*Purpose: Interleukin-6 (IL-6) is a proinflammatory cytokine involved in mediating both T-cell and B-cell responses in allograft rejection. In previous studies, we demonstrated that inhibition of IL-6 signaling using a mousenized anti IL-6 receptor antibody (MMR16-1) resulted in attenuation of donor specific antibody (DSA) responses. Here, we report a dosage-finding study using a novel mousenized anti IL-6 antibody (ALD518-P18) for DSA suppression.

*Methods: Study was conducted in a mouse model involving a C57BL/6 mouse as the recipient of a skin allograft from a C57BL/6-tg-HLA.A2 mouse. Donor-specific antibodies (DSA) were measured using an FACS based antibody binding assay. Concentrations of serum amyloid A (SAA), IL-6, IL-6 receptor and co-receptor GP130 were quantified in an ELISA.

*Results: An intensified dosing regimen (15-dose group) consisting of 15 daily IP injections of ALD518-P-18 (1mg/kg) starting at Day 0 through 14 post-transplantation failed to suppress de novo DSA responses despite robust SAA inhibition (p<0.01 vs. control). Serum IL-6 concentrations in the 15-dose group significantly elevated by 2.6-fold at day 1(280+31pg/ml, p<0.01 vs. control), 24-fold at day 7(1055+40pg/ml, p<0.01), and 188-fold (2127+43pg/ml, p<0.01) at day 14. In contrast, a regimen (3-dose group) consisting of 3 doses of ALD518-P18 (1mg/kg/IP injection) at Days 0, 2 and 7 significantly reduced DSA IgM (13.8+6.3MFI, p=0.005 vs. control; 38.7+7.6MFI) and IgG (144+104MFI, p=0.028 vs. control; 349+14.6MFI). A sub-therapeutic dose of CTLA4Ig (100ug abatacept per IP injection at days 0, 2 and 7) exhibited moderate suppression of DSA IgM (p<0.01 vs. control) and IgG (p<0.05 vs. control). The magnitudes of DSA suppression by low dose abatacept are comparable to that seen in 3-dose clazakizumab group. Combined group (3-doses of claza plus 3-doses of abatacept) showed a significant suppression of de novo DSA responses (n=4, 55.2+0.96MFI, p=0.0008 vs. control at Day 7). In addition, DSA IgG titers in combined therapy mice were significantly lower than that in low dose abatacept monotherapy mice (p=0.033), indicating synergisitc activities. ELISA shows that IL-6 concentrations at Day 14 in 3-dose claza group (853+71pg) were higher than the control group (55+38pg, p=0.0006) and low-dose abatacept group (16+5pg, p=0.0003), but significant lower when compared to 15-dose claza group (2127+43pg, p=0.0002).

*Conclusions: Targeting of IL-6 pathway with pharmaceutic antibody clazakizumab is an applicable approach to allogeneic desensitization in transplantation. The high level of IL-6 measured in ELISA represents accumulation of inactive claza-bound IL-6 complexes in the blood circulation, which is dose-dependent.

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