Purpose: Antibody-directed enzyme prodrug therapy is a two-stage treatment whereby a tumor-targeted antibody-enzyme complex localizes
in tumor for selective conversion of prodrug. The purpose of this ...study was to establish optimal variables for single administration
of MFECP1, a recombinant antibody-enzyme fusion protein of an anti–carcinoembryonic antigen single-chain Fv antibody and the
bacterial enzyme carboxypeptidase G2 followed by a bis-iodo phenol mustard prodrug. MFECP1 is manufactured in mannosylated
form to facilitate normal tissue elimination.
Experimental Design: Pharmacokinetic, biodistribution, and tumor localization studies were used to test the hypothesis that MFECP1 localizes in
tumor and clears from normal tissue via the liver. Firstly, safety of MFECP1 and a blood concentration of MFECP1 that would
avoid systemic prodrug activation were tested. Secondly, dose escalation of prodrug was done. Thirdly, the dose of MFECP1
and timing of prodrug administration were optimized.
Results: MFECP1 was safe and well tolerated, cleared rapidly via the liver, and was less immunogenic than previously used products.
Eighty-fold dose escalation from the starting dose of prodrug was carried out before dose-limiting toxicity occurred. Confirmation
of the presence of enzyme in tumor and DNA interstrand cross-links indicating prodrug activation were obtained for the optimal
dose and time point. A total of 28 of 31 patients was evaluable for response, the best response being a 10% reduction of tumor
diameter, and 11 of 28 patients had stable disease.
Conclusions: Optimal conditions for effective therapy were established. A study testing repeat treatment is currently being undertaken.
MFECP1 is a mannosylated antibody-enzyme fusion protein used in antibody-directed enzyme prodrug therapy (ADEPT). The antibody selectively targets tumor cells and the targeted enzyme converts a ...prodrug into a toxic drug. MFECP1 is obtained from expression in the yeast Pichia pastoris and produced to clinical grade. The P. pastoris-derived mannosylation of the fusion protein aids rapid normal tissue clearance required for successful ADEPT. The work presented provides evidence that MFECP1 is cleared by the endocytic and phagocytic mannose receptor (MR), which is known to bind to mannose-terminating glycans. MR-transfected fibroblast cells internalize MFECP1 as revealed by flow cytometry and confocal microscopy. Immunofluorescence microscopy shows that in vivo clearance in mice occurs predominantly by MR on liver sinusoidal endothelial cells, although MR is also expressed on adjacent Kupffer cells. In the spleen, MFECP1 is taken up by MR-expressing macrophages residing in the red pulp and not by dendritic cells which are found in the marginal zone and white pulp. Clearance can be inhibited in vivo by the MR inhibitor mannan as shown by increased enzyme activities in blood. The work improves understanding of interactions of MFECP1 with normal tissue, shows that glycosylation can be exploited in the design of recombinant anticancer therapeutics and opens the ways for optimizing pharmacokinetics of mannosylated recombinant therapeutics.
Purpose: Antibody-directed enzyme prodrug therapy (ADEPT) requires highly selective antibody-mediated delivery of enzyme to tumor.
MFE-CP, a multifunctional genetic fusion protein of antibody and ...enzyme, was designed to achieve this by two mechanisms. First
by using a high affinity and high specificity single chain Fv antibody directed to carcinoembryonic antigen. Second by rapid
removal of antibody-enzyme from normal tissues by virtue of post-translational mannosylation. The purpose of this paper is
to investigate these dual functions in an animal model of pharmacokinetics, pharmacodynamics, toxicity, and efficacy.
Experimental Design: MFE-CP was expressed in the yeast Pichia pastoris and purified via an engineered hexahistidine tag. Biodistribution and therapeutic effect of a single ADEPT cycle (1,000 units/kg
MFE-CP followed by 70 mg/kg ZD2767P prodrug at 6, 7, and 8 hours) and multiple ADEPT cycles (9-10 cycles within 21-24 days)
was studied in established human colon carcinoma xenografts, LS174T, and SW1222.
Results: Selective localization of functional enzyme in tumors and rapid clearance from plasma was observed within 6 hours, resulting
in tumor to plasma ratios of 1,400:1 and 339:1, respectively for the LS174T and SW1222 models. A single ADEPT cycle produced
reproducible tumor growth delay in both models. Multiple ADEPT cycles significantly enhanced the therapeutic effect of a single
cycle in the LS174T xenografts ( P = 0.001) and produced regressions in the SW1222 xenografts ( P = 0.0001), with minimal toxicity.
Conclusions: MFE-CP fusion protein, in combination with ZD2767P, provides a new and successful ADEPT system, which offers the potential
for multiple cycles and antitumor efficacy. These results provide a basis for the next stage in clinical development of ADEPT.
Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technology and the annual meeting of The Antibody Society, will be held in San Diego, CA on December 11-15, ...2016. Each of 14 sessions will include six presentations by leading industry and academic experts. In this meeting preview, the session chairs discuss the relevance of their topics to current and future antibody therapeutics development. Session topics include bispecifics and designer polyclonal antibodies; antibodies for neurodegenerative diseases; the interface between passive and active immunotherapy; antibodies for non-cancer indications; novel antibody display, selection and screening technologies; novel checkpoint modulators / immuno-oncology; engineering antibodies for T-cell therapy; novel engineering strategies to enhance antibody functions; and the biological Impact of Fc receptor engagement. The meeting will open with keynote speakers Dennis R. Burton (The Scripps Research Institute), who will review progress toward a neutralizing antibody-based HIV vaccine; Olivera J. Finn, (University of Pittsburgh School of Medicine), who will discuss prophylactic cancer vaccines as a source of therapeutic antibodies; and Paul Richardson (Dana-Farber Cancer Institute), who will provide a clinical update on daratumumab for multiple myeloma. In a featured presentation, a representative of the World Health Organization's INN expert group will provide a perspective on antibody naming. "Antibodies to watch in 2017" and progress on The Antibody Society's 2016 initiatives will be presented during the Society's special session. In addition, two pre-conference workshops covering ways to accelerate antibody drugs to the clinic and the applications of next-generation sequencing in antibody discovery and engineering will be held on Sunday December 11, 2016.
CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against ...established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology.
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•CD25 expression is largely restricted to Treg cells in mice and humans•FcγRIIb inhibits anti-CD25-mediated depletion of intra-tumoral Treg cells•Fc-optimized anti-CD25 efficiently depletes intra-tumoral Treg cells•Anti-CD25 synergizes with PD-1 blockade to reject established tumors
Anti-CD25 antibodies have displayed only modest therapeutic activity against established tumors. Arce Vargas et al. demonstrate that existing anti-CD25 antibodies fail to deplete intra-tumoral Treg cells due to upregulation of FcγRIIb within tumors. Fc-optimized anti-CD25 mediates effective depletion of tumor-infiltrating Treg cells and synergizes with PD-1 blockade to promote tumor eradication.
Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society, will be held in San Diego, CA in early December 2015. In this meeting preview, the chairs provide their thoughts on ...the importance of their session topics, which include antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, overcoming resistance to clinical immunotherapy, and building comprehensive IGVH-gene repertoires through discovering, confirming and cataloging new germline IGVH genes. The Antibody Society's special session will focus on "Antibodies to watch" in 2016, which are a subset of the nearly 50 antibodies currently in Phase 3 clinical studies. Featuring over 100 speakers in total, the meeting will commence with keynote presentations by Erica Ollmann Saphire (The Scripps Research Institute), Wayne A. Marasco (Dana-Farber Cancer Institute/Harvard Medical School), Joe W. Gray (Oregon Health & Science University), and Anna M. Wu (University of California Los Angeles), and it will conclude with workshops on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries and on computational antibody design.
The protocol describes a method for capture of secreted hexahistidine-tagged proteins using expanded-bed adsorption immobilized-metal affinity chromatography. The starting material for the procedure ...is any crude feedstock that contains a histidine (His)-tagged target protein. The protocol is exemplified using unclarified broth from Pichia pastoris fermentation as feedstock. The protocol can be used for laboratory studies or as part of a process for production of recombinant biotherapeutics to standards of good manufacturing practice. It takes approximately 5 h to purify proteins from 10 liters of feedstock and a further 5-6 h to sterilize and regenerate the column.
The Antibody Engineering and Therapeutics conference, which serves as the annual meeting of The Antibody Society, will be held in Huntington Beach, CA from Sunday December 8 through Thursday December ...12, 2013. The scientific program will cover the full spectrum of challenges in antibody research and development, and provide updates on recent progress in areas from basic science through approval of antibody therapeutics. Keynote presentations will be given by Leroy Hood (Institute of System Biology), who will discuss a systems approach for studying disease that is enabled by emerging technology; Douglas Lauffenburger (Massachusetts Institute of Technology), who will discuss systems analysis of cell communication network dynamics for therapeutic biologics design; David Baker (University of Washington), who will describe computer-based design of smart protein therapeutics; and William Schief (The Scripps Research Institute), who will discuss epitope-focused immunogen design.
In this preview of the conference, the workshop and session chairs share their thoughts on what conference participants may learn in sessions on: (1) three-dimensional structure antibody modeling; (2) identifying clonal lineages from next-generation data sets of expressed V
H
gene sequences; (3) antibodies in cardiometabolic medicine; (4) the effects of antibody gene variation and usage on the antibody response; (5) directed evolution; (6) antibody pharmacokinetics, distribution and off-target toxicity; (7) use of knowledge-based design to guide development of complementarity-determining regions and epitopes to engineer or elicit the desired antibody; (8) optimizing antibody formats for immunotherapy; (9) antibodies in a complex environment; (10) polyclonal, oligoclonal and bispecific antibodies; (11) antibodies to watch in 2014; and (12) polyreactive antibodies and polyspecificity.
The 25th anniversary of the Antibody Engineering & Therapeutics Conference, the Annual Meeting of The Antibody Society, will be held in Huntington Beach, CA, December 7-11, 2014. Organized by IBC ...Life Sciences, the event will celebrate past successes, educate participants on current activities and offer a vision of future progress in the field. Keynote addresses will be given by academic and industry experts Douglas Lauffenburger (Massachusetts Institute of Technology), Ira Pastan (National Cancer Institute), James Wells (University of California, San Francisco), Ian Tomlinson (GlaxoSmithKline) and Anthony Rees (Rees Consulting AB and Emeritus Professor, University of Bath). These speakers will provide updates of their work, placed in the context of the substantial growth of the industry over the past 25 years.
Purpose: In preclinical models, radioimmunotherapy with 131 I-A5B7 antiâcarcinoembryonic antigen (CEA) antibody ( 131 I-A5B7) combined with the vascular disruptive agent combretastatin-A4-phosphate ...(CA4P) produced cures unlike either agent
alone. We conducted a phase I trial determining the dose-limiting toxicity (DLT), maximum tolerated dose, efficacy, and mechanism
of this combination in patients with gastrointestinal adenocarcinomas.
Experimental Design: Patients had CEA of 10 to 1,000 μg/L, QTc â¤450 ms, no cardiac arrhythmia/ischaemia, and adequate hematology/biochemistry.
Tumor was suitable for blood flow analysis by dynamic contrast enhanced-magnetic resonance imaging (MRI). The starting dose
was 1,800 MBq/m 2 of 131 I-A5B7 on day 1 and 45 mg/m 2 CA4P given 48 and 72 hours post- 131 I-A5B7, then weekly for up to seven weeks.
Results: Twelve patients were treated, with mean age of 63 years (range, 32-77). Two of six patients at the first dose level had DLTs
(grade 4 neutropenia). The dose was reduced to 1,600 MBq/m 2 , and CA4P escalated to 54 mg/m 2 . Again, two of six patients had DLTs (neutropenia). Of ten assessable patients, three had stable disease and seven had progressive
disease. Single-photon emission computed tomography confirmed tumor antibody uptake in all 10 patients. DCE-MRI confirmed
falls in kinetic parameters (K trans /IAUGC 60 ) in 9 of 12 patients. The change of both pharmacokinetic parameters reached a level expected to produce efficacy in one patient
who had a minor response on computed tomography and a reduced serum tumor marker level.
Conclusions: This is believed to be the first trial reporting the combination of radioimmunotherapy and vascular disruptive agent; each
component was shown to function, and myelosuppression was dose-limiting. Optimal dose and timing of CA4P, and moderate improvements
in the performance of radioimmunotherapy seem necessary for efficacy.
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