Immune Inhibitory Receptors Research Articles

Abstract 1875: Novel cell-based bioassays for monoclonal antibody and bispecific molecules in PD-1 blockade monotherapy and combination therapy

Abstract Programmed death 1 (PD-1) is an immune checkpoint (IC) receptor that negatively regulates T-cell immune function. Blocking the interaction of PD-1 with its ligand PD-L1 unleashes the immune system and enhances anti-tumor responses. Immunotherapy using PD-1 blockade has led to a paradigm shift in cancer drug discovery, due to its durable effect against a wide variety of cancers. However, despite its tremendous clinical success in cancer treatment, a significant fraction of patients and tumor types remain unresponsive to this therapy. This has led to the development of combination therapies combining PD-1 checkpoint inhibitors with a broad range of clinically active treatments including those targeting other IC receptors. A major challenge in immunotherapy drug development is the lack of quantitative and reproducible functional assays as existing methods rely heavily on primary immune cells and are highly variable. Here, we report the development of a suite of cell line-based reporter bioassays for monoclonal antibody targeting human PD-1/PD-L1, or bispecific molecules target PD-1/PD-L1 and a co-stimulatory receptor (e.g., 4-1BB, OX40, ICOS) or an immune inhibitory receptor (e.g., CTLA-4, LAG-3, TIGIT). A mouse PD-1/PD-L1 assay and a human PD-1/PD-L2 assay are also developed and can be used in parallel with human PD-1/PD-L1 assay to address preclinical study and assay specificity. Each of these assays consist of two engineered cell lines: a T effector cell line that express a luciferase reporter driven by specific promoter/response elements responding to the intracellular signals mediated by the T cell receptor (TCR), PD-1 and the second IC receptor, and an artificial antigen presenting cells (aAPCs). Particularly, the combination bioassays are able to measure the synergetic effect of PD-1 blockade with a second IC inhibitor receptor blockade or a costimulatory receptor activation. These reporter-based assays reflect the mechanisms of action for the therapeutic drugs of interest and are sensitive and quantitative. They are prequalified according to ICH guideline and demonstrated the performance characteristics (specificity, precision, accuracy, and linearity) to be used as potency assay for product release and stability studies during immunotherapy drug development and manufacture. Citation Format: Jamison Grailer, Julia Gilden, Jun Wang, Denise Garvin, Pete Stecha, Jim Hartnett, Steven Edenson, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Novel cell-based bioassays for monoclonal antibody and bispecific molecules in PD-1 blockade monotherapy and combination therapy [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 1875.

Abstract 3433: Novel dual antagonist anti-LILRB1/LILRB2 antibodies reprogram myeloid cells and potentiate T cells

Abstract Background: Genome-wide association studies have identified HLA-G and LILRB1/2 loci associated with cancer risk. LILRB1 and LILRB2 are distinct inhibitory immune checkpoint receptors that recognize MHC-I ligands, particularly immunosuppressive HLA-G. LILRB1-mediated inhibition can lead to impairment of T-cell cytotoxicity and preventing the efficient engulfment of tumor cells by macrophages as a “don’t eat me” signal. LILRB2 is expressed primarily by myeloid cells, especially in the tumor microenvironment, as an important immunosuppressive signal to inhibit the stimulation of an immune response. A dual antagonist antibody targeting both LILRB1 and LILRB2 could be more effective for cancer immunotherapy than targeting either LILRB1 or LILRB2 individually. Methods: Mice were immunized with recombinant D1-D2 domain of LILRB1 and LILRB2 proteins. Resulted heavy chain and light chain variable regions of IgG were PCR amplified from splenocytes and bone marrow plasma cells. PCR amplicons were built into yeast libraries. Yeast displayed scFv were screened for LILRB1 and LILRB2 binding. The dual binders were converted to effector silent human IgG1 and evaluated for ability to block HLA-G binding to LILRB1/RB2. The dual binder and dual blocker were further assessed in series of assays using human T cells and various primary myeloid cell subsets, including human monocyte-derived macrophages (hMDMs), tumor-associated macrophages (TAMs), dendritic cells (DCs) and monocytes. Results: We have discovered a panel of anti-LILRB1/2 antibodies that bind to both LILRB1 and LILRB2 with high affinity and block the interactions to HLA-G efficiently. The anti-LILRB1/2 antibodies inhibit both LILRB1 and LILRB2 signaling and modulate the function of immune cells. Anti-LILRB1/LILRB2-mediatted LILRB1 blockade: 1) enhances phagocytosis of tumor cells by macrophages; 2) releases HLA-G mediated inhibition on TCR activation in Jurkat T cells over-expressing LILRB1; 3) promotes the activation of human primary T cells stimulated with allogenic DCs. Anti-LILRB1/LILRB2-mediatted LILRB2 blockad:1) enhances TNFα expression and inhibits IL10 expression in LPS-stimulated human PBMC; 2) reprograms both hMDMs and TAMs induced by tumor cells to polarize toward a more inflammatory phenotype, including enhanced T cell responses to stimulation by DCs and macrophages, increased expression of M1 markers and secreted proinflammatory cytokine TNFα, decreased expression of M2 markers and secreted anti-inflammatory cytokine IL10. Conclusions: We have generated dual antagonist anti-LILRB1/LILRB2 antibodies. The anti-LILRB1/LILRB2 can reprogram suppressive myeloid cells to a stimulatory state, enhance phagocytotic function of myeloid cells, and promote the activation of lymphoid cells. The data support further development of anti-LILRB1/LILRB2 as an immunotherapeutic for solid tumor malignancies. Citation Format: Guangan Hu, Quanju Zhao, Lai Shi, Nan Bing, Dong Zhang. Novel dual antagonist anti-LILRB1/LILRB2 antibodies reprogram myeloid cells and potentiate T cells [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 3433.

Abstract 6268: Discovery and characterization of the CD96 antibody GSK6097608, a high-affinity, antagonistic anti-CD96 antibody for cancer immunotherapy

Abstract The overall therapeutic benefit of blocking the first generation of immune checkpoint pathways (PD-1 and CTLA-4) has been demonstrated across multiple tumor types, yielding long term protection in some patients. However, most patients do not respond to these single-agent approaches. Thus, collaborative strategies that seek to engage novel pathways and cell types may provide therapeutic options for patients wherein pre-existing host and tumor microenvironment factors do not favor current immunotherapeutic agents or in tumors where adaptive resistance has occurred. In recent years, the CD226 (DNAX Accessory Molecule-1 [DNAM-1]) axis has emerged as a relevant regulatory node in for natural killer (NK) and T cells - particularly in the context of tumor immunology. Similar to the competitive interplay between CTLA-4/CD28 and B7 (CD80/86), inhibitory receptors within the axis (e.g., CD96 [TACTILE]) effectively compete with the co-stimulatory receptor CD226 for binding to shared ligands (e.g., CD155), thereby impairing the initiation and/or promotion of ongoing antitumor immune responses. Indeed, genetic or monoclonal antibody (mAb)-based co-inhibition of CD96 with other immune checkpoints has proven efficacious in several nonclinical tumor models. CD96 has been shown to impact both T cell and NK cell function, offering a level of versatility as a target for cancer immunotherapy. For example, in the setting of anti-PD-1 neoadjuvant treatment, significantly improved survival of pancreatic ductal adenocarcinoma (PDAC) tumor-bearing mice was observed following enhancement of NK cell activity by CD96 antibody treatment. Equally, pronounced effects on primary tumor growth following anti-PD-1, -TIGIT, and -CD96 mAb treatment in a colorectal carcinoma tumor model (CT26) was found to be dependent on CD8+ T cells. GSK6097608 is a clinical-stage fully-human immunoglobulin G1 (IgG1)κ mAb that targets the inhibitory immune receptor CD96. GSK6097608 was identified, in part, for its ability prevent and disrupt CD96:CD155 interactions, thereby promoting T and NK cell function. GSK6097608 demonstrated concentration-dependent rescue of human immune cell activity following exposure to plate-bound recombinant CD155 (cognate receptor/ligand); an effect that was improved with concomitant TIGIT blockade. Notably, functional activity was found to be dependent on intact Fc-FcγR co-engagement, as potentiation of primary human T and NK cell function was lost when GSK6097608 was grafted on an Fc-attenuated backbone. Here, we describe some of the biophysical and functional characteristics that support the rationale for clinical evaluation of GSK6097608. Citation Format: Yan Degenhardt, Jun Guan, Peter Morley, David Jones, Michael Conner, Stephen Eastman, Wei Wang, Andrew Sanderson, Anand Ravindran, Julie Krueger, Iris Roth, James Smothers, Jeremy D. Waight. Discovery and characterization of the CD96 antibody GSK6097608, a high-affinity, antagonistic anti-CD96 antibody for cancer immunotherapy [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 6268.

Abstract 610: The expression of the PVRIG/TIGIT pathway is dominant in the bone marrow of patients with multiple myeloma

Abstract Blocking inhibitory immune receptors has shown limited clinical benefit in patients with multiple myeloma (MM), warranting the discovery of alternative immune inhibitory pathways. Blockade of the immune checkpoint TIGIT was shown to enhance anti-tumor immunity in MM pre-clinical models. In addition to TIGIT, the DNAM1 axis includes the novel inhibitory receptor PVRIG. Both TIGIT and PVRIG deliver inhibitory signals to T and NK cells and compete with the co-activating receptor DNAM1 for binding to PVR and PVRL2, respectively. Accordingly, TIGIT and PVRIG co-blockade was shown to synergize in enhancing anti-tumor immunity in preclinical models. PVRL2 and PVR were shown to be expressed on MM plasma cells in the bone marrow (BM). In this study, we evaluated DNAM1 axis receptor expression in the BM of patients with MM. BM mononuclear cells derived from 21 patients with MM were analyzed for the expression of PD1 and DNAM1 axis molecules by flow cytometry. Patients classified with progressed disease (PD), or complete response (CR) were treated with multiple lines of therapies including targeted therapies, chemotherapies, proteasome inhibitors. PVRIG demonstrated the highest expression among all evaluated receptors on NK (88%), NKT (81%) and CD8+ T cells (79%), significantly higher than PD1 (p<0.0001) and TIGIT (P<0.001). TIGIT showed substantially increased expression on NK (64%, P<0.0001) and CD8+ T cells (58%, p<0.05) compared to PD1 (expressed on 12% of NK cells and 42% of CD8+ T cells). Importantly, 50% and 60% of CD8+ and NK cells, respectively, co-expressed TIGIT and PVRIG, and all examined cell populations showed increased levels of DNAM1 (>50%). Additionally, the expression of PVRIG ligand, PVRL2, on BM plasma cells from 6 patients with MM was demonstrated by immuno-histochemistry and flow cytometry. A fraction of CD8+ T cells were DNAM1- and positive for PVRIG/TIGIT/PD1 (22%), suggesting the accumulation of an exhausted CD8+ T cell population in the MM tumor microenvironment. PVRIG showed significantly higher (p<0.0001) expression on DNAM+ CD8+ T cells (81%), compared to TIGIT (43%) and PD1 (34%), supporting the potential of PVRIG blockade to enhance DNAM1 signaling and subsequent CD8+ T cell activation. Finally, patients with CR had a trend towards higher DNAM1 expression on CD8+ T cells (75%) compared to PD patients (54%, p=0.057). To conclude, DNAM1 axis receptors are dominantly expressed on MM BM lymphocytes, with PVRIG exhibiting the most prominent expression. The reduced expression of DNAM1 in patients with PD suggests a link between DNAM1 axis and clinical outcome. Our findings highlight for the first time the dominant expression of PVRIG, as well as TIGIT and suggest that combined blockade of these checkpoints may potentially benefit patients with MM, placing the DNAM1 axis as a promising therapeutic pathway in MM therapy. Citation Format: Masha Frenkel, Zoya Alteber, Ning Xu, Mingjie Li, Haiming Chen, Gady Cojocaru, James Berenson, Eran Ophir. The expression of the PVRIG/TIGIT pathway is dominant in the bone marrow of patients with multiple myeloma [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 610.

Mechanisms for Host Immune Evasion Mediated by Plasmodium falciparum-Infected Erythrocyte Surface Antigens.

Plasmodium falciparum infection causes the most severe form of malaria. It has been hypothesized that P. falciparum directly suppresses host immune responses because sufficient acquired immunity is often not induced even by repeated P. falciparum infections in malaria-endemic areas. It is known that many kinds of P. falciparum-derived proteins are expressed on the surface of P. falciparum-infected erythrocytes (IEs), and these proteins have long been thought to be a key to the elucidation of the host immune evasion mechanisms. Our recent studies have revealed that the P. falciparum-derived erythrocyte surface antigen, RIFIN, the largest multiple gene family protein in the P. falciparum genome, suppresses host immune cell activation through direct interaction with human inhibitory immune receptors. In this review, we will discuss the molecular mechanisms for host immune evasion by P. falciparum-infected erythrocyte surface antigens. In addition, we will discuss the recently identified host immune response to P. falciparum using specialized antibodies that target host-P. falciparum-derived molecule interactions.

Abstract 664: Immune inhibitory receptors ILT2 and ILT4 exhibit both distinct and overlapping biology in vitro and in vivo

Abstract 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.

Abstract 601: IO-108, A fully human therapeutic antibody blocking the myeloid checkpoint LILRB2/ILT4, promotes innate and adaptive anti-cancer immunity in preclinical studies

Abstract Tumor-associated myeloid cells inhibit anti-cancer immune responses systemically and in the tumor microenvironment (TME), thereby limiting the efficacy of T cell checkpoint inhibitors. However, the plasticity of myeloid cells may enable therapeutic intervention. The immune inhibitory receptor LILRB2 (also known as ILT4) is expressed primarily by myeloid cells (monocytes, macrophages, dendritic cells and neutrophils), and has emerged as a key myeloid checkpoint contributing to the tolerogenic activity of myeloid cells associated with cancer. LILRB2 has several ligands (classical and non-classical MHC-I, ANGPTL2/5, SEMA4A and CD1) and most of these are known to contribute to immune suppression in the TME. Using computational biology approaches, we found that high LILRB2 expression is associated with macrophage infiltration in many solid tumor types from TCGA. Therefore, LILRB2 is a compelling target to overcome immune suppressive activity of cancer-associated myeloid cells. IO-108 is a fully human IgG4 therapeutic antibody that binds to LILRB2 with high affinity and specificity. IO-108 binds to all myeloid cells in the solid TME and periphery. In vitro studies support that blockade of LILRB2 interaction with its various ligands is the anticipated mechanism of action of IO-108. The LILRB2 antagonist activity of IO-108 produces the desired pro-inflammatory (re)polarization of myeloid cells. IO-108 treatment results in increased pro-inflammatory responses and an enhanced antigen-presenting cell (APC) phenotype to multiple stimuli (e.g., T cell activators, STING and TLR agonists) in ex vivo assays. In addition, IO-108 precludes the anti-inflammatory myeloid cell phenotype resulting from “tumor conditioning” and promotes the differentiation of monocytes and immature dendritic cells (DC) into pro-inflammatory DC. IO-108 enhances the effect of PD-1 blocking antibodies in allogeneic mixed leukocyte reactions of CD4+ T cells and macrophages. Moreover, IO-108 polarized primary myeloid cells isolated from solid tumor patient blood and ovarian cancer-associated ascites towards a proinflammatory phenotype and attenuated their suppressive effect on autologous T cell proliferation and production of proinflammatory cytokines. In vivo, IO-108 inhibits tumor growth in mouse models, which is associated with immune cell activation. Importantly, IO-108 presents a favorable pharmacokinetic and safety profile in preclinical models. Collectively, the preclinical characterization of IO-108 enabled a comprehensive clinical biomarker plan and lends rationale to the clinical study (NCT05054348) of IO-108 as a novel immunotherapy for multiple solid tumor types, including those relapsed/refractory to standard of care therapies. Citation Format: Jing-Tyan Ma, Xiaoye Liu, Ryan Stafford, Krista McCutcheon, Heyu Chen, Tao Huang, Hongyu Tian, Kyu Hong, ChengCheng Zhang, Luke Chung, An Song, Paul Woodard, XCharlene Liao, MariaJose Costa. IO-108, A fully human therapeutic antibody blocking the myeloid checkpoint LILRB2/ILT4, promotes innate and adaptive anti-cancer immunity in preclinical studies [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 601.

Abstract 5203: Discovery and development of SLD2435, a novel humanized IgG1 bispecific antibody simultaneously targeting human TIGIT and PVRIG for cancer immunotherapy

Abstract Despite dramatic success for immune checkpoint inhibition in treating certain patient subsets, there remains high unmet medical need for the treatment of patients who are refractory to or relapse following treatment with current checkpoint inhibitors. TIGIT and PVRIG, are exciting novel targets for immune-oncology which function as immune inhibitory receptors expressed on both T and NK cells and have been reported to inhibit anti-tumor activity. SLD2435, a novel humanized IgG1 bispecific antibody simultaneously targeting human TIGIT and PVRIG that synergistically enhances both NK and T cell functions, demonstrates significant preclinical anti-tumor efficacy. Here, we reported the discovery, development and characterization of SLD2435 for potential use in cancer patients. Anti-human TIGIT mAb was generated from mouse hybridoma whereas anti-human PVRIG Ab was discovered from Alpaca as single-domain antibody (sdAb). Both Abs were identified based on the ability to block the interaction of TIGIT or PVRIG with their cognate ligands (PVR or PVRL2 respectively) and for the ability to enhance primary human NK cytotoxicity over tumor cell lines as well as to promote the function of antigen-specific CD8+ T cells. Lead anti-human PVRIG sdAb was then selected and fused to the N-terminal of lead anti-human TIGIT mAb with a peptide linker for SLD2435 construction followed by subsequent testing in multiple abovementioned assays. SLD2435 simultaneously bound to human TIGIT and PVRIG with sub-nanomolar affinity and showed cross-reactivity to respective cynomolgus targets together with strong avidity mediated receptor-ligand interaction blocking at both protein and cellular levels. In vitro functional studies revealed that SLD2435 induced potent primary human NK cell mediated cytotoxicity over WiDr tumor cells better than the combination of anti-PVRIG and anti-TIGIT mAbs. It could also trigger preferential depletion of TIGIT+/TIGIT+PVRIG+ Treg via ADCC. In addition, SLD2435 enhanced IFN-γ production of CMV antigen-specific CD8+ T cells culturing with PVR+PVRL2+HLA matched Colo205 tumor cells and had synergistic effect on T cell activation when further combined with an anti-PD-L1 mAb. In consistent with in vitro results, SLD2435 displayed significant anti-tumor efficacy in vivo in a HuPBMC A375 xenograft model. At last, SLD2435 demonstrated great developability profile under various stress tests. Taken together, these data support the further development of SLD2435 as a potential novel therapeutic agent for the treatment of cancer patients. Citation Format: Xiaofeng Zhao, Yayuan Fu, Xiaoqing Liu, Fengli Shan, Zhenna Gao, Mengyu Wang, Huiming Xu, Hai Huang, Yang Liu, Wenqing Yang, Zhuoxiao Cao, Renhong Tang. Discovery and development of SLD2435, a novel humanized IgG1 bispecific antibody simultaneously targeting human TIGIT and PVRIG for cancer immunotherapy [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 5203.

Abstract 605: MoA-based potency bioassays for immunotherapy programs targeting the TIGIT/CD112R/CD226 axis

Abstract Immune checkpoint (IC) receptors play critical roles in maintaining immune homeostasis and have emerged as proven drug targets for the treatment of various cancers and autoimmune disorders. Monoclonal antibodies (mAb) designed to block immune inhibitory receptors PD-1 and CTLA-4 have been approved and show unprecedented efficacy in cancer treatment. This led to rapid expansion of drug development to many more IC receptors including members of the T cell immunoreceptor with Ig and ITIM domains (TIGIT) - CD112R (PVRIG) - CD226 Axis. TIGIT binds to its ligands CD155 (PVR) and CD112 (PVRL2) while CD112R binds to CD112. Binding of TIGIT or CD112R to CD155/CD112 inhibits the co-stimulatory activity of the paired receptor CD226. Thus, therapeutic targeting of this axis can modulate T cell and NK cell activation and unleash anti-cancer immunity. One of the bottlenecks of immunotherapy drug development is the lack of quantitative and reproducible functional assays for mAb potency determination. Existing methods rely heavily on primary immune cells which is labor intensive and highly variable. To address this need, we developed a panel of cell-based reporter bioassays that can quantitatively measure the potencies of mAbs targeting TIGIT/CD155, TIGIT/CD112, and CD112R/CD112. Owing to positive clinical trial results of TIGIT blockade in combination with PD-1/PD-L1 blockade, we further developed a potency bioassay for bispecific antibodies simultaneously targeting TIGIT and PD-1/PD-L1. These bioassays each consist of an engineered T effector cell line that expresses T cell receptors and IC receptors of interest, and an engineered artificial antigen presenting cells (aAPC) expressing the corresponding ligands for the IC receptors. The T effector cell lines also express a luciferase reporter driven by a promoter specifically responding to TCR activation which can be inhibited by signaling from the IC receptors. Additionally, we developed bioassays that can measure potencies of agonist or antagonist mAbs targeting CD226. These bioassays are designed to reflect the mechanisms of action for the drug candidates designed for each IC receptor and the assay signals are specific. Most are prequalified according to ICH guidelines and show the precision, accuracy and linearity required for relative potency determination and product stability study under cGMP environments. Therefore, these MoA-based bioassays can serve as valuable tools for early drug discovery, potency determination and stability studies throughout the development of mAbs targeting the TIGIT/CD112R/CD226 axis. Citation Format: Jamison Grailer, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong, Zhi-Jie Jey Cheng. MoA-based potency bioassays for immunotherapy programs targeting the TIGIT/CD112R/CD226 axis [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 605.

First-in-human study of SRF388, a first-in-class IL-27 targeting antibody, as monotherapy and in combination with pembrolizumab in patients with advanced solid tumors.

2501 Background: The immunoregulatory cytokine IL-27 upregulates inhibitory immune checkpoint receptors (eg, PD-L1, TIGIT) and downregulates proinflammatory cytokines (eg, IFNγ, TNFα). SRF388 is a fully human IgG1 blocking antibody to IL-27 with potential to promote immune activation in the tumor microenvironment. A phase 1 study was conducted to establish the preliminary safety of SRF388 and to identify recommended phase 2 doses (RP2D) suitable for monotherapy and combination expansions (NCT04374877). Methods: The dose-escalation study (accelerated single patient followed by standard 3+3) enrolled patients (pts) with advanced treatment-refractory solid tumors. Upon RP2D selection, monotherapy and combination expansions opened for treatment-refractory clear cell renal cell cancer (ccRCC), hepatocellular cancer (HCC), and non-small cell lung cancer. SRF388 was administered IV every 4 weeks (wks) as monotherapy and every 3 wks with pembrolizumab. Tumor response was assessed by RECIST1.1. Results: The monotherapy dose-escalation enrolled 29 pts with doses ranging from 0.003 to 20 mg/kg. Median age was 64 years. Most pts were female (62%) with ECOG PS of 1 (72%). Approximately 80% had prior PD-(L)1 blockade, and 48% had ≥4 prior therapies. Treatment-related adverse events (TRAEs) occurred in 21%, and all were low grade. Fatigue was the only TRAE reported in ≥10% (n = 3). No dose-limiting toxicities (DLTs) or Grade ≥3 TRAEs were observed. Median time on study was 9 wks (range 0–59). One patient with highly treatment-refractory NSCLC experienced a confirmed partial response (PR) at 8 wks that was durable for 20 wks. Nine pts (31%) experienced disease stabilization at 8 wks, with 6 of 9 exhibiting durable disease control at 6 months. Of the 7 pts with ccRCC in the dose-escalation portion of the trial, 3 (43%) experienced durable disease control for ≥20 wks (range: 20-32). With doses up to 20 mg/kg, SRF388 PK remain linear with an estimated T1/2 of 10-12 days. PK characteristics and safety profile support dosing every 3 or 4 wks. Based on safety, tolerability, PK, peripheral pSTAT1 inhibition, and preliminary efficacy, 10 mg/kg was selected as the RP2D. Both the pembrolizumab safety cohort (n = 10) and Stage 1 of the ccRCC monotherapy expansion (n = 17) have fully enrolled. Of the 10 evaluable pts with ccRCC, 1 confirmed monotherapy PR has been reported, enabling Stage 2 initiation. Changes in several serum cytokines and chemokines were observed after SRF388 administration, including expected increase in circulating IL-27 levels. Conclusions: Results of IL-27 pathway blockade with a first-in-class therapeutic demonstrates that SRF388 has good tolerability with encouraging preliminary antitumor activity as a monotherapy. Updated data, including safety and clinical outcomes as well as correlative biomarker analyses, will be presented. Clinical trial information: NCT04374877.

Semi-automated validation and quantification of CTLA-4 in 90 different tumor entities using multiple antibodies and artificial intelligence

AbstractCTLA-4 is an inhibitory immune checkpoint receptor and a negative regulator of anti-tumor T-cell function. This study is aimed for a comparative analysis of CTLA-4+ cells between different tumor entities. To quantify CTLA-4+ cells, 4582 tumor samples from 90 different tumor entities as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Two different antibody clones (MSVA-152R and CAL49) were validated and quantified using a deep learning framework for automated exclusion of unspecific immunostaining. Comparing both CTLA-4 antibodies revealed a clone dependent unspecific staining pattern in adrenal cortical adenoma (63%) for MSVA-152R and in pheochromocytoma (67%) as well as hepatocellular carcinoma (36%) for CAL49. After automated exclusion of non-specific staining reaction (3.6%), a strong correlation was observed for the densities of CTLA-4+ lymphocytes obtained by both antibodies (r = 0.87; p < 0.0001). A high CTLA-4+ cell density was linked to low pT category (p < 0.0001), absent lymph node metastases (p = 0.0354), and PD-L1 expression in tumor cells or inflammatory cells (p < 0.0001 each). A high CTLA-4/CD3-ratio was linked to absent lymph node metastases (p = 0.0295) and to PD-L1 positivity on immune cells (p = 0.0026). Marked differences exist in the number of CTLA-4+ lymphocytes between tumors. Analyzing two independent antibodies by a deep learning framework can facilitate automated quantification of immunohistochemically analyzed target proteins such as CTLA-4.

Efficient expansion of mouse hematopoietic stem cells ex vivo by membrane anchored Angptl2

Hematopoietic stem cell (HSC) transplantation represents an important curative therapy for numerous hematological and immune diseases. Many efforts have been applied to achieve attainable ex vivo HSC expansion. We previously showed that angiopoietin-like proteins 2 (Angptl2) binds and activates the immune inhibitory receptor human leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) to support the expansion of HSC. However, soluble Angptl2 is unstable and the downstream signaling would be attenuated by ligand-binding triggered receptor endocytosis, compromising the potential of Angptl2 to expand HSCs. We proposed that membrane anchored Angptl2 will overcome these limitations. In this study, we constructed the C-terminal and N-terminal anchored membrane Angptl2 (Cm-Angptl2 and Nm-Angptl2) by adding a transmembrane domain at the C-terminal or an anchor sequence at the N-terminal respectively. Both forms of Angptl2 showed efficient expression on the surface of feeder cells. Nm-Angptl2, but not Cm-Angptl2, induces a potent activation of LILRB2 reporter, indicating the fibronectin (FBN) domain at the C-terminus of Angptl2 is essential to stimulate LILRB2 signaling. Compared to soluble Angptl2, Nm-Angptl2 displays higher activities to activate LILRB2 reporter, and to promote the expansion of mouse HSCs as determined by transplantation and limiting dilution assay. Our study revealed the importance of FBN domain for Angptl2 to activate LILRB2 and demonstrated that Nm-Angptl2 have enhanced activities than the soluble protein in LILRB2 activation and HSC expansion, providing a strategy to explore the mode of ligand induced receptor signaling, and an optimized approach to expand HSCs ex vivo.

CD200R1 Contributes to Successful Functional Reinnervation after a Sciatic Nerve Injury.

Activating and inhibitory immune receptors play a critical role in regulating systemic and central nervous system (CNS) immune and inflammatory processes. The CD200R1 immunoreceptor induces a restraining signal modulating inflammation and phagocytosis in the CNS under different inflammatory conditions. However, it remains unknown whether CD200R1 has a role in modulating the inflammatory response after a peripheral nerve injury, an essential component of the successful regeneration. Expression of CD200R1 and its ligand CD200 was analyzed during homeostasis and after a sciatic nerve crush injury in C57Bl/6 mice. The role of CD200R1 in Wallerian Degeneration (WD) and nerve regeneration was studied using a specific antibody against CD200R1 injected into the nerve at the time of injury. We found an upregulation of CD200R1 mRNA after injury whereas CD200 was downregulated acutely after nerve injury. Blockade of CD200R1 significantly reduced the acute entrance of both neutrophils and monocytes from blood after nerve injury. When long term regeneration and functional recovery were evaluated, we found that blockade of CD200R1 had a significant effect impairing the spontaneous functional recovery. Taken together, these results show that CD200R1 has a role in mounting a successful acute inflammatory reaction after injury, and contributes to an effective functional recovery.

Epigenetic regulation of T cell exhaustion.

Chronic antigen stimulation during viral infections and cancer can lead to T cell exhaustion, which is characterized by reduced effector function and proliferation, and the expression of inhibitory immune checkpoint receptors. Recent studies have demonstrated that T cell exhaustion results in wholescale epigenetic remodeling that confers phenotypic stability to these cells and prevents T cell reinvigoration by checkpoint blockade. Here, we review foundational technologies to profile the epigenome at multiple scales, including mapping the locations of transcription factors and histone modifications, DNA methylation and three-dimensional genome conformation. We discuss how these technologies have elucidated the development and epigenetic regulation of exhausted T cells and functional implications across viral infection, cancer, autoimmunity and engineered T cell therapies. Finally, we cover emerging multi-omic and genome engineering technologies, current and upcoming opportunities to apply these to T cell exhaustion, and therapeutic opportunities for T cell engineering in the clinic.

Lymphocytic Thyroiditis Transcriptomic Profiles Support the Role of Checkpoint Pathways and B Cells in Pathogenesis.

Background: Autoimmune thyroid diseases are the most common types of autoimmune diseases, but their physiopathology is still relatively unexplored. Genotype-tissue expression (GTEx) is a publicly available repository containing RNAseq data, including profiles from thyroid. Approximately 14.8% of these glands were affected by focal lymphocytic thyroiditis and 6.3% were annotated as Hashimoto. We interrogated these data to improve the characterization of infiltrating cells and to identify new molecular pathways active in autoimmune thyroiditis. Materials and Methods: Histological GTEx images of 336 thyroid samples were classified into three categories, that is, non-infiltrated thyroid, small focal infiltrated thyroid, and extensive lymphoid infiltrated thyroid. Differentially expressed genes among these categories were identified and subjected to in silico pathway enrichment analysis accordingly. CIBERSORTx deconvolution was used to characterize infiltrating cells. Results: As expected, most of the transcriptional changes were dependent on tissue infiltration. Upregulated genes in tissues include-in addition to lineage-specific B and T cell genes-a broad representation of inhibitory immune checkpoint receptors expressed by B and T lymphocytes. CIBERSORTx analysis identified 22 types of infiltrating cells showed that T cells predominate 3:1 over B cells in glands with small infiltrates, only by 1.7:1 in those with large infiltrates. Follicular helper and memory CD4 T cells were significantly more abundant in glands with large infiltrates (p < 0.0001), but the most prominent finding in these glands was an almost sixfold increase in the number of naive B cells (p < 0.0001). A predominance of M2 macrophages over M1 and M0 macrophages was observed in the three gland categories (p < 0.001). Conclusions: Analysis of transcriptomic RNA-seq profiles constitutes a rich source of information for the analysis of autoimmune tissues. High-resolution transcriptomic data analysis of thyroid glands indicates the following: (a) in all infiltrated glands, active autoimmune response coexists with suppressor counteracting mechanisms involving several inhibitory checkpoint receptor pairs, (b) glands with small infiltrates contain an unexpected relatively high proportion of B lymphocytes, and (c) in highly infiltrated glands, there is a distinct transcriptomic signature of active tertiary lymphoid organs. These results support the concept that the autoimmune response is amplified in the thyroid tissue.

Systemic Immune Dysfunction in Cancer Patients Driven by IL6 Induction of LAG3 in Peripheral CD8+ T Cells.

Many cancer patients do not develop a durable response to the current standard-of-care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (e.g., PD1 and LAG3). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This is associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warranted to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.

Immune Infiltration Characteristics and a Gene Prognostic Signature Associated With the Immune Infiltration in Head and Neck Squamous Cell Carcinoma.

Background: Immunotherapy has become the new standard of care for recurrent and metastatic head and neck squamous cell carcinoma (HNSCC), and PD-L1 is a widely used biomarker for immunotherapeutic response. However, PD-L1 expression in most cancer patients is low, and alternative biomarkers used to screen the population benefiting from immunotherapy are still being explored. Tumor microenvironment (TME), especially tumor immune-infiltrating cells, regulates the body’s immunity, affects the tumor growth, and is expected to be a promising biomarker for immunotherapy. Purpose: This article mainly discussed how the immune-infiltrating cell patterns impacted immunity, thereby affecting HNSCC patients’ prognosis. Method: The immune-infiltrating cell profile was generated by the CIBERSORT algorithm based on the transcriptomic data of HNSCC. Consensus clustering was used to divide groups with different immune cell infiltration patterns. Differentially expressed genes (DEGs) obtained from the high and low immune cell infiltration (ICI) groups were subjected to Kaplan–Meier and univariate Cox analysis. Significant prognosis-related DEGs were involved in the construction of a prognostic signature using multivariate Cox analysis. Results: In our study, 408 DEGs were obtained from high- and low-ICI groups, and 59 of them were significantly associated with overall survival (OS). Stepwise multivariate Cox analysis developed a 16-gene prognostic signature, which could distinguish favorable and poor prognosis of HNSCC patients. An ROC curve and nomogram verified the sensitivity and accuracy of the prognostic signature. The AUC values for 1 year, 2 years, and 3 years were 0.712, 0.703, and 0.700, respectively. TCGA-HNSCC cohort, GSE65858 cohort, and an independent GSE41613 cohort proved a similar prognostic significance. Notably, the prognostic signature distinguished the expression of promising immune inhibitory receptors (IRs) well and could predict the response to immunotherapy. Conclusion: We established a tumor immune cell infiltration (TICI)-based 16-gene signature, which could distinguish patients with different prognosis and help predict the response to immunotherapy.

Pneumonia Severity and Phase Linked to Virus-Specific T Cell Responses with Distinct Immune Checkpoints during pH1N1 Infection.

The detailed features and the longitudinal variation of influenza-specific T cell responses within naturally infected patients and the relationship with disease severity remain uncertain. In this study, we characterized the longitudinal influenza-specific CD4+ and CD8+ T cell responses, T cell activation, and migration-related cytokine/chemokine secretion in pH1N1-infected patients with or without viral pneumonia with human PBMCs. Both the influenza-specific CD4+ and CD8+ T cells presented higher responses in patients with severe infection than in mild ones, but with distinct longitudinal variations, phenotypes of memory markers, and immune checkpoints. At 7 ± 3 d after onset of illness, effector CD8+ T cells (CD45RA+CCR7-) with high expression of inhibitory immune receptor CD200R dominated the specific T cell responses. However, at 21 ± 3 d after onset of illness, effector memory CD4+ T cells (CD45RA-CCR7-) with high expression of PD1, CTLA4, and LAG3 were higher among the patients with severe disease. The specific T cell magnitude, T cell activation, and migration-related cytokines/chemokines possessed a strong connection with disease severity. Our findings illuminate the distinct characteristics of immune system activation during dynamic disease phases and its correlation with lung injury of pH1N1 patients.

The Role of Immune Regulatory Molecules in COVID-19.

As the fifth pandemic in the 21st century, coronavirus 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become the most prominent global concern in the last 2 years. Variable manifestations characterize SARS-CoV-2 infection. Despite the design and production of effective vaccines and their considerable effect on reducing the COVID-19 prevalence and mortality rate, no definitive cure for the disease has yet been found. Mutations may also affect the effectiveness of vaccines. The host immune response to the pathogen has a critical role in the course of the disease. Positive and negative signals often balance the immune system. Immune regulatory molecules, also known as immune checkpoint receptors, balance the immune responses. These molecules mainly have inhibitory functions and prevent hyperactivation of immune cells or trigger adverse signaling pathways. For a decade, the immune checkpoint blockade, as a therapeutic target for cancer immunotherapy, has been utilized. Some of the inhibitory receptors are recognized as exhaustion markers on T cells. The signaling pathway of these markers restricts the function of T cells against viral infection. Dysregulation of T cells was observed in SARS-CoV-2 infection and can modify proliferation, differentiation, cytokine production, and type of response. The pivotal role of immune inhibitory receptors in the function of acquired, cell-mediated, immune defense T cells makes them a fascinating subject to study. This review article summarized recent findings on immune regulatory molecules and their role in SARS-CoV-2 infection, hoping to find a way to design novel treatments.

Remodeling the tumor microenvironment via blockade of LAIR-1 and TGF-β signaling enables PD-L1-mediated tumor eradication.

Collagens in the extracellular matrix (ECM) provide a physical barrier to tumor immune infiltration, while also acting as a ligand for immune inhibitory receptors. Transforming growth factor-β (TGF-β) is a key contributor to shaping the ECM by stimulating the production and remodeling of collagens. TGF-β activation signatures and collagen-rich environments have both been associated with T cell exclusion and lack of responses to immunotherapy. Here, we describe the effect of targeting collagens that signal through the inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) in combination with blockade of TGF-β and programmed cell death ligand 1 (PD-L1). This approach remodeled the tumor collagenous matrix, enhanced tumor infiltration and activation of CD8+ T cells, and repolarized suppressive macrophage populations, resulting in high cure rates and long-term tumor-specific protection across murine models of colon and mammary carcinoma. The results highlight the advantage of direct targeting of ECM components in combination with immune checkpoint blockade therapy.