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Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3–fibronectin interaction represents a “stromal checkpoint” through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.

Supplementary Table from The Fibronectin–ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells

Supplementary Figure from The Fibronectin–ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells

Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3–fibronectin interaction represents a “stromal checkpoint” through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.

Suppressive myeloid cells inhibit antitumor immunity by preventing T cell responses. Immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. Our team recently identified fibronectin as a functional ligand for ILT3. The fibronectin-ILT3 interaction serves as a “stromal checkpoint” through which the extracellular matrix actively promotes myeloid cell suppression in the tumor microenvironment. We now report the pre-clinical characterization of NGM831, a humanized IgG1 (N297G) antibody that binds to ILT3 of human and cynomolgus monkey origin with high specificity and sub-nM affinity and blocks the interaction of ILT3 with both its reported ligands, fibronectin and ApoE. In a gene expression profiling experiment, treatment of tolerogenic dendritic cells with NGM831 in the presence of fibronectin decreased their expression of inhibitory and scavenger receptors (CD163, MRC1, MARCO, STAB1, LILRB2) and markers of dendritic cell immaturity (CD14, CD209, FOLR2) and increased their expression of genes involved in antigen presentation (TNFSF14, DCSTAMP, CD82, OLR1) and TH1 polarization (SPP1, TBX21), suggesting that these cells had been remodeled to a more stimulatory phenotype. Consistent with these observations, treatment of tolerogenic dendritic cells with NGM831 increased their production of pro-inflammatory cytokines in response to FcɣR ligation, restored their responsiveness to maturation stimuli such as lipopolysaccharide (LPS) and CD40 ligand (CD40L), and increased their production of chemokines that could induce the migration of other immune cells to sites of inflammation. In addition, NGM831 acted synergistically with pembrolizumab to increase the ability of dendritic cells to stimulate allogeneic T cells in mixed lymphocyte reactions. Taken together, these results demonstrate that NGM831 can block fibronectin-mediated immunosuppression and promote myeloid cell reprogramming. These data support the clinical development of the ILT3 antagonist antibody NGM831 alone and in combination with immune checkpoint blockade. NGM831 is expected to enter clinical trials for the treatment of advanced solid tumors in early 2022. Citation Format: Peirong Chen, Suzanne C. Crawley, Vicky Y. Lin, Varun Kapoor, Kevin J. Paavola, Hung-I Chen, Joshua S. Lichtman, Betty Li, Kalyani Mondal, Alan K. Kutach, Yan Wang, Daniel D. Kaplan, Julie M. Roda. Pre-clinical characterization of NGM831, an ILT3 antagonist antibody for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB216.

Background: Immune suppression in the tumor microenvironment is known to contribute to tumor immune evasion. ILT2 (aka LILRB1) and ILT4 (aka LILRB2) are distinct ITIM-containing immunosuppressive receptors that recognize the shared ligand MHC-I. Both ILT2 and ILT4 are highly expressed on tumor infiltrating myeloid cells, while ILT2 is also expressed on tumor-associated T and NK cells. Given their common expression on myeloid cells, we anticipated that ILT2 and ILT4 blockade would be redundant. However, assays showed distinct functional activities of ILT2 or ILT4, with limited overlap. Here we explore the relative contribution of ILT2 and ILT4 on in vitro immune activation and in vivo anti-tumor activity. Methods: Anti-ILT2 and anti-ILT4 antibodies were assessed on T cells, NK cells and various myeloid cell subsets, including monocytes, macrophages, myeloid-derived suppressor cells (MDSC) and dendritic cells. ILT2 and ILT4 do not have clear murine orthologs; thus, to characterize in vivo activity, we utilized humanized murine tumor models, including PBMC-reconstituted and CD34-reconstituted mice. Mice were engrafted with tumors and treated with anti-ILT2, anti-ILT4, dual antagonist NGM707 and/or anti-PD-1 antagonist antibodies. Results: The in vitro and in vivo biology of ILT2 and ILT4 was explored using mono-specific antagonist antibodies, as well as the ILT2/ILT4 dual antagonist NGM707. Surprisingly, ILT2- and ILT4-specific antibodies showed distinct activities on myeloid cell populations, despite expression of both receptors. Using assays designed to induce optimal MHC-I engagement, both ILT2 and ILT4 contributed to myeloid suppression. ILT2 blockade further enhanced macrophage phagocytosis, CD8+ T cell cytolytic activity and activation of NKG2C+ memory-like NK cells. Humanized mouse models were used to characterize the in vivo anti-tumor activity of ILT2 and ILT4 blockade. Consistent with our in vitro findings, we observed a distinct effect of ILT2 and ILT4 blockade on tumor growth inhibition. Combination with an anti-PD-1 antibody further enhanced tumor suppression. Conclusions: Despite high expression of ILT2 and ILT4 on myeloid cell populations, evidence from standard in vitro assays suggested that ILT2 and ILT4 have distinct functional activities. Here, we provide evidence that both ILT2 and ILT4 play key roles in myeloid immune suppression, and that blockade of these two receptors can be additive or synergistic. ILT2 blockade additionally enhanced T cell and NK cell function. Furthermore, in vivo studies delineated a role for both ILT2 and ILT4 blockade in promoting anti-tumor immune responses and demonstrated that blockade of ILT2 and ILT4 is complementary to PD-1 inhibition. These data support the clinical evaluation of the dual ILT2/ILT4 antagonist antibody NGM707 alone and in combination with PD-1 blockade. Citation Format: Jane Tian, Samir Qurashi, Christina Song, Lauren Rocha, Kalyani Mondal, Suzanne Crawley, Sara Medfisch, Peirong Krisney Chen, Julie Roda, Jon Sitrin, Carmence Ho, Jonathan Aguayo, Lee Rivera, Jiawei Huang, Vicky Lin, David Shen, Yan Wang, Hui Tian, Alan Kutach, James Sissons, Daniel D. Kaplan, Geoffrey W. Stone. Immune inhibitory receptors ILT2 and ILT4 exhibit both distinct and overlapping biology in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 664.

Type 1 Diabetes (T1D) is a chronic disease with potentially severe complications, and β-cell deficiency underlies this disease. Despite active research, no therapy to date has been able to induce β-cell regeneration in humans. Here, we discovered the β-cell regenerative effects of glucagon receptor antibodies (anti-GcgR). Treatment with anti-GcgR in mouse models of β-cell deficiency leads to reversal of hyperglycemia, increased plasma insulin and C-peptide levels, and restored β-cell mass. We demonstrate that both β-cell proliferation and α- to β-cell transdifferentiation contribute to anti-GcgR-induced β-cell regeneration. Interestingly, anti-GcgR-induced α-cell hyperplasia can be uncoupled from β-cell regeneration with treatment cessation and antibody elimination from the body. Importantly, we are able to show that anti-GcgR induced α- to β-cell transdifferentiation is also observed in non-human primates. Furthermore, anti-GcgR and anti-CD3 combination therapy reverses diabetes and increases β-cell mass in the non-obese diabetic (NOD) mouse model of T1D. Taken together, our novel findings suggest that the combination of anti-GcgR with an immune modulator may be an effective therapy for T1D.

Type 1 diabetes mellitus (T1D) is a chronic disease with potentially severe complications, and β-cell deficiency underlies this disease. Despite active research, no therapy to date has been able to induce β-cell regeneration in humans. Here, we discover the β-cell regenerative effects of glucagon receptor antibody (anti-GcgR). Treatment with anti-GcgR in mouse models of β-cell deficiency leads to reversal of hyperglycemia, increase in plasma insulin levels, and restoration of β-cell mass. We demonstrate that both β-cell proliferation and α- to β-cell transdifferentiation contribute to anti-GcgR-induced β-cell regeneration. Interestingly, anti-GcgR-induced α-cell hyperplasia can be uncoupled from β-cell regeneration after antibody clearance from the body. Importantly, we are able to show that anti-GcgR-induced β-cell regeneration is also observed in non-human primates. Furthermore, anti-GcgR and anti-CD3 combination therapy reverses diabetes and increases β-cell mass in a mouse model of autoimmune diabetes.

Background: Myeloid immune suppression in the tumor microenvironment is known to contribute to tumor immune evasion. ILT2 (aka LILRB1) and ILT4 (aka LILRB2) are distinct ITIM-containing immunosuppressive receptors that recognize shared MHC-I ligands. Both ILT2 and ILT4 are highly expressed on tumor infiltrating myeloid cells, while ILT2 is also expressed on tumor-associated T and NK cells. Furthermore, both ILT2 and ILT4 are upregulated in patients who do not respond to T cell checkpoint inhibitor therapy, implicating these receptors as potential resistance mechanisms. Given their regulation in human cancers and their potential to mediate immune suppression in a broad range of tumor-associated immune cells, ILT2 and ILT4 may represent promising targets for cancer immunotherapy. The complementary nature of ILT2 and ILT4 activities suggests a dual antagonist strategy is warranted. To this end, we have developed NGM707. NGM707 is a humanized monoclonal antibody with specificity for both ILT2 and ILT4, blocking their interaction with both classical MHC-I (HLA-A, HLA-B, HLA-C) as well as the non-classical MHC-I molecule HLA-G. We present here preclinical data on the pharmacology of NGM707. Methods: NGM707 was evaluated for ILT2 and ILT4 specificity, ligand blocking and functional activity. Activity was assessed on various myeloid cell subsets, including monocyte-derived macrophages, myeloid-derived suppressor cells (MDSC) and dendritic cells. ILT2 blockade by NGM707 was also assessed on NK cells and T cells. Results: In vitro and biochemical pharmacology studies demonstrated that NGM707 bound potently to human ILT2 (1.03 nM KD) and ILT4 (0.205 nM KD). NGM707 binding to ILT2 and ILT4 blocked interaction with their ligands, including classical MHC-I and non-classical MHC-I (HLA-G). Functional studies demonstrated that NGM707-mediated ILT2 and ILT4 blockade reprogrammed suppressive myeloid cells and enhanced their activity, including stimulation of allogeneic T cell responses. NGM707-mediated ILT2 blockade also enhanced NK- and CD8 T cell-mediated killing of tumor cells. NGM707 in combination with an anti-PD-1 antibody additively enhanced macrophage-mediated allogeneic stimulation of CD4 T cells, suggesting combined treatment with NGM707 and an anti-PD-1 antibody can lead to complementary immune-modulatory activities. Conclusions: NGM707 is a novel dual antagonist antibody targeting both ILT2 and ILT4. The preclinical data presented indicate that by blocking ILT4, NGM707 can reprogram suppressive myeloid cells to a stimulatory state, and that by blocking ILT2, NGM707 can stimulate the activation of both myeloid and lymphoid cells. These data support the clinical evaluation of NGM707 as a therapeutic for patients with solid tumor malignancies. Citation Format: Kalyani Mondal, Christina Song, Jane Tian, Carmence Ho, Lee B. Rivera, Jiawei Huang, Peirong Chen, Suzanne Crawley, Vicky Y. Lin, Jonathan Sitrin, Julie M. Roda, David Shen, Hui Tian, Yan Wang, Alan Kutach, Jeong Kim, James Sissons, Daniel D. Kaplan, Geoffrey W. Stone. Preclinical evaluation of NGM707, a novel anti-ILT2/anti-ILT4 dual antagonist monoclonal antibody [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 LB156.

Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these 'non-homeostatic' conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the 'emergency circuit' that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.

This corrects the article DOI: 10.1038/nature24042.

JAK3 kinase plays a critical role in several cytokine signaling pathways involved in immune cell development and function. The studies presented in this report were undertaken to elucidate the kinetic mechanism of the JAK3 kinase domain, investigate the role of activation loop phosphorylation in regulating its catalytic activity, and examine its inhibition by the anti-rheumatoid arthritis drug, tofacitinib. Phosphorylation of two Tyr residues in JAK3's activation loop has been reported to impact its kinase activity. The recombinant JAK3 kinase domain used in our studies was heterogeneous in its activation loop phosphorylation, with the non-phosphorylated protein being the dominant species. Kinetic analysis revealed similar kinetic parameters for the heterogeneously phosphorylated JAK3, JAK3 mono-phosphorylated on Tyr 980, and the activation loop mutant YY980/981FF. Bisubstrate and product inhibition kinetic results were consistent with both sequential random and sequential ordered kinetic mechanisms. Solvent viscosometric experiments showed perturbation of k cat , suggesting the phosphoryl transfer step is not likely rate limiting. This was supported by results from quench-flow experiments, where a rapid burst of product formation was observed. Kinetic analysis of JAK3 inhibition by tofacitinib indicated inhibition is time dependent, characterized by on- and off-rate constants of 1.4 ± 0.1 μM −1 s −1 and 0.0016 ± 0.0005 s −1 , respectively.

Relativistic quantum theories are equipped with a background Minkowski spacetime and non‐relativistic quantum theories with a Galilean space‐time. Traditional investigations have distinguished their distinct space‐time structures and have examined ways in which relativistic theories become sufficiently like Galilean theories in a low velocity approximation or limit. A different way to look at their relationship is to see that both kinds of theories are special cases of a certain five‐dimensional generalization involving no limiting procedures or approximations. When one compares them, striking features emerge that bear on philosophical questions, including the ontological status of the wave function and time reversal invariance.