RESUMO
In this work, we have generated novel Fc-comprising NK cell engagers (NKCEs) that bridge human NKp30 on NK cells to human epidermal growth factor receptor (EGFR) on tumor cells. Camelid-derived VHH single-domain Abs specific for human NKp30 and a humanized Fab derived from the EGFR-specific therapeutic Ab cetuximab were used as binding arms. By combining camelid immunization with yeast surface display, we were able to isolate a diverse panel of NKp30-specific VHHs against different epitopes on NKp30. Intriguingly, NKCEs built with VHHs that compete for binding to NKp30 with B7-H6, the natural ligand of NKp30, were significantly more potent in eliciting tumor cell lysis of EGFR-positive tumor cells than NKCEs harboring VHHs that target different epitopes on NKp30 from B7-H6. We demonstrate that the NKCEs can be further improved with respect to killing capabilities by concomitant engagement of FcγRIIIa and that soluble B7-H6 does not impede cytolytic capacities of all scrutinized NKCEs at significantly higher B7-H6 concentrations than observed in cancer patients. Moreover, we show that physiological processes requiring interactions between membrane-bound B7-H6 and NKp30 on NK cells are unaffected by noncompeting NKCEs still eliciting tumor cell killing at low picomolar concentrations. Ultimately, the NKCEs generated in this study were significantly more potent in eliciting NK cell-mediated tumor cell lysis than cetuximab and elicited a robust release of proinflammatory cytokines, both features which might be beneficial for antitumor therapy.
Assuntos
Citocinas , Receptor 3 Desencadeador da Citotoxicidade Natural , Humanos , Antígenos B7/metabolismo , Morte Celular , Cetuximab/farmacologia , Epitopos , Receptores ErbB , Células Matadoras Naturais , Ligantes , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismoRESUMO
Activating NK cell receptors represent promising target structures to elicit potent antitumor immune responses. In this study, novel immunoligands were generated that bridge the activating NK cell receptor NKp30 on NK cells with epidermal growth factor receptor (EGFR) on tumor cells in a bispecific IgG-like format based on affinity-optimized versions of B7-H6 and the Fab arm derived from cetuximab. To enhance NKp30 binding, the solitary N-terminal IgV domain of B7-H6 (ΔB7-H6) was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel ΔB7-H6-derived NK cell engagers revealed an up to 45-fold-enhanced affinity for NKp30 and significantly improved NK cell-mediated, EGFR-dependent killing of tumor cells compared with the NK cell engager based on the wild-type ΔB7-H6 domain. In this regard, potencies (EC50 killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of IFN-γ and TNF-α was significantly increased. Importantly, equipment of the ΔB7-H6-based NK cell engagers with a human IgG1 Fc part competent in Fc receptor binding resulted in an almost 10-fold superior killing of EGFR-overexpressing tumor cells compared with molecules either triggering FcγRIIIa or NKp30. Additionally, INF-γ and TNF-α release was increased compared with molecules solely triggering FcγRIIIa, including the clinically approved Ab cetuximab. Thus, incorporating affinity-matured ligands for NK cell-activating receptors might represent an effective strategy for the generation of potent novel therapeutic agents with unique effector functions in cancer immunotherapy.
Assuntos
Antígenos B7/metabolismo , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Neoplasias/imunologia , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/metabolismo , Antígenos B7/genética , Linhagem Celular Tumoral , Cetuximab/genética , Citocinas/metabolismo , Citotoxicidade Imunológica , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Engenharia Genética , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Mediadores da Inflamação/metabolismo , Células Matadoras Naturais/transplante , Ativação Linfocitária , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Neoplasias/terapia , Ligação Proteica , Transdução de SinaisRESUMO
Actinic keratosis (AK) are common lesions in light-skinned individuals that can potentially progress to cutaneous squamous cell carcinoma (cSCC). Both conditions may be associated with significant morbidity and constitute a major disease burden, especially among the elderly. To establish an evidence-based framework for clinical decision making, the guideline "actinic keratosis and cutaneous squamous cell carcinoma" was updated and expanded by the topics cutaneous squamous cell carcinoma in situ (Bowen's disease) and actinic cheilitis. The guideline is aimed at dermatologists, general practitioners, ear nose and throat specialists, surgeons, oncologists, radiologists and radiation oncologists in hospitals and office-based settings, as well as other medical specialties, policy makers and insurance funds involved in the diagnosis and treatment of patients with AK and cSCC. A separate guideline exists for patients and their relatives. In this part, we will address aspects relating to epidemiology and etiology, diagnostics, surgical and systemic treatment of cutaneous squamous cell carcinoma (cSCC), surveillance and prevention.
Assuntos
Doença de Bowen , Carcinoma de Células Escamosas , Ceratose Actínica , Neoplasias Cutâneas , Humanos , Idoso , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/epidemiologia , Carcinoma de Células Escamosas/prevenção & controle , Ceratose Actínica/diagnóstico , Ceratose Actínica/epidemiologia , Ceratose Actínica/prevenção & controle , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/prevenção & controle , Doença de Bowen/diagnóstico , Pele/patologiaRESUMO
Despite several therapeutic advances, patients with multiple myeloma (MM) require additional treatment options since no curative therapy exists yet. In search of a novel therapeutic antibody, we previously applied phage display with myeloma cell screening and developed TP15, a scFv targeting intercellular adhesion molecule 1 (ICAM-1/CD54). To more precisely evaluate the antibody's modes of action, fully human IgG1 antibody variants were generated bearing wild-type (MSH-TP15) or mutated Fc to either enhance (MSH-TP15 Fc-eng.) or prevent (MSH-TP15 Fc k.o.) Fc gamma receptor binding. Especially MSH-TP15 Fc-eng. induced potent antibody-dependent cell-mediated cytotoxicity (ADCC) against malignant plasma cells by efficiently recruiting NK cells and engaged macrophages for antibody-dependent cellular phagocytosis (ADCP) of tumor cells. Binding studies with truncated ICAM-1 demonstrated MSH-TP15 binding to ICAM-1 domain 1-2. Importantly, MSH-TP15 and MSH-TP15 Fc-eng. both prevented myeloma cell engraftment and significantly prolonged survival of mice in an intraperitoneal xenograft model. In the subcutaneous model MSH-TP15 Fc-eng. was superior to MSH-TP15, whereas MSH-TP15 Fc k.o. was not effective in both models - reflecting the importance of Fc-dependent mechanisms of action also in vivo. The efficient recruitment of immune cells and the potent anti-tumor activity of the Fc-engineered MSH-TP15 antibody hold significant potential for myeloma immunotherapy.
Assuntos
Mieloma Múltiplo , Animais , Humanos , Camundongos , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Imunoglobulina G , Molécula 1 de Adesão Intercelular/genética , Mieloma Múltiplo/tratamento farmacológico , Receptores de IgG/genéticaRESUMO
BACKGROUND: Psoriasis is an immune-mediated disease with a genetic background. Local psoriatic changes can be triggered by exogenous mechanical or irritant factors. Causative occupational factors have to be distinguished from the spontaneous course of psoriasis in occupational medical evaluations. The objective of this work is to demonstrate the medico-legal grounds for a homogenous assessment. PATIENTS AND METHODS: The presented recommendations were developed in a working group for Occupational and Environmental Dermatology (ABD) and the German contact dermatitis group (DKG) of the German Dermatologic Society (DDG) based on the German medico-legal framework. RESULTS: Causality between the insured activity and the appearance of psoriasis is a prerequisite for the recognition of the occupational nature of the disease. This is the case if the occupational activity is the exclusive or a legally essential contributing factor either for first manifestation or aggravation of the disease. A connection must be denied if everyday events are sufficient to trigger the psoriasis. From 1995 to 2010, 130 cases of psoriasis have been recognized as occupationally related by the German statutory accident insurance. CONCLUSIONS: The appraisal of psoriatic disease in the occupational medical evaluation is subject to the case assessment of the expert witness. In this position paper we present recommendations for a homologous basis for diagnosis, causality assessment, estimation of reduction in earning capacity and rehabilitation of occupational psoriasis.
Assuntos
Dermatite Ocupacional/diagnóstico , Dermatite Ocupacional/genética , Prova Pericial/legislação & jurisprudência , Dermatoses da Mão/diagnóstico , Dermatoses da Mão/genética , Psoríase/diagnóstico , Psoríase/genética , Algoritmos , Avaliação da Deficiência , Progressão da Doença , Predisposição Genética para Doença , Alemanha , Humanos , Seguro de Acidentes/legislação & jurisprudência , Exposição Ocupacional/efeitos adversos , Exposição Ocupacional/legislação & jurisprudência , Avaliação da Capacidade de Trabalho , Indenização aos Trabalhadores/legislação & jurisprudênciaRESUMO
In Germany over 2.5 million employees have an increased risk of skin cancer due to their occupational exposure to natural UV-irradiation. The medical consultation board "Occupational diseases" of the Ministry of Labor and Social affairs has investigated the association between occupational UV-irradiation and skin cancer risk and recommends to add the following new occupational disease into the appendix1 of the German ordinance on occupational diseases: "Squamous cell carcinoma and multiple actinic keratosis due to natural UV-irradiation". In this article we report in the view of the German Society of Occupational and Environmental Dermatology (ABD) and the German Statutory accident insurance (DGUV), whose criteria have to be fulfilled for the notification and recognition of an occupational skin cancer due to natural UV-irradiation.
Assuntos
Dermatologia/normas , Doenças Profissionais/diagnóstico , Doenças Profissionais/etiologia , Medicina do Trabalho/normas , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/etiologia , Raios Ultravioleta/efeitos adversos , Alemanha , Guias como Assunto , Humanos , Notificação de Abuso , Doenças Profissionais/classificação , Neoplasias Cutâneas/classificação , Luz Solar/efeitos adversosRESUMO
Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.
Assuntos
Anticorpos Monoclonais , Receptores de IgG , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Animais , Camundongos , Humanos , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Imunização , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/genética , Biblioteca de Peptídeos , Técnicas de Visualização da Superfície Celular , Hibridomas , Especificidade de Anticorpos , Feminino , Camundongos Endogâmicos BALB CRESUMO
Natural killer (NK) cells emerged as a promising effector population that can be harnessed for anti-tumor therapy. In this work, we constructed NK cell engagers (NKCEs) based on NKp30-targeting single domain antibodies (sdAbs) that redirect the cytotoxic potential of NK cells toward epidermal growth factor receptor (EGFR)-expressing tumor cells. We investigated the impact of crucial parameters such as sdAb location, binding valencies, the targeted epitope on NKp30, and the overall antibody architecture on the redirection capacity. Our study exploited two NKp30-specific sdAbs, one of which binds a similar epitope on NKp30 as its natural ligand B7-H6, while the other sdAb addresses a non-competing epitope. For EGFR-positive tumor targeting, humanized antigen-binding domains of therapeutic antibody cetuximab were used. We demonstrate that NKCEs bivalently targeting EGFR and bivalently engaging NKp30 are superior to monovalent NKCEs in promoting NK cell-mediated tumor cell lysis and that the architecture of the NKCE can substantially influence killing capacities depending on the NKp30-targeting sdAb utilized. While having a pronounced impact on NK cell killing efficacy, the capabilities of triggering antibody-dependent cellular phagocytosis or complement-dependent cytotoxicity were not significantly affected comparing the bivalent IgG-like NKCEs with cetuximab. However, the fusion of sdAbs can have a slight impact on the NK cell release of immunomodulatory cytokines, as well as on the pharmacokinetic profile of the NKCE due to unfavorable spatial orientation within the molecule architecture. Ultimately, our findings reveal novel insights for the engineering of potent NKCEs triggering the NKp30 axis.
Assuntos
Fator de Crescimento Epidérmico , Células Matadoras Naturais , Cetuximab/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Sítios de Ligação de Anticorpos , Receptores ErbB/metabolismo , Epitopos/metabolismoRESUMO
The majority of therapeutic antibodies, bispecific antibodies, and chimeric antigen receptor (CAR) T cells in cancer therapy are based on an antibody or antibody fragment that specifically binds a target present on the surface of a tumor cell. Suitable antigens that can be used for immunotherapy are ideally tumor-specific or tumor-associated and stably expressed on the tumor cell. The identification of new target structures to further optimize immunotherapies could be realized by comparing healthy and tumor cells using "omics" methods to select promising proteins. However, differences in post-translational modifications and structural alterations that can be present on the tumor cell surface are difficult to identify or even not accessible by these techniques. In this chapter, we describe an alternative approach to potentially identify antibodies targeting novel tumor-associated antigens (TAA) or epitopes by using cellular screening and phage display of antibody libraries. Isolated antibody fragments can be further converted into chimeric IgG or other antibody formats to investigate the anti-tumor effector functions and finally identify and characterize the respective antigen.
Assuntos
Bacteriófagos , Neoplasias , Humanos , Antígenos de Superfície , Biblioteca de Peptídeos , Neoplasias/terapia , Antígenos , Antígenos de NeoplasiasRESUMO
The activating receptor natural killer group 2, member D (NKG2D) represents an attractive target for immunotherapy as it exerts a crucial role in cancer immunosurveillance by regulating the activity of cytotoxic lymphocytes. In this study, a panel of novel NKG2D-specific single-chain fragments variable (scFv) were isolated from naïve human antibody gene libraries and fused to the fragment antigen binding (Fab) of rituximab to obtain [CD20×NKG2D] bibodies with the aim to recruit cytotoxic lymphocytes to lymphoma cells. All bispecific antibodies bound both antigens simultaneously. Two bibody constructs, [CD20×NKG2D#3] and [CD20×NKG2D#32], efficiently activated natural killer (NK) cells in co-cultures with CD20+ lymphoma cells. Both bibodies triggered NK cell-mediated lysis of lymphoma cells and especially enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by CD38 or CD19 specific monoclonal antibodies suggesting a synergistic effect between NKG2D and FcγRIIIA signaling pathways in NK cell activation. The [CD20×NKG2D] bibodies were not effective in redirecting CD8+ T cells as single agents, but enhanced cytotoxicity when combined with a bispecific [CD19×CD3] T cell engager, indicating that NKG2D signaling also supports CD3-mediated T cell activation. In conclusion, engagement of NKG2D with bispecific antibodies is attractive to directly activate cytotoxic lymphocytes or to support their activation by monoclonal antibodies or bispecific T cell engagers. As a perspective, co-targeting of two tumor antigens may allow fine-tuning of antibody cancer therapies. Our proposed combinatorial approach is potentially applicable for many existing immunotherapies but further testing in different preclinical models is necessary to explore the full potential.
Assuntos
Anticorpos Biespecíficos , Linfoma , Neoplasias , Humanos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Células Matadoras Naturais , Linfoma/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/metabolismo , Antígenos CD19RESUMO
P8-D6 is a novel dual inhibitor of human topoisomerase I (TOP1) and II (TOP2) with broad pro-apoptotic antitumor activity. NCI-60 screening revealed markedly improved cytotoxicity of P8-D6 against solid and leukemia cell lines compared with other single and dual topoisomerase inhibitors, for example, irinotecan, doxorubicin, or pyrazoloacridine. In this study, we investigated the capacity of P8-D6 to inhibit myeloma cell growth in vitro and in vivo Growth inhibition assays demonstrated significant anti-myeloma effects against different myeloma cell lines with IC50 values in the low nanomolar range. Freshly isolated plasma cells of patients with multiple myeloma were killed by P8-D6 with similar doses. P8-D6 activated caspase 3/7 and induced significant apoptosis of myeloma cells. Supportive effects of bone marrow stromal cells on IL6-dependent INA-6 myeloma cells were abrogated by P8-D6 and apoptosis occurred in a time- and dose-dependent manner. Of note, healthy donor peripheral blood mononuclear cells and human umbilical vein endothelial cells were not affected at concentrations toxic for malignant plasma cells. Treatment of myeloma xenografts in immunodeficient SCID/beige mice by intravenous and, notably, also oral application of P8-D6 markedly inhibited tumor growths, and significantly prolonged survival of tumor-bearing mice.
Assuntos
Mieloma Múltiplo/tratamento farmacológico , Naftalenos/uso terapêutico , Inibidores da Topoisomerase II/uso terapêutico , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Mieloma Múltiplo/patologia , Naftalenos/farmacologia , Inibidores da Topoisomerase II/farmacologiaRESUMO
To identify new antibodies for the treatment of plasma cell disorders including multiple myeloma (MM), a single-chain Fragment variable (scFv) antibody library was generated by immunizing mice with patient-derived malignant plasma cells. To enrich antibodies binding myeloma antigens, phage display with cellular panning was performed. After depleting the immune library with leukocytes of healthy donors, selection of antibodies was done with L-363 plasma cell line in two consecutive panning rounds. Monitoring the antibodies' enrichment throughout the panning by next-generation sequencing (NGS) identified several promising candidates. Initially, 41 unique scFv antibodies evolving from different B cell clones were selected. Nine of these antibodies strongly binding to myeloma cells and weakly binding to peripheral blood mononuclear cells (PBMC) were characterized. Using stably transfected Chinese hamster ovary cells expressing individual myeloma-associated antigens revealed that two antibodies bind CD38 and intercellular adhesion molecule-1 (ICAM-1), respectively, and 7 antibodies target yet unknown antigens. To evaluate the therapeutic potential of our new antibodies, in a first proof-of-concept study the CD38 binding scFv phage antibody was converted into a chimeric IgG1. Further analyses revealed that #5-CD38-IgG1 shared an overlapping epitope with daratumumab and isatuximab and had potent anti-myeloma activity comparable to the two clinically approved CD38 antibodies. These results indicate that by phage display and deep sequencing, new antibodies with therapeutic potential for MM immunotherapy can be identified.
Assuntos
Bacteriófagos , Plasmócitos , Animais , Células CHO , Cricetinae , Cricetulus , Sequenciamento de Nucleotídeos em Larga Escala , Imunoglobulina G , Fatores Imunológicos , Imunoterapia , Leucócitos Mononucleares , Camundongos , Biblioteca de PeptídeosRESUMO
Targeting CD19 represents a promising strategy for the therapy of B-cell malignancies. Although non-engineered CD19 antibodies are poorly effective in mediating complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), these effector functions can be enhanced by Fc-engineering. Here, we engineered a CD19 antibody with the aim to improve effector cell-mediated killing and CDC activity by exchanging selected amino acid residues in the Fc domain. Based on the clinically approved Fc-optimized antibody tafasitamab, which triggers enhanced ADCC and ADCP due to two amino acid exchanges in the Fc domain (S239D/I332E), we additionally added the E345K amino acid exchange to favor antibody hexamerization on the target cell surface resulting in improved CDC. The dual engineered CD19-DEK antibody bound CD19 and Fcγ receptors with similar characteristics as the parental CD19-DE antibody. Both antibodies were similarly efficient in mediating ADCC and ADCP but only the dual optimized antibody was able to trigger complement deposition on target cells and effective CDC. Our data provide evidence that from a technical perspective selected Fc-enhancing mutations can be combined (S239D/I332E and E345K) allowing the enhancement of ADCC, ADCP and CDC with isolated effector populations. Interestingly, under more physiological conditions when the complement system and FcR-positive effector cells are available as effector source, strong complement deposition negatively impacts FcR engagement. Both effector functions were simultaneously active only at selected antibody concentrations. Dual Fc-optimized antibodies may represent a strategy to further improve CD19-directed cancer immunotherapy. In general, our results can help in guiding optimal antibody engineering strategies to optimize antibodies' effector functions.