RESUMEN
PURPOSE: Metabolic imaging using [18F]FDG is the current standard for clinical PET; however, some malignancies (e.g., indolent lymphomas) show low avidity for FDG. The majority of B cell lymphomas express CD20, making it a valuable target both for antibody-based therapy and imaging. We previously developed PET tracers based on the humanised anti-CD20 antibody obinutuzumab (GA101). Preclinical studies showed that the smallest bivalent fragment, the cys-diabody (GAcDb, 54.5 kDa) with a peak uptake at 1-2 h post-injection and a biological half-life of 2-5 h, is compatible with short-lived positron emitters such as fluorine-18 (18F, t1/2 110 min), enabling same-day imaging. METHODS: GAcDb was radiolabeled using amine-reactive N-succinimidyl 4-[18F]-fluorobenzoate ([18F]SFB), or thiol-reactive N-[2-(4-[18F]-fluorobenzamido)ethyl]maleimide ([18F]FBEM) for site-specific conjugation to C-terminal cysteine residues. Both tracers were used for immunoPET imaging of the B cell compartment in human CD20 transgenic mice (hCD20TM). [18F]FB-GAcDb immunoPET was further evaluated in a disseminated lymphoma (A20-hCD20) syngeneic for hCD20TM and compared to [18F]FDG PET. Tracer uptake was confirmed by ex vivo biodistribution. RESULTS: The GAcDb was successfully 18F-radiolabeled using two different conjugation methods resulting in similar specific activities and without impairing immunoreactivity. Both tracers ([18F]FB-GAcDb and [18F]FBEM-GAcDb) specifically target human CD20-expressing B cells in transgenic mice. Fast blood clearance results in high contrast PET images as early as 1 h post injection enabling same-day imaging. [18F]FB-GAcDb immunoPET detects disseminated lymphoma disease in the context of normal tissue expression of hCD20, with comparable sensitivity as [18F]FDG PET but with added specificity for the therapeutic target. CONCLUSIONS: [18F]FB-GAcDb and [18F]FBEM-GAcDb could monitor normal B cells and B cell malignancies non-invasively and quantitatively in vivo. In contrast to [18F]FDG PET, immunoPET provides not only information about the extent of disease but also about presence and localisation of the therapeutic target.
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Anticuerpos/inmunología , Antígenos CD20/inmunología , Radioisótopos de Flúor , Linfoma de Células B/diagnóstico por imagen , Linfoma de Células B/patología , Tomografía de Emisión de Positrones/métodos , Animales , Humanos , Marcaje Isotópico , Linfoma de Células B/inmunología , Ratones , Ratones Transgénicos , Radioquímica , Factores de Tiempo , Distribución TisularRESUMEN
The noninvasive detection and quantification of CD8(+) T cells in vivo are important for both the detection and staging of CD8(+) lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS169.4.2.1) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8(+) T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8(+) T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for (64)Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both (64)Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice.
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Anticuerpos Monoclonales/inmunología , Linfocitos T CD8-positivos/citología , Fragmentos de Inmunoglobulinas/inmunología , Tomografía de Emisión de Positrones , Alelos , Animales , Especificidad de Anticuerpos/inmunología , Antígenos/química , Radioisótopos de Cobre/química , Epítopos/química , Citometría de Flujo , Inmunoterapia/métodos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones SCID , Ratas , Distribución TisularRESUMEN
Epithelial membrane protein-2 (EMP2) is upregulated in a number of tumors and therefore remains a promising target for mAb-based therapy. In the current study, image-guided therapy for an anti-EMP2 mAb was evaluated by PET in both syngeneic and immunodeficient cancer models expressing different levels of EMP2 to enable a better understanding of its tumor uptake and off target accumulation and clearance. The therapeutic efficacy of the anti-EMP2 mAb was initially evaluated in high- and low-expressing tumors, and the mAb reduced tumor load for the high EMP2-expressing 4T1 and HEC-1-A tumors. To create an imaging agent, the anti-EMP2 mAb was conjugated to p-SCN-Bn-deferoxamine (DFO) and radiolabeled with 89Zr. Tumor targeting and tissue biodistribution were evaluated in syngeneic tumor models (4T1, CT26, and Panc02) and human tumor xenograft models (Ramos, HEC-1-A, and U87MG/EMP2). PET imaging revealed radioactive accumulation in EMP2-positive tumors within 24 hours after injection, and the signal was retained for 5 days. High specific uptake was observed in tumors with high EMP2 expression (4T1, CT26, HEC-1-A, and U87MG/EMP2), with less accumulation in tumors with low EMP2 expression (Panc02 and Ramos). Biodistribution at 5 days after injection revealed that the tumor uptake ranged from 2 to approximately 16%ID/cc. The results show that anti-EMP2 mAbs exhibit EMP2-dependent tumor uptake with low off-target accumulation in preclinical cancer models. The development of improved anti-EMP2 Ab fragments may be useful to track EMP2-positive tumors for subsequent therapeutic interventions.
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Glicoproteínas de Membrana , Neoplasias , Tomografía de Emisión de Positrones , Radioisótopos , Circonio , Animales , Femenino , Humanos , Ratones , Anticuerpos Monoclonales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Glicoproteínas de Membrana/metabolismo , Neoplasias/diagnóstico por imagen , Neoplasias/metabolismo , Tomografía de Emisión de Positrones/métodos , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-mediated tumor delivery of cytokines can overcome limitations of systemic administration (toxicity, short half-lives). Previous work showed improved antitumor potency of anti-CD20-IFNα fusion proteins in preclinical mouse models of B-cell lymphoma. Although tumor targeting is mediated by the antibody part of the fusion protein, the cytokine component might strongly influence biodistribution and pharmacokinetics, as a result of its affinity, size, valency, and receptor distribution. Here, we used immunoPET to study the in vivo biodistribution and tumor targeting of the anti-CD20 rituximab-murine IFNα1 fusion protein (Rit-mIFNα) and compared it with the parental mAb (rituximab, Rit). Rit-mIFNα and Rit were radiolabeled with zirconium-89 (89Zr, t1/2 78.4 hours) and injected into C3H mice bearing syngeneic B-cell lymphomas (38C13-hCD20). Dynamic [(2 hours post injection (p.i.)] and static (4, 24, and 72 hours) PET scans were acquired. Ex vivo biodistribution was performed after the final scan. Both 89Zr-Rit-mIFNα and 89Zr-Rit specifically target hCD20-expressing B-cell lymphoma in vivo. 89Zr-Rit-mIFNα showed specific uptake in tumors (7.6 ± 1.0 %ID/g at 75 hours p.i.), which was significantly lower than 89Zr-Rit (38.4 ± 9.9 %ID/g, P < 0.0001). ImmunoPET studies also revealed differences in the biodistribution, 89Zr-Rit-mIFNα showed rapid blood clearance and high accumulation in the liver compared with 89Zr-Rit. Importantly, immunoPET clearly revealed a therapeutic effect of the single 89Zr-Rit-mIFNα dose, resulting in smaller tumors and fewer lymph node metastases compared with mice receiving 89Zr-Rit. Mice receiving 89Zr-Rit-mIFNα had enlarged spleens, suggesting that systemic immune activation contributes to therapeutic efficacy in addition to the direct antitumoral activity of IFNα. In conclusion, immunoPET allows the noninvasive tracking and quantification of the antibody-cytokine fusion protein and helps understand the in vivo behavior and therapeutic efficacy.
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Linfoma de Células B , Radioisótopos , Animales , Línea Celular Tumoral , Humanos , Linfoma de Células B/tratamiento farmacológico , Ratones , Ratones Endogámicos C3H , Tomografía de Emisión de Positrones/métodos , Radioisótopos/uso terapéutico , Distribución Tisular , Circonio/uso terapéuticoRESUMEN
PURPOSE: Radioimmunotherapy uses tumor-specific antibodies to deliver therapeutic radionuclides, but hematological toxicity due to the long serum half-life of intact antibodies remains a challenge. We evaluated a smaller antibody fragment, the minibody, with faster kinetics and a potentially improved therapeutic index. PROCEDURES: The anti-prostate stem cell antigen (PSCA) minibody (A11 Mb) was radiolabeled with iodine-124 ([124I]I-A11 Mb) or conjugated with deferoxamine (DFO) and labeled with zirconium-89 ([89Zr]Zr-DFO-A11 Mb) for surrogate immunoPET to profile pharmacokinetics in a human prostate cancer xenograft model. Subsequently, minibodies labeled with two therapeutic beta emitters, directly iodinated [131I]I-A11 Mb (non-residualizing) and 177Lu chelated using DTPA ([177Lu]Lu-DTPA-A11 Mb) (residualizing), were compared for in vitro antigen-specific cytotoxicity. Full biodistribution studies (in 22Rv1-PSCA tumor bearing and hPSCA knock-in mice) were conducted for dosimetry calculations. Finally, the lead candidate [131I]I-A11 Mb was evaluated in a radioimmunotherapy experiment. Escalating single doses (3.7, 11, or 37 MBq) and saline control were administered to 22Rv1-PSCA tumor bearing mice and anti-tumor effects (tumor volume) and toxicity (body weight) were monitored. RESULTS: Minibodies radiolabeled with therapeutic beta emitters [131I]I-A11 Mb and [177Lu]Lu-DTPA-A11 Mb exhibited comparable tumor cell growth inhibition in vitro. In vivo surrogate immunoPET imaging using [89Zr]Zr-DFO-A11 Mb showed activity retention in liver and kidney up to 72 h, while [124I]I-A11 Mb cleared from liver, kidney, and blood by 48 h. Based on full biodistribution and dosimetry calculations, administering 37 MBq [131I]I-A11 Mb was predicted to deliver a favorable dose to the tumor (35 Gy), with a therapeutic index of 22 (tumor:bone marrow). For [177Lu]Lu-DTPA-A11 Mb, the kidneys would be dose-limiting, and the maximum tolerated activity (7.4 MBq) was not predicted to deliver an effective radiation dose to tumor. Radioimmunotherapy with a single dose of [131I]I-A11 Mb showed dose-dependent tumor inhibition with minimal off-target toxicity and improved median survival (19 and 24 days, P < 0.001) compared with untreated mice (12 days). CONCLUSIONS: These findings show the potential of the anti-PSCA minibody for targeted radioimmunotherapy with minimal toxicity, and the application of immunoPET and dosimetry for personalized treatment.
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Antígenos de Neoplasias/metabolismo , Radioisótopos de Yodo/química , Lutecio/química , Proteínas de Neoplasias/metabolismo , Ácido Pentético/química , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/terapia , Radioinmunoterapia , Radioisótopos/química , Animales , Anticuerpos Monoclonales/farmacocinética , Línea Celular Tumoral , Proliferación Celular , Relación Dosis-Respuesta en la Radiación , Proteínas Ligadas a GPI/metabolismo , Masculino , Ratones , Ácido Pentético/farmacocinética , Neoplasias de la Próstata/inmunología , Radiometría , Análisis de Supervivencia , Distribución TisularRESUMEN
PURPOSE: A great challenge in the diagnosis and treatment of prostate cancer is distinguishing between indolent or local disease and aggressive or metastatic disease. Antibody-based positron emission tomography (immuno-PET) as a cancer-specific imaging modality could improve diagnosis of primary disease, aid the detection of metastases to regional lymph nodes as well as to distant sites (e.g., bone), and monitor response to therapy. PROCEDURE: In search for a more physiologically relevant disease model, a human prostate stem cell antigen knock-in (hPSCA KI) mouse model was generated. The use of a syngeneic prostate cancer cell line transduced to express human PSCA (RM-9-hPSCA) enabled the evaluation of anti-PSCA immuno-PET in immunocompetent mice and in the context of normal tissue expression of PSCA. Two PSCA-specific humanized antibody fragments, A11 minibody and A2 cys-diabody, were radiolabeled with positron emitters iodine-124 and zirconium-89, respectively ([124I]A11 Mb and [89Zr]A2cDb), and used for immuno-PET in wild-type, hPSCA KI and tumor-bearing mice. RESULTS: The hPSCA KI mice express PSCA at low levels in the normal prostate, bladder and stomach, reproducing the expression pattern seen in humans. [124I]A11 Mb immuno-PET detected increased levels of PSCA expression in the stomach, and because I-124 is non-residualizing, very little activity was seen in organs of clearance (liver, kidney, spleen). However, due to the longer half-life of the 80 kDa protein, blood activity (and thus urine activity) at 20 h postinjection remains high. The smaller 50 kDa [89Zr]A2cDb cleared faster, resulting in lower blood and background activity, despite the use of a residualizing radiometal. Importantly, [89Zr]A2cDb immuno-PET showed antigen-specific targeting of PSCA-expressing tumors and minimal nonspecific uptake in PSCA-negative controls. CONCLUSION: Tracer biodistribution was not significantly impacted by normal tissue expression of PSCA. [89Zr]A2cDb immuno-PET yielded high tumor-to-blood ratio at early time points. Rapid renal clearance of the 50 kDa tracer resulted in an unobstructed view of the pelvic region at 20 h postinjection that would allow the detection of cancer in the prostate.
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Antígenos de Neoplasias/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/diagnóstico por imagen , Radioisótopos , Células Madre/citología , Circonio , Animales , Antígenos de Neoplasias/genética , Línea Celular Tumoral , Cruzamientos Genéticos , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Radioisótopos de Yodo , Masculino , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Tomografía Computarizada por Tomografía de Emisión de Positrones , Próstata , Neoplasias de la Próstata/metabolismo , Distribución TisularRESUMEN
Positron emission tomography (PET) molecular imaging is a powerful tool for interrogating physiological and biochemical processes to understand the biology of disease and advance therapeutic developments. Near-infrared fluorescence (NIRF) optical imaging has become increasingly popular for intraoperative staging to enable cellular resolution imaging of tumor margins during surgical resection. In addition, engineered antibody fragments have emerged as promising molecular imaging agents given their exquisite target selectivity, rapid systemic clearance and site-selective chemical modification. We report a tri-functional platform for construction of a modular antibody fragment that can rapidly be labeled with radionuclides or fluorophores for PET or NIRF molecular imaging of prostate stem cell antigen (PSCA).
RESUMEN
Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with 18F-labeled trans-cyclooctene (18F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with 18F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and 18F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery.
Asunto(s)
Carbocianinas/química , Ciclooctanos/química , Radioisótopos de Flúor/química , Microscopía Fluorescente , Tomografía de Emisión de Positrones , Neoplasias de la Próstata/diagnóstico por imagen , Animales , Anticuerpos/química , Antígenos de Neoplasias/sangre , Cisteína/química , Fluorescencia , Colorantes Fluorescentes , Proteínas Ligadas a GPI/sangre , Humanos , Fragmentos de Inmunoglobulinas , Masculino , Ratones , Proteínas de Neoplasias/sangre , Trasplante de Neoplasias , Imagen Óptica , Radiofármacos , Distribución TisularRESUMEN
Selective inhibition of tumor necrosis factor (TNF) signaling through the proinflammatory axis of TNF-receptor 1 (TNFR1) while leaving pro-survival and regeneration-promoting signals via TNFR2 unaffected is a promising strategy to circumvent limitations of complete inhibition of TNF action by the approved anti-TNF drugs. A previously developed humanized antagonistic TNFR1-specific antibody, ATROSAB, showed potent inhibition of TNFR1-mediated cellular responses. Because the parental mouse antibody H398 possesses even stronger inhibitory potential, we scrutinized the specific binding parameters of the two molecules and revealed a faster dissociation of ATROSAB compared to H398. Applying affinity maturation and re-engineering of humanized variable domains, we generated a monovalent Fab derivative (13.7) of ATROSAB that exhibited increased binding to TNFR1 and superior inhibition of TNF-mediated TNFR1 activation, while lacking any agonistic activity even in the presence of cross-linking antibodies. In order to improve its pharmacokinetic properties, several Fab13.7-derived molecules were generated, including a PEGylated Fab, a mouse serum albumin fusion protein, a half-IgG with a dimerization-deficient Fc, and a newly designed Fv-Fc format, employing the knobs-into-holes technology. Among these derivatives, the Fv13.7-Fc displayed the best combination of improved pharmacokinetic properties and antagonistic activity, thus representing a promising candidate for further clinical development.
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Anticuerpos Monoclonales/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/antagonistas & inhibidores , Anticuerpos de Cadena Única/inmunología , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Neutralizantes/inmunología , Afinidad de Anticuerpos , Humanos , Ratones , Ingeniería de Proteínas/métodosRESUMEN
An all-electronic, droplet-based batch microfluidic device, operated using the electrowetting on dielectric (EWOD) mechanism was developed for on-demand synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), the most commonly used 18F-prosthetic group for biomolecule labeling. In order to facilitate the development of peptides, and proteins as new diagnostic and therapeutic agents, we have diversified the compact EWOD microfluidic platform to perform the three-step radiosynthesis of [18F]SFB starting from the no carrier added [18F]fluoride ion. In this report, we established an optimal microliter droplet reaction condition to obtain reliable yields and synthesized [18F]SFB with sufficient radioactivity for subsequent conjugation to the anti-PSCA cys-diabody (A2cDb) and for small animal imaging. The three-step, one-pot radiosynthesis of [18F]SFB radiochemistry was adapted to a batch microfluidic platform with a reaction droplet sandwiched between two parallel plates of an EWOD chip, and optimized. Specifically, the ratio of precursor to base, droplet volume, reagent concentration, reaction time, and evaporation time were found be to be critical parameters. [18F]SFB was successfully synthesized on the EWOD chip in 39 ± 7% (n = 4) radiochemical yield in a total synthesis time of â¼120 min ([18F]fluoride activation, [18F]fluorination, hydrolysis, and coupling reaction, HPLC purification, drying and reformulation). The reformulation and stabilization step for [18F]SFB was important to obtain a high protein labeling efficiency of 33.1 ± 12.5% (n = 3). A small-animal immunoPET pilot study demonstrated that the [18F]SFB-PSCA diabody conjugate showed specific uptake in the PSCA-positive human prostate cancer xenograft. The successful development of a compact footprint of the EWOD radiosynthesizer has the potential to empower biologists to produce PET probes of interest themselves in a standard laboratory.
RESUMEN
PURPOSE: The inability to intraoperatively distinguish primary tumor, as well as lymphatic spread, increases the probability of positive surgical margins, tumor recurrence, and surgical toxicity. The goal of this study was to develop a tumor-specific optical probe for real-time fluorescence-guided surgery. EXPERIMENTAL DESIGN: A humanized antibody fragment against PSCA (A11 minibody, A11 Mb) was conjugated with a near-infrared fluorophore, IRDye800CW. The integrity and binding of the probe to PSCA were confirmed by gel electrophoresis, size-exclusion chromatography, and flow cytometry, respectively. The ability of the probe to detect tumor-infiltrated lymph nodes and metastatic lesions was evaluated in 2 xenograft models, as well as in transgenic mice expressing human PSCA (hPSCA). An invasive intramuscular model was utilized to evaluate the efficacy of the A11 Mb-IRDye800CW-guided surgery. RESULTS: A11 Mb was successfully conjugated with IRDye800CW and retained specific binding to PSCA. In vivo imaging showed maximal signal-to-background ratios at 48 hours. The A11 Mb-IRDye800CW specifically detected PSCA-positive primary tumors, tumor-infiltrated lymph nodes, and distant metastases with high contrast. Fluorescence guidance facilitated more complete tumor resection, reduced tumor recurrence, and improved overall survival, compared with conventional white light surgery. The probe successfully identified primary orthotopic tumors and metastatic lesions in hPSCA transgenic mice. CONCLUSIONS: Real-time fluorescence image-guided surgery with A11 Mb-IRDye800CW enabled detection of lymph node metastases and positive surgical margins, facilitated more complete tumor removal, and improved survival, compared with white light surgery. These results may be translatable into clinical practice to improve surgical and patient outcomes.
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Antígenos de Superficie/genética , Glutamato Carboxipeptidasa II/genética , Indoles/farmacología , Neoplasias de la Próstata/diagnóstico por imagen , Cirugía Asistida por Computador , Animales , Antígenos de Superficie/aislamiento & purificación , Línea Celular Tumoral , Modelos Animales de Enfermedad , Fluorescencia , Regulación Neoplásica de la Expresión Génica/genética , Glutamato Carboxipeptidasa II/aislamiento & purificación , Xenoinjertos , Humanos , Rayos Infrarrojos , Masculino , Márgenes de Escisión , Ratones , Imagen Óptica , Próstata/cirugía , Prostatectomía/métodos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Espectroscopía Infrarroja CortaRESUMEN
Endoglin has been identified as a promising cell surface antigen for vascular targeting approaches in cancer therapy, e.g. employing antibody molecules as targeting moieties. However, in vivo analysis of such strategies in mouse models requires antibodies recognizing endoglin on mouse endothelial cells. Here we describe the isolation of single-chain Fv fragments (scFvs) from phage display libraries, which bind to the extracellular region of mouse endoglin. One of these clones, scFv mE12, showed strong (K(d)=11 nM) and selective binding to purified endoglin and also to the endoglin-expressing mouse endothelioma cell line eEnd.2. This antibody recognized a linear epitope located in the N-terminal region (aa 27-361) of endoglin. Cell binding was further increased by generating a bivalent scFv-Fc fusion protein composed of scFv mE12 and the human gamma1 Fc part. Moreover, scFv mE12 was endowed with an additional cysteine residue in the linker region and applied for the generation of anti-endoglin scFv immunoliposomes capable of selectively binding to endoglin-expressing cells. Thus, anti-mouse endoglin scFv mE12 should be useful to analyze vascular targeting strategies in mice.
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Sistemas de Liberación de Medicamentos , Epítopos/inmunología , Regiones Constantes de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Especificidad de Anticuerpos/inmunología , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Endoglina , Epítopos/genética , Humanos , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/uso terapéutico , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/uso terapéutico , Cadenas gamma de Inmunoglobulina/genética , Cadenas gamma de Inmunoglobulina/inmunología , Cadenas gamma de Inmunoglobulina/uso terapéutico , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
Inadequate diagnostic methods for prostate cancer lead to over- and undertreatment, and the inability to intraoperatively visualize positive margins may limit the success of surgical resection. Prostate cancer visualization could be improved by combining the complementary modalities of immuno-positron emission tomography (immunoPET) for preoperative disease detection, and fluorescence imaging-guided surgery (FIGS) for real-time intraoperative tumor margin identification. Here, we report on the evaluation of dual-labeled humanized anti-prostate stem cell antigen (PSCA) cys-minibody (A11 cMb) for immunoPET/fluorescence imaging in subcutaneous and orthotopic prostate cancer models. Methods: A11 cMb was site-specifically conjugated with the near-infrared fluorophore Cy5.5 and radiolabeled with 124I or 89Zr. 124I-A11 cMb-Cy5.5 was used for successive immunoPET/fluorescence imaging of prostate cancer xenografts expressing high or moderate levels of PSCA (22Rv1-PSCA and PC3-PSCA). 89Zr-A11 cMb-Cy5.5 dual-modality imaging was evaluated in an orthotopic model. Ex vivo biodistribution at 24 h was used to confirm the uptake values, and tumors were visualized by post-mortem fluorescence imaging. Results: A11 cMb-Cy5.5 retained low nanomolar affinity for PSCA-positive cells. Conjugation conditions were established (dye-to-protein ratio of 0.7:1) that did not affect the biodistribution, pharmacokinetics, or clearance of A11 cMb. ImmunoPET using dual-labeled 124I-A11 cMb-Cy5.5 showed specific targeting to both 22Rv1-PSCA and PC3-PSCA s.c. xenografts in nude mice. Ex vivo biodistribution confirmed specific uptake to PSCA-expressing tumors with 22Rv1-PSCA:22Rv1 and PC3-PSCA:PC3 ratios of 13:1 and 5.6:1, respectively. Consistent with the immunoPET, fluorescence imaging showed a strong signal from both 22Rv1-PSCA and PC3-PSCA tumors compared with non-PSCA expressing tumors. In an orthotopic model, 89Zr-A11 cMb-Cy5.5 immunoPET was able to detect intraprostatically implanted 22Rv1-PSCA cells. Importantly, fluorescence imaging clearly distinguished the prostate tumor from surrounding seminal vesicles. Conclusion: Dual-labeled A11 cMb specifically visualized PSCA-positive tumor by successive immunoPET/fluorescence, which can potentially be translated for preoperative whole-body prostate cancer detection and intraoperative surgical guidance in patients.
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Antígenos de Neoplasias/análisis , Inmunoglobulinas/administración & dosificación , Imagen Molecular/métodos , Proteínas de Neoplasias/análisis , Imagen Óptica/métodos , Tomografía de Emisión de Positrones/métodos , Neoplasias de la Próstata/diagnóstico por imagen , Animales , Carbocianinas/administración & dosificación , Modelos Animales de Enfermedad , Colorantes Fluorescentes/administración & dosificación , Proteínas Ligadas a GPI/análisis , Xenoinjertos , Humanos , Masculino , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias de la Próstata/cirugía , Radiofármacos/administración & dosificación , Cirugía Asistida por Computador/métodosRESUMEN
Pancreatic cancer has a high mortality rate due to late diagnosis and the tendency to invade surrounding tissues and metastasize at an early stage. A molecular imaging agent that enables both presurgery antigen-specific PET (immuno-PET) and intraoperative near-infrared fluorescence (NIRF) guidance might benefit diagnosis of pancreatic cancer, staging, and surgical resection, which remains the only curative treatment. Methods: We developed a dual-labeled probe based on A2 cys-diabody (A2cDb) targeting the cell-surface prostate stem cell antigen (PSCA), which is expressed in most pancreatic cancers. Maleimide-IRDye800CW was site-specifically conjugated to the C-terminal cys-tag (A2cDb-800) without impairing integrity or affinity (half-maximal binding, 4.3 nM). Direct radioiodination with 124I (124I-A2cDb-800) yielded a specific activity of 159 ± 48 MBq/mg with a radiochemical purity exceeding 99% and 65% ± 4.5% immunoreactivity (n = 3). In vivo specificity for PSCA-expressing tumor cells and biodistribution of the dual-modality tracer were evaluated in a prostate cancer xenograft model and compared with single-labeled 124I-A2cDb. Patient-derived pancreatic ductal adenocarcinoma xenografts (PDX-PDACs) were grown subcutaneously in NSG mice and screened for PSCA expression by immuno-PET. Small-animal PET/CT scans of PDX-PDAC-bearing mice were obtained using the dual-modality 124I-A2cDb-800 followed by postmortem NIRF imaging with the skin removed. Tumors and organs were analyzed ex vivo to compare the relative fluorescent signals without obstruction by other organs. Results: Specific uptake in PSCA-positive tumors and low nonspecific background activity resulted in high-contrast immuno-PET images. Concurrent with the PET studies, fluorescent signal was observed in the PSCA-positive tumors of mice injected with the dual-tracer 124I-A2cDb-800, with low background uptake or autofluorescence in the surrounding tissue. Ex vivo biodistribution confirmed comparable tumor uptake of both 124I-A2cDb-800 and 124I-A2cDb. Conclusion: Dual-modality imaging using the anti-PSCA cys-diabody resulted in high-contrast immuno-PET/NIRF images of PDX-PDACs, suggesting that this imaging agent might offer both noninvasive whole-body imaging to localize PSCA-positive pancreatic cancer and fluorescence image-guided identification of tumor margins during surgery.
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Antígenos de Neoplasias/inmunología , Rayos Infrarrojos , Proteínas de Neoplasias/inmunología , Imagen Óptica/métodos , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Anticuerpos de Cadena Única/inmunología , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Proteínas Ligadas a GPI/inmunología , Radioisótopos de Yodo , Masculino , Ratones , Estadificación de Neoplasias , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Anticuerpos de Cadena Única/farmacocinética , Distribución TisularRESUMEN
Inflammatory bowel diseases (IBDs) in humans are characterized in part by aberrant CD4-positive (CD4+) T-cell responses. Currently, identification of foci of inflammation within the gut requires invasive procedures such as colonoscopy and biopsy. Molecular imaging with antibody fragment probes could be used to noninvasively monitor cell subsets causing intestinal inflammation. Here, GK1.5 cys-diabody (cDb), an antimouse CD4 antibody fragment derived from the GK1.5 hybridoma, was used as a PET probe for CD4+ T cells in the dextran sulfate sodium (DSS) mouse model of IBD. Methods: The DSS mouse model of IBD was validated by assessing changes in CD4+ T cells in the spleen and mesenteric lymph nodes (MLNs) using flow cytometry. Furthermore, CD4+ T cell infiltration in the colons of colitic mice was evaluated using immunohistochemistry. 89Zr-labeled GK1.5 cDb was used to image distribution of CD4+ T cells in the abdominal region and lymphoid organs of mice with DSS-induced colitis. Region-of-interest analysis was performed on specific regions of the gut to quantify probe uptake. Colons, ceca, and MLNs were removed and imaged ex vivo by PET. Imaging results were confirmed by ex vivo biodistribution analysis. Results: An increased number of CD4+ T cells in the colons of colitic mice was confirmed by anti-CD4 immunohistochemistry. Increased uptake of 89Zr-maleimide-deferoxamine (malDFO)-GK1.5 cDb in the distal colon of colitic mice was visible in vivo in PET scans, and region-of-interest analysis of the distal colon confirmed increased activity in DSS mice. MLNs from colitic mice were enlarged and visible in PET images. Ex vivo scans and biodistribution confirmed higher uptake in DSS-treated colons (DSS, 1.8 ± 0.40; control, 0.45 ± 0.12 percentage injected dose [%ID] per organ, respectively), ceca (DSS, 1.1 ± 0.38; control, 0.35 ± 0.09 %ID per organ), and MLNs (DSS, 1.1 ± 0.58; control, 0.37 ± 0.25 %ID per organ). Conclusion:89Zr-malDFO-GK1.5 cDb detected CD4+ T cells in the colons, ceca, and MLNs of colitic mice and may prove useful for further investigations of CD4+ T cells in preclinical models of IBD, with potential to guide development of antibody-based imaging in human IBD.
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Linfocitos T CD4-Positivos/inmunología , Colitis/diagnóstico por imagen , Colitis/inmunología , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Animales , Colitis/patología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
PURPOSE: Molecular imaging of CD4+ T cells throughout the body has implications for monitoring autoimmune disease and immunotherapy of cancer. Given the key role of these cells in regulating immunity, it is important to develop a biologically inert probe. GK1.5 cys-diabody (cDb), a previously developed anti-mouse CD4 antibody fragment, was tested at different doses to assess its effects on positron emission tomography (PET) imaging and CD4+ T cell viability, proliferation, CD4 expression, and function. PROCEDURES: The effect of protein dose on image contrast (lymphoid tissue-to-muscle ratio) was assessed by administering different amounts of 89Zr-labeled GK1.5 cDb to mice followed by PET imaging and ex vivo biodistribution analysis. To assess impact of GK1.5 cDb on T cell biology, GK1.5 cDb was incubated with T cells in vitro or administered intravenously to C57BL/6 mice at multiple protein doses. CD4 expression and T cell proliferation were analyzed with flow cytometry and cytokines were assayed. RESULTS: For immunoPET imaging, the lowest protein dose of 2 µg of 89Zr-labeled GK1.5 cDb resulted in significantly higher % injected dose/g in inguinal lymph nodes (ILN) and spleen compared to the 12-µg protein dose. In vivo administration of GK1.5 cDb at the high dose of 40 µg caused a transient decrease in CD4 expression in spleen, blood, lymph nodes, and thymus, which recovered within 3 days postinjection; this effect was reduced, although not abrogated, when 2 µg was administered. Proliferation was inhibited in vivo in ILN but not the spleen by injection of 40 µg GK1.5 cDb. Concentrations of GK1.5 cDb in excess of 25 nM significantly inhibited CD4+ T cell proliferation and interferon-γ production in vitro. Overall, using low-dose GK1.5 cDb minimized biological effects on CD4+ T cells. CONCLUSIONS: Low-dose GK1.5 cDb yields high-contrast immunoPET images with minimal effects on T cell biology in vitro and in vivo and may be a useful tool for investigating CD4+ T cells in the context of preclinical disease models. Future approaches to minimizing biological effects may include the creation of monovalent fragments or selecting anti-CD4 antibodies which target alternative epitopes.
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Anticuerpos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Cisteína/metabolismo , Tomografía de Emisión de Positrones , Animales , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/citología , Proliferación Celular , Citocinas/metabolismo , Femenino , Tejido Linfoide/diagnóstico por imagen , Ratones Endogámicos C57BL , Distribución TisularRESUMEN
Purpose: The B-cell antigen CD20 provides a target for antibody-based positron emission tomography (immunoPET). We engineered antibody fragments targeting human CD20 and studied their potential as immunoPET tracers in transgenic mice (huCD20TM) and in a murine lymphoma model expressing human CD20.Experimental Design: Anti-CD20 cys-diabody (cDb) and cys-minibody (cMb) based on rituximab and obinutuzumab (GA101) were radioiodinated and used for immunoPET imaging of a murine lymphoma model. Pairwise comparison of obinutuzumab-based antibody fragments labeled with residualizing (89Zr) versus non-residualizing (124I) radionuclides by region of interest analysis of serial PET images was conducted both in the murine lymphoma model and in huCD20TM to assess antigen modulation in vivoResults:124I-GAcDb and 124I-GAcMb produced high-contrast immunoPET images of B-cell lymphoma and outperformed the respective rituximab-based tracers. ImmunoPET imaging of huCD20TM showed specific uptake in lymphoid tissues. The use of the radiometal 89Zr as alternative label for GAcDb and GAcMb yielded greater target-specific uptake and retention compared with 124I-labeled tracers. Pairwise comparison of 89Zr- and 124I-labeled GAcDb and GAcMb allowed assessment of in vivo internalization of CD20/antibody complexes and revealed that CD20 internalization differs between malignant and endogenous B cells.Conclusions: These obinutuzumab-based PET tracers have the ability to noninvasively and quantitatively monitor CD20-expression and have revealed insights into CD20 internalization upon antibody binding in vivo Because they are based on a humanized mAb they have the potential for direct clinical translation and could improve patient selection for targeted therapy, dosimetry prior to radioimmunotherapy, and prediction of response to therapy. Clin Cancer Res; 23(23); 7242-52. ©2017 AACR.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos CD20/inmunología , Linfoma de Células B/diagnóstico por imagen , Linfoma de Células B/tratamiento farmacológico , Tomografía de Emisión de Positrones/métodos , Animales , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales Humanizados/farmacocinética , Antígenos CD20/genética , Antígenos CD20/metabolismo , Línea Celular Tumoral , Endocitosis/efectos de los fármacos , Endocitosis/inmunología , Femenino , Humanos , Fragmentos de Inmunoglobulinas/metabolismo , Fragmentos de Inmunoglobulinas/uso terapéutico , Radioisótopos de Yodo/metabolismo , Radioisótopos de Yodo/farmacocinética , Linfoma de Células B/genética , Ratones Endogámicos BALB C , Ratones SCID , Ratones Transgénicos , Radioisótopos/metabolismo , Radioisótopos/farmacocinética , Distribución Tisular , Circonio/metabolismo , Circonio/farmacocinéticaRESUMEN
The rapidly advancing field of cancer immunotherapy is currently limited by the scarcity of noninvasive and quantitative technologies capable of monitoring the presence and abundance of CD8(+) T cells and other immune cell subsets. In this study, we describe the generation of (89)Zr-desferrioxamine-labeled anti-CD8 cys-diabody ((89)Zr-malDFO-169 cDb) for noninvasive immuno-PET tracking of endogenous CD8(+) T cells. We demonstrate that anti-CD8 immuno-PET is a sensitive tool for detecting changes in systemic and tumor-infiltrating CD8 expression in preclinical syngeneic tumor immunotherapy models including antigen-specific adoptive T-cell transfer, agonistic antibody therapy (anti-CD137/4-1BB), and checkpoint blockade antibody therapy (anti-PD-L1). The ability of anti-CD8 immuno-PET to provide whole body information regarding therapy-induced alterations of this dynamic T-cell population provides new opportunities to evaluate antitumor immune responses of immunotherapies currently being evaluated in the clinic.
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Linfocitos T CD8-positivos/diagnóstico por imagen , Linfocitos T CD8-positivos/inmunología , Neoplasias del Colon/diagnóstico por imagen , Neoplasias del Colon/terapia , Inmunoterapia Adoptiva/métodos , Tomografía de Emisión de Positrones/métodos , Radioisótopos/administración & dosificación , Radiofármacos/administración & dosificación , Circonio/administración & dosificación , Animales , Anticuerpos Biespecíficos , Antígenos CD8 , Neoplasias del Colon/inmunología , Deferoxamina/administración & dosificación , Deferoxamina/química , Deferoxamina/inmunología , Modelos Animales de Enfermedad , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Inmunoconjugados/inmunología , Linfocitos Infiltrantes de Tumor/diagnóstico por imagen , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Radioisótopos/química , Radiofármacos/química , Radiofármacos/inmunología , Circonio/químicaRESUMEN
PURPOSE: The inability to visualize cancer during prostatectomy contributes to positive margins, cancer recurrence, and surgical side effects. A molecularly targeted fluorescent probe offers the potential for real-time intraoperative imaging. The goal of this study was to develop a probe for image-guided prostate cancer surgery. EXPERIMENTAL DESIGN: An antibody fragment (cys-diabody, cDb) against prostate stem cell antigen (PSCA) was conjugated to a far-red fluorophore, Cy5. The integrity and binding of the probe to PSCA was confirmed by gel electrophoresis, size exclusion, and flow cytometry, respectively. Subcutaneous models of PSCA-expressing xenografts were used to assess the biodistribution and in vivo kinetics, whereas an invasive intramuscular model was utilized to explore the performance of Cy5-cDb-mediated fluorescence guidance in representative surgical scenarios. Finally, a prospective, randomized study comparing surgical resection with and without fluorescent guidance was performed to determine whether this probe could reduce the incidence of positive margins. RESULTS: Cy5-cDb demonstrated excellent purity, stability, and specific binding to PSCA. In vivo imaging showed maximal signal-to-background ratios at 6 hours. In mice carrying PSCA(+) and negative (-) dual xenografts, the mean fluorescence ratio of PSCA(+/-) tumors was 4.4:1. In surgical resection experiments, residual tumors <1 mm that were missed on white light surgery were identified and resected using fluorescence guidance, which reduced the incidence of positive surgical margins (0/8) compared with white light surgery alone (7/7). CONCLUSIONS: Fluorescently labeled cDb enables real-time in vivo imaging of prostate cancer xenografts in mice, and facilitates more complete tumor removal than conventional white light surgery alone.
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Anticuerpos Monoclonales/farmacología , Fragmentos de Inmunoglobulinas/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/cirugía , Cirugía Asistida por Computador , Animales , Antígenos de Neoplasias/metabolismo , Modelos Animales de Enfermedad , Colorantes Fluorescentes , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Humanos , Masculino , Ratones , Proteínas de Neoplasias/metabolismo , Imagen Óptica/métodos , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/metabolismo , Cirugía Asistida por Computador/métodos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
INTRODUCTION: This work describes the development and characterization of two antibody fragments that specifically target the α(v)ß(6) integrin, a non-covalent diabody and a disulfide-stabilized cys-diabody. The diabodies were analyzed for their ability to bind both immobilized and cell surface-bound α(v)ß(6). Radiolabeling was done using non-site-specific and site-specific conjugation approaches with N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]-SFB) and the bifunctional chelator 1,4,7-triazacyclononane-triacetic acid maleimide (NOTA-maleimide) and copper-64 ([(64)Cu]), respectively. The affects of each radiolabeling method on RCY, RCP, and immunoreactivity were analyzed for the [(18)F]-FB-α(v)ß(6) diabody, [(18)F]-FB-α(v)ß(6) cys-diabody, and the [(64)Cu]-NOTA-α(v)ß(6) cys-diabody. METHODS: Diabodies were constructed from the variable domains of the humanized 6.3G9 anti-α(v)ß(6) intact antibody. The anti-α(v(ß(6) cys-diabody was engineered with C-terminal cysteines to enable covalent dimerization and site-specific modification. Biochemical characterization included SDS-PAGE, Western blot, and electrospray ionization to confirm MW, and flow cytometry and ELISA experiments were used to determine binding affinity and specificity to α(v)ß(6). The diabodies were radiolabeled with [(18)F]-SFB and in addition, the anti-α(v)ß(6) cys-diabody was also radiolabeled site-specifically using NOTA-maleimide and [(64)Cu]. Immunoreactivities were confirmed using in vitro cell binding to DX3Puroß(6) (α(v)ß(6)+) and DX3Puro (α(v)ß(6)-)cell lines. RESULTS: The diabodies were purified from cell culture supernatants with purities >98%. Subnanomolar binding affinity towards αvß6 was confirmed by ELISA (diabody IC(50)=0.8 nM, cys-diabody IC(50)=0.6 nM) and flow cytometry revealed high specificity only to the DX3Puroß(6) cell line for both diabodies. RCYs were 22.6%±3.6% for the [(18)F]-FB-α(v)ß(6) diabody, 8.3%±1.7% for the [(18)F]-FB-α(v)ß(6) cys-diabody and 43.5%±5.5% for the [(64)Cu]-NOTA-α(v)ß(6) cys-diabody. In vitro cell binding assays revealed excellent specificity and retention of immunoreactivity ([(18)F]-FB-α(v)ß(6) diabody=58.7%±6.7%, [(18)F]-FB-α(v)ß(6) cys-diabody=80.4%±4.4%, [(64)Cu]-NOTA-α(v)ß(6) cys-diabody=59.4%±0.6%) regardless of the radiolabeling method used. CONCLUSIONS: Two novel diabodies with excellent binding affinity and specificity for the α(v)ß(6) integrin in vitro were developed. Radiolabeling of the diabodies with fluorine-18 ([(18)F]) and [(64)Cu] revealed advantages and disadvantages with regards to methodologies and RCYs, however immunoreactivities were well preserved regardless of radiolabeling approach.