Fc receptor-mediated endocytosis of monomeric IgG1 by human mononuclear phagocytes was evaluated under conditions where aggregated IgG and insulin readily undergo receptor-mediated internalization. U937 cells or normal human peripheral blood monocytes were incubated at 37°C in the absence of free radioligand after having first bound 1251-1gG1 at O°C. To determine the amount of cell-associated IgG1 internalized after varying periods of 370C incubation, surface-bound IgG1 was removed by sequential exposure of cells at 0°C to a nonspecific proteinase for 1 h and to acetic acid at pH 3.2 for 3 min. The failure to develop a proteinase-and acid-resistant fraction, similar to that seen over time at 37°C in parallel experiments with 1251-insulin and 1251-aggregated IgG, and the lack of degradation of the IgG1 released into the medium from the same cells over time show that these cells do not endocytose and degrade monomeric IgG by an Fc receptor-specific mechanism and suggest that constitutive recycling without degradation is unlikely to be occurring. These data fulfill one prediction of the hypothesis that receptor-receptor interaction triggers Fc receptormediated endocytosis.The molecular mechanism whereby IgG immune complexes bound to Fc receptors (FcRs) 1 initiate the process of endocytosis is largely unknown. One hypothesis advanced to explain this event is that endocytosis is triggered by receptor-receptor interaction resulting from cross-linking of two or more FcRs by multivalent ligands. The testable predictions of this hypothesis, as it relates to endocytosis by mononuclear phagocytes, are (a) that monomeric ligand is not endocytosed, whereas (b) dimeric or small oligomeric ligand complexes are endocytosed. Although endocytosis of oligomeric IgG complexes has been documented (21, 35), the fate of FcR-bound monomeric IgG remains unclear. Two recent studies suggest that monomeric IgG is internalized by an FcR-mediated mechanism in murine and human mononuclear phagocytes (21,35). This finding has been used to argue that a "conveyer belt" model (6) may best describe ligand internalization and that receptor cross-linking may not be necessary as a trigger t Abbreviations used in this paper: AggHGG, aggregated human IgG; BBD buffer, balanced salt solution with 1 mg/ml bovine serum albumin and 100 U/ml deoxyribonuclease; FcR, Fc receptor; TCA, trichloroacetic acid. for endocytosis (35). However, the design of experiments to test monomeric IgG endocytosis is complicated by several factors. First, the FcR may recycle under certain circumstances (28,29). Additionally, monomeric IgG may become aggregated under conditions that favor endocytosis (3,7,17) and may thereby be endocytosed as an oligomeric complex. Another problem is high nonspecific uptake of IgG (2) causing insensitivity of the assay. Thus, small amounts of FcR-mediated endocytosis and degradation by phagocytic cells incubated at 37"C with high concentrations of monomeric radioligand are difficult to ascribe with certainty to monomeric IgG uptake (21,3...
Studies have demonstrated that the clinical benefit of PD-1 blockade can be further improved by combination with an αCTLA-4 mAb in some indications. However, this increased activity is commensurate with significant immune related adverse events (irAE's). Therefore, novel approaches are required to uncouple toxicity from anti-tumour efficacy and realise the full potential of this combination. MEDI5752 is a monovalent bispecific human IgG1 monoclonal antibody (mAb) with an engineered fragment crystallisable (Fc) domain to reduce Fc effector function, that specifically binds two clinically validated negative T cell regulators; PD-1 (programmed cell death 1) and CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). MEDI5752 has been designed to suppress the PD-1 pathway and provide modulated CTLA-4 inhibition to uncouple CTLA-4 dependent peripheral toxicity from tumour efficacy. PD-1 expression is a defining feature of tumour infiltrating lymphocytes (TILs). We show that MEDI5752 can saturate CTLA-4 on PD-1+ cells at orders of magnitude lower concentrations than required to saturate CTLA-4 on PD-1- cells. Moreover, our data demonstrate that monovalent targeting of CTLA-4 with MEDI5752 is significantly less potent (15 fold) than bivalent targeting with a parental αCTLA-4 mAb in reporter assays. In contrast, the switch to monovalent targeting of PD-1 has limited effect on potency (within 3-fold compared to a parental αPD-1 mAb) in a PD-1/L1 reporter assay. Together these data demonstrate the potential for MEDI5752 to inhibit CTLA-4 on TILs whilst sparing peripheral T cell populations and reducing toxicity. Furthermore, profiling of MEDI5752 in a range of primary T cell activation assays reveals equivalent activity to a combination of parental PD-1 and CTLA-4 antibodies. MEDI5752 is rapidly internalised upon target binding with kinetics similar to the parental αCTLA-4 mAb reflecting the rapid recycling of this receptor. However, in contrast to an αCTLA-4 mAb (or an αPD-1 mAb), MEDI5752, by tethering CTLA-4 to PD-1, leads to the internalisation and subsequent degradation of PD-1. This novel mechanism of action further differentiates MEDI5752 from a combination of mAb's targeting PD-1 and CTLA-4. MEDI5752 is a novel monovalent bispecific which may provide an improved therapeutic index when compared to a combination of bivalent αPD-1 and αCTLA-4 mAb's, and could provide benefit in cancer indications. Citation Format: Simon J. Dovedi, Yariv Mazor, Matthew Elder, Sumati Hasani, Bo Wang, Suzanne Mosely, Desmond Jones, Anna Hansen, Chuning Yang, Yanli Wu, Ikbel Achour, Nick Durham, Gareth Browne, Thomas Murray, James Hair, Michelle Morrow, Godfrey Rainey, Maria Jure Kunkel, John Gooya, Daniel Freeman, Ronald Herbst, Robert Wilkinson. MEDI5752: A novel bispecific antibody that preferentially targets CTLA-4 on PD-1 expressing T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2776.
Summary Ten patients with small cell lung cancer were treated with high dose human lymphoblastoid interferon (50-100 megaunitsm-2) for 5 days, followed by low dose interferon (3 megaunits m-2) for 3 weeks. At the end of treatment, and one month later, there was no evidence of either complete or partial response. The treatment produced fever, anorexia and weight loss, with transient leucopenia and thrombocytopenia; there was evidence of a non-cholestatic elevation of serum alanine aminotransferase, with clinical deterioration in the condition of three patients presenting with hyponatraemia. A transient hypocalcaemia during high dose therapy was also noted. It seems that lymphoblastoid interferon as a single agent is unlikely to have a role in the treatment of small cell lung cancer, and that its administration as employed in this study is associated with considerable toxicity.
The success of immune checkpoint inhibitors CTLA4 and PD-1 monoclonal antibody, both FDA approved cancer therapies, specifically counteracts inhibitory pathways to activate antigen-specific T cells. However, their success is limited by the fact that not all patients respond to immunotherapy and a variety of adverse events due to non specific and systemic T cell activation limits their administration. A newly identified B7 family receptor, CD28H/TMIGD2, is constitutively expressed on T, pDCs and innate lymphoid cells including NK and ILCs. CD28H is also detected in TILs including the CD8+ tissue resident memory T cells (TRM), a T cell subset that correlates with better prognosis and response to immune checkpoint inhibitor therapy. Functionally, CD28H provides costimulatory function to T cells in the context of TCR signaling events during activation. We engineered a bispecific antibody (Bis mAb) targeting CD28H and PDL1 aimed to augment T cell costimulation and NK activation. The CD28H-PDL1 Bis mAb potentiates T cell proliferative and cytokine responses in antigen-specific human T cell assays and induces human NK -mediated redirected killing of PDL1+ tumor cells. In T cells, the mechanism of action of the Bis mAb requires intracellular signaling domain of the CD28H which downmodulates SHP phosphorylation upon CD3 activation. Intriguingly, we found that the Bis mAb increases induction of CD8 TRM cells in vitro. In human TILs, where CD28H is mostly expressed on CD8 TRM in tumors, the Bis mAb enables higher cytokine responses over PDL1 mAb alone. Given the recent relevance of TRM cells as an important pool of anti-tumor T cell immunity, the rationale for targeting this population via CD28H in the context of blocking PDL1 may prove to be a therapeutic tool for enhancing responses to checkpoint inhibitor therapy. Citation Format: Madhu Ramaswamy, Taeil Kim, Desmond Jones, Hormas Ghadially, Tamer Mahmoud, Andrew Garcia, Susan Wilson, Jeffrey Riggs, Darren Schofield, GIanluca Carlesso. A bispecific antibody targeting CD28H and PDL-1 is a novel and potent immunomodulator of T cell responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4438.
Background: 4-1BB, also called TNF receptor super family member 9 or CD137, is mainly expressed on the surface of activated T, NK, and mononuclear cells. 4-1BB is activated by its ligand 4-1BBL or an activating 4-1BB monoclonal antibody, and stimulates T cell activation, proliferation and cytokine production. This co-stimulatory signal can also multiply antigen presenting cells and result in enhanced cell factor secretion. Experiments show that 4-1BB co-stimulation regulates T cell and antigen presenting cell function for antitumor immunity, providing new targets for tumor immunological therapies. Methods: Here, we developed a cohort of 4-1BB specific antibodies using the classic hybridoma technology. Then, we utilized humanized 4-1BB mice (B-h-4-1BB) and implanted syngeneic tumors subcutaneously, followed by treating mice with purified testing antibodies. Via this approach, we are able to discern several clones that effectively inhibited tumor growth without prior knowledge of their in vitro activities. These clones were further selected for humanization. The cohort of recombinant humanized 4-1BB antibodies were screened in B-h4-1BB mice. Results: A cohort of 4-1BB specific antibodies were successfully generated and purified. These purified antibodies were screened by their efficacy to stimulate anti-tumor activity in live animals using Biocytogen's humanized mouse platform. Top clones were humanized and screened by using B-h4-1BB mice. Both the Fv and Fc portion of the lead antibody were optimized using the B-h4-1BB mice, leading to the humanized form of the antibodies. Conclusions: We adopted an in vivo screen approach to discover candidates of 4-1BB specific antibodies that have potent anti-tumor activity. We discovered the leads with the most potent anti-tumor activity.Legal entity responsible for the study: Biocytogen Boston Corp.
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