RESUMEN
PURPOSE: Interest in targeted alpha-therapy has surged due to α-particles' high cytotoxicity. However, the widespread clinical use of this approach could be limited by on-/off-target toxicities. Here, we investigated the inverse electron-demand Diels-Alder ligation between an 225Ac-labeled tetrazine radioligand and a trans-cyclooctene-bearing anti-CA19.9 antibody (5B1) for pretargeted α-radioimmunotherapy (PRIT) of pancreatic ductal adenocarcinoma (PDAC). This alternative strategy is expected to reduce nonspecific toxicities as compared with conventional radioimmunotherapy (RIT).Experimental Design: A side-by-side comparison of 225Ac-PRIT and conventional RIT using a directly 225Ac-radiolabeled immunoconjugate evaluates the therapeutic efficacy and toxicity of both methodologies in PDAC murine models. RESULTS: A comparative biodistribution study of the PRIT versus RIT methodology underscored the improved pharmacokinetic properties (e.g., prolonged tumor uptake and increased tumor-to-tissue ratios) of the PRIT approach. Cerenkov imaging coupled to PRIT confirmed the in vivo biodistribution of 225Ac-radioimmunoconjugate but-importantly-further allowed for the ex vivo monitoring of 225Ac's radioactive daughters' redistribution. Human dosimetry was extrapolated from the mouse biodistribution and confirms the clinical translatability of 225Ac-PRIT. Furthermore, longitudinal therapy studies performed in subcutaneous and orthotopic PDAC models confirm the therapeutic efficacy of 225Ac-PRIT with the observation of prolonged median survival compared with control cohorts. Finally, a comparison with conventional RIT highlighted the potential of 225Ac-PRIT to reduce hematotoxicity while maintaining therapeutic effectiveness. CONCLUSIONS: The ability of 225Ac-PRIT to deliver a radiotherapeutic payload while simultaneously reducing the off-target toxicity normally associated with RIT suggests that the clinical translation of this approach will have a profound impact on PDAC therapy.
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Actinio/química , Actinio/farmacología , Química Clic , Inmunoconjugados/química , Inmunoconjugados/farmacología , Radiofármacos/química , Radiofármacos/farmacología , Actinio/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/diagnóstico por imagen , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/terapia , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Humanos , Inmunoconjugados/uso terapéutico , Ratones , Imagen Molecular , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Radiometría , Radiofármacos/uso terapéutico , Distribución Tisular , Investigación Biomédica Traslacional , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias PancreáticasRESUMEN
PURPOSE: Tumor-specific molecular imaging is an important tool for assessing disease burden and treatment response. CA19.9 is an important tumor-specific marker in several malignancies, including urothelial carcinoma. [89Zr]DFO-HuMab-5B1 (MVT-2163) is a CA19.9-specific antibody-based construct that has been validated in preclinical animal models of lung, colorectal, and pancreatic malignancies for positron emission tomography (PET) imaging and is currently in a phase I trial for pancreatic cancer (NCT02687230). Here, we examine whether [89Zr]DFO-HuMab-5B1 may be useful in defining urothelial malignancies. PROCEDURES: Surface expression of CA19.9 was confirmed in the human bladder cancer line HT 1197. The radioimmunoconjugate [89Zr]DFO-HuMab-5B1 was injected into mice bearing HT 1197 xenografts, and followed by PET imaging, ex vivo experiments including biodistribution, histology and autoradiography, and analysis of blood samples for shed antigen levels were performed. RESULTS: [89Zr]DFO-HuMab-5B1 specifically accumulates in HT 1197 engrafted tumors when imaged with PET. Ex vivo biodistribution of organs and autoradiography of engrafted tumors confirm our construct's specific tumor binding. The target antigen CA19.9 was not found to be shed in vitro or in vivo. CONCLUSIONS: [89Zr]DFO-HuMab-5B1 can be used to delineate urothelial carcinomas by PET imaging and may provide tumor-specific information prior to, during, and after systemic therapies.
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Modelos Biológicos , Tomografía de Emisión de Positrones , Radioisótopos/química , Neoplasias de la Vejiga Urinaria/diagnóstico por imagen , Circonio/química , Animales , Anticuerpos Monoclonales/metabolismo , Autorradiografía , Antígeno CA-19-9/metabolismo , Línea Celular Tumoral , Humanos , Ratones Desnudos , Suero/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CA19.9 is one of the most commonly occurring and highest density antigens in >90% of pancreatic cancers, making it an excellent target for monoclonal antibody (mAb)-based imaging and therapy applications. Preloading of unlabeled antibodies to enhance targeting of a radiolabeled mAb has been previously described both for imaging and radioimmunotherapy studies for other targets. We investigated the effect of preloading with the unmodified anti-CA19.9 antibody 5B1 on the uptake and contrast of the PET tracer 89Zr-5B1 in subcutaneous and orthotopic murine models of pancreatic cancer utilizing Capan-2 xenografts, known to both express CA19.9 and shed antigen into circulation. Biodistribution and PET imaging studies with 89Zr-5B1 alone showed high levels in the liver, spleen, and lymph nodes of mice with subcutaneous Capan-2 tumor xenografts when administered without preinjection of 5B1. When unlabeled 5B1 was administered prior to 89Zr-5B1, the tracer significantly enhanced image contrast and tumor to tissue ratios in the same model, and the improvement was related to the time interval between the injections. Moreover, tumors were clearly delineated in an orthotopic pancreatic cancer model using our optimized approach. Taken together, these data suggest that preloading with 5B1 can improve 89Zr-5B1 imaging of disease in a Capan-2 mouse model and that exploration of preloading may have clinical utility for ongoing clinical investigations.
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Anticuerpos Monoclonales/química , Antígeno CA-19-9/química , Neoplasias Pancreáticas/diagnóstico , Radioisótopos/química , Radiofármacos/química , Circonio/química , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Desnudos , Tomografía de Emisión de Positrones/métodos , Distribución TisularRESUMEN
The pretargeting system based on the inverse electron demand Diels-Alder reaction (IEDDA) between trans-cyclooctene (TCO) and tetrazine (Tz) combines the favorable pharmacokinetic properties of radiolabeled small molecules with the affinity and specificity of antibodies. This strategy has proven to be an efficient method for the molecularly targeted delivery of pharmaceuticals, including isotopes for radiological imaging. Despite encouraging results from in vivo PET imaging studies, this promising system has yet to be thoroughly evaluated for pretargeted radioimmunotherapy (PRIT). Toward that end, we synthesized two novel 177Lu-labeled tetrazine-bearing radioligands. Next, we compared the usefulness of our ligands for PRIT when paired with TCO-modified 5B1-a human, anti-CA19.9 mAb-in preclinical murine models of pancreatic cancer. The exemplary ligand, 177Lu-DOTA-PEG7-Tz, showed rapid (4.6 ± 0.8% ID/g at 4 hours) and persistent (16.8 ± 3.9% ID/g at 120 hours) uptake in tumors while concurrently clearing from blood and nontarget tissues. Single-dose therapy studies using 5B1-TCO and varying amounts of 177Lu-DOTA-PEG7-Tz (400, 800, and 1,200 µCi) showed that our system elicits a dose-dependent therapeutic response in mice bearing human xenografts. Furthermore, dosimetry calculations suggest that our approach is amenable to clinical applications with its excellent dosimetric profile in organs of clearance (i.e., liver and kidneys) as well as in dose-limiting tissues, such as red marrow. This study established that a pretargeted methodology utilizing the IEDDA reaction can rapidly and specifically deliver a radiotherapeutic payload to tumor tissue, thus illustrating its excellent potential for clinical translation. Mol Cancer Ther; 16(1); 124-33. ©2016 AACR.
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Antineoplásicos/química , Antineoplásicos/farmacología , Química Clic , Radioinmunoterapia , Radiofármacos/química , Radiofármacos/farmacología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Ratones , Estructura Molecular , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Polietilenglicoles/química , Tomografía de Emisión de Positrones , Radiometría , Distribución Tisular , Carga Tumoral/efectos de los fármacos , Carga Tumoral/efectos de la radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: 5B1 is a fully human, monoclonal antibody that has shown promise for the PET imaging of cancers expressing carbohydrate antigen 19.9 (CA19.9)--a carbohydrate prevalent in cells with aberrant glycosylation and an established effector of metastasis. The long physiologic half-life of the antibody and interference from circulating CA19.9 may increase the time required to generate quality images as well as the risk of radiation exposure to healthy tissues during repeated PET imaging. Pretargeting methodologies are an effective approach to expeditiously acquire PET images, but in this case, the pretargeting approach is complicated by the internalization of 5B1 by CA19.9-expressing cells. We sought to adapt and optimize a pretargeting strategy that exploits the bioorthogonal reaction between transcyclooctene (TCO) and tetrazine (Tz) to overcome these complications. METHODS: 5B1 was modified with TCO, and a novel NOTA-PEG7-Tz radioligand was synthesized with the goal of improving on a previously reported analog. BxPC3 and Capan-2 cells were evaluated for their ability to internalize anti-CA19.9 antibodies using a fluorometric assay, and xenografts of the same lines were used for in vivo studies. The pretargeting approach was optimized, and the 2 radioligands were compared using biodistribution and PET imaging in murine models of pancreatic cancer. RESULTS: BxPC3 and Capan-2 cells were shown to rapidly internalize anti-CA19.9 monoclonal antibodies, including 5B1. (64)Cu-NOTA-PEG7-Tz showed improved in vivo pharmacokinetics relative to (64)Cu-NOTA-Tz using 5B1-TCO as the targeting vector. PET imaging and biodistribution studies showed that injecting the radioligand 72 h after the administration of 5B1-TCO resulted in the best uptake (8.2 ± 1.7 percentage injected dose per gram at 20 h after injection) and tumor-to-background activity concentration ratios. Dosimetry calculations revealed that the pretargeting system produced a greater than 25-fold reduction in total body radiation exposure relative to (89)Zr-desferrioxamine-5B1. PET/CT imaging in an orthotopic Capan-2 xenograft model--which secretes large amounts of CA19.9 and more rapidly internalizes anti-CA19.9 antibodies--showed that this approach is viable even in the difficult circumstances presented by a circulating antigen and internalized targeting vector. CONCLUSION: The 5B1-TCO and (64)Cu-NOTA-PEG7-Tz system evaluated in these studies can delineate CA19.9-positive xenografts in murine models of pancreatic cancer despite the challenges posed by the combination of circulating antigen and internalization of the 5B1-TCO.
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Anticuerpos Antineoplásicos , Antígenos de Neoplasias , Neoplasias Pancreáticas/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Animales , Línea Celular Tumoral , Radioisótopos de Cobre , Deferoxamina/síntesis química , Deferoxamina/farmacocinética , Compuestos Heterocíclicos con 1 Anillo/síntesis química , Compuestos Heterocíclicos con 1 Anillo/farmacocinética , Humanos , Ratones , Trasplante de Neoplasias , Dosis de Radiación , Radioisótopos , Radiofármacos/síntesis química , Distribución Tisular , CirconioRESUMEN
A first-of-its-kind (18)F pretargeted PET imaging approach based on the bioorthogonal inverse electron demand Diels-Alder (IEDDA) reaction between tetrazine (Tz) and trans-cyclooctene (TCO) is presented. As proof-of-principle, a TCO-bearing immunoconjugate of the anti-CA19.9 antibody 5B1 and an Al[(18)F]NOTA-labeled tetrazine radioligand were harnessed for the visualization of CA19.9-expressing BxPC3 pancreatic cancer xenografts. Biodistribution and (18)F-PET imaging data clearly demonstrate that this methodology effectively delineates tumor mass with activity concentrations up to 6.4 %ID/g at 4 h after injection of the radioligand.
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Ciclooctanos/química , Radioisótopos de Flúor/química , Inmunoconjugados/química , Neoplasias Pancreáticas/diagnóstico , Tomografía de Emisión de Positrones/métodos , Animales , Antígenos de Carbohidratos Asociados a Tumores/análisis , Química Clic/métodos , Reacción de Cicloadición/métodos , Ciclooctanos/farmacocinética , Radioisótopos de Flúor/farmacocinética , Humanos , Inmunoconjugados/farmacocinética , Ratones , Páncreas/patología , Neoplasias Pancreáticas/patología , Radiofármacos/química , Radiofármacos/farmacocinéticaRESUMEN
Molecular imaging agents for preoperative positron emission tomography (PET) and near-infrared fluorescent (NIRF)-guided delineation of surgical margins could greatly enhance the diagnosis, staging, and resection of pancreatic cancer. PET and NIRF optical imaging offer complementary clinical applications, enabling the noninvasive whole-body imaging to localize disease and identification of tumor margins during surgery, respectively. We report the development of PET, NIRF, and dual-modal (PET/NIRF) imaging agents, using 5B1, a fully human monoclonal antibody that targets CA19.9, a well-established pancreatic cancer biomarker. Desferrioxamine (DFO) and/or a NIRF dye (FL) were conjugated to the heavy-chain glycans of 5B1, using a robust and reproducible site-specific (ss) labeling methodology to generate three constructs ((ss)DFO-5B1, (ss)FL-5B1, and (ss)dual-5B1) in which the immunoreactivity was not affected by the conjugation of either label. Each construct was evaluated in a s.c. xenograft model, using CA19.9-positive (BxPC3) and -negative (MIAPaCa-2) human pancreatic cancer cell lines. Each construct showed exceptional uptake and contrast in antigen-positive tumors with negligible nonspecific uptake in antigen-negative tumors. Additionally, the dual-modal construct was evaluated in an orthotopic murine pancreatic cancer model, using the human pancreatic cancer cell line, Suit-2. The (ss)dual-5B1 demonstrated a remarkable capacity to delineate metastases and to map the sentinel lymph nodes via tandem PET-computed tomography (PET/CT) and NIRF imaging. Fluorescence microscopy, histopathology, and autoradiography were performed on representative sections of excised tumors to visualize the distribution of the constructs within the tumors. These imaging tools have tremendous potential for further preclinical research and for clinical translation.
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Antígeno CA-19-9/inmunología , Inmunoconjugados/inmunología , Imagen Multimodal/métodos , Neoplasias Pancreáticas/metabolismo , Tomografía de Emisión de Positrones/métodos , Espectroscopía Infrarroja Corta/métodos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Línea Celular Tumoral , Deferoxamina/química , Modelos Animales de Enfermedad , Femenino , Colorantes Fluorescentes/química , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Ratones Noqueados , Ratones Desnudos , Microscopía Fluorescente , Estructura Molecular , Neoplasias Pancreáticas/diagnóstico , Radioisótopos/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Distribución Tisular , Trasplante Heterólogo , Circonio/químicaRESUMEN
UNLABELLED: Despite their considerable advantages, many circulating biomarkers have well-documented limitations. One prominent shortcoming in oncology is a high frequency of false-positive indications for malignant disease in upfront diagnosis. Because one common cause of false positivism is biomarker production from benign disorders in unrelated host tissues, we hypothesized that probing the sites of biomarker secretion with an imaging tool could be a broadly useful strategy to deconvolute the meaning of foreboding but inconclusive circulating biomarker levels. METHODS: In preparation to address this hypothesis clinically, we developed (89)Zr-5B1, a fully human, antibody-based radiotracer targeting tumor-associated CA19.9 in the preclinical setting. RESULTS: (89)Zr-5B1 localized to multiple tumor models representing diseases with undetectable and supraphysiologic serum CA19.9 levels. Among these, (89)Zr-5B1 detected orthotopic models of pancreatic ductal adenocarcinoma, an elusive cancer for which the serum assay is measured in humans but with limited specificity in part because of the frequency of CA19.9 secretion from benign hepatic pathologies. CONCLUSION: In this report, a general strategy to supplement some of the shortcomings of otherwise highly useful circulating biomarkers with immunoPET is described. To expedite the clinical validation of this model, a human monoclonal antibody to CA19.9 (a highly visible but partially flawed serum biomarker for several cancers) was radiolabeled and evaluated, and the compelling preclinical evidence suggests that the radiotracer may enhance the fidelity of diagnosis and staging of pancreatic ductal adenocarcinoma, a notoriously occult cancer.
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Antígenos de Carbohidratos Asociados a Tumores/sangre , Biomarcadores de Tumor/sangre , Neoplasias/sangre , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Radioisótopos , Circonio , Animales , Anticuerpos Monoclonales/farmacocinética , Línea Celular Tumoral , Transformación Celular Neoplásica , Femenino , Humanos , Ratones , Neoplasias/patologíaRESUMEN
PURPOSE: The carbohydrate antigen sialyl-Lewis(a) (sLe(a)), also known as CA19.9, is widely expressed on epithelial tumors of the gastrointestinal tract and breast and on small-cell lung cancers. Since overexpression of sLe(a) appears to be a key event in invasion and metastasis of many tumors and results in susceptibility to antibody-mediated lysis, sLe(a) is an attractive molecular target for tumor therapy. EXPERIMENTAL DESIGN: We generated and characterized fully human monoclonal antibodies (mAb) from blood lymphocytes from individuals immunized with a sLe(a)-KLH vaccine. RESULTS: Several mAbs were selected based on ELISA and FACS including two mAbs with high affinity for sLe(a) (5B1 and 7E3, binding affinities 0.14 and 0.04 nmol/L, respectively) and further characterized. Both antibodies were specific for Neu5Acα2-3Galß1-3(Fucα1-4)GlcNAcß as determined by glycan array analysis. Complement-dependent cytotoxicity against DMS-79 cells was higher (EC(50) 0.1 µg/mL vs. 1.7 µg/mL) for r7E3 (IgM) than for r5B1 (IgG1). In addition, r5B1 antibodies showed high level of antibody-dependent cell-mediated cytotoxicity activity on DMS-79 cells with human NK cells or peripheral blood mononuclear cells. To evaluate in vivo efficacy, the antibodies were tested in a xenograft model with Colo205 tumor cells engrafted into SCID (severe combined immunodeficient mice) mice. Treatment during the first 21 days with four doses of r5B1 (100 µg per dose) doubled the median survival time to 207 days, and three of five animals survived with six doses. CONCLUSION: On the basis of the potential of sLe(a) as a target for immune attack and their affinity, specificity, and effector functions, 5B1and 7E3 may have clinical utility.
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Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos , Antígeno CA-19-9/inmunología , Citotoxicidad Inmunológica , Neoplasias Experimentales/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Línea Celular Tumoral , Ensayo de Actividad Hemolítica de Complemento , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Leucocitos Mononucleares/inmunología , Ratones , Ratones SCID , Células T Asesinas Naturales/inmunología , Neoplasias Experimentales/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dutch-belted and New Zealand White rabbits were passively immunized with AVP-21D9, a human monoclonal antibody to protective antigen (PA), at the time of Bacillus anthracis spore challenge using either nasal instillation or aerosol challenge techniques. AVP-21D9 (10 mg/kg) completely protected both rabbit strains against lethal infection with Bacillus anthracis Ames spores, regardless of the inoculation method. Further, all but one of the passively immunized animals (23/24) were completely resistant to rechallenge with spores by either respiratory challenge method at 5 weeks after primary challenge. Analysis of the sera at 5 weeks after primary challenge showed that residual human anti-PA levels decreased by 85 to 95%, but low titers of rabbit-specific anti-PA titers were also measured. Both sources of anti-PA could have contributed to protection from rechallenge. In a subsequent study, bacteriological and histopathology analyses revealed that B. anthracis disseminated to the bloodstream in some naïve animals as early as 24 h postchallenge and increased in frequency with time. AVP-21D9 significantly reduced the dissemination of the bacteria to the bloodstream and to various organs following infection. Examination of tissue sections from infected control animals, stained with hematoxylin-eosin and the Gram stain, showed edema and/or hemorrhage in the lungs and the presence of bacteria in mediastinal lymph nodes, with necrosis and inflammation. Tissue sections from infected rabbits dosed with AVP-21D9 appeared comparable to corresponding tissues from uninfected animals despite lethal challenge with B. anthracis Ames spores. Concomitant treatment with AVP-21D9 at the time of challenge conferred complete protection in the rabbit inhalation anthrax model. Early treatment increased the efficacy progressively and in a dose-dependent manner. Thus, AVP-21D9 could offer an adjunct or alternative clinical treatment regimen against inhalation anthrax.
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Carbunco/prevención & control , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Toxinas Bacterianas/inmunología , Modelos Animales de Enfermedad , Pulmón/microbiología , Administración por Inhalación , Animales , Carbunco/microbiología , Carbunco/patología , Carbunco/transmisión , Bacillus anthracis/patogenicidad , Bacillus anthracis/fisiología , Humanos , Pulmón/patología , Conejos , Esporas Bacterianas/inmunologíaRESUMEN
Prevention of inhalation anthrax requires early and extended antibiotic therapy, and therefore, alternative treatment strategies are needed. We investigated whether a human monoclonal antibody (AVP-21D9) to protective antigen (PA) would protect mice, guinea pigs, and rabbits against anthrax. Control animals challenged with Bacillus anthracis Ames spores by the intranasal route died within 3 to 7 days. AVP-21D9 alone provided minimal protection against anthrax in the murine model, but its efficacy was notably better in guinea pigs. When Swiss-Webster mice, challenged with five 50% lethal doses (LD50s) of anthrax spores, were given a single 16.7-mg/kg of body weight AVP-21D9 antibody dose combined with ciprofloxacin (30 mg/kg/day for 6 days) 24 h after challenge, 100% of the mice were protected for more than 30 days, while ciprofloxacin or AVP-21D9 alone showed minimal protection. Similarly, when AVP-21D9 antibody (10 to 50 mg/kg) was combined with a low, nonprotective dose of ciprofloxacin (3.7 mg/kg/day) and administered to guinea pigs for 6 days, synergistic protection against anthrax was observed. In contrast, a single dose of AVP-21D9 antibody (1, 5, 10, or 20 mg/kg) but not 0.2 mg/kg alone completely protected rabbits against challenge with 100 LD50s of B. anthracis Ames spores, and 100% of the rabbits survived rechallenge. Further, administration of AVP-21D9 (10 mg/kg) to rabbits at 0, 6, and 12 h after challenge with anthrax spores resulted in 100% survival; however, delay of antibody treatment by 24 and 48 h reduced survival to 80% and 60%, respectively. Serological analysis of sera from various surviving animals 30 days postprimary infection showed development of a species-specific PA enzyme-linked immunosorbent assay antibody titer that correlated with protection against reinfection. Taken together, the effectiveness of human anti-PA antibody alone or in combination with low ciprofloxacin levels may provide the basis for an improved strategy for prophylaxis or treatment following inhalation anthrax infection.