James S. Huston

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.

Positron emission tomography (PET) imaging of neuroblastoma and melanoma with 64Cu-SarAr immunoconjugates

The advancement of positron emission tomography (PET) depends on the development of new radiotracers that will complement 18F-FDG. Copper-64 (64Cu) is a promising PET radionuclide, particularly for antibody-targeted imaging, but the high in vivo lability of conventional chelates has limited its clinical application. The objective of this work was to evaluate the novel chelating agent SarAr (1-N-(4-aminobenzyl)-3, 6,10,13,16,19-hexaazabicyclo[6.6.6] eicosane-1,8-diamine) for use in developing a new class of tumor-specific 64Cu radiopharmaceuticals for imaging neuroblastoma and melanoma. The anti-GD2 monoclonal antibody (mAb) 14.G2a, and its chimeric derivative, ch14.18, target disialogangliosides that are overexpressed on neuroblastoma and melanoma. Both mAbs were conjugated to SarAr using carbodiimide coupling. Radiolabeling with 64Cu resulted in >95% of the 64Cu being chelated by the immunoconjugate. Specific activities of at least 10 μCi/μg (1 Ci = 37 GBq) were routinely achieved, and no additional purification was required after 64Cu labeling. Solid-phase radioimmunoassays and intact cell-binding assays confirmed retention of bioactivity. Biodistribution studies in athymic nude mice bearing s.c. neuroblastoma (IMR-6, NMB-7) and melanoma (M21) xenografts showed that 15–20% of the injected dose per gram accumulated in the tumor at 24 hours after injection, and only 5–10% of the injected dose accumulated in the liver, a lower value than typically seen with other chelators. Uptake by a GD2-negative tumor xenograft was significantly lower (<5% injected dose per gram). MicroPET imaging confirmed significant uptake of the tracer in GD-2-positive tumors, with minimal uptake in GD-2-negative tumors and nontarget tissues such as liver. The 64Cu-SarAr-mAb system described here is potentially applicable to 64Cu-PET imaging with a broad range of antibody or peptide-based imaging agents.

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 GD2, 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 GD2‐specific chimeric antigen receptor (CAR) comprising an anti‐GD2 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 GD2 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 GD2‐specific antibody or anti‐idiotypic antibody occupying the CAR’s cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD2‐specific NK cells was also found against primary NB cells and GD2 expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.

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

INTRODUCTIONnUse 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.nnnMETHODSnThe 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnThe 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).

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.

The burgeoning antibody landscape

The current Antibody Special Issue (Ab SI) comes during a period of dramatic growth in the antibody field. The Antibody Society was founded by its Board of Directors in 2007 and it soon developed a relationship with Protein Engineering, Design and Selection as the Society’s antibody engineering journal. This annual Ab SI was initiated in 2009 and has been a reflection of the field’s growth in general. Last year the PEDS Ab SI Commentary was an important contribution from Andrew Bradbury and Andreas Plückthun (2015a) that detailed their proposal for solving the problem of reproducibility in reagent antibodies by moving toward a norm where reagent antibodies are fully sequenced and have properly characterized specificity and affinity. This proposal (Bradbury and Plückthun, 2015a,b,c) has since evolved into an international working group that will meet in late September, 2016, at Asilomar, California, to arrive at common objectives to follow on the path toward this important goal for biomedical research. A summary of objectives defined by this Asilomar Meeting (jointly sponsored by The Antibody Society and the Global Biological Standards Institute) will be presented in The Antibody Society session of our Annual Meeting (11–15 December 2016) in San Diego on 14 December (refer to www.AntibodySociety.org for more details). Reference may also be made to other recent reports (Baker, 2015; Freedman et al., 2016).

267. Enhancement of T-Cell Immunity by CEA Delivery to Dendritic Cells with Adenovirus Retargeted Via sCAR-Fibritin-CD40L

Dendritic cells (DC) are excellent targets for antigen-specific immune intervention. However, several attempts to transfer genes of interest to DC by Adenovirus (Ad) have been disappointing, due to limited expression of the relevant receptors and limited uptake of the Ad into DC. Although successful gene transduction to DC has been achieved with very high doses of Ad, associated toxicity was too high for clinical use. Increased transduction efficiency with relatively lower adenoviral dose was previously documented by retargeting Ad to specific cell types using either a two-component targeting moiety or genetically modified adenovirus.