Ritsuko Sawada

Human Monoclonal Antibody AVP-21D9 to Protective Antigen Reduces Dissemination of the Bacillus anthracis Ames Strain from the Lungs in a Rabbit Model

ABSTRACT 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.

Human Monoclonal Antibodies to Sialyl-Lewisa (CA19.9) with Potent CDC, ADCC, and Antitumor Activity

Purpose: The carbohydrate antigen sialyl-Lewisa (sLea), 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 sLea appears to be a key event in invasion and metastasis of many tumors and results in susceptibility to antibody-mediated lysis, sLea 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 sLea–KLH vaccine. Results: Several mAbs were selected based on ELISA and FACS including two mAbs with high affinity for sLea (5B1 and 7E3, binding affinities 0.14 and 0.04 nmol/L, respectively) and further characterized. Both antibodies were specific for Neu5Acα2–3Galβ1–3(Fucα1–4)GlcNAcβ as determined by glycan array analysis. Complement-dependent cytotoxicity against DMS-79 cells was higher (EC50 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 sLea as a target for immune attack and their affinity, specificity, and effector functions, 5B1and 7E3 may have clinical utility. Clin Cancer Res; 17(5); 1024–32. ©2011 AACR.

Applying PET to Broaden the Diagnostic Utility of the Clinically Validated CA19.9 Serum Biomarker for Oncology

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 89Zr-5B1, a fully human, antibody-based radiotracer targeting tumor-associated CA19.9 in the preclinical setting. Results: 89Zr-5B1 localized to multiple tumor models representing diseases with undetectable and supraphysiologic serum CA19.9 levels. Among these, 89Zr-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.

Pretargeted Immuno-PET of Pancreatic Cancer: Overcoming Circulating Antigen and Internalized Antibody to Reduce Radiation Doses

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. 64Cu-NOTA-PEG7-Tz showed improved in vivo pharmacokinetics relative to 64Cu-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 89Zr-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 64Cu-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.

Site-specifically labeled CA19.9-targeted immunoconjugates for the PET, NIRF, and multimodal PET/NIRF imaging of pancreatic cancer

Significance Pancreatic cancer will soon be the second leading cause of cancer deaths annually, yet no adequate molecular imaging tools exist to aid in the staging, monitoring, and treatment of the disease. Here we describe the development and preclinical evaluation of three unique immunoconjugates for positron emission tomography, near-infrared fluorescent optical imaging, and multimodal imaging of pancreatic ductal adenocarcinoma (PDAC). The probes were developed using a site-specific, chemoenzymatic methodology that is robust, reproducible, and modular. By targeting CA19.9, the most abundant antigen in >90% of PDAC tumors, we were able to obtain high-quality images in multiple murine models of PDAC, suggesting these constructs could be the core of a molecular imaging toolkit aimed at improving outcomes for patients with PDAC. 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 (ssDFO-5B1, ssFL-5B1, and ssdual-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 ssdual-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.

Abstract 4993: Novel fully human anti-GD2 monoclonal antibodies with potent therapeutic activity against neuroblastoma, sarcoma and melanoma

Background: Gangliosides such as GD2, GD3 and GM2 are promising targets for antibody-mediated cancer therapy since they are expressed at high levels on the surface of several cancers, including neuroblastomas, sarcomas and melanomas. Anti-GD2 monoclonal antibodies have shown promising clinical outcomes in neuroblastoma and a chimeric anti-GD2 antibody (Unituxin™) was recently approved by FDA. However, murine-derived antibodies discovered so far show adverse effects that limit their clinical utility. Fully human antibodies derived from immunized patients might be able to overcome these limitations, since epitope specificity has evolved in the human tissue environment. Here we describe the characterization and functional evaluation of two potential development candidates. Methods: PBMCs from patients immunized with GD2 lactone-keyhole limpet hemocyanin (GD2L-KLH) conjugate vaccine were utilized to isolate fully human monoclonal antibodies (humAbs). HumAbs were selected for 1) specificity against GD2 initially by ELISA and cell surface binding by flow cytometry 2) by glycan array and affinity assays and 3) by effector functions in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) assays. The therapeutic potential of lead antibodies was further evaluated in xenograft models in SCID mice using GD2 positive CHLA255 neuroblastoma as well as with TC71 and SaoS2 sarcoma cell lines. Results: Antibodies that bind specific to purified GD2 by ELISA were expressed as recombinant antibodies in CHO cells. Many recovered antibodies showed cross-reactivity with several gangliosides in ELISA assays. Two antibodies with high specificity for GD2, 1B7 and 31F9 were selected for further studies. Binding to native antigen expressed on the cell surface of different cell lines was confirmed by FACS analysis. 31F9 was monospecific for GD2 (affinity by Surface Plasmon Resonance was ∼4 nM) while 1B7 showed dual specificity for GD2 and GM2 with affinities of ∼1 nM for GD2 and ∼ 370 nM for GM2, respectively. These two antibodies induced CDC ranged between 35% and 80%, and ADCC with human PBMCs between 40% and 65% on CHLA255, TC71 and SaoS2 cell lines. Survival following IV or SC challenge with SaoS2 and TC71 cells (respectively) was at least doubled following six IP treatments with 200 mcg of either 1B7 or 31F9 twice weekly. Inhibition of CHLA255 tumor growth was less dramatic. Conclusions: Vaccinated patients produce antigen-specific IgG antibodies that were further analyzed on the molecular level. Antibodies with single and dual specificity (GD2 and GM2) were recovered and shown to be very active in functional assays. Based on these favorable in vitro and in vivo studies, 1B7 and 31F9 humAbs merit further development efforts to evaluate potential utility for treatment of GD2 positive cancers. Citation Format: Govind Ragupathi, Xiaohong Wu, Ritsuko Sawada, Philip O. Livingston, Wolfgang W. Scholz. Novel fully human anti-GD2 monoclonal antibodies with potent therapeutic activity against neuroblastoma, sarcoma and melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4993.

Abstract B25: Development of 5B1, an anti-CA19.9 monoclonal antibody, as a near-infrared fluorescent probe for intraoperative imaging of pancreatic cancer

Background: For patients diagnosed with pancreatic ductal adenocarcinoma (PDAC), resection offers the best chance of survival. However, for those 20% of patients who qualify for resection, the 5-year survival rate is only 25% due to the high incidence of undiscovered metastases. Along with identifying metastases, properly defining the margin between tumor and healthy tissue is also a crucial determinant of the success of resection. The intraoperative use of near-infrared fluorescence (NIRF) imaging agents to facilitate the identification of previously undetected metastases and to define proper resection margins has the potential to improve the outcomes in patients. Monoclonal antibodies (mABs) are one promising platform for the development of NIRF imaging agents due to their high-specificity and low background in healthy tissues. CA19.9 is a well-established biomarker for PDAC, and 5B1, a fully human, anti-CA19.9 antibody has already shown promise both as a potential therapeutic and as a platform for development of a positron emission tomography agent. Based on previous studies we investigated 5B1’s potential as a NIRF imaging agent for use in an intraoperative setting. Methods: IRDye800CW-NHS and IRDye650-NHS were conjugated to 5B1 and a mouse IgG via lysine residues to generate 5B1-FL800, 5B1-FL650, and IgG-FL800. Each construct and the free acid of the IR800Dye were characterized using in vitro binding assays with two CA19.9-positive cell pancreatic cancer derived lines (BxPC3 and L3.6pl) and a negative cell line (PanC-1). The in vivo dose needed achieve an optimal signal to background ratio was determined in female, athymic nude mice with varying doses of 5B1-FL800. The potential of the constructs for use as NIRF agents for image-guided tumor identification and surgery was examined in a subcutaneous xenograft model using the previously mentioned cell lines as well as an L3.6pl orthotopic pancreas xenograft model. Images were obtained at 24, 48, 96, and 144 hours (750nm excitation/800nm emission). Post mortem, NIRF image-guided surgery was performed to identify and remove the tumor tissue and the relative uptake was compared to healthy tissue. Immunohistochemistry (IHC) as well as hematoxylin and eosin staining (HE May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B25.

Abstract C53: Potent CDC, ADCC, and antitumor activity of human monoclonal antibodies to Sialyl Lewisa.

The carbohydrate antigen sialyl-Lewis A (sLe a ), also known as CA19.9, is widely expressed on epithelial tumors of the gastrointestinal tract, on breast cancer cells, and also on small cell lung cancer cells. sLe a serves as a ligand for epithelial leukocyte adhesion molecules and over-expression of sLe a appears to be a key event in invasion and metastasis of many tumor cells. Since tumor cells expressing sLe a are highly susceptible to antibody mediated lysis mechanisms, sLe a presents an attractive molecular target for tumor therapy in a minimal disease setting. Hybridomas were generated with human B cells isolated from individuals vaccinated with MSKCC9s cancer vaccine. Antibody producing cells were identified by ELISA using sLe a -HSA and synthetic sLe a -PAA-biotin conjugates. Binding to the native antigen expressed on the cell surface was tested by FACS analysis on sLe a positive DMS-79 cells and others and the biological activity was measured in CDC and ADCC assays. The binding affinity was determined by Surface Plasmon Resonance and the specificity of the carbohydrate binding was evaluated by ELISA and by glycan array analysis (CFG, Array 4.1). Recombinant antibodies were generated in CHO cells and purified on Protein A (IgG) or hydroxyapatite (IgM), respectively. We report the discovery and initial characterization of fully human antibodies that were generated from blood lymphocytes from individuals immunized with sLe a -KLH vaccine. Two antibodies were selected for further studies based on the apparent high affinity, which was estimated by BiaCore at 0.14 nM for 5B1 (IgG/λ) and 0.04 nM for 7E3 (IgM/κ). Both antibodies were highly specific for Neu5Acα2–3Galβ1–3 (Fucα1–4) GlcNAcβ and did not bind to sialyl-Lewisx, Lewis a , and other related carbohydrates. Both antibodies have been expressed as fully functional human recombinant antibodies in CHO cells. Complement dependent cytotoxicity (CDC) against DMS-79 cells was approximately 60% and 70–90% for r5B1 and r7E3, respectively. Moreover, r5B1 antibodies showed approximately 50% ADCC of DMS-79 cells with human NK cells (at 5:1 ratio) and 80–90% ADCC with human peripheral blood mononuclear cells with two different blood donors (at 100:1 E/T ratio). These antibodies were tested in two xenograft models: 1) Treatment of animals with 5B1 on the day of engraftment with DMS-79 cells in a subcutaneous model completely prevented tumor growth. 2) Delayed treatment with various doses of 5B1 showed dose dependent protection up to complete cure in SCID mice engrafted (IV) with Colo205 cells. 7E3 antibodies did not show higher protection despite increased apparent affinity. The specificity and potency of 5B1 antibodies in CDC and ADCC assays translates into good activity in xenograft models. Further studies are warranted to explore the activity against additional cancer specific cell lines in animal models and to pursue preclinical development of 5B1 antibodies. Funding support by NCI #R42CA128362. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C53.

Abstract B122: Human monoclonal antibodies to sialyl‐Lewis‐A with potent CDC and ADCC activity

The carbohydrate antigen sialyl‐Lewis A (sLea) is widely expressed on epithelial tumors of the gastrointestinal tract, on breast cancer cells, and also on small cell lung cancer cells, but is expressed minimally or not at all on normal tissues. sLea serves as a ligand for epithelial leukocyte adhesion molecules and higher expression of sLea was observed in patients with greater node involvement. Since over‐expression of sLea appears to be a key event in invasion and metastasis of many tumor cells and tumor cells expressing sLea are highly susceptible to antibody mediated lysis mechanisms, sLea presents an attractive molecular target for tumor therapy in a minimal disease setting. Here we report the discovery and initial characterization of fully human antibodies that were generated from blood lymphocytes from individuals immunized with sLea vaccine at MSKCC. Antibodies were identified by ELISA using sLea‐HSA conjugates and binding to the native antigen was verified by FACS analysis on DMS‐79 cells. Two antibodies were selected for further studies based on the apparent high affinity, which was estimated by Biacore at 0.14 nM for 5B1 (IgG/λ) and 0.04 nM for 7E3 (IgM/κ). These antibodies did not bind to sialyl‐Lewis X, Lewis A and other related carbohydrates. Both antibodies have been expressed as fully functional human recombinant antibodies in CHO cells. Complement dependent cytotoxicity (CDC) against DMS‐79 cells was approximately 60% and 70–90% for r5B1 and r7E3, respectively. Moreover, r5B1 antibodies showed approximately 50% ADCC of DMS‐79 cells with human NK cells (at 5:1 ratio) and 80–90% ADCC with human peripheral blood mononuclear cells with two different blood donors (at 100:1 ratio). These results are very encouraging and we believe that further studies to scale up and test these antibodies in various tumor challenge models are warranted. Since sLea is widely expressed on human cancers, such antibodies could eventually find utility in approximately half of the new cancer cases occurring each year. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B122.