RESUMO
Targeted alpha-particle therapy (TAT) might be a relevant therapeutic strategy to circumvent resistance to conventional therapies in the case of HER2-positive metastatic cancer. Single-domain antibody fragments (sdAb) are promising vehicles for TAT because of their excellent in vivo properties, high target affinity, and fast clearance kinetics. This study combines the cytotoxic α-particle emitter bismuth-213 (213Bi) and HER2-targeting sdAbs. The in vitro specificity, affinity, and cytotoxic potency of the radiolabeled complex were analyzed on HER2pos cells. Its in vivo biodistribution through serial dissections and via Cherenkov and micro-single-photon emission computed tomography (CT)/CT imaging was evaluated. Finally, the therapeutic efficacy and potential associated toxicity of [213Bi]Bi-DTPA-2Rs15d were evaluated in a HER2pos tumor model that manifests peritoneal metastasis. In vitro, [213Bi]Bi-DTPA-2Rs15d bound HER2pos cells in a HER2-specific way. In mice, high tumor uptake was reached already 15 min after injection, and extremely low uptake values were observed in normal tissues. Co-infusion of gelofusine resulted in a 2-fold reduction in kidney uptake. Administration of [213Bi]Bi-DTPA-2Rs15d alone and in combination with trastuzumab resulted in a significant increase in median survival. We describe for the very first time the successful labeling of an HER2-sdAb with the α-emitter 213Bi, and after intravenous administration, revealing high in vivo stability and specific accumulation in target tissue and resulting in an increased median survival of these mice especially in combination with trastuzumab. These results indicate the potential of [213Bi]Bi-DTPA-sdAb as a new radioconjugate for TAT, alone and as an add-on to trastuzumab for the treatment of HER2pos metastatic cancer.
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Bismuto/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Radioisótopos/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Anticorpos de Domínio Único/farmacologia , Animais , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Cricetulus , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Ovarianas/metabolismo , Receptor ErbB-2/metabolismo , Distribuição Tecidual , Trastuzumab/farmacologiaRESUMO
The blockade of immune checkpoints (ICPs), such as cytotoxic T lymphocyte associated protein-4 (CTLA-4) and programmed death-1 (PD-1) and its ligand (PD-L1), has propelled the field of immuno-oncology into its current era. Drugs targeting these ICPs have improved clinical outcome in a number of patients with solid and hematological cancers. Nonetheless, some patients have no benefit from these ICP-blocking therapies. This observation has instigated research into alternative pathways that are responsible for the escape of cancer cells from anti-cancer immune responses. From this research, a number of molecules have emerged as promising therapeutic targets, including lymphocyte activating gene-3 (LAG-3), a next-generation ICP. We will review the current knowledge on the biological activity of LAG-3 and linked herewith its expression on activated immune cells. Moreover, we will discuss the prognostic value of LAG-3 and how LAG-3 expression in tumors can be monitored, which is an aspect that is of utmost importance, as the blockade of LAG-3 is actively pursued in clinical trials.
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Antígenos CD/metabolismo , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias/imunologia , Animais , Antígenos CD/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Humanos , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Introduction: Multiple myeloma (MM) remains incurable, despite the advent of chimeric antigen receptor (CAR)-T cell therapy. This unfulfilled potential can be attributed to two untackled issues: the lack of suitable CAR targets and formats. In relation to the former, the target should be highly expressed and reluctant to shedding; two characteristics that are attributed to the CS1-antigen. Furthermore, conventional CARs rely on scFvs for antigen recognition, yet this withholds disadvantages, mainly caused by the intrinsic instability of this format. VHHs have been proposed as valid scFv alternatives. We therefore intended to develop VHH-based CAR-T cells, targeting CS1, and to identify VHHs that induce optimal CAR-T cell activation together with the VHH parameters required to achieve this. Methods: CS1-specific VHHs were generated, identified and fully characterized, in vitro and in vivo. Next, they were incorporated into second-generation CARs that only differ in their antigen-binding moiety. Reporter T-cell lines were lentivirally transduced with the different VHH-CARs and CAR-T cell activation kinetics were evaluated side-by-side. Affinity, cell-binding capacity, epitope location, in vivo behavior, binding distance, and orientation of the CAR-T:MM cell interaction pair were investigated as predictive parameters for CAR-T cell activation. Results: Our data show that the VHHs affinity for its target antigen is relatively predictive for its in vivo tumor-tracing capacity, as tumor uptake generally decreased with decreasing affinity in an in vivo model of MM. This does not hold true for their CAR-T cell activation potential, as some intermediate affinity-binding VHHs proved surprisingly potent, while some higher affinity VHHs failed to induce equal levels of T-cell activation. This could not be attributed to cell-binding capacity, in vivo VHH behavior, epitope location, cell-to-cell distance or binding orientation. Hence, none of the investigated parameters proved to have significant predictive value for the extent of CAR-T cell activation. Conclusions: We gained insight into the predictive parameters of VHHs in the CAR-context using a VHH library against CS1, a highly relevant MM antigen. As none of the studied VHH parameters had predictive value, defining VHHs for optimal CAR-T cell activation remains bound to serendipity. These findings highlight the importance of screening multiple candidates.
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Imunoterapia Adotiva , Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Anticorpos de Domínio Único , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Humanos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Domínio Único/imunologia , Imunoterapia Adotiva/métodos , Animais , Linhagem Celular Tumoral , Camundongos , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Anticorpos de Cadeia Única/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted radionuclide therapy (TRT) using targeting moieties labeled with α-particle-emitting radionuclides (α-TRT) is an intensely investigated treatment approach as the short range of α-particles allows effective treatment of local lesions and micrometastases. However, profound assessment of the immunomodulatory effect of α-TRT is lacking in literature. Methods: Using flow cytometry of tumors, splenocyte restimulation, and multiplex analysis of blood serum, we studied immunologic responses ensuing from TRT with an antihuman CD20 single-domain antibody radiolabeled with 225Ac in a human CD20 and ovalbumin expressing B16-melanoma model. Results: Tumor growth was delayed with α-TRT and increased blood levels of various cytokines such as interferon-γ, C-C motif chemokine ligand 5, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1. Peripheral antitumoral T-cell responses were detected on α-TRT. At the tumor site, α-TRT modulated the cold tumor microenvironment (TME) to a more hospitable and hot habitat for antitumoral immune cells, characterized by a decrease in protumoral alternatively activated macrophages and an increase in antitumoral macrophages and dendritic cells. We also showed that α-TRT increased the percentage of programmed death-ligand 1 (PD-L1)-positive (PD-L1pos) immune cells in the TME. To circumvent this immunosuppressive countermeasure we applied immune checkpoint blockade of the programmed cell death protein 1-PD-L1 axis. Combination of α-TRT with PD-L1 blockade potentiated the therapeutic effect, however, the combination aggravated adverse events. A long-term toxicity study revealed severe kidney damage ensuing from α-TRT. Conclusion: These data suggest that α-TRT alters the TME and induces systemic antitumoral immune responses, which explains why immune checkpoint blockade enhances the therapeutic effect of α-TRT. However, further optimization is warranted to avoid adverse events.
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Melanoma Experimental , Anticorpos de Domínio Único , Animais , Humanos , Anticorpos de Domínio Único/farmacologia , Antígeno B7-H1/metabolismo , Microambiente Tumoral , Inibidores de Checkpoint Imunológico/farmacologia , Imunomodulação , Melanoma Experimental/radioterapia , Imunidade , Linhagem Celular TumoralRESUMO
Macrophages play an important role throughout the body. Antiinflammatory macrophages expressing the macrophage mannose receptor (MMR, CD206) are involved in disease development, ranging from oncology to atherosclerosis and rheumatoid arthritis. [68Ga]Ga-NOTA-anti-CD206 single-domain antibody (sdAb) is a PET tracer targeting CD206. This first-in-human study, as its primary objective, evaluated the safety, biodistribution, and dosimetry of this tracer. The secondary objective was to assess its tumor uptake. Methods: Seven patients with a solid tumor of at least 10 mm, an Eastern Cooperative Oncology Group score of 0 or 1, and good renal and hepatic function were included. Safety was evaluated using clinical examination and blood sampling before and after injection. For biodistribution and dosimetry, PET/CT was performed at 11, 90, and 150 min after injection; organs showing tracer uptake were delineated, and dosimetry was evaluated. Blood samples were obtained at selected time points for blood clearance. Metabolites in blood and urine were assessed. Results: Seven patients were injected with, on average, 191 MBq of [68Ga]Ga-NOTA-anti-CD206-sdAb. Only 1 transient adverse event of mild severity was considered to be possibly, although unlikely, related to the study drug (headache, Common Terminology Criteria for Adverse Events grade 1). The blood clearance was fast, with less than 20% of the injected activity remaining after 80 min. There was uptake in the liver, kidneys, spleen, adrenals, and red bone marrow. The average effective dose from the radiopharmaceutical was 4.2 mSv for males and 5.2 mSv for females. No metabolites were detected. Preliminary data of tumor uptake in cancer lesions showed higher uptake in the 3 patients who subsequently progressed than in the 3 patients without progression. One patient could not be evaluated because of technical failure. Conclusion: [68Ga]Ga-NOTA-anti-CD206-sdAb is safe and well tolerated. It shows rapid blood clearance and renal excretion, enabling high contrast-to-noise imaging at 90 min after injection. The radiation dose is comparable to that of routinely used PET tracers. These findings and the preliminary results in cancer patients warrant further investigation of this tracer in phase II clinical trials.
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Neoplasias , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Masculino , Feminino , Humanos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Radioisótopos de Gálio , Distribuição Tecidual , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Radiometria , Macrófagos/metabolismoRESUMO
Targeted radionuclide therapy (TRT) using probes labeled with Lutetium-177 (177Lu) represents a new and growing type of cancer therapy. We studied immunologic changes in response to TRT with 177Lu labeled anti-human CD20 camelid single domain antibodies (sdAb) in a B16-melanoma model transfected to express human CD20, the target antigen, and ovalbumin, a surrogate tumor antigen. High-dose TRT induced melanoma cell death, calreticulin exposure, and ATP-release in vitro. Melanoma-bearing mice received fractionated low and high-dose TRT via tumor targeting anti-human CD20 sdAbs, as opposed to control sdAbs. Tumor growth was delayed with both doses. Low- and high-dose TRT increased IL10 serum levels. Low-dose TRT also decreased CCL5 serum levels. At the tumor, high-dose TRT induced a type I IFN gene signature, while low-dose TRT induced a proinflammatory gene signature. Low- and high-dose TRT increased the percentage of PD-L1pos and PD-L2pos myeloid cells in tumors with a marked increase in alternatively activated macrophages after high-dose TRT. The percentage of tumor-infiltrating T cells was not changed, yet a modest increase in ovalbumin-specific CD8pos T-cells was observed after low-dose TRT. Contradictory, low and high-dose TRT decreased CD4pos Th1 cells in addition to double negative T cells. In conclusion, these data suggest that low and high-dose TRT induce distinct immunologic changes, which might serve as an anchoring point for combination therapy.
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Melanoma Experimental , Anticorpos de Domínio Único , Animais , Antígenos CD20 , Linhagem Celular Tumoral , Modelos Animais de Doenças , Lutécio , Melanoma Experimental/patologia , Camundongos , Ovalbumina , Radioisótopos/uso terapêuticoRESUMO
Cancer is a heterogeneous disease, requiring treatment tailored to the unique phenotype of the patient's tumor. Monoclonal antibodies (mAbs) and variants thereof have enabled targeted therapies to selectively target cancer cells. Cancer cell-specific mAbs have been used for image-guided surgery and targeted delivery of radionuclides or toxic agents, improving classical treatment strategies. Cancer cell-specific mAbs can further inhibit tumor cell growth or can stimulate immune-mediated destruction of cancer cells, a feature that has also been achieved through mAb-mediated manipulation of immune cells and pathways. Drawbacks of mAbs and their variants, together with the discovery of camelid heavy chain-only antibodies and the many advantageous features of their variable domains, referred to as VHHs, single domain antibodies or nanobodies (Nbs), resulted in the exploration of Nbs as an alternative targeting moiety. We therefore review the state-of-the-art as well as novel exploitation strategies of Nbs for targeted cancer therapy.
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Neoplasias , Anticorpos de Domínio Único , Anticorpos Monoclonais , Humanos , Neoplasias/tratamento farmacológico , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/uso terapêuticoRESUMO
Immune checkpoint blockade (ICB) of the PD-1 pathway revolutionized the survival forecast for advanced non-small cell lung cancer (NSCLC). Yet, the majority of PD-L1+ NSCLC patients are refractory to anti-PD-L1 therapy. Recent observations indicate a pivotal role for the PD-L1+ tumor-infiltrating myeloid cells in therapy failure. As the latter comprise a heterogenous population in the lung tumor microenvironment, we applied an orthotopic Lewis Lung Carcinoma (LLC) model to evaluate 11 different tumor-residing myeloid subsets in response to anti-PD-L1 therapy. While we observed significantly reduced fractions of tumor-infiltrating MHC-IIlow macrophages and monocytes, serological levels of TNF-α restored in lung tumor-bearing mice. Notably, we demonstrated in vivo and in vitro that anti-PD-L1 therapy mediated a monocyte-specific production of, and response to TNF-α, further accompanied by their significant upregulation of CD80, VISTA, LAG-3, SIRP-α and TIM-3. Nevertheless, co-blockade of PD-L1 and TNF-α did not reduce LLC tumor growth. A phenomenon that was partly explained by the observation that monocytes and TNF-α play a Janus-faced role in anti-PD-L1 therapy-mediated CTL stimulation. This was endorsed by the observation that monocytes appeared crucial to effectively boost T cell-mediated LLC killing in vitro upon combined PD-L1 with LAG-3 or SIRP-α blockade. Hence, this study enlightens the biomarker potential of lung tumor-infiltrated monocytes to define more effective ICB combination strategies.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Antígeno B7-H1/metabolismo , Humanos , Inibidores de Checkpoint Imunológico , Fatores Imunológicos/uso terapêutico , Imunoterapia , Neoplasias Pulmonares/patologia , Camundongos , Monócitos , Microambiente Tumoral , Fator de Necrose Tumoral alfa/uso terapêuticoRESUMO
Immunotherapy has improved the treatment of malignant skin cancer of the melanoma type, yet overall clinical response rates remain low. Combination therapies could be key to meet this cogent medical need. Because epigenetic hallmarks represent promising combination therapy targets, we studied the immunogenic potential of a dual inhibitor of histone methyltransferase G9a and DNA methyltransferases (DNMTs) in the preclinical B16-OVA melanoma model. Making use of tumor transcriptomic and functional analyses, methylation-targeted epigenetic reprogramming was shown to induce tumor cell cycle arrest and apoptosis in vitro coinciding with transient tumor growth delay and an IFN-I response in immune-competent mice. In consideration of a potential impact on immune cells, the drug was shown not to interfere with dendritic cell maturation or T-cell activation in vitro. Notably, the drug promoted dendritic cell and, to a lesser extent, T-cell infiltration in vivo, yet failed to sensitize tumor cells to programmed cell death-1 inhibition. Instead, it increased therapeutic efficacy of TCR-redirected T cell and dendritic cell vaccination, jointly increasing overall survival of B16-OVA tumor-bearing mice. The reported data confirm the prospect of methylation-targeted epigenetic reprogramming in melanoma and sustain dual G9a and DNMT inhibition as a strategy to tip the cancer-immune set-point towards responsiveness to active and adoptive vaccination against melanoma.
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Melanoma Experimental , Neoplasias Cutâneas , Animais , Metilação de DNA , Histonas/metabolismo , Camundongos , Modelos Teóricos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/terapia , VacinaçãoRESUMO
Immune checkpoint inhibitors targeting the programmed cell death-1 (PD-1) and its ligand PD-L1 have proven to be efficient cancer therapies in a subset of patients. From all the patients with various cancer types, only 20% have a positive response. Being able to distinguish patients that do express PD-1/PD-L1 from patients that do not allows patients to benefit from a more personalized and efficient treatment of tumor lesion(s). Expression of PD-1 and PD-L1 is typically assessed via immunohistochemical detection in a tumor biopsy. However, this method does not take in account the expression heterogeneity within the lesion, nor the possible metastasis. To visualize whole-body PD-L1 expression by PET imaging, we developed a nanobody-based radio-immunotracer targeting PD-L1 site-specifically labeled with gallium-68. The cysteine-tagged nanobody was site-specifically conjugated with a maleimide (mal)-NOTA chelator and radiolabeling was tested at different nanobody concentrations and temperatures. Affinity and specificity of the tracer, referred to as [68Ga]Ga-NOTA-mal-hPD-L1 Nb, were assayed by surface plasmon resonance and on PD-L1POS or PD-L1NEG 624-MEL cells. Xenografted athymic nude mice bearing 624-MEL PD-L1POS or PD-L1NEG tumors were injected with the tracer and ex vivo biodistribution was performed 1 h 20 min post-injection. Ideal 68Ga-labeling conditions were found at 50 °C for 15 min. [68Ga]Ga-NOTA-mal-hPD-L1 Nb was obtained in 80 ± 5% DC-RCY with a RCP > 99%, and was stable in injection buffer and human serum up to 3 h (>99% RCP). The in vitro characterization showed that the NOTA-functionalized Nb retained its affinity and specificity. Ex vivo biodistribution revealed a tracer uptake of 1.86 ± 0.67% IA/g in the positive tumors compared with 0.42 ± 0.04% IA/g in the negative tumors. Low background uptake was measured in the other organs and tissues, except for the kidneys and bladder, due to the expected excretion route of Nbs. The data obtained show that the site-specific 68Ga-labeled NOTA-mal-hPD-L1 Nb is a promising PET radio-immunotracer due to its ease of production, stability and specificity for PD-L1.
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Recent advances in the field of immune-oncology led to the discovery of next-generation immune checkpoints (ICPs). Lymphocyte activation gene-3 (LAG-3), being the most widely studied among them, is being explored as a target for the treatment of cancer patients. Several antagonistic anti-LAG-3 antibodies are being developed and are prime candidates for clinical application. Furthermore, validated therapies targeting cytotoxic T-lymphocyte-associated protein-4, programmed cell-death protein-1, or programmed cell-death ligand-1 showed that only subsets of patients respond. This finding highlights the need for better tools for patient selection and monitoring. The potential of molecular imaging to detect ICPs noninvasively in cancer is supported by several preclinical and clinical studies. Here, we report on a single-domain antibody to evaluate whole-body LAG-3 expression in various syngeneic mouse cancer models using nuclear imaging. Methods: SPECT/CT scans of tumor-bearing mice were performed 1 h after injection with radiolabeled single-domain antibody. Organs and tumors of mice were isolated and evaluated for the presence of the radiolabeled tracer and LAG-3-expressing immune cells using a γ-counter and flow cytometry respectively. PD-1/LAG-3-blocking antibodies were injected in MC38-bearing mice. Results: The radiolabeled single-domain antibody detected LAG-3 expression on tumor-infiltrating lymphocytes (TILs) as soon as 1 h after injection in MC38, MO4, and TC-1 cancer models. The single-domain antibody tracer visualized a compensatory upregulation of LAG-3 on TILs in MC38 tumors of mice treated with PD-1-blocking antibodies. When PD-1 blockade was combined with LAG-3 blockade, a synergistic effect on tumor growth delay was observed. Conclusion: These findings consolidate LAG-3 as a next-generation ICP and support the use of single-domain antibodies as tools to noninvasively monitor the dynamic evolution of LAG-3 expression by TILs, which could be exploited to predict therapy outcome.
Assuntos
Linfócitos do Interstício Tumoral , Receptor de Morte Celular Programada 1 , Anticorpos de Domínio Único , HumanosRESUMO
Monoclonal antibodies that target the inhibitory immune checkpoint axis consisting of programmed cell death protein 1 (PD-1) and its ligand, PD-L1, have changed the immune-oncology field. We identified K2, an anti-human PD-L1 single-domain antibody fragment, that can enhance T cell activation and tumor cell killing. In this study, the potential of different K2 formats as immune checkpoint blocking medicines was evaluated using a gene-based delivery approach. We showed that 2K2 and 3K2, a bivalent and trivalent K2 format generated using a 12 GS (glycine-serine) linker, were 313- and 135-fold more potent in enhancing T cell receptor (TCR) signaling in PD-1POS cells than was monovalent K2. We further showed that bivalent constructs generated using a 30 GS linker or disulfide bond were 169- and 35-fold less potent in enhancing TCR signaling than was 2K2. 2K2 enhanced tumor cell killing in a 3D melanoma model, albeit to a lesser extent than avelumab. Therefore, an immunoglobulin (Ig)G1 antibody-like fusion protein was generated, referred to as K2-Fc. K2-Fc was significantly better than avelumab in enhancing tumor cell killing in the 3D melanoma model. Overall, this study describes K2-based immune checkpoint medicines, and it highlights the benefit of an IgG1 Fc fusion to K2 that gains bivalency, effector functions, and efficacy.
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Immune checkpoints, such as programmed death-ligand 1 (PD-L1), limit T-cell function and tumor cells use this ligand to escape the anti-tumor immune response. Treatments with monoclonal antibodies blocking these checkpoints have shown long-lasting responses, but only in a subset of patients. This study aims to develop a Nanobody (Nb)-based probe in order to assess human PD-L1 (hPD-L1) expression using positron emission tomography imaging, and to compare the influence of two different radiolabeling strategies, since the Nb has a lysine in its complementarity determining region (CDR), which may impact its affinity upon functionalization. The Nb has been conjugated with the NOTA chelator site-specifically via the Sortase-A enzyme or randomly on its lysines. [68Ga]Ga-NOTA-(hPD-L1) Nbs were obtained in >95% radiochemical purity. In vivo tumor targeting studies at 1 h 20 post-injection revealed specific tumor uptake of 1.89 ± 0.40%IA/g for the site-specific conjugate, 1.77 ± 0.29%IA/g for the random conjugate, no nonspecific organ targeting, and excretion via the kidneys and bladder. Both strategies allowed for easily obtaining 68Ga-labeled hPD-L1 Nbs in high yields. The two conjugates were stable and showed excellent in vivo targeting. Moreover, we proved that the random lysine-conjugation is a valid strategy for clinical translation of the hPD-L1 Nb, despite the lysine present in the CDR.
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Anticorpos Anti-Idiotípicos/imunologia , Antígeno B7-H1/imunologia , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/farmacologia , Linhagem Celular Tumoral , Humanos , Marcação por Isótopo , Neoplasias/imunologia , Neoplasias/patologia , Compostos Radiofarmacêuticos/farmacologia , Distribuição Tecidual/efeitos dos fármacosRESUMO
Neuropilin-1 (NRP-1) is a co-receptor for semaphorins and vascular endothelial growth factor (VEGF) family members that can be expressed on cancer cells and tumor-infiltrating myeloid, endothelial and lymphoid cells. It has been linked to a tumor-promoting environment upon interaction with semaphorin 3A (Sema3A). Nanobodies (Nbs) targeting NRP-1 were generated for their potential to hamper the NRP-1/Sema3A interaction and their impact on colorectal carcinoma (CRC) development was evaluated in vivo through the generation of anti-NRP-1-producing CRC cells. We observed that tumor growth was significantly delayed and survival prolonged when the anti-NRP-1 Nbs were produced in vivo. We further analyzed the tumor microenvironment and observed that the pro-inflammatory MHC-IIhigh/trophic MHC-IIlow macrophage ratio was increased in tumors that produce anti-NRP-1 Nbs. This finding was corroborated by an increase in the expression of genes associated with MHC-IIhigh macrophages and a decrease in the expression of MHC-IIlow macrophage-associated genes in the macrophage pool sorted from anti-NRP-1 Nb-producing tumors. Moreover, we observed a significantly higher percentage of tumor-associated antigen-specific CD8+ T cells in tumors producing anti-NRP-1 Nbs. These data demonstrate that an intratumoral expression of NRP-1/Sema3A blocking biologicals increases anti-tumor immunity.
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HER2-targeted therapies have drastically improved the outcome for breast cancer patients. However, when metastasis to the brain is involved, current strategies fail to hold up to the same promise. Camelid single-domain antibody-fragments (sdAbs) have been demonstrated to possess favorable properties for detecting and treating cancerous lesions in vivo using different radiolabeling methods. Here we evaluate the anti-HER2 sdAb 2Rs15d, coupled to diagnostic γ- and therapeutic α- and ß--emitting radionuclides for the detection and treatment of HER2pos brain lesions in a preclinical setting. 2Rs15d was radiolabeled with 111In, 225Ac and 131I using DTPA- and DOTA-based bifunctional chelators and Sn-precursor of SGMIB respectively and evaluated in orthotopic tumor-bearing athymic nude mice. Therapeutic efficacy as well as systemic toxicity were determined for 131I- and 225Ac-labeled sdAbs and compared to anti-HER2 monoclonal antibody (mAb) trastuzumab in two different HER2pos tumor models. Radiolabeled 2Rs15d showed high and specific tumor uptake in both HER2pos SK-OV-3-Luc-IP1 and HER2pos MDA-MB-231Br brain lesions, whereas radiolabeled trastuzumab was unable to accumulate in intracranial SK-OV-3-Luc-IP1 tumors. Administration of [131I]-2Rs15d and [225Ac]-2Rs15d alone and in combination with trastuzumab showed a significant increase in median survival in 2 tumor models that remained largely unresponsive to trastuzumab treatment alone. Histopathological analysis revealed no significant early toxicity. Radiolabeled sdAbs prove to be promising vehicles for molecular imaging and targeted radionuclide therapy of metastatic lesions in the brain. These data demonstrate the potential of radiolabeled sdAbs as a valuable add-on treatment option for patients with difficult-to-treat HER2pos metastatic cancer.
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Targeted therapy and immunotherapy have become mainstream in cancer treatment. However, only patient subsets benefit from these expensive therapies, and often responses are short-lived or coincide with side effects. A growing modality in precision oncology is the development of theranostics, as this enables patient selection, treatment and monitoring. In this approach, labeled compounds and an imaging technology are used to diagnose patients and select the best treatment option, whereas for therapy, related compounds are used to target cancer cells or the tumor stroma. In this context, nanobodies and nanobody-directed therapeutics have gained interest. This interest stems from their high antigen specificity, small size, ease of labeling and engineering, allowing specific imaging and design of therapies targeting antigens on tumor cells, immune cells as well as proteins in the tumor environment. This review provides a comprehensive overview on the state-of-the-art regarding the use of nanobodies as theranostics, and their importance in the emerging field of personalized medicine.
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Neoplasias/imunologia , Neoplasias/terapia , Anticorpos de Domínio Único/imunologia , Animais , Humanos , Imunoterapia/métodos , Medicina de Precisão/métodos , Nanomedicina Teranóstica/métodosRESUMO
Dendritic cell [DC] vaccines can induce durable clinical responses, at least in a fraction of previously treated, late stage cancer patients. Several preclinical studies suggest that shielding programmed death-ligand 1 [PD-L1] on the DC surface may be an attractive strategy to extend such clinical benefits to a larger patient population. In this study, we evaluated the use of single domain antibody [sdAb] K2, a high affinity, antagonistic, PD-L1 specific sdAb, for its ability to enhance DC mediated T-cell activation and benchmarked it against the use of the monoclonal antibodies [mAbs], MIH1, 29E.2A3 and avelumab. Similar to mAbs, sdAb K2 enhanced antigen-specific T-cell receptor signaling in PD-1 positive (PD-1pos) reporter cells activated by DCs. We further showed that the activation and function of antigen-specific CD8 positive (CD8pos) T cells, activated by DCs, was enhanced by inclusion of sdAb K2, but not mAbs. The failure of mAbs to enhance T-cell activation might be explained by their low efficacy to bind PD-L1 on DCs when compared to binding of PD-L1 on non-immune cells, whereas sdAb K2 shows high binding to PD-L1 on immune as well as non-immune cells. These data provide a rationale for the inclusion of sdAb K2 in DC-based immunotherapy strategies.
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The tumor microenvironment of numerous prevalent cancer types is abundantly infiltrated with tumor-associated macrophages (TAMs). Macrophage mannose receptor (MMR or CD206) expressing TAMs have been shown to be key promoters of tumor progression and major opponents of successful cancer therapy. Therefore, depleting MMR+ TAMs is an interesting approach to synergize with current antitumor therapies. We studied the potential of single-domain antibodies (sdAbs) specific for MMR to target proteins to MMR+ TAMs. Anti-MMR sdAbs were genetically coupled to a reporter protein, mWasabi (wasabi green, WG), generating sdAb "drug" fusion proteins (SFPs), referred to as WG-SFPs. The resulting WG-SFPs were highly efficient in targeting MMR+ macrophages both in vitro and in vivo. As we showed that second mitochondria-derived activator of caspase (SMAC) mimetics modulate MMR+ macrophages, we further coupled the anti-MMR sdAb to an active form of SMAC, referred to as tSMAC. The resulting tSMAC-SFPs were able to bind and upregulate caspase3/7 activity in MMR+ macrophages in vitro. In conclusion, we report the proof-of-concept of an elegant approach to conjugate anti-MMR sdAbs to proteins, which opens new avenues for targeted manipulation of MMR+ tumor-promoting TAMs.
Assuntos
Sistemas de Liberação de Medicamentos , Lectinas Tipo C/metabolismo , Macrófagos/efeitos dos fármacos , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Anticorpos de Domínio Único/administração & dosagem , Animais , Proteínas Reguladoras de Apoptose/administração & dosagem , Proteínas Reguladoras de Apoptose/farmacologia , Feminino , Células HEK293 , Humanos , Macrófagos/metabolismo , Receptor de Manose , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/administração & dosagem , Proteínas Mitocondriais/farmacologia , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Domínio Único/farmacologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
The PD-1:PD-L1 immune checkpoint axis is central in the escape of cancer cells from anticancer immune responses. Monoclonal antibodies (mAbs) specific for PD-L1 have been approved for treatment of various cancer types. Although PD-L1 blockade has proven its merit, there are still several aspects that require further attention to fully capitalize on its potential. One of these is the development of antigen-binding moieties that enable PD-L1 diagnosis and therapy. We generated human PD-L1 binding single domain antibodies (sdAbs) and selected sdAb K2, a sdAb with a high affinity for PD-L1, as a lead compound. SPECT/CT imaging in mice following intravenous injection of Technetium-99m (99mTc)-labeled sdAb K2 revealed high signal-to-noise ratios, strong ability to specifically detect PD-L1 in melanoma and breast tumors, and relatively low kidney retention, which is a unique property for radiolabeled sdAbs. We further showed using surface plasmon resonance that sdAb K2 binds to the same epitope on PD-L1 as the mAb avelumab, and antagonizes PD-1:PD-L1 interactions. Different human cell-based assays corroborated the PD-1:PD-L1 blocking activity, showing enhanced T-cell receptor signaling and tumor cell killing when PD-1POS T cells interacted with PD-L1POS tumor cells. Taken together, we present sdAb K2, which specifically binds to human PD-L1, as a new diagnostic and therapeutic agent in cancer management.
RESUMO
Immune checkpoint inhibition (ICI) is a promising cancer therapy, which has progressed rapidly from a preclinical concept to clinical implementation. Commonly considered targets in ICI are CTLA-4, PD-1/PD-L1, and LAG-3, and the list grows. As ICI is generally only beneficial for a subset of patients, there is a need to select patients that are eligible for therapy as well as to monitor therapy response. There is growing interest to do this noninvasively, by molecular imaging with target-specific tracers. To this day, noninvasive imaging has focused on CTLA-4 and PD-1/PD-L1, while there is no noninvasive tool available to accurately assess LAG-3 expression in vivo. In this proof-of-concept study, we developed nanobodies, the smallest functional fragments from camelid heavy chain-only antibodies, to noninvasively evaluate mouse LAG-3 expression using single photon emission computed tomography (SPECT)/CT imaging. The in vitro characterization of 114 nanobodies led to the selection of nine nanobodies binding to mouse LAG-3. The injection of 99mTechnetium-labeled nanobodies in healthy mice showed specific uptake in immune peripheral organs like the spleen and lymph nodes, which was not observed in LAG-3 gene knock-out mice. Moreover, nanobody uptake could be visualized using SPECT/CT and correlated to the presence of LAG-3 as assessed in flow cytometry and immunohistochemistry. SPECT/CT scans of tumor bearing mice further confirmed the diagnostic potential of the nanobodies. These findings substantiate the approach to use nanobodies as a tool to image inhibitory immune checkpoints in the tumor environment.