Objective Dysregulated APRIL/BAFF signaling is implicated in the pathogenesis of multiple autoimmune diseases, including systemic lupus erythematosus and lupus nephritis. We undertook this study to develop and evaluate a high‐affinity APRIL/BAFF antagonist to overcome the clinical limitations of existing B cell inhibitors. Methods A variant of TACI‐Fc generated by directed evolution showed enhanced binding for both APRIL and BAFF and was designated povetacicept (ALPN‐303). Povetacicept was compared to wild‐type (WT) TACI‐Fc and related molecules in vitro and in vivo. Results Povetacicept inhibited APRIL and BAFF more effectively than all evaluated forms of WT TACI‐Fc and selective APRIL and BAFF inhibitors in cell‐based reporter assays and primary human B cell assays, mediating potent suppression of B cell proliferation, differentiation, and immunoglobulin (Ig) secretion. In mouse immunization models, povetacicept significantly reduced serum immunoglobulin titers and antibody‐secreting cells more effectively than anti‐CD20 monoclonal antibodies, WT TACI‐Fc, or APRIL and BAFF inhibitors. In the NZB × NZW mouse lupus nephritis model, povetacicept significantly enhanced survival and suppressed proteinuria, anti–double‐stranded DNA antibody titers, blood urea nitrogen, glomerulonephritis, and renal immunoglobulin deposition. In the bm12 mouse lupus model, povetacicept significantly reduced splenic plasmablasts, follicular helper T cells, and germinal center B cells. In non‐human primates, povetacicept was well tolerated, exhibited high serum exposure, and significantly decreased serum IgM, IgA, and IgG levels after a single dose. Conclusion Enhanced APRIL and BAFF inhibition by povetacicept led to greater inhibition of B cell populations critical for autoantibody production compared to WT TACI‐Fc and CD20‐, APRIL‐, or BAFF‐selective inhibitors. Potent, dual inhibition by povetacicept has the potential to significantly improve clinical outcomes in autoantibody‐related autoimmune diseases. image

ObjectiveCD28 and inducible T cell costimulator (ICOS) appear to have nonredundant roles in T cell activation and adaptive immunity. We undertook this study to characterize in vitro and in vivo the therapeutic potential of acazicolcept (ALPN‐101), an Fc fusion protein of a human variant ICOS ligand (ICOSL) domain designed to inhibit both CD28 and ICOS costimulation, in inflammatory arthritis.MethodsAcazicolcept was compared in vitro with inhibitors of either the CD28 or ICOS pathways (abatacept and belatacept [CTLA‐4Ig], prezalumab [anti‐ICOSL monoclonal antibody]) in receptor binding and signaling assays, and in a collagen‐induced arthritis (CIA) model. Acazicolcept was also compared in cytokine and gene expression assays of peripheral blood mononuclear cells (PBMCs) from healthy donors or rheumatoid arthritis (RA) or psoriatic arthritis (PsA) patients stimulated with artificial antigen‐presenting cells (APCs) expressing CD28 and ICOS ligands*.ResultsAcazicolcept bound CD28 and ICOS, prevented ligand binding, and inhibited human T cell functional interactions, matching or exceeding the activity of CD28 or ICOS costimulatory single‐pathway inhibitors tested individually or in combination. Acazicolcept administration significantly reduced disease in the CIA model and more potently than abatacept. Acazicolcept also inhibited proinflammatory cytokine production from stimulated PBMCs in cocultures with artificial APCs and demonstrated unique effects on gene expression distinct from those induced by abatacept, prezalumab, or a combination of both.ConclusionBoth CD28 and ICOS signaling play critical roles in inflammatory arthritis. Therapeutic agents such as acazicolcept that coinhibit both ICOS and CD28 signaling may mitigate inflammation and/or disease progression in RA and PsA more effectively than inhibitors of either pathway alone.image

Dehydroepiandrosterone sulfate (DHEAS), one of the most abundant neurosteroids synthesized de novo in the nervous system, has well characterized effects on memory and cognitive performances. However, little is known about the underlying synaptic mechanisms. In this study, we investigated the effects of chronic administration of DHEAS (20mg/kg for 7days) on the plasticity of Schaffer collateral-CA1 synapses by applying an optical recording technique on the hippocampal slices stained with voltage-sensitive dyes. We report here that chronically administered DHEAS significantly facilitated the induction of frequency-dependent LTP, termed DHEAS-facilitated LTP. While tetanus of at least 50 pulses (at 100Hz) were required to induce LTP in control rats, only 20 pulses were needed in DHEAS-treated animals. In contrast DHEA, the non-sulfated form of DHEAS, had no facilitating effect on the induction of LTP. We found that chronically administered DHEAS did not alter the presynaptic glutamate release in response to both single pulse and tetanic stimulation, suggesting that certain alterations happened in postsynaptic neurons. Co-administration of the sigma 1 (σ1) receptor antagonists, haloperidol or NE100, with DHEAS completely inhibited the DHEAS-facilitated LTP. However, acute administration of σ1 receptor antagonists to the slices did not affect the induction of DHEAS-facilitated LTP, suggesting that σ1 receptor is a key target of chronic actions of DHEAS but is not involved in the induction of DHEAS-facilitated LTP. Our findings provide evidence that chronically administered DHEAS plays a priming role in inducing a facilitated synaptic plasticity probably via a chronic activation of σ1 receptor in rat hippocampal CA1 pyramidal cells.

Abstract Introduction: Checkpoint inhibition (CPI) has been shown to be an effective anti-tumor therapy, but CPI alone is frequently insufficient to control tumor growth, and costimulatory signals may also be required to produce clinically significant anti-tumor responses. PD1-PDL1 are established CPI targets and TMIGD2 is an inhibitory receptor expressed on T cells that is engaged by its cognate ligand HHLA2 on tumor cells, leading to inhibition of T cell responses. Novel biologics combining CD28 costimulation and CPI may yield promising tumor antigen-specific therapeutic candidates. Methods: Variants of CD86 with increased CD28 affinity were engineered using our directed evolution platform. PD1 and TMIGD2 variants were also engineered for increased affinity to PDL1 and HHLA2, respectively. Fusion proteins were generated including either PD1 or TMIGD2 domains, an effectorless Fc domain, and an engineered CD86 domain to generate proteins to provide target-dependent costimulation (TDC) and evaluated in multiple in vitro T cell stimulation assays. Costimulatory effects were confirmed by evaluating T cell proliferation, cytokine release, and tumor cell killing. Fusion proteins were also tested in vivo in a mouse tumor model using MC38 cells expressing human PDL1 or HHLA2, and in a humanized tumor model where an HPV+ squamous cell carcinoma cell line expressing either PDL1 or HHLA2 was implanted into NSG mice. Tumor-bearing NSG mice were given primary human T cells expressing an HPV peptide-specific TCR, with or without the corresponding fusion protein, and tumor growth was measured over time. Results: PD1-CD86 and TMIGD2-CD86 TDC proteins enhanced T cell costimulation in multiple in vitro T cell response assays, and costimulation was dependent on target cell lines expressing PDL1 or HHLA2, respectively. There was no effect on T cell responses if the target cells did not express the relevant target protein. PD1 and TMIGD2 domains alone had little effect on T cell responses, indicating these TDC proteins were driving enhanced responses. PD1-CD86 and TMIGD2-CD86 TDC proteins enhanced anti-tumor responses in vivo in a syngeneic MC38 implantation model only when MC38 cells expressed human PDL1 or HHLA2, respectively. Both proteins also enhanced antitumor responses in a humanized tumor implantation system using an HPV+ tumor cell line expressing the corresponding target antigen in the presence of human T cells expressing an HPV-specific TCR, but failed to control tumor growth when tumor cells lacked expression of the target protein. Conclusions: Tumor antigen-specific antitumor therapy can be achieved with fusion proteins that combine engineered CPI and CD28 costimulatory domains. Such novel biologics may provide promising approaches to enhancing the efficacy of CPI monotherapies and to address checkpoint inhibitor-resistant tumors. Citation Format: Steven D. Levin, Mark F. Maurer, Chelsea Gudgeon, Siddarth Chandrasekaran, Daniel Ardourel, Daniel Demonte, Joseph Kuijper, Martin Wolfson, Logan Garrett, Kayla N. Kleist, Sherri Mudri, Hieu Nguyen, Michelle Seaberg, Rachel Wang, Jing Yang, Katherine E. Lewis, Stacey R. Dillon, Mark Rixon, Stanford L. Peng. Engineered variant domain fusion proteins provide checkpoint inhibition and tumor antigen dependent CD28 costimulation resulting in potent anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1740.

Abstract Background T cell costimulation has been strongly implicated in the pathogenesis of IBD, yet CD28 costimulatory pathway inhibitors (e.g. abatacept, CTLA4-Fc) have not proven clinically efficacious, implicating an alternative costimulatory pathway. ICOS is a costimulatory receptor highly related to CD28, upregulated upon T cell activation and mediating costimulatory signals in post-activation T cells - suggesting ICOS may be more relevant in active disease. In contrast, CD28 predominates in naïve T cells and is less critical in activated, effector and/or memory T cells. ALPN-101 is an Fc fusion protein of a human inducible T cell costimulator ligand (ICOSL) variant immunoglobulin domain (vIgDTM) engineered to inhibit simultaneously the CD28 and ICOS pathways. It has been shown to have potent in vitro immunosuppressive activity and in vivo efficacy in models of disease for which implication of CD28 and ICOS has been reported (e.g. aGvHD, inflammatory arthritis, Sjögren’s, lupus, MS). Its safety, tolerability, and dose-dependent pharmacokinetics/dynamics are under study in a Ph1 healthy volunteer study. Here, we evaluate ALPN-101 in vitro using PBMC from Crohn’s and ulcerative colitis patients demonstrating superior suppression of T cell activation and cytokine release and show its efficacy to both prevent and treat disease in a mouse T cell transfer model of chronic colitis. Methods Primary cell assays were performed with PBMC stimulated with K562 cells (CD80+, CD86+, ICOSL+, anti-CD3 (OKT3) +) to evaluate suppression of cytokine release and compare to single pathway inhibition. ALPN-101 was assessed in the CD4+CD45RBhigh T cell-induced colitis model either singly dosed on Day 0 or 14 or repeat dosed 2x/week starting at Day 0 or 14 through Day 41, respectively. Serum cytokine and flow analysis of blood was performed throughout the study. Clinical presence of colitis was assessed using a disease activity index based on weight loss and stool consistency. At end of study, colons were measured and assessed histologically. Results ALPN-101 suppressed cytokine release (IFNγ, IL-2) from healthy or IBD patient PBMCs superior to single pathway inhibitors. In vivo, preventively or therapeutically, a single dose of ALPN-101 was efficacious to significantly improve multiple colitis readouts. Repeat dosing completely prevented onset of colitis. ALPN-101-treated mice gained weight and had colon weight-to-length ratios similar to the no-colitis cohort and demonstrated significant suppression of T cells and pro-inflammatory cytokines (e.g. TNFα, IL-12/23, IL-6). Conclusion Dual pathway inhibitor ALPN-101 is superior to single pathway inhibition in human in vitro and mouse in vivo translational studies and may be a novel therapeutic candidate for the treatment of IBD. Clinical trials for ALPN-101 in multiple inflammatory diseases are planned and underway. Consistent with clinical findings, histological analysis confirms efficacy of ALPN-101 in reducing colitis. All ALPN-101 treated groups had lower histological scores compared to the Fc control treated group (B). Mice treated from Day 0 to the end of the study had no pathological colon findings (C), 4/12 mice treated from Day 14 to end of study had no signs of colitis (D). None of the mice in these groups had their muscularis layer of colon affected by inflammation. Mice treated with a single dose at day 0 or day 14 had milder colitis than Fc control-treated mice, although the differences were not statistically significant.

Background/Purpose: Despite many recent advances in immunotherapy, graft versus host disease (GvHD), which reflects immune-mediated attack of recipient tissue by donor T cells, remains the major cause of morbidity and nonrelapse mortality after hematopoietic stem cell transplantation (HCT). The immunoglobulin superfamily (IgSF) is a large, diverse family of proteins that includes several key T cell-expressed members such as CD28, inducible T cell costimulator (ICOS), PD-1, and CTLA-4. CD28 and ICOS in particular play critical roles in T cell activation and adaptive immunity. Blockade of either pathway suppresses GvHD in syngeneic and xenograft mouse models; however, these two pathways are nonredundant and monotherapies directed against either pathway alone do not appear to be completely efficacious. Therefore, novel molecules that co-antagonize both CD28 and ICOS may provide more effective therapies for the prevention or treatment of GvHD. Methods: We used our proprietary variant Ig domain (vIgDTM) platform, which consists of directed evolution of select IgSF proteins, to create tailored ICOSL-Fc fusion proteins that can bind both ICOS and CD28 with high affinity, and co-inhibit both pathways. Mutant ICOSL-Fc molecules were evaluated in vitro with allogeneic mixed lymphocyte reactions (MLR) by co-culturing human pan T cells with activated human monocyte-derived dendritic cells. Lead candidate molecules were subsequently tested in multiple in vivo mouse models, including ovalbumin-induced delayed type hypersensitivity (DTH) and human xenograft PBMC-NSGTM GvHD. Results: ICOSL-Fc fusion proteins containing variant ICOSL domains significantly attenuate T cell activation in vitro when compared to wild-type (WT) ICOSL or belatacept, a clinically available CTLA4-Ig therapeutic, as assessed by suppressed proliferation and cytokine production in MLR, and ear swelling in DTH. In the human PBMC-NSG GvHD model, treatment with ICOSL-Fc significantly protected mice from the effects of xenogeneic T cell activation in vivo, with treated animals exhibiting greatly enhanced survival and reduced disease scores compared to WT ICOSL and belatacept. The level of protection in this model roughly correlated with the potency of the molecules in the in vitroMLR assay. Conclusion: The vIgD therapeutic platform has broad potential to generate potent biologics for the treatment of serious inflammatory conditions. Efficacy in vitro and in vivo of ICOSL-Fc vIgDs exceeds that of WT ICOSL-Fc or belatacept, correlating with their engineered increased affinities for cognate ligand (ICOS) and CD28. ICOSL-Fc vIgDs are therefore promising, novel therapeutic candidates for the prevention and/or treatment of GvHD. Preclinical development to enable clinical studies is underway.

Background/Purpose: ALPN-101 is a potent dual inhibitor of the ICOS and CD28 T cell costimulatory pathways designed for therapeutic application in inflammatory diseases. CD28 and ICOS bind CD80/CD86 and ICOS ligand (ICOSL), respectively, and play critical roles in T cell activation and adaptive immunity. ALPN-101 has previously been demonstrated to have potent efficacy - superior to wild type ICOSL-Fc - in models of graft versus host disease (GvHD), a disease reflecting immune-mediated attack of recipient tissue by donor T cells. Here, we examined the efficacy of a single dose of ALPN-101 or repeat dosing with different dose levels in GvHD. We also explored the potential therapeutic benefit of ALPN-101 in another T cell-driven inflammatory disease, hemophagocytic lymphohistiocytosis (HLH), a spectrum of disorders of the immune system characterized by the excessive production of cytokines by activated T cells and macrophages accumulating in organs such as the liver, spleen, bone marrow, and brain, which mediate significant tissue damage.

This invention concerns a family of chimericantibodies with high affinities to a high molecular weight, tumor-associated Sialylated glycoprotein antigen (TAG-72) of human origin. These antibodies have (1) high affinity animal V, and V, Sequences which mediate TAG-72 binding and (2) human C and C, regions. They are thought to produce signifi cantly fewer Side-effects when administered to human patients by virtue of their human C, and C, antibody domains. The nucleotide and amino acid Sequences of VOTAGV, CC46 V, CC49, CC83 V, and CC92 V, and CC49, CC83 V, and CC92 V, idiotype sequences are disclosed, as well as in Vivo methods of treatment and diagnostic assay using these chimeric antibodies.

Significance Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. It is the sole high-affinity receptor for IgG and plays a significant role in immunotherapy. To date, there is no structural information available on how the receptor recognizes its antibody ligands, however. Consequently, the mechanism of its high-affinity IgG binding remains unclear. We report the first structure of the high-affinity Fc receptor in complex with IgG-Fc. The structural work reveals a direct receptor recognition of Fc glycan as a major factor in receptor affinity. This is the first example of Fc receptor making direct glycan contact through protein residues. The results have implications for the use of glycan engineering in immunotherapy.