The ionic charge of copper-64 complexes conjugated to an engineered antibody affects biodistribution.

The development of biomolecules as imaging probes requires radiolabeling methods that do not significantly influence their biodistribution. Sarcophagine (Sar) chelators form extremely stable complexes with copper and are therefore a promising option for labeling proteins with (64)Cu. However, initial studies using the first-generation sarcophagine bifunctional chelator SarAr to label the engineered antibody fragment ch14.18-ΔCH2 (MW 120 kDa) with (64)Cu showed high tracer retention in the kidneys, presumably because the high local positive charge on the Cu(II)-SarAr moiety resulted in increased binding of the labeled protein to the negatively charged basal cells of the glomerulus. To test this hypothesis, ch14.18-ΔCH2 was conjugated with a series of Sar derivatives of decreasing positive charge and three commonly used macrocyclic polyaza polycarboxylate (PAC) bifunctional chelators (BFC). The immunoconjugates were labeled with (64)Cu and injected into mice, and PET/CT images were obtained at 24 and 48 h postinjection (p.i.). At 48 h p.i., ex vivo biodistribution was assessed. In addition, to demonstrate the potential of metastasis detection using (64)Cu-labeled ch14.18-ΔCH2, a preclinical imaging study of intrahepatic neuroblastoma tumors was performed. Reducing the positive charge on the Sar chelators decreased kidney uptake of Cu-labeled ch14.18-ΔCH2 by more than 6-fold, from >45 to <6% ID/g, whereas the uptake in most other tissues, including liver, was relatively unchanged. However, despite this dramatic decrease, the renal uptake of the PAC BFCs was generally lower than that of the Sar derivatives, as was the liver uptake. Uptake of (64)Cu-labeled ch14.18-ΔCH2 in neuroblastoma hepatic metastases was detected using PET.

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin.

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.

Avidity-mediated enhancement of in vivo tumor targeting by single-chain Fv dimers.

Radiolabeled single-chain Fv (sFv) molecules display highly specific tumor retention in the severe combined immunodeficient (SCID) mouse model; however, the absolute quantity of sFv retained in the tumors is diminished by the rapid renal elimination resulting from the small size of the sFv molecules (Mr 27,000) and by dissociation of the monovalent sFv from tumor-associated antigen. We previously reported significant improvement in tumor retention without a loss of targeting specificity on converting monovalent sFv into divalent [(sFv')2] dimers, linked by a disulfide bond between COOH-terminal cysteinyl peptides engineered into the sFv'. However, our data for enhanced dimer localization in tumors could not distinguish between the contributions of enhanced avidity and increased systemic retention associated with the larger size of 54 kDa [(sFv')2] dimers relative to 27-kDa sFv. In this investigation, we have compared tumor targeting of divalent anti-c-erbB-2/HER2/neu 741F8-1 (sFv')2 homodimers with monovalent 741F8/26-10 (sFv')2 heterodimers (Mr 54,000) and 741F8 sFv monomers (741F8 sFv has binding specificity for erbB-2/HER2/neu and 26-10 sFv specificity for digoxin and related cardiac glycosides). These studies allowed us to distinguish the dominant effect of valency over molecular weight in accounting for the superior tumor retention of 741F8-1 (sFv')2 homodimers. Each of the radioiodinated species was administered i.v. to SCID mice bearing SK-OV-3 human tumor xenografts and tumor localization at 24 hours post i.v. injection was determined for 125I-741F8-1 (sFv')2 (3.57 %ID/g), 125I-741F8/26-10 (sFv')2 (1.13 %ID/g), and 125I-741F8-1 sFv (1.25 %ID/g). These findings substantiate that the improved tumor retention of (sFv')2 homodimers over sFv monomers results from the availability of dual binding sites rather than from the slower systemic clearance of homodimers.

Antibody Engineering & Therapeutics 2016: The Antibody Society's annual meeting, December 11-15, 2016, San Diego, CA.

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.

The INNs and outs of antibody nonproprietary names.

An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a -mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies.

Structure of a single-chain Fv bound to the 17 N-terminal residues of huntingtin provides insights into pathogenic amyloid formation and suppression.

Huntington's disease is triggered by misfolding of fragments of mutant forms of the huntingtin protein (mHTT) with aberrant polyglutamine expansions. The C4 single-chain Fv antibody (scFv) binds to the first 17 residues of huntingtin [HTT(1-17)] and generates substantial protection against multiple phenotypic pathologies in situ and in vivo. We show in this paper that C4 scFv inhibits amyloid formation by exon1 fragments of huntingtin in vitro and elucidate the structural basis for this inhibition and protection by determining the crystal structure of the complex of C4 scFv and HTT(1-17). The peptide binds with residues 3-11 forming an amphipathic helix that makes contact with the antibody fragment in such a way that the hydrophobic face of this helix is shielded from the solvent. Residues 12-17 of the peptide are in an extended conformation and interact with the same region of another C4 scFv:HTT(1-17) complex in the asymmetric unit, resulting in a ß-sheet interface within a dimeric C4 scFv:HTT(1-17) complex. The nature of this scFv-peptide complex was further explored in solution by high-resolution NMR and physicochemical analysis of species in solution. The results provide insights into the manner in which C4 scFv inhibits the aggregation of HTT, and hence into its therapeutic potential, and suggests a structural basis for the initial interactions that underlie the formation of disease-associated amyloid fibrils by HTT.

Antibody Engineering & Therapeutics 2015: The Antibody Society's annual meeting December 7-10, 2015, San Diego, CA.

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.

Antibody engineering and therapeutics conference. The annual meeting of the antibody society, Huntington Beach, CA, December 7-11, 2014.

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.

Antibody engineering and therapeutics, The Annual Meeting of the Antibody Society: December 8-12, 2013, Huntington Beach, CA.

The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates.

IBC's 23rd Antibody Engineering and 10th Antibody Therapeutics Conferences and the Annual Meeting of The Antibody Society: December 2-6, 2012, San Diego, CA.

Now in its 23rd and 10th years, respectively, the Antibody Engineering and Antibody Therapeutics conferences are the Annual Meeting of The Antibody Society. The scientific program covers the full spectrum of challenges in antibody research and development from basic science through clinical development. In this preview of the conferences, the chairs provide their thoughts on sessions that will allow participants to track emerging trends in (1) the development of next-generation immunomodulatory antibodies; (2) the complexity of the environment in which antibodies must function; (3) antibody-targeted central nervous system (CNS) therapies that cross the blood brain barrier; (4) the extension of antibody half-life for improved efficacy and pharmacokinetics (PK)/pharmacodynamics (PD); and (5) the application of next generation DNA sequencing to accelerate antibody research. A pre-conference workshop on Sunday, December 2, 2012 will update participants on recent intellectual property (IP) law changes that affect antibody research, including biosimilar legislation, the America Invents Act and recent court cases. Keynote presentations will be given by Andreas Plückthun (University of Zürich), who will speak on engineering receptor ligands with powerful cellular responses; Gregory Friberg (Amgen Inc.), who will provide clinical updates of bispecific antibodies; James D. Marks (University of California, San Francisco), who will discuss a systems approach to generating tumor targeting antibodies; Dario Neri (Swiss Federal Institute of Technology Zürich), who will speak about delivering immune modulators at the sites of disease; William M. Pardridge (University of California, Los Angeles), who will discuss delivery across the blood-brain barrier; and Peter Senter (Seattle Genetics, Inc.), who will present his vision for the future of antibody-drug conjugates. For more information on these meetings or to register to attend, please visit www.IBCLifeSciences.com/AntibodyEng or call 800-390-4078. Members of The Antibody Society and mAbs journal subscribers receive a 20% discount for meeting registration. To obtain this discount, email kdostie@ibcusa.com. mAbs is the official therapeutics journal of The Antibody Society and offers a discounted subscription to Society members for $49.

Imaging cancer using PET--the effect of the bifunctional chelator on the biodistribution of a (64)Cu-labeled antibody.

INTRODUCTION: Use of copper radioisotopes in antibody radiolabeling is challenged by reported loss of the radionuclide from the bifunctional chelator used to label the protein. The objective of this study was to investigate the relationship between the thermodynamic stability of the (64)Cu-complexes of five commonly used bifunctional chelators (BFCs) and the biodistribution of an antibody labeled with (64)Cu using these chelators in tumor-bearing mice. METHODS: The chelators [S-2-(aminobenzyl)1,4,7-triazacyclononane-1,4,7-triacetic acid (p-NH(2)-Bn-NOTA): 6-[p-(bromoacetamido)benzyl]-1, 4, 8, 11-tetraazacyclotetradecane-N, N', N'', N'''-tetraacetic acid (BAT-6): S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododocane tetraacetic acid (p-NH(2)-Bn-DOTA): 1,4,7,10-tetraazacyclododocane-N, N', N", N"'-tetraacetic acid (DOTA): and 1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine (SarAr)] were conjugated to the anti-GD2 antibody ch14.18, and the modified antibody was labeled with (64)Cu and injected into mice bearing subcutaneous human melanoma tumors (M21) (n = 3-5 for each study). Biodistribution data were obtained from positron emission tomography images acquired at 1, 24 and 48 hours post-injection, and at 48 hours post-injection a full ex vivo biodistribution study was carried out. RESULTS: The biodistribution, including tumor targeting, was similar for all the radioimmunoconjugates. At 48 h post-injection, the only statistically significant differences in radionuclide uptake (p < 0.05) were between blood, liver, spleen and kidney. For example, liver uptake of [(64)Cu]ch14.18-p-NH(2)-Bn-NOTA was 4.74 ± 0.77 per cent of the injected dose per gram of tissue (%ID/g), and for [(64)Cu]ch14.18-SarAr was 8.06 ± 0.77 %ID/g. Differences in tumor targeting correlated with variations in tumor size rather than which BFC was used. CONCLUSIONS: The results of this study indicate that differences in the thermodynamic stability of these chelator-Cu(II) complexes were not associated with significant differences in uptake of the tracer by the tumor. However, there were significant differences in tracer concentration in other tissues, including those involved in clearance of the radioimmunoconjugate (e.g., liver and spleen).

SEEDbodies: fusion proteins based on strand-exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies.

Bispecific antibodies and asymmetric Fc fusion proteins offer opportunities for important advances in therapeutics. Bivalent IgG depends upon in vivo dimerization of its heavy chains, mediated by homodimeric association of its C(H)3 domains. We have developed a heterodimeric Fc platform that supports the design of bispecific and asymmetric fusion proteins by devising strand-exchange engineered domain (SEED) C(H)3 heterodimers. These derivatives of human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers that are composed of alternating segments of human IgA and IgG C(H)3 sequences. The resulting pair of SEED C(H)3 domains preferentially associates to form heterodimers when expressed in mammalian cells. SEEDbody (Sb) fusion proteins consist of [IgG1 hinge]-C(H)2-[SEED C(H)3], that may be genetically linked to one or more fusion partners. This investigation reports on the generation of mono-Fab-Sb and Sb-IL2 monocytokine as models. They were expressed at high levels in NS/0 cells, purified on recombinant protein A resin and were well-behaved in solution. When administered intravenously to mice, Sb pharmacokinetics exhibited the long serum half-life extensions typical of comparable Fc-containing immunofusion and IgG1 controls.