/pharmacology , Immunoglobulin Fab Fragments/therapeutic use , Mice , Mice, SCID , Neoplasms/metabolism , Protein Binding , Xenograft Model Antitumor AssaysABSTRACT
Overexpression of the epidermal growth factor (EGF) receptor (EGFR) in cancer cells correlates with tumor malignancy and poor prognosis for cancer patients. For this reason, the EGFR has become one of the main targets of anticancer therapies. Structural data obtained in the last few years have revealed the molecular mechanism for ligand-induced EGFR dimerization and subsequent signal transduction, and also how this signal is blocked by either monoclonal antibodies or small molecules. Nimotuzumab (also known as h-R3) is a humanized antibody that targets the EGFR and has been successful in the clinics. In this work, we report the crystal structure of the Fab fragment of Nimotuzumab, revealing some unique structural features in the heavy variable domain. Furthermore, competition assays show that Nimotuzumab binds to domain III of the extracellular region of the EGFR, within an area that overlaps with both the surface patch recognized by Cetuximab (another anti-EGFR antibody) and the binding site for EGF. A computer model of the Nimotuzumab-EGFR complex, constructed by docking and molecular dynamics simulations and supported by mutagenesis studies, unveils a novel mechanism of action, with Nimotuzumab blocking EGF binding while still allowing the receptor to adopt its active conformation, hence warranting a basal level of signaling.