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1.
J Am Chem Soc ; 143(27): 10131-10142, 2021 07 14.
Artículo en Inglés | MEDLINE | ID: mdl-34180666

RESUMEN

Synthesis of ligand-functionalized nanomaterials with control over size, shape, and ligand orientation facilitates the design of targeted nanomedicines for therapeutic purposes. DNA nanotechnology has emerged as a powerful tool to rationally construct two- and three-dimensional nanostructures, enabling site-specific incorporation of protein ligands with control over stoichiometry and orientation. To efficiently target cell surface receptors, exploration of the parameters that modulate cellular accessibility of these nanostructures is essential. In this study, we systematically investigate tunable design parameters of antibody-functionalized DNA nanostructures binding to therapeutically relevant receptors, including the programmed cell death protein 1, the epidermal growth factor receptor, and the human epidermal growth factor receptor 2. We show that, although the native affinity of antibody-functionalized DNA nanostructures remains unaltered, the absolute number of bound surface receptors is lower compared to soluble antibodies due to receptor accessibility by the nanostructure. We explore structural determinants of this phenomenon to improve efficiency, revealing that receptor binding is mainly governed by nanostructure size and DNA handle location. The obtained results provide key insights in the ability of ligand-functionalized DNA nanostructures to bind surface receptors and yields design rules for optimal cellular targeting.


Asunto(s)
Comunicación Celular , ADN/química , ADN/metabolismo , Nanoestructuras , Animales , Células CHO , Cricetulus , Sistemas de Liberación de Medicamentos , Humanos , Proteínas de Punto de Control Inmunitario , Ligandos , Nanotubos , Unión Proteica
2.
Chembiochem ; 18(24): 2390-2394, 2017 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-28994180

RESUMEN

Overexpression of (mutated) receptor tyrosine kinases is a characteristic of many aggressive tumors, and induction of receptor uptake has long been recognized as a therapeutic modality. A conjugate of a synthetically produced cell-penetrating peptide (CPP), corresponding to amino acids 38-59 of human lactoferrin, and the recombinant llama single-domain antibody (VHH) 7D12, which binds the human epidermal growth factor receptor (EGFR), was generated by sortase A mediated transpeptidation. The conjugate blocks EGF-mediated EGFR activation with higher efficacy than that of both modalities alone; a phenomenon that is caused by both effective receptor blockade and internalization. Thus, the VHH-CPP conjugate shows a combination of activities that implement a highly powerful new design principle to block receptor activation by its clearance from the cell surface.


Asunto(s)
Péptidos de Penetración Celular/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Inmunoconjugados/farmacología , Péptidos de Penetración Celular/inmunología , Endocitosis , Humanos , Inmunoconjugados/uso terapéutico , Lactoferrina/inmunología , Fragmentos de Péptidos/inmunología
3.
Bioconjug Chem ; 28(2): 539-548, 2017 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-28045502

RESUMEN

Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation. First, sortase A mediated transPEGylation is used for introduction of a chemical click moiety. The resulting clickable VHHs are then used for conjugation to other groups employing the Cu+-independent strain-promoted alkyne-azide cycloadition (SPAAC) reaction. Using this approach, tail-to-tail bispecific VHHs and VHH-targeted nanoparticles are generated without affecting VHH functionality. Furthermore, this approach allows the bioconjugation of multiple moieties to VHHs for simple and convenient production of VHH-based theranostics.


Asunto(s)
Camélidos del Nuevo Mundo/inmunología , Inmunoconjugados/química , Cadenas Pesadas de Inmunoglobulina/química , Nanopartículas/química , Polietilenglicoles/química , Anticuerpos de Dominio Único/química , Alquinos/química , Aminoaciltransferasas/metabolismo , Animales , Azidas/química , Proteínas Bacterianas/metabolismo , Química Clic/métodos , Reacción de Cicloadición/métodos , Cisteína Endopeptidasas/metabolismo , Inmunoconjugados/inmunología , Inmunoconjugados/metabolismo , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Polietilenglicoles/metabolismo , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/metabolismo
4.
Bioconjug Chem ; 27(7): 1697-706, 2016 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-27306828

RESUMEN

The use of a bioorthogonal reaction for the selective cleavage of tumor-bound antibody-drug conjugates (ADCs) would represent a powerful new tool for ADC therapy, as it would not rely on the currently used intracellular biological activation mechanisms, thereby expanding the scope to noninternalizing cancer targets. Here we report that the recently developed inverse-electron-demand Diels-Alder pyridazine elimination reaction can provoke rapid and self-immolative release of doxorubicin from an ADC in vitro and in tumor-bearing mice.


Asunto(s)
Liberación de Fármacos , Inmunoconjugados/química , Animales , Línea Celular Tumoral , Química Clic , Doxorrubicina/química , Femenino , Humanos , Inmunoconjugados/sangre , Inmunoconjugados/farmacocinética , Cinética , Ratones , Ratones Endogámicos BALB C , Piridazinas/química
5.
Acta Neuropathol ; 130(1): 131-44, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25862637

RESUMEN

MET has gained interest as a therapeutic target for a number of malignancies because of its involvement in tumorigenesis, invasion and metastasis. At present, a number of inhibitors, both antibodies against MET or its ligand hepatocyte growth factor, and small molecule MET tyrosine kinase inhibitors are in clinical trials. We here describe a novel variant of MET that is expressed in 6% of high-grade gliomas. Characterization of this mutation in a glioma cell line revealed that it consists of an intronic deletion, resulting in a splice event connecting an intact splice donor site in exon 6 with the next splice acceptor site being that of exon 9. The encoded protein lacks parts of the extracellular IPT domains 1 and 2, encoded by exons 7 and 8, resulting in a novel pseudo-IPT and is named MET(Δ7-8). MET(Δ7-8) is located predominantly in the cytosol and is constitutively active. The auto-activating nature of MET(Δ7-8), in combination with a lack of transmembrane localization, renders MET(Δ7-8) not targetable using antibodies, although the protein is efficiently deactivated by MET-specific tyrosine kinase inhibitors. Testing of MET-expressing tumors for the presence of this variant may be important for treatment decision making.


Asunto(s)
Glioma/genética , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Eliminación de Secuencia , Anilidas/farmacología , Animales , Anticuerpos/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patología , Línea Celular Tumoral , Femenino , Glioma/tratamiento farmacológico , Glioma/metabolismo , Glioma/patología , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Masculino , Ratones , Clasificación del Tumor , Trasplante de Neoplasias , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Piridinas/farmacología , ARN Mensajero/metabolismo , Sarcoma/genética , Sarcoma/metabolismo , Sarcoma/patología
6.
Molecules ; 20(7): 12076-92, 2015 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-26147581

RESUMEN

Membrane type-1 matrix metalloproteinase (MT1-MMP or MMP-14) plays an important role in adverse cardiac remodelling. Here, we aimed to develop radiolabeled activatable cell penetrating peptides (ACPP) sensitive to MT1-MMP for the detection of elevated MT1-MMP levels in adverse cardiac remodelling. Three ACPP analogs were synthesized and the most potent ACPP analog was selected using MT1-MMP sensitivity and enzyme specificity assays. This ACPP, called ACPP-B, showed high sensitivity towards MT1-MMP, soluble MMP-2, and MT2-MMP, while limited sensitivity was measured for other members of the MMP family. In in vitro cell assays, radiolabeled ACPP-B showed efficient cellular uptake upon activation. A pilot in vivo study showed increased uptake of the radiolabeled probe in regions of infarcted myocardium compared to remote myocardium, warranting further in vivo evaluation.


Asunto(s)
Péptidos de Penetración Celular/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Sondas Moleculares , Radioisótopos/metabolismo , Animales , Línea Celular Tumoral , Péptidos de Penetración Celular/farmacocinética , Humanos , Técnicas In Vitro , Masculino , Ratones , Especificidad por Sustrato , Distribución Tisular
7.
Mol Pharm ; 11(9): 3090-6, 2014 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-25077373

RESUMEN

Radioimmunotherapy (RIT) of solid tumors is hampered by low tumor-to-nontumor (T/NT) ratios of the radiolabeled monoclonal antibodies resulting in low tumor doses in patients. Pretargeting technologies can improve the effectiveness of RIT in cancer therapy by increasing this ratio. We showed that a pretargeting strategy employing in vivo chemistry in combination with clearing agents, proceeds efficiently in tumor-bearing mice resulting in high T/NT ratios. A dosimetry study indicated that the chemical pretargeting technology, which centered on the bioorthogonal Diels-Alder click reaction between a radiolabeled tetrazine probe and a trans-cyclooctene-oxymethylbenzamide-tagged CC49 antibody (CC49-TCO(1)), can match the performance of clinically validated high-affinity biological pretargeting approaches in mice ( Rossin J Nucl Med. 2013 , 54 , 1989 - 1995 ). Nevertheless, the increased protein surface hydrophobicity of CC49-TCO(1) led to a relatively rapid blood clearance and concomitant reduced tumor uptake compared to native CC49 antibody. Here, we present the in vivo evaluation of a TCO-oxymethylacetamide-tagged CC49 antibody (CC49-TCO(2)), which is highly reactive toward tetrazines and less hydrophobic than CC49-TCO(1). CC49-TCO(2) was administered to healthy mice to determine its blood clearance and the in vivo stability of the TCO. Next, pretargeting biodistribution and SPECT studies with CC49-TCO(2), tetrazine-functionalized clearing agent, and radiolabeled tetrazine were carried out in nude mice bearing colon carcinoma xenografts (LS174T). CC49-TCO(2) had an increased circulation half-life, a 1.5-fold higher tumor uptake, and a 2.6-fold improved in vivo TCO stability compared to the more hydrophobic TCO-benzamide-CC49. As a consequence, and despite the 2-fold lower reactivity of CC49-TCO(2) toward tetrazines compared with CC49-TCO(1), administration of radiolabeled tetrazine afforded a significantly increased tumor accumulation and improved T/NT ratios in mice pretargeted with CC49-TCO(2). In conclusion, the TCO-acetamide derivative represents a large improvement in in vivo Diels-Alder pretargeting, possibly enabling application in larger animals and eventually humans.


Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/radioterapia , Ciclooctanos/química , Ciclooctanos/uso terapéutico , Inmunoconjugados/química , Inmunoconjugados/farmacología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Carcinoma/tratamiento farmacológico , Carcinoma/inmunología , Carcinoma/radioterapia , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Reacción de Cicloadición/métodos , Ciclooctanos/inmunología , Femenino , Semivida , Humanos , Inmunoconjugados/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Radioinmunoterapia/métodos , Radiometría/métodos , Radiofármacos/química , Radiofármacos/inmunología , Radiofármacos/farmacología , Distribución Tisular/inmunología
8.
Mol Pharm ; 11(5): 1415-23, 2014 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-24641497

RESUMEN

The noninvasive imaging of matrix metalloproteinases (MMPs) activity in postischemic myocardial tissue holds great promise to predict cardiac function post-myocardial infarction. Consequently, development of MMP specific molecular imaging probes for noninvasive visualization and quantification of MMP activity is of great interest. A novel MMP imaging strategy is based on activatable cell-penetrating peptide probes (ACPP) that are sensitive to the proteolytic activity of MMP-2 and -9. The MMP-mediated activation of these ACPPs drives probe accumulation at the target site. The aim of this study was the development and characterization of radiolabeled MMP-2/9 sensitive ACPPs to assess MMP activity in myocardial remodeling in vivo. Specifically, a short and long-circulating MMP activatable cell-penetrating imaging probe (ACPP and Alb-ACPP, respectively; the latter is an ACPP modified with an albumin binding ligand that prolongs blood clearance) were successfully synthesized and radiolabeled. Subsequently, their biodistributions were determined in vivo in a Swiss mouse model of myocardial infarction. Both peptide probes showed a significantly higher uptake in infarcted myocardium compared to remote myocardium. The biodistribution for dual-isotope radiolabeled probes, which allowed us to discriminate between uncleaved ACPP and activated ACPP, showed increased retention of activated ACPP and activated Alb-ACPP in infarcted myocardium compared to remote myocardium. The enhanced retention correlated to gelatinase levels determined by gelatin zymography, whereas no correlation was found for the negative control: an MMP-2/9 insensitive non-ACPP. In conclusion, radiolabeled MMP sensitive ACPP probes enable to assess MMP activity in the course of remodeling post-myocardial infarction in vivo. Future research should evaluate the feasibility and the predictive value of the ACPP strategy for assessing MMP activity as a main player in postinfarction myocardial remodeling in vivo.


Asunto(s)
Metaloproteinasa 2 de la Matriz/química , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/química , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/química , Metaloproteinasas de la Matriz/metabolismo , Sondas Moleculares , Péptidos , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Sondas Moleculares/química , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Péptidos/química
9.
Biomedicines ; 12(3)2024 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-38540255

RESUMEN

Therapeutic antibodies (Abs) which act on a broader range of epitopes may provide more durable protection against the genetic drift of a target, typical of viruses or tumors. When these Abs exist concurrently on the targeted antigen, several mechanisms of action (MoAs) can be engaged, boosting therapeutic potency. This study selected combinations of four and five Abs with non- or partially overlapping epitopes to the SARS-CoV-2 spike glycoprotein, on or outside the crucial receptor binding domain (RBD), to offer resilience to emerging variants and trigger multiple MoAs. The combinations were derived from a pool of unique-sequence scFv Ab fragments retrieved from two SARS-CoV-2-naïve human phage display libraries. Following recombinant expression to full-length human IgG1 candidates, a biolayer interferometric analysis mapped epitopes to bins and confirmed that up to four Abs from across the bins can exist simultaneously on the spike glycoprotein trimer. Not all the bins of Abs interfered with the spike protein binding to angiotensin converting enzyme 2 (ACE2) in competitive binding assays, nor neutralized the pseudovirus or authentic virus in vitro, but when combined in vivo, their inclusion resulted in a much stronger viral clearance in the lungs of intranasally challenged hamsters, compared to that of those treated with mono ACE2 blockers. In addition, the Ab mixtures activated in vitro reporter cells expressing Fc-gamma receptors (FcγRs) involved in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The best four-Ab combination neutralized seventeen variants of concern from Wuhan-Hu1 to Omicron BA.4/BA.5 in vitro.

10.
Mol Pharm ; 10(11): 4309-21, 2013 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-24099178

RESUMEN

Fibrin targeting is an attractive strategy for nuclear imaging of thrombosis, atherosclerosis and cancer. Recently, FibPep, an (111)In-labeled fibrin-binding peptide, was established as a tracer for fibrin SPECT imaging and was reported to allow sensitive detection of minute thrombi in mice using SPECT. In this study, we developed EPep, a novel (111)In-labeled fibrin-binding peptide containing the fibrin-binding domain of the clinically verified EP-2104R peptide, and sought to compare the potential of EPep and FibPep as tracers for fibrin SPECT imaging. In vitro, both EPep and FibPep showed high stability in serum, but were less stable in liver and kidney homogenate assays. Both peptide probes displayed comparable affinities toward human and mouse derived fibrin (Kd ≈ 1 µM), and similarly to FibPep, EPep showed fast blood clearance, low nontarget uptake and high thrombus uptake (6.8 ± 1.2% ID g(-1)) in a mouse carotid artery thrombosis model. Furthermore, EPep showed a similar affinity toward rat derived fibrin (Kd ≈ 1 µM), displayed high thrombus uptake in a rat carotid artery thrombosis model (0.74 ± 0.39% ID g(-1)), and allowed sensitive detection of thrombosis in rats using SPECT. In contrast, FibPep displayed a significantly lower affinity toward rat derived fibrin (Kd ≈ 14 µM) and low uptake in rat thrombi (0.06 ± 0.02% ID g(-1)) and did not allow clear visualization of carotid artery thrombosis in rats using SPECT. These results were confirmed ex vivo by autoradiography, which showed a 7-fold higher ratio of activity in the thrombus over the contralateral carotid artery for EPep in comparison to FibPep. These findings suggest that the FibPep binding fibrin epitope is not fully homologous between humans and rats, and that preclinical rat models of disease should not be employed to gauge the clinical potential of FibPep. In conclusion, both peptides showed approximately similar metabolic stability and affinity toward human and mouse derived fibrin, and displayed high thrombus uptake in a mouse carotid artery thrombosis model. Therefore, both EPep and FibPep are promising fibrin targeted tracers for translation into clinical settings to serve as novel tools for molecular imaging of fibrin.


Asunto(s)
Péptidos , Tomografía Computarizada de Emisión de Fotón Único/métodos , Animales , Trombosis de las Arterias Carótidas/diagnóstico , Fibrina/química , Humanos , Radioisótopos de Indio , Péptidos/química , Ratas , Trombosis/diagnóstico
11.
EJNMMI Res ; 13(1): 100, 2023 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-37985555

RESUMEN

BACKGROUND: CD103 is an integrin specifically expressed on the surface of cancer-reactive T cells. The number of CD103+ T cells significantly increases during successful immunotherapy and might therefore be an attractive biomarker for noninvasive PET imaging of immunotherapy response. Since the long half-life of antibodies preclude repeat imaging of CD103+ T cell dynamics early in therapy, we therefore here explored PET imaging with CD103 Fab fragments radiolabeled with a longer (89Zr) and shorter-lived radionuclide (68Ga). METHODS: Antihuman CD103 Fab fragment Fab01A was radiolabeled with 89Zr or 68Ga, generating [89Zr]Zr-hCD103.Fab01A and [68Ga]Ga-hCD103.Fab01A, respectively. In vivo evaluation of these tracers was performed in male nude mice (BALB/cOlaHsd-Foxn1nu) with established CD103-expressing CHO (CHO.CD103) or CHO-wildtype (CHO.K1) xenografts, followed by serial PET imaging and ex vivo bio-distribution. RESULTS: [89Zr]Zr-hCD103.Fab01A showed high tracer uptake in CD103+ xenografts as early as 3 h post-injection. However, the background signal remained high in the 3- and 6-h scans. The background was relatively low at 24 h after injection with sufficient tumor uptake. [68Ga]Ga-hCD103.Fab01Ashowed acceptable uptake and signal-to-noise ratio in CD103+ xenografts after 3 h, which decreased at subsequent time points. CONCLUSION: [89Zr]Zr-hCD103.Fab01A demonstrated a relatively low background and high xenograft uptake in scans as early as 6 h post-injection and could be explored for repeat imaging during immunotherapy in clinical trials. 18F or 64Cu could be explored as alternative to 68Ga in optimizing half-life and radiation burden of the tracer.

12.
Mol Ther Nucleic Acids ; 33: 599-616, 2023 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-37637207

RESUMEN

IL-12 is a potent cytokine for cancer immunotherapy. However, its systemic delivery as a recombinant protein has shown unacceptable toxicity in the clinic. Currently, the intratumoral injection of IL-12-encoding mRNA or DNA to avoid such side effects is being evaluated in clinical trials. In this study, we aimed to improve this strategy by further favoring IL-12 tethering to the tumor. We generated in vitro transcribed mRNAs encoding murine single-chain IL-12 fused to diabodies binding to CSF1R and/or PD-L1. These targeted molecules are expressed in the tumor microenvironment, especially on myeloid cells. The binding capacity of chimeric constructs and the bioactivity of IL-12 were demonstrated in vitro and in vivo. Doses as low as 0.5 µg IL-12-encoding mRNA achieved potent antitumor effects in subcutaneously injected B16-OVA and MC38 tumors. Treatment delivery was associated with increases in IL-12p70 and IFN-γ levels in circulation. Fusion of IL-12 to the diabodies exerted comparable efficacy against bilateral tumor models. However, it achieved tethering to myeloid cells infiltrating the tumor, resulting in nearly undetectable systemic levels of IL-12 and IFN-γ. Overall, tethering IL-12 to intratumoral myeloid cells in the mRNA-transferred tumors achieves similar efficacy while reducing the dangerous systemic bioavailability of IL-12.

13.
Biomolecules ; 12(10)2022 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-36291540

RESUMEN

T cell engager (TCE) antibodies have emerged as promising cancer therapeutics that link cytotoxic T-cells to tumor cells by simultaneously binding to CD3E on T-cells and to a tumor-associated antigen (TAA) expressed by tumor cells. We previously reported a novel bispecific format, the IgG-like Fab x sdAb-Fc (also known as half-IG_VH-h-CH2-CH3), combining a conventional antigen-binding fragment (Fab) with a single domain antibody (sdAb). Here, we evaluated this Fab x sdAb-Fc format as a T-cell redirecting bispecific antibody (TbsAbs) by targeting mEGFR on tumor cells and mCD3E on T cells. We focused our attention specifically on the hinge design of the sdAb arm of the bispecific antibody. Our data show that a TbsAb with a shorter hinge of 23 amino acids (TbsAb.short) showed a significantly better T cell redirected tumor cell elimination than the TbsAb with a longer, classical antibody hinge of 39 amino acids (TbsAb.long). Moreover, the TbsAb.short form mediated better T cell-tumor cell aggregation and increased CD69 and CD25 expression levels on T cells more than the TbsAb.long form. Taken together, our results indicate that already minor changes in the hinge design of TbsAbs can have significant impact on the anti-tumor activity of TbsAbs and may provide a new means to improve their potency.


Asunto(s)
Anticuerpos Biespecíficos , Neoplasias , Anticuerpos de Dominio Único , Humanos , Anticuerpos Biespecíficos/química , Neoplasias/terapia , Inmunoglobulina G , Aminoácidos , Muerte Celular
14.
J Immunother Cancer ; 10(12)2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36600560

RESUMEN

PURPOSE: CD103, an integrin specifically expressed on the surface of cancer-reactive T cells, is significantly increased during successful immunotherapy across human malignancies. In this study, we describe the generation and zirconium-89 (89Zr) radiolabeling of monoclonal antibody (mAb) clones that specifically recognize human CD103 for non-invasive immune positron-emission tomography (PET) imaging of T cell infiltration as potential biomarker for effective anticancer immune responses. EXPERIMENTAL DESIGN: First, to determine the feasibility of anti-CD103 immuno-PET to visualize CD103-positive cells at physiologically and clinically relevant target densities, we developed an 89Zr-anti-murine CD103 PET tracer. Healthy, non-tumor bearing C57BL/6 mice underwent serial PET imaging after intravenous injection, followed by ex vivo biodistribution. Tracer specificity and macroscopic tissue distribution were studied using autoradiography combined with CD103 immunohistochemistry. Next, we generated and screened six unique mAbs that specifically target human CD103 positive cells. Optimal candidates were selected for 89Zr-anti-human CD103 PET development. Nude mice (BALB/cOlaHsd-Foxn1nu) with established CD103 expressing Chinese hamster ovary (CHO) or CHO wild-type xenografts were injected with 89Zr-anti-human CD103 mAbs and underwent serial PET imaging, followed by ex vivo biodistribution. RESULTS: 89Zr-anti-murine CD103 PET imaging identified CD103-positive tissues at clinically relevant target densities. For human anti-human CD103 PET development two clones were selected based on strong binding to the CD103+ CD8+ T cell subpopulation in ovarian cancer tumor digests, non-overlapping binding epitopes and differential CD103 blocking properties. In vivo, both 89Zr-anti-human CD103 tracers showed high target-to-background ratios, high target site selectivity and a high sensitivity in human CD103 positive xenografts. CONCLUSION: CD103 immuno-PET tracers visualize CD103 T cells at relevant densities and are suitable for future non-invasive assessment of cancer reactive T cell infiltration.


Asunto(s)
Neoplasias , Tomografía de Emisión de Positrones , Humanos , Ratones , Animales , Cricetinae , Distribución Tisular , Ratones Desnudos , Células CHO , Ratones Endogámicos C57BL , Cricetulus , Tomografía de Emisión de Positrones/métodos , Anticuerpos Monoclonales/metabolismo
15.
J Immunol Methods ; 489: 112914, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33197470

RESUMEN

In addition to their known implication in allergy studies, IgE antibodies are becoming an increasingly interesting antibody class in cancer research. However, large-scale purification of IgE antibodies still poses substantial challenges, as they cannot be purified using techniques commonly used for other immunoglobulins such as protein A or protein G chromatography. Here, we have developed and optimised a gentle and simple IgE purification method based on thiophilic interaction chromatography (TIC). IgE binds to the thiophilic resin in presence of 1.2 M ammonium sulfate and is eluted in low salt concentration. Monomericity of purified antibodies ranged between 54 and 73%. Preparative size-exclusion chromatography was thereafter performed to further improve the purity, which reached >95% in the final product. The overall recovery was around 30%. The purification method was tested on both hybridoma-produced and recombinantly produced IgE antibodies with reproducible results. In addition, the antigen binding activity of purified IgE antibodies was preserved, as shown by binding ELISA. Purification by TIC is cheap, gentle in terms of pH to preserve IgE folding and function, and universal as any IgE antibody can be purified irrespective of the species of origin or affinity. Potentially, it could be used for purification of other antibody isotypes as well, when gentle conditions are required.


Asunto(s)
Hibridomas/química , Inmunoglobulina E/aislamiento & purificación , Animales , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Hibridomas/inmunología , Inmunoglobulina E/química , Inmunoglobulina E/inmunología , Ratones , Células Tumorales Cultivadas
16.
J Immunol Methods ; 499: 113173, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34699840

RESUMEN

Tumor necrosis factor receptor 2 (TNFR2) has gained much research interest in recent years because of its potential pivotal role in autoimmune disease and cancer. However, its function in regulating different immune cells is not well understood. There is a need for well-characterized reagents to selectively modulate TNFR2 function, thereby enabling definition of TNFR2-dependent biology in human and mouse surrogate models. Here, we describe the generation, production, purification, and characterization of a panel of novel antibodies targeting mouse TNFR2. The antibodies display functional differences in binding affinity and potency to block TNFα. Furthermore, epitope binding showed that the anti-mTNFR2 antibodies target different domains on the TNFR2 protein, associated with varying capacity to enhance CD8+ T-cell activation and costimulation. Moreover, the anti-TNFR2 antibodies demonstrate binding to isolated splenic mouse Tregs ex vivo and activated CD8+ cells, reinforcing their potential use to establish TNFR2-dependent immune modulation in translational models of autoimmunity and cancer.


Asunto(s)
Anticuerpos/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Animales , Células CHO , Cricetulus , Femenino , Ratones , Ratas , Ratas Sprague-Dawley
17.
J Cancer Res Clin Oncol ; 146(12): 3111-3122, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32989604

RESUMEN

PURPOSE: Bispecific antibodies (BsAbs) have emerged as a leading drug class for cancer therapy and are becoming increasingly of interest for therapeutic applications. As of April 2020, over 123 BsAbs are under clinical evaluation for use in oncology (including the two marketed BsAbs Blinatumomab and Catumaxomab). The majority (82 of 123) of BsAbs under clinical evaluation can be categorized as bispecific immune cell engager whereas a second less well-discussed subclass of BsAbs targets two tumor-associated antigens (TAAs). In this review, we summarize the clinical development of dual TAAs targeting BsAbs and provide an overview of critical considerations when designing dual TAA targeting BsAbs. METHODS: Herein the relevant literature and clinical trials published in English until April 1st 2020 were searched using PubMed and ClinicalTrials.gov database. BsAbs were considered to be active in clinic if their clinical trials were not terminated, withdrawn or completed before 2018 without reporting results. Data missed by searching ClinicalTrials.gov was manually curated. RESULTS: Dual TAAs targeting BsAbs offer several advantages including increased tumor selectivity, potential to concurrently modulate two functional pathways in the tumor cell and may yield improved payload delivery. CONCLUSIONS: Dual TAAs targeting BsAbs represent a valuable class of biologics and early stage clinical studies have demonstrated promising anti-tumor efficacy in both hematologic malignancies and solid tumors.


Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antígenos de Neoplasias/inmunología , Neoplasias/terapia , Anticuerpos Biespecíficos/inmunología , Antígenos de Neoplasias/efectos de los fármacos , Humanos , Neoplasias/inmunología , Neoplasias/patología
18.
J Immunol Methods ; 483: 112811, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32569598

RESUMEN

Due to the technical innovations in generating bispecific antibodies (BsAbs) in recent years, BsAbs have become important reagents for diagnostic and therapeutic applications. However, the difficulty of producing a heterodimer consisting of two different arms with high yield and purity constituted a major limitation for their application in academic and clinical settings. Here, we describe a novel Fc-containing BsAb format (Fab × sdAb-Fc) composed of a conventional antigen-binding fragment (Fab), and a single domain antibody (sdAb), which avoids heavy-light chain mis-pairing during antibody assembly. In this study, the Fab x sdAb-Fc BsAbs were efficiently produced by three widely used heavy-heavy chain heterodimerization methods: Knobs-into-holes (KIH), Charge-pairs (CP) and controlled Fab-arm exchange (cFAE), respectively. The novel Fab x sdAb-Fc format provided a rapid and efficient strategy to generate BsAb with high purity and a unique possibility to further purify desired BsAbs from undesired antibodies based on molecular weight (MW). Compared to conventional BsAb formats, the advantages of Fab x sdAb-Fc format may thus provide a straightforward opportunity to apply bispecific antibody principles to research and development of novel targets and pathways in diseases such as cancer and autoimmunity.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Receptores ErbB/inmunología , Glutamato Carboxipeptidasa II/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Glicoproteínas de Membrana/inmunología , Anticuerpos de Dominio Único/inmunología , Animales , Anticuerpos Biespecíficos/biosíntesis , Anticuerpos Biespecíficos/genética , Especificidad de Anticuerpos , Células CHO , Cricetulus , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glutamato Carboxipeptidasa II/genética , Glutamato Carboxipeptidasa II/metabolismo , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Fragmentos Fc de Inmunoglobulinas/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Peso Molecular , Mutación , Prueba de Estudio Conceptual , Multimerización de Proteína , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/genética
19.
Eur J Pharm Biopharm ; 124: 63-72, 2018 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-29274374

RESUMEN

Photodynamic therapy (PDT) is an emerging method to treat light-accessible malignancies. To increase specificity and allow dose reduction, conjugates of photosensitizers (PS) with antibodies against tumor-associated antigens have been developed for photoimmunotherapy (PIT). However, so far it is unclear whether cellular internalization of these conjugates after binding affects PIT efficacy. The use of low molecular weight llama single domain antibodies (VHHs, nanobodies) for PIT is preferred above full size antibodies because of better tumor penetration. Therefore, we functionalized the VHH 7D12, directed against the epidermal growth factor receptor (EGFR), with a PS (IRDye700DX). To assess the impact of cellular internalization on activity, the VHHs were additionally conjugated to a cell-penetrating peptide (VHH[PS]-CPP). Here we show that upon illumination with near-infrared (NIR) light, both VHH[PS] and VHH[PS]-CPP conjugates specifically induce cell death of EGFR expressing cancer cell lines and of EGFR-expressing cells derived from surgically obtained ascites from patients with high-grade serous ovarian cancer. However, VHH[PS] conjugates were significantly more effective compared to internalizing VHH[PS]-CPP suggesting that cell surface association is required for optimal therapeutic activity.


Asunto(s)
Receptores ErbB/metabolismo , Inmunoconjugados/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Anticuerpos de Dominio Único/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Relación Dosis-Respuesta a Droga , Composición de Medicamentos , Endocitosis , Receptores ErbB/inmunología , Femenino , Humanos , Inmunoconjugados/química , Inmunoconjugados/inmunología , Inmunoconjugados/metabolismo , Nanomedicina/métodos , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Tecnología Farmacéutica/métodos
20.
Eur J Pharm Biopharm ; 113: 229-239, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28034806

RESUMEN

Epithelial ovarian cancer is characterized by a high mortality rate and is in need for novel therapeutic avenues to improve patient outcome. The tumor's extracellular matrix ("stroma") offers new possibilities for targeted drug-delivery. Recently we identified highly sulfated chondroitin sulfate (CS-E) as a component abundantly present in the ovarian cancer extracellular matrix, and as a novel target for anti-cancer therapy. Here, we report on the functionalization of drug-loaded lyophilisomes (albumin-based biocapsules) to specifically target the stroma of ovarian carcinomas with the potential to eliminate cancer cells. To achieve specific targeting, we conjugated single chain antibodies reactive with CS-E to lyophilisomes using a two-step approach comprising sortase-mediated ligation and bioorthogonal click chemistry. Antibody-functionalized lyophilisomes specifically targeted the ovarian cancer stroma through CS-E. In a CS-E rich micro-environment in vitro lyophilisomes induced cell death by extracellular release of doxorubicin which localized to the nucleus. Immunohistochemistry identified CS-E rich stroma in a variety of solid tumors other than ovarian cancer, including breast, lung and colon cancer indicating the potential versatility of matrix therapy and the use of highly sulfated chondroitin sulfates in cancer stroma as a micro-environmental hook for targeted drug delivery.


Asunto(s)
Antineoplásicos/administración & dosificación , Matriz Extracelular/efectos de los fármacos , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Carcinoma Epitelial de Ovario , Sistemas de Liberación de Medicamentos , Matriz Extracelular/metabolismo , Femenino , Humanos , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología
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