Background/Purpose: The activation of T helper cells requires cognate recognition of antigen-derived peptides presented by Major Compatibility Complex Class II (MHC II) molecules, which are encoded by the human leukocyte antigen gene complex (HLA)-DR, HLA-DP and HLA-DQ genes. Polymorphisms in the HLA-DR beta chain 1 gene HLADRB1 are highly associated with many autoimmune diseases. The HLA-DRB1 ‘Shared Epitope’ is the single most significant genetic risk factor for Rheumatoid Arthritis. Blockade of the interaction of HLA-DR with the TCR might inhibit activation of autoreactive T cells while preserve protective antigen presentation by other HLA molecules.

Methods: HLA-DR selective binding Fabs were identified from phage display libraries and cloned as IgG2sigma/kappa antibodies. The affinities of the anti-HLA-DR antibodies for HLA-DR1 or DR4 complexes were measured by Surface Plasmon Resonance (SPR). To evaluate the potency of anti-HLA-DR mabs in vitro, peptide-specific T cell hybridoma lines were cocultured with APCs expressing HLA-DR1 or DR4. In addition, a mixed lymphocyte reaction (MLR) assay was established via coculture of human CD4+ T cells with bone marrow-derived dendritic cells (BMDC) from human HLA-DR4 transgenic (Tg) mice. To evaluate the efficacy of anti-HLA-DR mabs in vivo, human HLA-DR4 Tg mice were immunized with hemagglutinin (HA) peptide and HA-specific T cell expansion was assessed by ex vivo stimulation.

Results: DR4B127 is an anti-HLA-DR monoclonal antibody with a ‘silent’ Fc (lacking effector function). It selectively binds to HLA-DR1 or DR4 in the presence of various antigen peptides (KD=9~146 nM) but does not binds to HLA-DP or HLA-DQ. Its binding epitope does not overlap with the MHC II/TCR interfaces. However, interaction of MHC II/CD4 could be affected. Unlike other anti-HLA-DR antibodies (e.g. L243), the binding of DR4B127 does not cause apoptosis of primary human B cells in vitro. Target-mediated internalization is observed for DR4B127 and other HLA-DR-binding antibodies in vitro, and internalization efficiency is correlated with binding affinity. DR4B127 dose-dependently inhibits HLA-DR1- or HLA-DR4-restricted, HA peptide-dependent or HLA-DR4-restricted, type II collagen (CII) peptide-dependent activation of T cell hybridoma cells. DR4B127 also inhibits a human T cell/ HLA-DR4 Tg mouse BMDC MLR.  DR4B127 demonstrated a short half-life with t_1/2 < 24h in both HLA-DR4 Tg mice and cyno monkeys.  DR4B127 does not deplete B cells in HLA-DR4 Tg mice in vivo. However, quick loss of target engagement is observed as over 90% of total HLA-DR epitopes on B cells are not covered by antibody after 24h. Sustained exposure of DR4B127 by repeated dosing is sufficient to inhibit HA-specific T cell expansion in vivo, while single dose treatment is not efficacious.

Conclusion: DR4B127 is differentiated from other anti-HLA-DR mabs, demonstrating a unique binding epitope and non-depleting profile. Although short half-life and transient target engagement preclude further development as a therapeutic for autoimmune indications, DR4B127 may have utility as a research tool for selective uptake into HLA-DR-expressing cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/blockade-of-antigen-specific-t-cell-activation-by-a-non-depleting-anti-hla-dr-monoclonal-antibody-with-a-unique-binding-epitope/