Molecular Insights of Nickel Binding to Therapeutic Antibodies as a Possible New Antibody Superantigen

The binding of nickel by immune proteins can manifest as Type IV contact dermatitis (Ni-specific T cells mediated) and less frequently as Type I hypersensitivity with both mechanisms remaining unknown to date. Since there are reports of patients co-manifesting the two hypersensitivities, a common mechanism may underlie both the TCR and IgE nickel binding. Focusing on Trastuzumab and Pertuzumab IgE variants as serendipitous investigation models, we found Ni-NTA interactions independent of Her2 binding to be due to glutamine stretches. These stretches are both Ni-inducible and in fixed pockets at the antibody complementarity-determining regions (CDRs) and framework regions (FWRs) of both the antibody heavy and light chains with influence from the heavy chain constant region. Comparisons with TCRs structures revealed similar interactions, demonstrating the possible underlying mechanism in selecting for Ni-binding IgEs and TCRs respectively. With the elucidation of the interaction, future therapeutic antibodies could also be sagaciously engineered to utilize such nickel binding for biotechnological purposes.

Fitting Together the Evolutionary Puzzle pieces of the Immunoglobulin T Gene from Antarctic Fishes

Cryonotothenioidea is the main group of fishes that thrive in the extremely cold Antarctic environment, thanks to the acquisition of peculiar morphological, physiological and molecular adaptations. We have previously disclosed that IgM, the main immunoglobulin isotype in teleosts, display typical cold-adapted features. Recently, we have analyzed the gene encoding the heavy chain constant region (CH) of the IgT isotype from the Antarctic teleost Trematomus bernacchii (family Nototheniidae), characterized by the near-complete deletion of the CH2 domain. Here, we aimed to track the loss of the CH2 domain along notothenioid phylogeny and to identify its ancestral origins. To this end, we obtained the IgT gene sequences from several species belonging to the Antarctic families Nototheniidae, Bathydraconidae and Artedidraconidae. All species display a CH2 remnant of variable size, encoded by a short Cτ2 exon, which retains functional splicing sites and therefore is included in the mature transcript. We also considered representative species from the three non-Antarctic families: Eleginopsioidea (Eleginops maclovinus), Pseudaphritioidea (Pseudaphritis urvillii) and Bovichtidae (Bovichtus diacanthus and Cottoperca gobio). Even though only E. maclovinus, the sister taxa of Cryonotothenioidea, shared the partial loss of Cτ2, the other non-Antarctic notothenioid species displayed early molecular signatures of this event. These results shed light on the evolutionary path that underlies the origins of this remarkable gene structural modification.

SCIDOT-06. TOWARDS EXCLUSIVELY LOCAL THERAPY OF GLIOBLASTOMA - ENGINEERING IL-12Fc WITH SUPERIOR TISSUE RETENTION AND MINIMAL SYSTEMIC EXPOSURE AFTER CNS ADMINISTRATION

Intratumoral application of IL-12 overrides the prevailing glioblastoma (GB) associated immunosuppression and can trigger efficient anti-tumor responses. With clinical studies currently testing local expression of IL-12 in brain tumors, concerns on IL-12 systemic toxicity via leakage resurface. The fusion to an immunoglobulin heavy chain constant region (“Fc tag”) should increase tissue retention of IL-12. However, export of IL-12Fc into the blood via the neonatal Fc receptor (FcRn) could nevertheless lead to systemic exposure. Subsequent systemic recycling via FcRn could lead to gradually increasing and eventually toxic serum concentrations. We analysed the effect of the Fc-tag on IL-12 tissue retention and evaluated whether FcRn also is involved in brain export and systemic recycling of IL-12Fc upon local delivery. Human or murine IL-12Fc was delivered in GB-bearing or naïve wt or FcRn-humanized mice (mFcRn-/- hFcRn tg) continuously via mini osmotic pumps or as bolus via convection-enhanced delivery (CED). Brain and blood concentration levels were assessed via ELISA. FcRn affinity of IL-12Fc mutants was assessed via ELISA and surface plasmon resonance. We observed much higher tissue retention of IL-12Fc compared to unmodified IL-12, but also an FcRn-dependent gradual increase of IL-12Fc serum levels. Testing a battery of amino acid substitutions at the FcRn interaction interface, we discovered unique substitutions that largely abolish brain export and systemic accumulation while preserving IL-12Fc functionality, leading to an over 100-fold higher brain to blood ratio than unmodified IL-12. We currently test the impact on efficacy of local IL-12Fc and checkpoint brain tumor treatment in vivo. Achieving high local concentrations at low to absent systemic exposure is an important prerequisite for a large therapeutic window for local treatment of neurologic diseases. The novel Fc-modifications present a promising platform for reducing systemic leakage of Fc-containing therapeutics in the context of continuous or intermittent CNS delivery beyond brain cancer therapy.

Effect of calorific intake on proteomic composition of colostrum in dairy cows

The amount of concentrated feed supplied to a dairy cow affects milk yield. However, there is no evidence of a relationship between the colostrum proteomic composition and energy intake. We supplied 30 heifers (4–24 months old, two groups of 15 heifers each) with either a normal diet and high-energy diet to investigate the correlation between energy intake and colostrum protein composition. Colostrum milk proteins were analysed on the day of calving and on the third day following calving using two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting (PMF). Five proteins were identified as differentially expressed between the two feeding groups in the colostrum on the day of calving. The levels of αS2-casein precursor and β-casein was higher in the colostrum from the high-energy diet group (HEG), whereas the levels of IgG3 heavy chain constant region, non-classical MHC class I antigen isoform X2, and β-casein A2 variant were higher in the normal-diet group (NEG) colostrum. Twelve differential proteins were identified on the third day: β-lactoglobulin, αS2-casein, zinc-α2-glycoprotein, lactoferrin, fibrinogen gamma-B chain isoform X1, non-classical MHC class I antigen isoform X2, complement C3, gelsolin isoform A precursor, vitamin D-binding protein isoform X1, immunoglobulin gamma 1 heavy chain constant region, IgG3 heavy chain constant region and polymeric immunoglobulin receptor. All were present at higher levels in the normal-diet group colostrum than in the high-energy diet group colostrum, although the milk yield from mature cows was lower in the normal-diet group. In conclusion, a high-energy diet can enhance milk production; however, the levels of immune-related factors are higher in the colostrum of cows fed a normal diet.