RESUMEN
PURPOSE: FAP is a membrane-bound protease under investigation as a pan-cancer target, given its high levels in tumors but limited expression in normal tissues. FAP-2286 is a radiopharmaceutical in clinical development for solid tumors that consists of two functional elements: a FAP-targeting peptide and a chelator used to attach radioisotopes. Preclinically, we evaluated the immune modulation and anti-tumor efficacy of FAP-2287, a murine surrogate for FAP-2286, conjugated to the radionuclide lutetium-177 (177Lu) as a monotherapy and in combination with a PD-1 targeting antibody. METHODS: C57BL/6 mice bearing MCA205 mouse FAP-expressing tumors (MCA205-mFAP) were treated with 177Lu-FAP-2287, anti-PD-1, or both. Tumor uptake of 177Lu- FAP-2287 was assessed by SPECT/CT scanning, while therapeutic efficacy was measured by tumor volume and survival. Immune profiling of tumor infiltrates was evaluated through flow cytometry, RNA expression, and immunohistochemistry analyses. RESULTS: 177Lu-FAP-2287 rapidly accumulated in MCA205-mFAP tumors leading to significant tumor growth inhibition (TGI) and longer survival time. Significant TGI was also observed from anti-PD-1 and the combination. In flow cytometry analysis of tumors, 177Lu-FAP-2287 increased CD8+ T cell infiltration which was maintained in the combination with anti-PD-1. The increase in CD8+ T cells was accompanied by an induction of STING-mediated type I interferon response and higher levels of co-stimulatory molecules such as CD86. CONCLUSION: In a preclinical model, FAP-targeted radiotherapy enhanced anti-PD-1-mediated TGI by modulating the TME and increasing the recruitment of tumor-infiltrating CD8+ T cells. These findings provide a rationale for clinical studies of combined 177Lu-FAP-2286 radiotherapy and immune checkpoint inhibition in FAP-positive tumors.
Asunto(s)
Linfocitos T CD8-positivos , Inhibidores de Puntos de Control Inmunológico , Animales , Ratones , Microambiente Tumoral , Línea Celular Tumoral , Ratones Endogámicos C57BL , FibroblastosRESUMEN
PURPOSE: Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor microenvironment of many solid cancers. FAP-2286 is a FAP-binding peptide coupled to a radionuclide chelator that is currently being investigated in patients as an imaging and therapeutic agent. The potency, selectivity, and efficacy of FAP-2286 were evaluated in preclinical studies. METHODS: FAP expression analysis was performed by immunohistochemistry and autoradiography on primary human cancer specimens. FAP-2286 was assessed in biochemical and cellular assays and in in vivo imaging and efficacy studies, and was further evaluated against FAPI-46, a small molecule-based FAP-targeting agent. RESULTS: Immunohistochemistry confirmed elevated levels of FAP expression in multiple tumor types including pancreatic, breast, and sarcoma, which correlated with FAP binding by FAP-2286 autoradiography. FAP-2286 and its metal complexes demonstrated high affinity to FAP recombinant protein and cell surface FAP expressed on fibroblasts. Biodistribution studies in mice showed rapid and persistent uptake of 68Ga-FAP-2286, 111In-FAP-2286, and 177Lu-FAP-2286 in FAP-positive tumors, with renal clearance and minimal uptake in normal tissues. 177Lu-FAP-2286 exhibited antitumor activity in FAP-expressing HEK293 tumors and sarcoma patient-derived xenografts, with no significant weight loss. In addition, FAP-2286 maintained longer tumor retention and suppression in comparison to FAPI-46. CONCLUSION: In preclinical models, radiolabeled FAP-2286 demonstrated high tumor uptake and retention, as well as potent efficacy in FAP-positive tumors. These results support clinical development of 68Ga-FAP-2286 for imaging and 177Lu-FAP-2286 for therapeutic use in a broad spectrum of FAP-positive tumors.
Asunto(s)
Radioisótopos de Galio , Sarcoma , Adulto , Animales , Línea Celular Tumoral , Fibroblastos , Células HEK293 , Humanos , Ratones , Cintigrafía , Distribución Tisular , Microambiente TumoralRESUMEN
The membrane protein carbonic anhydrase IX (CAIX) is highly expressed in many hypoxic or von Hippel-Lindau tumor suppressor-mutated tumor types. Its restricted expression in healthy tissues makes CAIX an attractive diagnostic and therapeutic target. DPI-4452 is a CAIX-targeting cyclic peptide with a DOTA cage, allowing radionuclide chelation for theranostic purposes. Here, we report CAIX expression in multiple tumor types and provide in vitro and in vivo evaluations of 68Ga-labeled DPI-4452 ([68Ga]Ga-DPI-4452) and 177Lu-labeled DPI-4452 ([177Lu]Lu-DPI-4452). Methods: CAIX expression was assessed by immunohistochemistry with a panel of tumor and healthy tissues. The molecular interactions of complexed and uncomplexed DPI-4452 with CAIX were assessed by surface plasmon resonance and cell-binding assays. In vivo characterization of radiolabeled and nonradiolabeled DPI-4452 was performed in HT-29 colorectal cancer (CRC) and SK-RC-52 clear cell renal cell carcinoma (ccRCC) human xenograft mouse models and in healthy beagle dogs. Results: Overexpression of CAIX was shown in several tumor types, including ccRCC, CRC, and pancreatic ductal adenocarcinoma. DPI-4452 specifically and selectively bound CAIX with subnanomolar affinity. In cell-binding assays, DPI-4452 displayed comparably high affinities for human and canine CAIX but a much lower affinity for murine CAIX, demonstrating that the dog is a relevant species for biodistribution studies. DPI-4452 was rapidly eliminated from the systemic circulation of beagle dogs. The highest uptake of [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 was observed in the small intestine and stomach, 2 organs known to express CAIX. Uptake in other organs (e.g., kidneys) was remarkably low. In HT-29 and SK-RC-52 xenograft mouse models, both [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 showed tumor-selective uptake; in addition, [177Lu]Lu-DPI-4452 significantly reduced tumor growth. These results demonstrated the theranostic potential of DPI-4452. Conclusion: DPI-4452 selectively targets CAIX. [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 localized to tumors and were well tolerated in mice. [177Lu]Lu-DPI-4452 demonstrated strong tumor growth inhibition in 2 xenograft mouse models. Thus, the 2 agents potentially provide a theranostic approach for selecting and treating patients with CAIX-expressing tumors such as ccRCC, CRC, and pancreatic ductal adenocarcinoma.
Asunto(s)
Anhidrasa Carbónica IX , Radioisótopos de Galio , Lutecio , Radioisótopos , Anhidrasa Carbónica IX/metabolismo , Humanos , Animales , Ratones , Radioisótopos/uso terapéutico , Línea Celular Tumoral , Distribución Tisular , Ligandos , Antígenos de Neoplasias/metabolismo , Nanomedicina Teranóstica , Medicina de Precisión , Femenino , PerrosRESUMEN
Cancer/testis antigens (CTA) are expressed in cancers and testis or placenta only and, therefore are considered promising targets for cancer immunotherapy and diagnosis. One family of CTA is the MAGEA family which comprises 13 members and was shown to be expressed synchronously with members from the CSAG (TRAG-3) family of CTA. The MAGEA genes are arranged in 4 subclusters located on the X chromosome. Subcluster III exposes a remarkable gene organization with an inverted repeat (IR) DNA structure of a triplicated couplet of a MAGEA gene and a CSAG gene. Analyzing the mRNA expression pattern of all genes of the MAGEA and CSAG family of cancer/testis genes, we show that the MAGEA and CSAG genes encoded in the large IR are expressed coordinately and independent from the MAGEAs encoded outside the IR. These results reinforce our hypothesis that the large MAGEA/CSAG-IR DNA structure has an impact on the regulation of gene expression.
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Antígenos de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Testículo/metabolismo , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Cromosomas Humanos X/genética , Análisis por Conglomerados , Metilación de ADN , Humanos , Masculino , Melanoma/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Identification of molecular features that determine peptide interaction with major histocompatibility complex I (MHC I) is essential for vaccine development. We have developed a concept for peptide design by combining an agent-based artificial ant system with artificial neural networks. A jury of feedforward networks classifies octapeptides that are recognized by mouse MHC I protein H-2K(b). Prediction accuracy yielded a correlation coefficient of 0.94. Peptides were designed in machina by the artificial ant system and tested in vitro for their MHC I stabilizing effect. The behavior of the search agents during the design process was controlled by the jury network. The experimentally determined prediction accuracy was 89% for the designed stabilizing and 95% for the non-stabilizing peptides. Novel H-2K(b) stabilizing peptides were conceived that reveal extensions of known residue motifs. The combined network-agent system recognized context dependencies of residue positions. A diverse set of novel sequences exhibiting substantial activity was generated.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Péptidos/química , Péptidos/metabolismo , Ingeniería de Proteínas , Modelos Teóricos , Redes Neurales de la ComputaciónRESUMEN
The human X chromosome consists of a high number of large inverted repeat (IR) DNA sequences which fulfill all requirements for formation of cruciform DNA structures. Such alternative DNA structures are suggested to have a great impact in altering the chromatin architecture and function. Our comprehensive analysis of the corresponding orthologous nucleotide sequences of an IR sequence from Homo sapiens and Pan troglodytes revealed that most of the nucleotide differences between the two species are symmetrical to the apex of the IR, and that the spacer region of the orthologous IRs are in reverse orientation. We provide evidence that this IR forms a large non-B DNA structure containing two Holliday junctions, allowing intrastrand nucleotide pairing of the arms and interstrand pairing of the spacer region of the IR. This structure would extrude into a large double-cruciform DNA structure providing the molecular basis of translocation events and regulation of gene expression.
Asunto(s)
Cromosomas Humanos X/química , Cromosomas Humanos X/genética , ADN/química , ADN/genética , Conformación de Ácido Nucleico , Pan troglodytes/genética , Cromosoma X/química , Cromosoma X/genética , Animales , Antígenos de Neoplasias/genética , Secuencia de Bases , ADN Cruciforme/química , ADN Cruciforme/genética , Humanos , Modelos Moleculares , Filogenia , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
The identification of tumor-associated T cell epitopes has contributed significantly to the understanding of the interrelationship of tumor and immune system and is instrumental in the development of therapeutic vaccines for the treatment of cancer. Most of the known epitopes have been identified with prediction algorithms that compute the potential capacity of a peptide to bind to HLA class I molecules. However, naturally expressed T cell epitopes need not necessarily be strong HLA binders. To overcome this limitation of the available prediction algorithms we established a strategy for the identification of T cell epitopes that include suboptimal HLA binders. To this end, an artificial neural network was developed that predicts HLA-binding peptides in protein sequences by taking the entire sequence context into consideration rather than computing the sum of the contribution of the individual amino acids. Using this algorithm, we predicted seven HLA A*0201-restricted potential T cell epitopes from known melanoma-associated Ags that do not conform to the canonical anchor motif for this HLA molecule. All seven epitopes were validated as T cell epitopes and three as naturally processed by melanoma tumor cells. T cells for four of the new epitopes were found at elevated frequencies in the peripheral blood of melanoma patients. Modification of the peptides to the canonical sequence motifs led to improved HLA binding and to improved capacity to stimulate T cells.