Mixed Lymphocyte Reaction Assay Research Articles

Abstract 598: A novel allosteric CBL-B inhibitor with differentiated immune enhancing activity in preclinical models

Abstract E3 ligase Casitas B-Lineage Lymphoma Proto-Oncogene B (CBL-B) is a key negative modulator of T-cell receptor and co-stimulatory regulation. CBL-B inhibition lowers the threshold of antigen-specific T cell activation, even in absence of co-stimulatory signaling or the presence of an immune suppressive environment. Genetic ablation of Cbl-b or functional inactivation of its E3 ligase activity in mice or primary human T cells enhances immune-mediated tumor growth control. Therefore, CBL-B inhibition may address the suboptimal response to current immunotherapies due to low inflammation, no/low co-stimulation signal or a high immune suppressive environment. We have identified a series of potent allosteric CBL-B inhibitors via the application of our proprietary Smart Allostery࣪ platform. Herein we demonstrate one of our lead CBL-B inhibitors showed superior activity compared to a competitor CBL-B inhibitor (NX-519) in both biochemical and cellular assays. At similar exposures in a syngeneic mouse tumor model (CT26), our CBL-B inhibitor also demonstrated an increase in tumor growth inhibition relative to NX-519. Mechanistically, our CBL-B inhibitor not only enhanced T cell activation, but also re-invigorated exhaustive T cells and caused resistance to T regulatory cell-mediated suppression, both in a concentration-dependent manner. We further demonstrated differentiation of CBL-B inhibition versus anti-PD1 and anti-CTLA4 in the mixed lymphocyte reaction (MLR) assay. Our CBL-B inhibitor significantly promoted T cell proliferation where anti-PD1 alone and anti-CTLA4 alone did not. Both anti-PD1 and CBL-B inhibition increased IFNγ production in this MLR assay, but that effect was further enhanced in combination. Together, the pre-clinical data presented here provided evidence of a differentiated profile of our allosteric CBL-B inhibitors to enhance key parameters associated with T cell activation and reduce susceptibility to immune suppression, supporting their further progression toward clinical development. Citation Format: Yilin Qi, Jun Kuai, Yingzhi Bi, Huadong Sun, Deborah G. Conrady, Rajiv G. Govindaraj, Graham Hone, Rajiah A. Denny, Ken Carson, Timothy Reilly, Geraldine Harriman, Fang Wang. A novel allosteric CBL-B inhibitor with differentiated immune enhancing activity in preclinical models [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 598.

Abstract 2576: ATG-037, a highly potent small molecule CD73 inhibitor has superior activity of reversing immunosuppression in higher-AMP environments compared with anti-CD73 antibodies

Abstract Background: CD73 is a cell surface enzyme which is highly expressed in the tumor microenvironment (TME). Degradation of adenosine monophosphate (AMP) into adenosine by CD73 results in the generation of an immunosuppressed and pro-angiogenic niche within the TME that promotes the onset and progression of cancer. Targeting CD73 has resulted in favourable anti-tumor effects in preclinical models and in clinic. One of the key mechanisms of therapeutic activity by CD73 inhibition is restoring the normal function of effector T cells from adenosine inhibition. ATG-037 is a highly potent and selective oral small molecule inhibitor of CD73. This study compared the ability of ATG-037 and clinical CD73 antibodies in reverting immunosuppression in higher-AMP environments in vitro. Methods: The activity of ATG-037 and CD73 antibodies (Hu101-28, MEDI9447) in inhibiting enzyme function of cell surface CD73 was evaluated by measuring the ATP-dependent oxidation of luciferin, which is inhibited by AMP, using Cell Titer Glo assay. Reversal of AMP/adenosine-mediated immune suppression of human T cells by CD73 inhibitors was determined by measuring T cell function in the presence of different concentrations of exogenous AMP. Markers for T cell proliferation, activation and cytotoxicity were assessed by flow cytometry, and the cytokine levels were measured by ELISA. Results: ATG-037, Hu101-28 and MEDI9447 inhibited the enzyme activity of cell surface-expressed CD73 on human A375 cells under 100 µM AMP with a half-maximal inhibitory concentration (IC50) of 0.36 nM,20.94 nM and 3.46 nM, respectively. ATG-037 demonstrated complete inhibition of CD73 activity, whereas MEDI9447 did not reach complete CD73 inhibition. T cell proliferation and activation induced by CD3/CD28 dynabeads were suppressed by 100 µM of extracellular AMP and the suppression was relieved by the addition of ATG-037 with half-maximal effective concentration (EC50) of 21.6 nM for CD8+ T cell proliferation, 27 nM for T cell activation, 2 nM for granzyme B expression, and 6.5 nM for T cell IFN-γ production. While Hu101-28 and MEDI9447 fail to restore the T cell function. In a mixed lymphocyte reaction assay, ATG-037, but not Hu101-28 or MEDI9447 reversed AMP-dependent T cell suppression. In addition, ATG-037 rescued immune cell activation in high AMP (500 µM, 1000 µM) environments. Conclusion: ATG-037 demonstrated potent and complete CD73 enzyme inhibition and stronger ability in restoring T cell function from high-level AMP mediated suppression, compared with clinical anti-CD73 antibodies. These data implicate the potential therapeutic advantages of small molecule CD73 inhibitors over blocking antibodies. Citation Format: Gang Bian, Jiahui Hua, Linjie Tian, Kevin Lynch, Jay Mei, Bo Shan, Bing Hou. ATG-037, a highly potent small molecule CD73 inhibitor has superior activity of reversing immunosuppression in higher-AMP environments compared with anti-CD73 antibodies [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 2576.

Abstract 4169: A bifunctional anti-PDL-1 antibody/IL-10 fusion protein targeting exhausted T cells for cancer immunotherapy

Abstract Background: Interleukin-10 (IL-10) has demonstrated utility in aiding CD8 T cell-mediated tumor control in mouse models but phase 2 clinical trials with pegylated IL-10 have so far not been entirely successful. More recent studies revealed that another half-life-extended form of IL-10 (IL10-Fc) can rejuvenate terminally-exhausted CD8+ T cells to boost anti-tumor activity. We investigated whether combination of a PDL-1-blocking antibody with a C-terminal IL-10 fusion may enhance anti-tumor immunity, and possibly achieve synergistic effects. Methods: Recombinant proteins were produced using an expiCHO system (Thermo Fisher). PDL-1 blocking activity was evaluated by ELISA, PD-1/PDL-1 signaling (Eurofins) and mixed lymphocyte reaction assays. IL-10 activity was evaluated by MC/9 cell proliferation and CD8 T cell activation and effector assays. Mouse CT26 colon cancer and EMT6 breast cancer studies employed the services of Oneness Biotech and Crown Bioscience, respectively. Results: To develop a bifunctional anti-PDL-1 antibody/IL-10 fusion protein, anti-human PDL-1 antibodies were first developed by screening an in-house human variable domain phage display library. Three different antibodies were then C-terminally-fused with human IL-10 polypeptide. All three showed high binding affinity to PDL-1 and blocked PD-1 signaling through PD-1/PDL-1 interaction in a dose-dependent manner. In CD4 T cell/dendritic cell co-cultures, the lead anti-PDL1/IL10 protein enhanced IL-2 and IFNγ production, and it enhanced IFNγ and granzyme B production from activated CD8 T cells as well as proliferation of MC/9 cells in an IL-10-dependent manner. Also, a mouse ‘surrogate’ version (BF11, using avelumab anti-PDL-1) showed superior efficacy compared to either IL10-Fc or anti-PDL1/TGFbR in a mouse CT26 colon tumor model, and correlated with increased serum levels of CXCL9 and IL-18. BF11 also allowed greater EMT6 tumor control than anti-PDL-1 alone or anti-PDL1/TGFbR fusion protein. Such BF11-treated mice also showed long-lasting immunity against a secondary EMT6 tumor challenge. This ability of BF11 to enhance tumor control was in part dependent on increased activity of tumor-resident cells as co-treatment with FTY720, which inhibits lymphocyte egress from secondary lymphoid organs, did not appear to reduce anti-tumor activity of BF11 in some, albeit not all, EMT6-tumor-bearing mice. Conclusions: Anti-PDL1/IL10 fusion protein not only targets PDL-1 for checkpoint blockade but also activates the IL-10 pathway to boost T cell response, presumably including revitalization of exhausted T cells as recently reported. Effective anti-tumor activities of a mouse surrogate fusion protein, BF11, are demonstrated in CT26 and EMT6 tumor models. Taken together, our findings support this dual mechanism-strategy to potentiate anti-tumor immunity. Citation Format: Huey-Wen Hsiao, Chih-Lun Hsiao, Pandelakis Koni, Hung-Kai Chen. A bifunctional anti-PDL-1 antibody/IL-10 fusion protein targeting exhausted T cells 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 4169.

Abstract 2828: A novel synthetic stealth receptor that redirects host immune cell alloreactivity and potentiates functional persistence of adoptively transferred off-the-shelf cell-based cancer therapy

Abstract Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of hematologic malignancies and have shown significant potential in solid tumor indications. However, logistical complexities associated with patient-specific CAR T-cell therapies often limit broad accessibility. Many of these challenges can be overcome with an allogeneic cellular product, but immune cell-mediated rejection of allogeneic cellular therapies remains a significant concern. Both allogeneic and autologous cell therapies currently rely on lymphodepleting conditioning to modulate the immune system and create greater access to homeostatic cytokines. However, protracted lympho-conditioning has been associated with poor immune reconstitution and increased susceptibility to opportunistic infections. Therefore, an ideal allogeneic cell therapy would be able to avoid immune rejection while reducing or eliminating the need for chemotherapeutic conditioning to deplete host lymphocytes. To address many of these challenges, we engineered our novel alloimmune defense receptor (ADR) that targets 41BB+ alloreactive immune cells while providing a CD3z signaling boost into our off-the-shelf iPSC derived NK cells expressing anti-CD19 CAR (CAR iNK). The ability of ADR+ CAR iNK cells to resist alloimmune rejection was tested by co-culturing ADR+ CAR iNK cells with allogeneic PBMCs from ten donors in a mixed lymphocyte reaction assay. Notably, ADR+ CAR iNK cells co-cultured with allogeneic PBMCs persisted to similar levels as the PBMC-free culture while ADR negative CAR iNK cells were eliminated when co-cultured with allogeneic PBMCs. Furthermore, all PBMC donors screened in ADR+ CAR iNK cell co-cultures showed ablation of reactive 41BB+ NK and T cells, with non-activated T cells remaining intact. CAR iNK cells +/- ADR were then compared in a Nalm6 disseminated in vivo model for anti-tumor efficacy. ADR+ CAR iNK cells and their ADR negative counterparts were found to equivalently control tumor. Building on this tumor model, we co-infused allogeneic T cells to mimic an immuno-competent setting. The data demonstrated that ADR negative CAR iNK cells were depleted and were unable to control tumor growth while significant levels of allogeneic T cells persisted. In contrast, ADR+ CAR iNK cells were able to resist allogeneic T cell attack, control tumor, and persist when compared to the ADR negative control. Collectively, our preliminary data suggest that ADR-armed CAR iNK cells withstand immune cell-mediated rejection with uncompromised effector function. We are actively developing models to confirm our initial finding that ADR+ effector cells also benefit from their engagement with alloreactive cells in immuno-competent settings to promote enhanced anti-tumor responses, proliferation, and persistence. Citation Format: Alan M. Williams, Ken Hayama, Yijia Pan, Brian Groff, Rina Mbofung, Lauren Fong, Nicholas Brookhouser, Berhan Mandefro, Ramzey Abujarour, Tom Lee, Quirin Hammer, Karl-Johan Malmberg, Maksim Mamonkin, Ryan Bjordahl, Jode Goodridge, Bahram Valamehr. A novel synthetic stealth receptor that redirects host immune cell alloreactivity and potentiates functional persistence of adoptively transferred off-the-shelf cell-based cancer 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 2828.

Abstract 4261: CD27 a new immuno-oncology target shaping innate and adaptive anti-tumor immune responses

Abstract CD27 is a member of the TNF-receptor superfamily, highly expressed on CD4+ and CD8+ T cells as well as on NK and NKT cells. It plays a key role on T cell proliferation and differentiation after stimulation with its ligand CD70. The co-stimulatory signal of CD27 on T cell is mediated via the NFκB pathway but also via the phosphatidylinositol 3 kinase and the protein kinase B. CD27/CD70 co-stimulation has the potential to boost immunity by T-cell activation, clonal expansion and enhanced differentiation into antigen specific cytotoxic and memory T cells. CD27/CD70 also influences the innate immune response via a direct activation of the NK cells and a subsequent secretion of interferon-gamma (IFN-γ). Therefore, CD27 signaling promotes cytotoxic T cell based anti-tumor immunity. With its central role in an immunological response, CD27 is a promising target for antitumor therapy. Previous works have demonstrated the efficacy of an agonistic CD27 antibody in controlling tumor growth and metastasis in different mice models including melanoma, renal cell carcinoma, breast cancer and lymphomas. This anti-tumor effect is mediated in part by an effective recruitment of IFN-γ producing CD8+ T cells within the tumor. Moreover, CD27 stimulation of Tumor Infiltrating Lymphocytes (TILs) can lower their threshold of activation and provide a broader repertoire of Ag-reactive T cells within the tumor. We have selected a lead therapeutic antibody from our library of 147 fully human anti-CD27 monoclonal antibodies generated in the Trianni mice. After confirming its binding potency and selectivity as well as its cross-reactivity with Non-Human Primate (NHP)-CD27 but not with the mouse-CD27, this lead candidate demonstrated strong agonistic proprieties. This was shown by its ability to induce a strong NFκB signal as well as to induce T cell proliferation and activation with secretion of pro-inflammatory cytokines. This antibody demonstrated agonistic proprieties without cross-linking confirming its potency. T cell activation observed after treatment with our anti-CD27 antibody only occurs in the presence of TCR engagement, preventing the risk of spontaneous activation of naïve T cells in vivo. The ability of our CD27 monoclonal antibody to increase an immune response was confirmed in a Mixed Lymphocyte Reaction assay with multiple donors. The role played by the NK cells and their activation via CD27 antibody was also demonstrated. To evaluate the anti-tumor functions of our lead antibody as a single agent or in combination with other immuno-therapies in vivo we have used human CD27 transgenic mice. We have demonstrated in the MB49 bladder tumor model as well as the EG7 thymoma model that our lead antibody induces a strong single agent anti-tumor activity and these tumors were totally controlled in combination with an anti-PD1 antibody. We are now analyzing the pharmacokinetic and pharmacodynamic of our antibody as well as its safety in a NHP model Citation Format: Thierry Guillaudeux, Yulia Ovechkina, Shaarwari Sridhar, David Peckham, Jessica Cross, Nathan Eyde, Emily Frazier, Neda Kabi, Remington Lance, Kurt Lustig, Mei Xu, Tarcha Eric, Shawn Iadonato. CD27 a new immuno-oncology target shaping innate and adaptive anti-tumor immune responses [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 4261.

Abstract 5525: An Fc-competent bispecific antibody targeting PD-L1 and TIGIT induces strong immune responses and potent anti-tumor efficacy

Abstract Background: TIGIT (T-cell immunoglobulin and ITIM domain), which is primarily expressed on activated and 'exhausted' T and NK cells, is one of the most promising 'next generation' immune checkpoint target. Engagement of TIGIT to its ligands (i.e., PVR and PVRL2) leads to inhibitory signaling in T cells and promotes the functional exhaustion of tumor-infiltrating T lymphocytes. Anti-TIGIT monoclonal antibodies have shown clinical benefit when combined with anti-PD-L1 agents in NSCLC. Here, we describe our novel anti-PD-L1 × TIGIT bispecific antibody (PD-L1 × TIGIT biAb) that blocks both the PD-L1/PD-1 and TIGIT/PVR/PVRL2 pathways and has the potential to exhibit equal clinical benefit compared to current combination therapies. Methods: PD-L1 × TIGIT biAb was engineered with a fully-human IgG targeting TIGIT in a g1-Fc backbone, fused to a VHH at the C-terminus targeting PD-L1. Binding affinities and specificity testing were studied by flow cytometry and biolayer interferometry. The co-binding of the PD-L1 × TIGIT biAb to TIGIT and PD-L1 was detected by ELISA. The immunomodulatory functions of the PD-L1 × TIGIT biAb were evaluated using luciferase reporter cell assays and mixed lymphocyte reaction (MLR) assays in vitro, and human PBMC models in vivo. Results: The PD-L1 × TIGIT biAb binds with high affinity to the extracellular domain of human TIGIT and PD-L1 and can bind to TIGIT and PD-L1 simultaneously. In a competition assay, the PD-L1 × TIGIT biAb efficiently blocked the interaction between TIGIT and PVR/PVRRL2, and likewise PD-L1 to PD-1. The PD-L1 × TIGIT biAb induced higher luciferase signals than the anti-TIGIT or anti-PD-L1 mAbs alone in a luciferase reporter-based cell system and enhanced IFN-γ production in an MLR assay. In vivo, the PD-L1 × TIGIT biAb demonstrates similar anti-tumor efficacy to the combination of anti-TIGIT and anti-PD-L1 mAbs, which is stronger than the single-agents alone. We have also completed GLP-toxicity studies that have shown excellent safety. Conclusion: We have discovered a novel PD-L1 × TIGIT biAb, which induces strong immune responses in vitro and in vivo, supporting its clinical development for the treatment of human cancers. Clinical trials shall be initiating in early 2022. Citation Format: Shuang Dai, Weifeng Huang, Zhijun Yuan, Shaogang Peng, Jiayi Si, Chao Wang, Yao Yan, Xiaoniu Miao, Yingda Xu, Joanne Sun, Xiaolin Liu, Andy Tsun, Tianhang Zhai. An Fc-competent bispecific antibody targeting PD-L1 and TIGIT induces strong immune responses and potent anti-tumor efficacy [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 5525.

Abstract 596: IOH-001, a novel CD47/PD-L1 bispecific antibody, enhances anti-tumor activity in solid tumors

Abstract Background: The survived cancer cells after cytotoxic chemotherapy are known to promote immune evasion phenotype by increasing the expression of PD-L1 and CD47. Targeting of CD47 and PD-L1 is predicted to overcome immune evasion by coordinately restoring each innate and adaptive immunity. IOH-001, a CD47/PD-L1 dual-targeting bispecific antibody, has been shown to activate immune cells in tumor microenvironment by blocking PD-1/PD-L1 signals that inhibit cytotoxic T-cells, while also blocking interaction with CD47/SIRPa between cancer cells and macrophages. We have investigated whether IOH-001 inhibits tumor growth via co-targeting tumor cells or not. Methods: To assess the function of IOH-001, a series of in vitro functional assays including cell surface binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis assays and mixed lymphocyte reaction (MLR) assay were performed compared with the parent antibody. In vivo efficacy of IOH-001 was tested in colon cancer mouse models with human genes KI. Results: IOH-001 has been shown to be bound strongly to various types of PD-L1/CD47-expressing cancer cells including the ones in solid and hematological cancers. In most cancer cells, IOH-001 was demonstrated to have a lower EC50 than the parental antibodies and to be bound in a dose-dependent manner as well. Interestingly, IOH-001 also has shown the selectivity to bind only to cancer cells even under the conditions of co-culturing RBC and cancer cells. IOH-001 has induced phagocytosis of cancer cells by human blood CD14+ monocyte-derived macrophages. Since IOH-001 is an IgG1 type antibody, ADCC and IFN-r expression have been increased compared with the parental antibodies. Consistent with the in vitro data, IOH-001 has more strongly suppressed tumor growth than the combination treatment of the parental antibodies in a dose-dependent manner in syngeneic animal models. Moreover, the tumor of the CR mouse has been identified not to be re-generated in the re-challenge model. Conclusion: IOH-001, dual-blockage of anti-CD47 and anti-PD-L1, has shown the benefits in treating some solid tumors. Bispecific antibody IOH-001 is more likely to work better in targeting tumor cells than the combination of anti-CD47 and anti-PD-L1. Preclinical efficacy results of IOH-001 provide a strong rationale for assessing therapeutic potential in clinical studies. Citation Format: Jeong-kook Kim, A-Ra Jeon, Jihyun Park, Ji Yea Choi, Ji Eun Park, Sun Kwang Song, Ji Hye Choi, Heewook Shin, Ji Hye Lee, Ji Hye Yun, Yoen Hee Ahn, Heung Tae Kim. IOH-001, a novel CD47/PD-L1 bispecific antibody, enhances anti-tumor activity in 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 596.

Abstract 3499: M1069 as dual A2A/A2B adenosine receptor antagonist counteracts immune-suppressive mechanisms of adenosine and reduces tumor growth in vivo

Abstract Under physiological conditions, the extracellular concentrations of adenosine are low, however, levels dramatically increase under metabolically stressful conditions, including inflammation and cancer. The regulatory functions of adenosine are mediated through four members of the adenosine receptor family: A1, A2A, A2B, and A3. While A2A was considered the major contributor to adenosine-mediated suppression of T cell, natural killer cell, and myeloid cell functions, A2B has also recently emerged as a potential modulator of these processes. Both A2A and A2B signal through the same Gs-mediated activation of adenylate cyclase, which can allow the lower affinity A2B receptor to compensate for the inhibition of A2A in an adenosine-rich tumor microenvironment (TME). A2B receptors are also reported to support tumor growth independent of A2A through the Gq subunit of G-protein-coupled receptor-mediated production of vascular endothelial growth factor (VEGF) by myeloid and tumor cells. Therefore, simultaneous targeting of both the A2A and A2B receptors may provide a higher potential for cancer immunotherapy in an adenosine-rich TME, where A2B can act in compensatory or complementary means to A2A. M1069 is a small-molecule, dual antagonist of the A2A and A2B adenosine receptors with a selectivity of >100 fold against the A1 and A3 receptors. In assays with primary human T cells, M1069 caused a dose-dependent suppression of 5′-N-ethylcarboxamide adenosine (stable analog of adenosine)-stimulated cyclic adenosine monophosphate (cAMP) and phosphorylated cAMP- response element binding protein (pCREB) induction and rescue of interleukin (IL)-2 production (A2A readout). M1069 also suppressed VEGF production from human macrophages (A2B readout) in adenosine-rich settings. M1069 exhibited superior suppression of protumorigenic cytokine secretion, including CXCL1, CXCL5 and granulocyte-colony stimulating factor, and the rescue of IL12 secretion from adenosine-differentiated dendritic cells, as compared to an A2Aselective antagonist. In addition, in a one-way mixed lymphocyte reaction assay, adenosine-differentiated dendritic cells treated with M1069 demonstrated superior T cell activation compared to adenosine-differentiated dendritic cells treated with an A2A-selective antagonist. These findings were further corroborated with the results from in vivo studies in a murine CD73hi/adenosine-rich 4T1 syngeneic breast tumor model, in which M1069, but not an A2A-selective antagonist, reduced tumor growth as a monotherapy and enhanced anti-tumor activity with chemotherapeutic agents. In summary, M1069 is a potent, dual A2A/A2B adenosine receptor antagonist, which is expected to counteract immune-suppressive mechanisms in the presence of high concentrations of adenosine and enhance the anti-tumor activity of chemotherapies. Citation Format: Rinat Zaynagetdinov, Kai Schiemann, Kalyan Nallaparaju, Natalya Belousova, Armine Matevossian, Zhouxiang Chen, Giorgio Kradjian, Meghana Pandya, Nemisha Dawra, Eva-Maria Krauel, Elissaveta Petrova, Oliver Poeschke, David Fischer, Marc Lecomte, Andree Blaukat, Bayard Huck, Jacques Moisan. M1069 as dual A2A/A2B adenosine receptor antagonist counteracts immune-suppressive mechanisms of adenosine and reduces tumor growth 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 3499.

Abstract 619: Functional evaluation of immuno-oncology drug candidates in customized and fine-tuned T cell bioassays to assess therapeutic potential

Abstract Re-activation of tumor-reactive T cells with so-called immune checkpoint inhibitors (ICIs) has translated into significant clinical breakthroughs. Specifically, antibodies directed against CTLA-4 and PD-L1/PD-1 have yielded long-term remission and cure in many solid as well as hematological malignancies. However, as not all cancer types respond to current ICI therapies, the quest for novel strategies to re-enable anti-tumor immunity by blocking immune checkpoints or by activating prominent co-stimulatory receptors is crucial. The field of cancer immunology has developed rapidly over the past decades, with numerous studies adding up to the complexity of immune-related interactions taking place in the tumor microenvironment. Compounds or combinations of compounds designed to target traditional or novel pathways require early evaluation of effectiveness on in vitro assays using primary immune cells or representative mouse models. On top of classical T cell bioassays, expanding on current methods and the development novel strategies to assess the modulation of cancer-related immunological networks is a continuously important process. One classical approach is the functional evaluation of compounds in a mixed lymphocyte reaction (MLR). Here, the potency of drug candidates to promote physiological T cell responses can be evaluated. Antigen-specific immune responses can be further assessed in Cytomegalovirus (CMV) recall- or in Staphylococcal enterotoxin B (SEB) activation assays. Additionally, it might also be beneficial to generate tumor-associated antigen-specific T cell pools or clones to be used in functional assays. The classical MLR assay could be expanded on by e.g., inclusion of specific tumor cell populations or T regulatory (Treg) cells, as evaluating reduced cancer cell viability, or reversing loss of immune effector cell functionality can be of interest. Furthermore, real-time evaluation of cancer cell killing by T cells upon addition of drug candidates could also provide pivotal information on the compound’s functional dynamics. With our deepening understanding of cancer-related immunological networks it becomes clear that different drug candidates may require customized and fine-tuned T cell bioassays to assess their full therapeutic potential. Moreover, quality controlled primary immune cells are an essential factor for the robustness of these assays and to yield reproducible and consistent results. Collectively, taking in mind these aspects will ease decision making and accelerate the drug discovery pipeline. Citation Format: Thibaut J. Janss, Martijn Vlaming, Simon Lefevre, Johan Arnold, Ellen Boelen, Sofie Pattijn. Functional evaluation of immuno-oncology drug candidates in customized and fine-tuned T cell bioassays to assess therapeutic potential [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 619.

Evaluation of checkpoint inhibitor therapies using a mixed lymphocyte reaction (MLR) assay

Abstract Checkpoint inhibitors block the ‘off’ signals that are engaged when T cells interact with antigen-presenting cells, such as dendritic cells (DCs). This increases T cell activation and enhances the anti-tumor response. Here we demonstrate the use of advanced flow cytometry to evaluate these immunotherapies in vitro by co-culturing immune cells from two donors in an MLR assay. Assays were set up as either one-way or two-way MLR co-cultures. One-way assays combined isolated DCs from one donor with CD4+ T cells from another. Two-way assays mixed PBMCs from two donors. Cells and checkpoint inhibitors were plated in 96 or 384-well formats for 3–6 days. Samples taken during the time course were analyzed for cell subsets and cytokine concentrations using the iQue® Advanced Flow Cytometry Platform. Expression of activation marker CD25 on T cells was 3-fold greater after 6 days in co-culture with DCs (62 ± 2 %) compared to T cells in monoculture (22 ± 9 %). The addition of an anti-PD1 checkpoint inhibitor induced a further, concentration dependent increase in CD25 expression. This was accompanied by increased production of cytokines indicative of activation and inflammation, such as TNFα and CCL2. Throughout the study, there were large differences in sensitivity to drug response between donor pairs. For example, IFNγ release increased 13.5-fold upon addition of anti-CTLA4 (17 ng/mL) with one donor pair, compared to only 1.3-fold with another. This highlights the need to characterize drug activity using multiple immune cell donor pairs. These data exemplify the use of advanced flow cytometry to generate pharmacological data for checkpoint inhibitor activity on T cells in MLR, with the potential to profile libraries of novel therapeutics in minimal time.

Anti-CD80/86 antibodies inhibit inflammatory reaction and improve graft survival in a high-risk murine corneal transplantation rejection model

We investigated the effects of anti-CD80/86 antibodies in a murine high-risk corneal transplantation rejection model. A mixed lymphocyte reaction (MLR) assay was conducted with anti-CD80/86 antibodies. Inflammatory cytokine levels in the culture supernatant were measured using an enzyme-linked immunosorbent assay. Interferon (IFN)-γ-producing CD4+ T cell frequencies in the MLR were assessed using flow cytometry. In vivo, high-risk corneal allograft survival and IFN-γ-producing CD4+ T cell frequencies in corneal grafts were assessed with intraperitoneal injection of anti-CD80/86 antibodies compared to phosphate-buffered saline (PBS). RNA-sequencing was performed on corneal grafts 2 weeks post-transplantation. Anti-CD80/86 antibodies significantly decreased T-cell proliferation, IFN-γ+-producing CD4+ T cell frequencies, and IFN-γ, interleukin (IL)-1β, IL-2, IL-10, and tumor necrosis factor-α production in the MLR compared to PBS injection. Intraperitoneal injection of anti-CD80/86 antibodies significantly prolonged corneal graft survival and decreased IFN-γ+-producing CD4+ T cell frequencies compared to PBS injection. Gene set enrichment analysis showed that the gene sets mainly enriched in the control group were related to allograft rejection and inflammatory response compared to PBS injection. Anti-CD80/86 antibodies significantly prolonged corneal graft survival by inhibiting T-cell proliferation and inflammatory response.

Imaging flow cytometry challenges the usefulness of classically used extracellular vesicle labeling dyes and qualifies the novel dye Exoria for the labeling of mesenchymal stromal cell–extracellular vesicle preparations

Background aimsExtracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is assumed to be important for EVs in mediating intercellular communication processes, labeling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of EV target cells and tissues. However, the accuracy and specificity of commonly utilized labeling dyes have not been sufficiently analyzed. MethodsBy combining recent advances in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant mesenchymal stromal cell (MSC)-EV preparations, the authors explored the EV labeling efficacy of various fluorescent dyes, specifically carboxyfluorescein diacetate succinimidyl ester, calcein AM, PKH67, BODIPY TR ceramide (Thermo Fisher Scientific, Darmstadt, Germany) and a novel lipid dye called Exoria (Exopharm Limited, Melbourne, Australia). ResultsThe authors’ analyses qualified Exoria as the only dye that specifically labeled EVs within the MSC-EV preparations. Furthermore, the authors demonstrated that Exoria labeling did not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labeled EVs were differentially taken up by different immune cell types. ConclusionsOverall, the results qualify Exoria as an appropriate dye for the labeling of EVs derived from the authors’ MSC-EV preparations. This study also demonstrates the need for the development of next-generation EV characterization tools that are able to localize and confirm the specificity of EV labeling.

P064 IMU-838, a Small Molecule DHODH Inhibitor in Phase 2 Clinical Trial for Ulcerative Colitis, Shows Potent Anti-inflammatory Activity in Cell-Culture-Based and In Vivo Systems

Abstract Background IMU-838 (vidofludimus calcium) is a well-tolerated and orally available small molecule inhibitor of dihydroorotate dehydrogenase (DHODH) and is currently in phase 2 clinical development for ulcerative colitis (UC). Here, we investigated the effect of IMU-838 on different immune cell subsets and analysed the mechanism of action in more detail in T cells. Methods The effect of IMU-838 on regulatory macrophages (Mregs) was investigated by adding IMU-838 or anti-TNFα antibodies (infliximab, adalimumab), alone or in combination, in a mixed lymphocyte reaction assay, determining Mregs (CD206+CD14+) by FACS analysis. To investigate the effect of IMU-838 on human T and B cells, PBMC were stimulated with PHA (48h) or Oligonucleotide 2006-PTO (70h), respectively. An affinity-dependent effect of IMU-838 was assessed on CD8 T cells from OT-I and OT-III mice, containing a high and a low affinity T cell receptor (TCR), respectively, for OVA257-264 peptide. These cells were stimulated with ovalbumin (OVA) peptide loaded splenocytes or anti-CD3/anti-CD28 for 3 days. Oxidative phosphorylation (OXPHOS) and glycolysis in murine T cells was investigated by the Seahorse/Agilent technique in murine CD4 and CD8 T cells activated with anti-CD3/anti-CD28 with or without IMU-838. Results IMU-838 slightly induced Mregs, but strongly and dose-dependently decreased TNFα and IL-6 secretion. Anti-TNFα antibodies alone strongly induced Mregs, but also increased IL-6 levels. When IMU-838 was added, Mregs induction was even higher, and IL-6 was reduced. Besides reducing proinflammatory cytokines secreted by macrophages, IMU-838 also inhibits proliferation of activated B and T cells, as well as inflammatory T cell cytokine secretion, IL-17A, IL-17F and IFNγ, and increased apoptosis up to 3-fold compared to vehicle control. By investigating IMU-838’s role on affinity related activity status of T cells, it was shown that IMU-838 strongly inhibited cell proliferation in the peptide stimulated high affinity, but not in the low affinity, CD8 T cells. Peptide stimulated high affinity T cells show an upregulation of OXPHOS and glycolysis pathways, which are involved in the generation of the cell’s energy supply, compared to low affinity cells. IMU-838 strongly inhibited OXPHOS and glycolysis in both CD4 and CD8 T cells. Conclusion IMU-838 reduces the proinflammatory immune cell response by inducing Mregs, reducing pro-inflammatory cytokine secretion and reducing immune cell proliferation. DHODH is only important in cells that received a strong stimulus and are highly metabolically active. Therefore, IMU-838 will only specifically target these cells and will still allow for a normal immune response, representing a huge safety advantage for treatment of IBD patients.

Allogeneic Bone-Marrow Mesenchymal Stem Cell with Moldable Cryogel for Craniofacial Bone Regeneration.

Allogeneic bone-marrow mesenchymal stem cells (BMSCs) can promote bone regeneration and substitute for autologous BMSCs if autologous sources are unavailable, but the efficacy of bone regeneration by allogeneic BMSCs is still inconsistent. A Lewis rat cranium defect model was used to investigate the efficacy of bone regeneration between autologous and allogeneic BMSCs in gelatin-nanohydroxyapatite cryogel scaffolds. BMSCs from Wistar rats served as the allogeneic cell lineage. The full-thickness cranium defects were treated by either blank control, cryogel only, allogeneic BMSC-seeded cryogel, or autologous BMSC-seeded cryogel (n = 5). Bone regeneration was monitored by micro-computed tomography and examined histologically at week 12. In addition, we assessed the immune responses in vitro by mixed lymphocyte reaction (MLR) assay and CD4+ immunochemistry staining ex vivo. The MLR showed that allogeneic BSMCs elicited a weak immune response on day 14 that progressively attenuated by day 28. In vivo, the bone regeneration in allogeneic BMSCs was inferior at week 4, but progressively matched the autologous BMSCs by week 12. Our results suggest that allogeneic BMSCs can serve as an alternative source for bone regeneration.

133P Novel fully human agonist antibodies against the T-cell costimulatory receptor CD27 shape adaptive anti-tumor immunity

133P Novel fully human agonist antibodies against the T-cell costimulatory receptor CD27 shape adaptive anti-tumor immunity

A Novel Stealth Strategy That Activates Adoptively Transferred Allogeneic Immune Cells and Avoids Rejection for Off-the-Shelf Cell-Based Cancer Therapy

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of hematologic malignancies, however, logistical complexities associated with patient-specific CAR T-cell therapies often limit broad accessibility to patients. Many of these challenges can be overcome with an allogeneic cellular product that is available off-the-shelf, and overcoming immune cell-mediated rejection of allogeneic cell therapy is an area of significant research. Conditioning chemotherapies, which are commonly administered with CAR T-cell therapy, suppress a patient's immune system and may create a suitable window of activity for allogeneic cell therapies to elicit clinical response. However, protracted lympho-conditioning has been associated with poor immune reconstitution and increased susceptibility to opportunistic infections. Deletion of human leukocyte antigen (HLA) surface expression is known to abrogate T-cell alloreactivity, but deletion of class I HLA must be combined with other immune-modulating strategies to avoid NK cell-mediated recognition.To this end, allogeneic models combining class I HLA deletion with NK cell inhibitory molecules, such as HLA-E and CD47, have been shown to abrogate NK cell reactivity in mouse models. However, since HLA-E is the canonical activator of NKG2C, a dominant activating receptor found on human NK cells, and since the ligand for CD47, SIRPα, is known to be expressed on macrophages and dendritic cells and not on human NK cells, the observed effects of these immune-modulating strategies may not translate into the patient-treatment setting. In assessment of their protective effects in a defined human system, we found that HLA-E or CD47 overexpression on class I HLA null human cells offer only partial protection in evading various human NK cell compartments. We found that class I HLA-null K562 cells engineered to over-express CD47 were ineffective in inhibiting NK cells (0 to 7% inhibition). Separately, K562 cells engineered to over-express HLA-E, while effective in inhibiting NKG2A+ NK cells (90.2% +/- 3.7% inhibition), were unable to completely inhibit CD56 dim NK cells (33.2% +/- 29.6% inhibition) and not only failed, but instead activated, NKG2C+ NK cells (167% +/- 69% activation). Our data highlight the limitations of engineered CD47 and HLA-E modalities in suppressing broad populations of NK cells in clinically relevant settings.We therefore evaluated expression of the alloimmune defense receptor (ADR) that uniquely targets alloreactive immune cells (Mo et al. Nat Biotechnol 2021). We have shown that the expression of ADR has the potential to evade host immune cells without the need for further genetic editing such as class I HLA deletion. To determine its applicability for off-the-shelf cell therapy, ADR expression was engineered into induced pluripotent stem cells (iPSCs) and iPSC-derived CAR-NK (CAR-iNK) cells were generated. CAR-iNK cells carrying the ADR modality (ADR+ CAR-iNK cells) showed normal patterns of differentiation (>99% CD56+ with co-expression of NK cell receptors such as NKG2D, NKp30 and NKp46), suggesting that ADR expression did not disrupt hematopoiesis or the expansion of iNK cells. Additionally, ADR+ and ADR-negative CAR-iNK cells produced similar cytotoxicity against tumor cells. We next tested the ability of ADR to provide resistance to alloimmune rejection by coculturing ADR+ CAR-iNK cells with allogeneic pBMCs in a mixed lymphocyte reaction (MLR) assay. Notably, ADR+ CAR-iNK cells maintained durable persistence throughout the entire duration of the MLR assay and suppressed the expansion of alloreactive T- and NK-cells in comparison to the control arm (Figure 1).Collectively, initial preclinical studies suggest that ADR-modified CAR-iNK cells resist host immune cell rejection, while eliciting a durable anti-tumor response. Our preliminary data show evidence toward a promising off-the-shelf solution for elimination of broad pools of alloreactive T- and NK- effector cells in the clinical setting without the need for lympho-depleting conditioning or genetic editing strategies. [Display omitted] DisclosuresWilliams: Fate Therapeutics: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. Malmberg: Merck: Research Funding; Vycellix: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Mamonkin: Beam Therapeutics: Other: Licensing payments; Fate Therapeutics: Other: Licensing payments; Allogene Therapeutics: Consultancy, Other: Licensing payments; Xenetic Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.

Effect of the Neddylation Inhibitor Pevonedistat on Normal Hematopoietic Stem Cell Subsets and Immune Cell Composition

Introduction: Antitumor activity of the neddylation inhibitor pevonedistat has been documented in several hematologic and non-hematologic malignancies. Unexpectedly, Zhou et al (PNAS, 2016) discovered a dose-dependent biphasic effect of pevonedistat in solid tumor cell lines. While micromolar concentrations inhibited tumor cell growth, low nanomolar concentrations significantly increased cell proliferation and tumor stem cell self-renewal both in vitro and in vivo. The effect of low-dose pevonedistat has not yet been explored in the field of hematopoietic stem cell transplantation. Therefore, we evaluated how pevonedistat affects the viabiilty, growth and proportions of CD34 + cell subpopulations. In view of the emerging role of neddylation in the regulation of both innate and adaptive immunity, we also investigated the influence of pevonedistat on T-cell activation to explore a potentially beneficial effect on posttransplant immune complications.Methods and Results: Using the WST-1 assay we confirmed the biphasic effect of pevonedistat on normal mobilized CD34 + cells. Incubation for 72 h with 0.1 µM pevonedistat significantly increased metabolic activity as a surrogate parameter for proliferation, while 1.0 µM pevonedistat showed a cytotoxic effect. We explored the underlying mechanism for the low-dose effect. Since Zhou et al. previously showed that pevonedistat can promote tumor stem cell proliferation by inducing EGFR homodimerization, we used a proximity ligation assay and found that 0.1 µM pevonedistat induced EGFR homodimerization in normal mobilized CD34 + cells, too. In addition to homodimerization, we also looked at phosphorylation at Tyr1068, a marker of EGFR activation. By flow cytometry, we showed that phosphorylation was increased by 0.01 µM and 0.1 µM pevonedistat. Using an ELISA-based transcription assay, we also observed a biphasic effect of pevonedistat on c-Myc expression, which is regarded as a marker of ‘stemness’. Incubation with pevonedistat for 72 hrs at 0.01 and 0.1 µM stimulated expression of c-Myc, whereas incubation at 1.0 µM downregulated c-Myc.Fractions of hematopoietic stem and progenitor cell (HSPC) subpopulations were measured in CD34 + cells from cord blood after incubation with 0.01, 0.1 and 1.0 µM pevonedistat. Flow cytometry was performed using antibodies against CD34, CD45RA and CD133, as well as 7-AAD for testing cell viability. Exposure to pevonedistat for 72 hrs at 0.1 µM caused an increase in the number of CD34 + cells compared to vehicle-treated CD34+ cells at 72 h as well as compared to initial number of CD34+ cells, whereas 1.0 µM caused a significant decrease. The absolute number of multipotent progenitors (MPP) (CD34 +CD133 +CD45RA -) remained relatively stable at all concentrations, while lympho-myeloid progenitors (LMPP) (CD34+CD133+CD45RA+) and late progenitors (LP) (CD34+CD133-CD45RA+) increased slightly with 0.1 µM pevonedistat compared with controls. However, a significant decrease in LMPP and LP cell numbers was observed at 1.0 µM.Different concentrations of pevonedistat were tested for their capability to modulate allogeneically stimulated T cell activation in a multi-donor mixed lymphocyte reaction (mdMLR) assay in vitro. Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (MSC-EV) were used as internal immuno-modulatory and non-immuno-modulatory controls in the assay. After 5 days, alterations in the immune cell composition were analyzed by flow cytometry. Pevonedistat was not toxic for MNCs in the mdMLR. However, it decreased the number of activated (CD25high CD54+) CD4+ cells and CD8+ cells.Conclusions: One of the problems in the post-transplant period is a rapid decline in MPP numbers, associated with increased risk of engraftment failure. We showed that low-dose pevonedistat (0.1 µM) is capable of increasing the number of CD34 + cells in vitro while keeping the absolute number of MPPs stable. This finding, together with the observed increase in c-Myc expression, suggests that pevonedistat may help to preserve ‘stemness’ of CD34+ donor cells, thus supporting engraftment of hematopoietic stem and progenitor cells. Furthermore, the immunosuppressive effects revealed by mdMLR suggest that low-dose pevonedistat may also play a useful immunomodulatory role in the post-transplant setting to potentially reduce the risk of graft-versus-host disease. [Display omitted] DisclosuresMajidi: Takeda: Research Funding. Germing: Jazz Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding; Celgene: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Honoraria. Zeiser: Incyte, Mallinckrodt, Novartis: Honoraria, Speakers Bureau. Gattermann: Celgene: Honoraria; Takeda: Research Funding; Novartis: Honoraria.

IL-10 Signaling Elicited by Nivolumab-Induced Activation of the MAP Kinase Pathway Does Not Fully Contribute to Nivolumab-Modulated Heterogeneous T Cell Responses.

Immune checkpoint inhibitor (ICI) therapy has revolutionized anti-cancer treatment for many late-stage cancer patients. However, ICI therapy has thus far demonstrated limited efficacy for most patients, and it remains unclear why this is so. Interleukin 10 (IL-10) is a cytokine that has been recognized as a central player in cancer biology with its ability to inhibit anti-tumor T cell responses. Recent studies suggest that IL-10 might also exert some intrinsic anti-tumor T cell responses, and clinical studies using recombinant IL-10 alone or in combination with ICI are underway. This paradoxical effect of IL-10 and its underlying mechanisms impacting ICI-modulated T cell responses remain poorly understood. In this study, using an in vitro mixed lymphocyte reaction assay, we found that treatment with ICIs such as the anti-programmed cell death receptor-1 (PD-1) mAb nivolumab elicits a strong expression of IL-10. While neutralization of IL-10 signaling with an anti-IL-10 specific mAb significantly decreases the production of IFN-γ by T cells in a cohort of donor cells, the opposite effect was observed in other donor cells. Similarly, neutralization of IL-10 signaling significantly decreases the expression of T cell activation markers Ki67 and CD25, as well as the production of Granzyme B in a cohort of donor cells, whereas the opposite effect was observed in others. Furthermore, we found that nivolumab and IL-10 differentially modulate the signal transducer and activator of transcription 3 (STAT3) and AKT serine–threonine kinase pathways. Finally, we found that nivolumab activates the mitogen-activated protein kinase (MAPK) pathway, which in turn is responsible for the observed induction of IL-10 production by nivolumab. These findings provide new insights into the mechanisms underlying anti-PD-1-modulated T cell responses by IL-10, which could lead to the discovery of novel combination treatments that target IL-10 and immune checkpoint molecules.

SAR study of small molecule inhibitors of the programmed cell death-1/programmed cell death-ligand 1 interaction.

The development of small molecule inhibitors of programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) has drawn research interest for the treatment of cancer. Recently, we reported the discovery of a novel dimeric core small molecule PD-1/PD-L1 inhibitor. In an effort to discover more potent inhibitors, we further explored the dimeric core scaffold. Our investigations of the structure-activity-relationship revealed that introduction of lipophilic substituents onto one of the di-alkoxylated phenyl rings improved binding affinities to PD-L1, and inhibitory activities of PD-1/PD-L1 in cellular assays. Furthermore, conversion of the ether linker part to an olefin linker not only improved binding affinity but also led to slow dissociation binding kinetics. We also explored more potent, as well as downsized, scaffolds. Compounds bearing a linear chain in place of one of the di-alkoxylated phenyl rings exhibited good binding affinity with improved ligand efficiency (LE). Representative compounds demonstrated potent inhibitory activities of PD-1/PD-L1 in the submicromolar range in cellular assays as well as cellular function in the mixed lymphocyte reaction (MLR) assay with efficacy comparable to anti-PD-1 antibody. Our results provide applicable information for the design of more potent inhibitors targeting PD-1/PD-L1 pathway.

B cell depletion with anti-CD20 mAb exacerbates anti-donor CD4+ T cell responses in highly sensitized transplant recipients

Pretransplant desensitization with rituximab has been applied to preformed donor-specific anti-human leukocyte antigen antibody (DSA)-positive recipients for elimination of preformed DSA. We investigated the impact of pretransplant desensitization with rituximab on anti-donor T cell responses in DSA-positive transplant recipients. To monitor the patients’ immune status, mixed lymphocyte reaction (MLR) assays were performed before and after desensitization with rituximab. Two weeks after rituximab administration, the stimulation index (SI) of anti-donor CD4+ T cells was significantly higher in the DSA-positive recipients than in the DSA-negative recipients. To investigate the mechanisms of anti-donor hyper responses of CD4+ T cells after B cell depletion, highly sensitized mice models were injected with anti-CD20 mAb to eliminate B cells. Consistent with clinical observations, the SI values of anti-donor CD4+ T cells were significantly increased after anti-CD20 mAb injection in the sensitized mice models. Adding B cells isolated from untreated sensitized mice to MLR significantly inhibited the enhancement of anti-donor CD4+ T cell response. The depletion of the CD5+ B cell subset, which exclusively included IL-10-positive cells, from the additive B cells abrogated such inhibitory effects. These findings demonstrate that IL-10+ CD5+ B cells suppress the excessive response of anti-donor CD4+ T cells responses in sensitized recipients.