Comparative Preclinical Evaluation of HER2-Targeting ABD-Fused Affibody® Molecules 177Lu-ABY-271 and 177Lu-ABY-027: Impact of DOTA Position on ABD Domain.

Radiolabeled Affibody-based targeting agent 177Lu-ABY-027, a fusion of an anti-HER2 Affibody molecule with albumin binding domain (ABD) site-specifically labeled at the C-terminus, has demonstrated a promising biodistribution profile in mice; binding of the construct to albumin prevents glomerular filtration and significantly reduces renal uptake. In this study, we tested the hypothesis that site-specific positioning of the chelator at helix 1 of ABD, at a maximum distance from the albumin binding site, would further increase the strength of binding to albumin and decrease the renal uptake. The new construct, ABY-271 with DOTA conjugated at the back of ABD, has been labelled with 177Lu. Targeting properties of 177Lu-ABY-271 and 177Lu-ABY-027 were compared directly. 177Lu-ABY-271 specifically accumulated in SKOV-3 xenografts in mice. The tumor uptake of 177Lu-ABY-271 exceeded uptake in any other organ 24 h and later after injection. However, the renal uptake of 177Lu-ABY-271 was two-fold higher than the uptake of 177Lu-ABY-027. Thus, the placement of chelator on helix 1 of ABD does not provide desirable reduction of renal uptake. To conclude, minimal modification of the design of Affibody molecules has a strong effect on biodistribution, which cannot be predicted a priori. This necessitates extensive structure-properties relationship studies to find an optimal design of Affibody-based targeting agents for therapy.

A novel adjuvant G3 induces both Th1 and Th2 related immune responses in mice after immunization with a trivalent inactivated split-virion influenza vaccine.

A preferred adjuvant should promote both Th1 and Th2 responses. However, most adjuvants in common use are biased towards a Th2-driven response. Therefore, the ability of a novel saponin-based adjuvant G3 to inducing balanced Th1 and Th2 responses in BALB/c mice immunized with a split trivalent seasonal influenza vaccine was evaluated in comparison to that of the adjuvant Al(OH)3. Clear differences in the IgG profiles induced by G3, Al(OH)3 or non-adjuvanted vaccine were recorded. Both adjuvants enhanced high and similar levels of the Th2 associated IgG1 subtype compared to mice given vaccine alone. Only G3 enhanced the IgG2a subclass reflecting a Th1 response, whereas Al(OH)3 even abrogated the IgG2a production. Accordingly, G3 enhanced the production of IL-2 and IFN-γ and also of IL-2/IFN-γ double secreting cells, emphasizing the strong Th1 driving effect of G3. Only Al(OH)3 increased splenocyte production of IL-17. Taken together, the results indicate a strong propensity for G3 to induce both Th1 and Th2 driven immune responses.

An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection.

The FluoroSpot assay, a variant of ELISpot utilizing fluorescent detection, has so far been used primarily for assessment of T cells, where simultaneous detection of several cytokines has allowed a more qualitative analysis of functionally distinct T cells. The potential to measure multiple analytes also presents several advantages when analyzing B cells. Our aim was to develop a B-cell FluoroSpot assay adaptable to studies of a variety of antigens. The assay utilizes anti-IgG antibodies immobilized in 96-well filter membrane plates. During cell culture, IgG antibodies secreted by antibody-secreting cells (ASCs) are captured in the vicinity of each of these cells and the specificity of single ASCs is defined using antigens for detection. The antigens were labeled with biotin or peptide tags enabling secondary detection with fluorophore-conjugated streptavidin or tag-specific antibodies. The assay, utilizing up to four different tag systems and fluorophores simultaneously, was evaluated using hybridomas and immunized splenocytes as ASCs. Assay variants were developed that could: i) identify multiple ASCs with different antigen specificities; ii) detect ASCs showing cross-reactivity with different but related antigens; and iii) define the antigen-specificity and, by including anti-IgG subclass detection reagents, simultaneously determine the IgG subclass of antibodies secreted by ASCs. As demonstrated here, the B-cell FluoroSpot assay using tag-based detection systems provides a versatile and powerful tool to investigate antibody responses by individual cells that can be readily adapted to studies of a variety of antigen-specific ASCs.

Measurement of human latent Transforming Growth Factor-ß1 using a latency associated protein-reactive ELISA.

Human Transforming Growth Factor (TGF)-ß1, one of three TGF-ß isoforms, is a pleotropic cytokine critical for many physiological and immunological processes. TGF-ß1 is secreted in a latent form, linked to Latency Associated Protein (LAP). Analysis of Latent TGF-ß1 by TGF-ß1 ELISA requires dissociation of TGF-ß1 from LAP, e.g. by acidification of samples. The ELISA then measures total TGF-ß1, equivalent to dissociated Latent TGF-ß1 plus any free TGF-ß1 present prior to acidification. Evolutionary conservation of TGF-ß1 across mammals also renders TGF-ß1 ELISAs reactive with TGF-ß1 in bovine serum often used in human cell cultures. To enable a direct analysis of Latent TGF-ß1, monoclonal antibodies were made against LAP from human Latent TGF-ß1 and used to develop a LAP ELISA detecting Latent TGF-ß1. The ELISA did not react with LAP from human Latent TGF-ß2 or 3, respectively, nor with Latent TGF-ß in bovine serum. EDTA-containing plasma from healthy subjects (n=20) was analyzed by conventional TGF-ß1 ELISA and LAP ELISA. By TGF-ß1 ELISA, total TGF-ß1 were detected in all samples (median 133 pM, range 34-348 pM); low levels of free TGF-ß1 found in 8/20 non-acidified samples showed that >98.5% of the total TGF-ß1 derived from Latent TGF-ß1. Latent TGF-ß1 found in non-acidified samples by LAP ELISA (median 154 pM, range 48-403 pM) was comparable in molar levels to, and correlated with, total TGF-ß1 (r(s) 0.96, p<0.0001). A similar agreement between the total TGF-ß1 and the LAP ELISA was found with citrate- and heparin-containing plasma. The LAP ELISA facilitates analysis of Latent TGF-ß1 without sample acidification and is not compromised by the presence of bovine serum in human cell supernatants.