Perforin Release Research Articles

Abstract 1389: In vitro killing assays for evaluation of immuno-oncology drug candidates

Abstract The need to better understand the tumor microenvironment (TME) dictates the characterization of the cell types involved, the roles they play and how they respond to treatment. To develop novel cancer immunotherapies, in vitro primary immune cell bioassays offer an early assessment of their effects on the various players of the TME. Cytotoxic cells are key players in the anti-tumor immune response. CD8+ T cells, also known as cytotoxic T lymphocytes, recognize and kill cells presenting antigens bound to MHC class I molecules, such as neoantigens present on the tumor cell surface. Activation of CD8+ T cells requires at least two signals from antigen presenting cells: binding of their T cell receptor (TCR) to the appropriate peptide presented on MHC I and co-stimulation by B7 binding to CD28. However, the TME possesses several mechanisms to reduce immune cell activity, such as the expression of checkpoint inhibitors dampening the immune system. Natural killer (NK) cells represent another type of cytotoxic cells, with their innate immune activity dictating an important role in anti-tumor immunity. NK cells induce apoptosis via the release of perforin and granzymes from granules, eliminating cells lacking expression MHC I, including cancer cells that have lost expression of MHC I. However, also the activity of NK cells can be hampered in the TME, notably by the PD1/PDL1 interaction.Increasing the activity of CD8+ T and NK cells as a therapeutic anti-tumor strategy has become an important point of attention. Such strategies can have several shapes, with Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of IgG1 isotypes being one of the mechanisms that can be exploited by immunoglobulin-based therapeutics. Another way to increase the killing of tumor cells is to re-direct T cells to tumor cells by employing bi-specific antibodies targeting specific tumor associated antigens. Using this strategy, T cell activation can be concentrated in the TME, thereby also avoiding off-tumor activity. Real-time in vitro assessment of the potential enhanced killing capacity of CD8+ T and NK cells elicited by test molecules could further provide pivotal information on the functional dynamics of test molecules. Using high quality primary human immune cells, in vitro bioassays were developed with improved robustness and reproducibility to screen for the potential to enhance tumor cell killing by novel therapeutics. The ability to evaluate the compounds capacity to increase or induce a cytotoxic activity profile and thereby facilitate the anti-tumor immune response is essential in the early drug development process. Citation Format: Johan Arnold, Thibaut J. Janss, Simon Lefevre, Martijn Vlaming, Ellen Boelen, Sofie Pattijn. In vitro killing assays for evaluation of immuno-oncology drug candidates [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 1389.

A Sampling of Highlights from the Literature

What We're Reading| June 03 2022 A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors Author & Article Information Online Issn: 2326-6074 Print Issn: 2326-6066 ©2022 American Association for Cancer Research2022American Association for Cancer Research Cancer Immunol Res (2022) 10 (6): 671. https://doi.org/10.1158/2326-6066.CIR-10-6-WWR Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record June 3 2022 Citation A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors. Cancer Immunol Res 1 June 2022; 10 (6): 671. https://doi.org/10.1158/2326-6066.CIR-10-6-WWR Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Perforin pores can be repaired in cancer cells by ESCRT proteins (from Fig. 8 of Rudd-Schmidt et al. Nature Communications 2019) CTLs can destroy cancer cells through polarized release of perforins, which puncture the cancer cell's plasma membrane, and granzymes, which enter through the holes created by the perforins, initiating apoptosis. Using multiple high-resolution imaging techniques to visualize the cancer cell–CTL synapse over time, Ritter et al. find that endosomal sorting complexes required for transport (ESCRT) proteins are recruited within cancer cells to the synapse upon CTL release of perforin. Cancer-cell susceptibility to CTL-mediated killing is increased if ESCRT function in the cancer cells is blocked. The data identify ESCRT-mediated repair of perforin-induced pores as a potential mechanism by which cancer cells resist CTL-mediated killing. Ritter AT, …, Mellman I. Science 2022 April 21;376:377–382. IFNγ signaling increases CAR T-cell cytotoxicity against solid tumors (from LeCornichon via Wikimedia Commons) Chimeric-antigen... You do not currently have access to this content.

A phase 1/2 dose escalation/expansion study of OR2805 alone or in combination in subjects with advanced solid tumors.

TPS2693 Background: M2-like macrophages are thought to promote an immunoinhibitory environment and prevent activation and proliferation of T cells, leading to resistance to checkpoint inhibitor (CPI) therapy. OR2805 is a fully human monoclonal antibody identified in a patient who had an exceptional response to CPI. OR2805 recognizes CD163, an immunosuppressive scavenger receptor highly expressed on M2 tumor-associated macrophages (TAMs). OR2805 treatment reprograms M2 macrophages to take on an immunostimulatory, M1-like phenotype. In macrophage/T cell coculture assays, OR2805 treatment relieved the suppressive effect of M2c macrophages as demonstrated by increased T-cell proliferation and stimulation of IFN-γ and perforin release. OR2805 restored the IFN-γ production of exhausted T cells and showed a synergistic effect on cocultures treated in combination with CPI. OR2805 monotherapy demonstrated significant anti-tumor activity in lung cancer xenograft models in NSG-SGM3 mice. Relieving the immune suppression of CD163-expressing TAMs by OR2805 to improve anti-tumor T-cell responses will be evaluated alone and in combination with CPI therapy. Methods: This phase 1/2, open-label, multicenter, dose-escalation/expansion study is assessing the safety, tolerability and preliminary activity of OR2805 alone and in combination with anti-PD-1 or chemotherapy in up to 325 adults (≥18 years) with histologically or cytologically confirmed metastatic or locally advanced solid tumors. The study is divided into 3 parts. Part A will enroll subjects with advanced solid tumors of any histology to describe the preliminary safety profile of OR2805 and determine the recommended phase 2 dose (RP2D) of OR2805 alone and in combination with an anti-PD-1 monoclonal antibody. Part B will further characterize the safety and preliminary antitumor activity of OR2805 either alone or in combination with an anti-PD-1 or chemotherapy in previously treated non-small cell lung cancer (NSCLC), melanoma and liposarcoma, as well as in previously untreated NSCLC in combination with an anti-PD-1 antibody. Part C will evaluate OR2805 monotherapy in relapsed squamous cell carcinoma of the head and neck and other tumor types with an emphasis on correlative biomarker analysis. OR2805 will be infused on Day 1 of 21-day cycles. Primary endpoints are adverse events, laboratory abnormalities, dose-limiting toxicities, and dose-level safety and activity. Secondary endpoints are objective response rates, duration of response, complete response, progression-free survival, overall survival, PK, and antidrug antibodies. Exploratory biomarkers of OR2805-mediated pharmacodynamic effects and immune stimulation will be explored. The study was opened September 2021 and is enrolling at sites in the United States with planned expansion within the US and to Europe. Clinical trial information: Clinical trial information: NCT05094804.

ESCRT-mediated membrane repair protects tumor-derived cells against T cell attack.

Cytotoxic T lymphocytes (CTLs) and natural killer cells kill virus-infected and tumor cells through the polarized release of perforin and granzymes. Perforin is a pore-forming toxin that creates a lesion in the plasma membrane of the target cell through which granzymes enter the cytosol and initiate apoptosis. Endosomal sorting complexes required for transport (ESCRT) proteins are involved in the repair of small membrane wounds. We found that ESCRT proteins were precisely recruited in target cells to sites of CTL engagement immediately after perforin release. Inhibition of ESCRT machinery in cancer-derived cells enhanced their susceptibility to CTL-mediated killing. Thus, repair of perforin pores by ESCRT machinery limits granzyme entry into the cytosol, potentially enabling target cells to resist cytolytic attack.

Impact of adaptive natural killer cells, KLRC2 genotype and cytomegalovirus reactivation on late mortality in patients with severe COVID‐19 lung disease

ObjectiveSARS‐CoV‐2 infection results in severe lung disease in up to 50% of hospitalised patients. The aetiopathogenesis in a subset of such patients, who continue to have progressive pulmonary disease following virus clearance, remains unexplored.MethodsWe investigated the role of NKG2C+/NKG2A− adaptive natural killer (ANK) cells, KLRC2 genotype and cytomegalovirus (CMV) reactivation in 22 such patients.ResultsThe median duration of virus positivity was 23 days, and the median duration of hospitalisation was 48 days. The overall survival at 60 days in this group was 50%. Older age and comorbidities impacted survival negatively. CMV viraemia was documented in 11 patients, with a survival of 25% vs 80% in those without viraemia with viral load correlating with mortality. Both NK and T cells were markedly depressed in all patients at day 15. However, only persistently low ANK cells at 30 days along with an inversely high NKG2C−/NKG2A+ inhibitory NK cells significantly correlated with high CMV viraemia and mortality, irrespective of KLRC2 genotype. However, day 30 ANK cells were significantly lower in the KLRC2 deletion group. The release of IFN‐γ and perforin was severely compromised in all patients at day +15, with significant improvement in the survivors at day +30, but not in those with adverse outcome.ConclusionPatients with progressive lung disease even after negative SARS‐CoV‐2 status, with persistently reduced and functionally compromised ANK cells, are more likely to have CMV reactivation and an adverse outcome, independent of KLRC2 genotype.

Progesterone induced blocking factor in health and disease

The foetus expressing paternal antigens ought to be “rejected” by the maternal immune system. However, the immunological relationship of the mother and the foetus does not follow the rules of transplantation immunology. Maternal immune functions are re-adjusted during pregnancy, to create a tolerant environment for the developing foetus. Progesterone and its downstream mediator; the progesterone induced blocking factor (PIBF) are important in this process. The mRNA transcribed from the PIBF1 gene contains 18 exons, and codes for a 90 kDa protein. The 90 kDa form is associated with the centrosome and plays a role in cell cycle regulation, while smaller isoforms produced by alternative spicing are secreted, and bind to the glycosylphosphatidylinositol (GPI) anchored PIBF receptor. Upon ligation, the former forms a heterodimer with the alpha chain of the interleukin-4 (IL-4) receptor and activates the Janus kinase/signal transducers and activators of transcription (Jak/STAT) pathway, via which, PIBF induces increased production of T helper2 (Th2) cytokines. PIBF regulates natural killer (NK) cytotoxicity, by inhibiting perforin release from the cytoplasmic granules of NK cells. During normal human pregnancy, the serum concentrations of PIBF increase with gestational age, and lower than normal serum levels predict spontaneous pregnancy termination. Depletion of PIBF during the peri-implantation period in mice, results in lower implantation and increased resorption rates, together with increased decidual and peripheral NK activity, downregulation of the genes implicated in T cell activation in CD4+ cells, and Th1 differentiation of the T cells. PIBF is expressed in rapidly proliferating immature cells as well as several tumours, and regulates invasion. The PIBF gene has been identified in the chromosomal region 13q21-q22—which is a common site for somatic deletions in a variety of malignant tumours. These data suggest that PIBF might be involved in tumorigenesis.

Abstract 3199: In vitro killing assays for immuno oncology candidates

Abstract The need to better understand the tumor microenvironment (TME) dictates the characterization of the cell types involved, the roles they play and how they respond to treatment. To develop better cancer immunotherapies, in vitro primary immune cell bioassays offer an early assessment of their effects on the various players of the TME. Cytotoxic cells are key players in the anti-tumor immune response. CD8+ T cells, also known as cytotoxic T lymphocytes, recognize and kill cells presenting antigens bound to MHC class I molecules, such as neoantigen present on the tumor cells. Activation of CD8+ T cells requires at least two signals from antigen presenting cells: binding of their T cell receptor (TCR) to the appropriate peptide presented on MHC I and co-stimulation by B7 binding to CD28. However, tumor environment developed different mechanisms to reduce this immune cell killing such as checkpoint inhibitors expression. In that case, the immune system will be dampened. A second type of cytotoxic cells, named Natural killer cells, are an innate immune cell type that plays an important role in anti-cancer immunity. NK cells target cells lacking MHC I expression, including cancer cells that have lost expression of MHC I. NK cells induce apoptosis in cancer cells via release of perforin and granzymes from granules, like CD8+ T cells. But here too, NK cells can be broken down by the tumor cells, notably by the PD1/PDL1 interaction. Increasing their efficacy as a therapeutic strategy has made the development of new therapeutics enhancing their anti-tumor response a priority. Those new therapeutics can have several shapes. The Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the IgG1 isotypes is one of the mechanisms that can be applied by immunoglobulin-based therapeutics. Another way to increase the killing of cancer cells is to target tumor antigens expressed by those cells using for example bispecific molecules that will enhance on the other side the T cell activation. Using primary immune cells, in vitro bioassays were developed to better screen the potential effect of new therapeutics on immune cell killing activity. Their ability to increase or induce a cytotoxic activity and facilitate the anti-tumor immune response can as a result be assessed early in the drug development process. Citation Format: Thibaut J. Janss, Juliette Lamy, Johan Arnold, Ellen Boelen, Sofie Pattijn. In vitro killing assays for immuno oncology candidates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3199.

Combinatorial immunotherapy of N-803 (IL-15 superagonist) and dinutuximab with ex vivo expanded natural killer cells significantly enhances in vitro cytotoxicity against GD2+ pediatric solid tumors and in vivo survival of xenografted immunodeficient NSG mice

BackgroundChildren with recurrent and/or metastatic osteosarcoma (OS), neuroblastoma (NB) and glioblastoma multiforme (GBM) have a dismal event-free survival (<25%). The majority of these solid tumors highly express GD2. Dinutuximab, an...

CF33-hNIS-antiPDL1 virus primes pancreatic ductal adenocarcinoma for enhanced anti-PD-L1 therapy.

Immunotherapeutic strategies that combine oncolytic virus (OV) and immune checkpoint inhibitors have the potential to overcome treatment resistance in pancreatic ductal adenocarcinoma (PDAC), one of the least immunogenic solid tumors. Oncolytic viral chimera, CF33-hNIS-antiPDL1 genetically modified to express anti-human PD-L1 antibody and CF33-hNIS-Δ without the anti-PD-L1 gene, were used to investigate the immunogenic effects of OVs and virus-delivered anti-PD-L1 in PDAC in vitro. Western blot, flow cytometry, and immunofluorescence microscopy were used to evaluate the effects of CF33-hNIS-Δ and IFNγ on PD-L1 upregulation in AsPC-1 and BxPC-3 cells, and CF33-hNIS-antiPDL1 production of anti-PD-L1 and surface PD-L1 blockade of AsPC-1 and BxPC-3 with or without cocultured activated T cells. The cytosolic and cell surface levels of PD-L1 in PDAC cell lines varied; only BxPC-3 showed high cell surface expression. Treatment of these cells with CF33-hNIS-Δ and IFNγ significantly upregulated PD-L1 expression and translocation of PD-L1 from the cytosol onto the cell surface. Following coculture of activated T cells and BxPC-3 with CF33-hNIS-antiPDL1, the cell surface PD-L1 blockade on BxPC-3 cells by virus-delivered anti-PD-L1 antibody increased granzyme B release and prevented virus-induced decrease of perforin release from activated CD8+ T cells. Our results suggest that CF33-IOVs can prime immune checkpoint inhibition of PDAC and enhance antitumor immune killing.

Acute lymphoblastic leukemia-derived exosome inhibits cytotoxicity of natural killer cells by TGF-β signaling pathway.

This study was conducted to explore whether acute lymphoblastic leukemia (ALL)-derived exosomes affect natural killer (NK) cells. Exosomes were isolated and identified from Jurkat cells and co-cultured with NK cells. Then, the cytotoxicity, viability, and release of perforin and granzyme B in NK92-MI cells were measured. PCR arrays were used to detect gene expression alterations in the transforming growth factor (TGF)-β pathway of NK92-MI cells treated or not treated with exosomes. The morphology and size of the exosomes isolated from Jurkat cells showed typical characteristics of exosomes, and the expression of cluster of differentiation 63 was detected. Jurkat-derived exosomes were internalized by NK92-MI cells, further inhibiting the proliferation and cytotoxicity of NK92-MI cells. An enzyme-linked immunosorbent assay revealed that the release of perforin and granzyme B from NK92-MI cells decreased after co-culture with exosomes. Similarly, western blot and immunofluorescence staining verified that Jurkat-derived exosomes inhibited the expression of granzyme B and perforin. Furthermore, Jurkat-derived exosomes enhanced the signaling of the TGF-β pathway in NK92-MI cells via the MDS1 and EVI1 complex loci and homeodomain interacting protein kinase 2. In conclusion, we found that ALL-derived exosomes inhibit the biological function of NK cells and provide support for the immunotherapy of ALL.

Innate immune cells in the tumor microenvironment.

The tumor immune microenvironment (TIME) is a complex ecosystem that contains adaptive and innate immune cells that have tumor-promoting and anti-tumor effects. There is still much to learn about the diversity, plasticity, and functions of innate immune cells in the TIME and their roles in determining the response to immunotherapies. Experts discuss recent advances in our understanding of their biology in cancer as well as outstanding questions and potential therapeutic avenues.

Bone marrow-derived mesenchymal stem cells inhibit CD8+ T cell immune responses via PD-1/PD-L1 pathway in multiple myeloma.

High expression of the inhibitory receptor programmed cell death ligand 1 (PD-L1) on tumor cells and tumor stromal cells have been found to play a key role in tumor immune evasion in several human malignancies. However, the expression of PD-L1 on bone marrow mesenchymal stem cells (BMSCs) and whether the programmed cell death 1 (PD-1)/PD-L1 signal pathway is involved in the BMSCs versus T cell immune response in multiple myeloma (MM) remains poorly defined. In this study, we explored the expression of PD-L1 on BMSCs from newly diagnosed MM (NDMM) patients and the role of PD-1/PD-L1 pathway in BMSC-mediated regulation of CD8+ T cells. The data showed that the expression of PD-L1 on BMSCs in NDMM patients was significantly increased compared to that in normal controls (NC) (18·81±1·61 versus 2·78±0·70%; P<0·001). Furthermore, the PD-1 expression on CD8+ T cells with NDMM patients was significantly higher than that in normal controls (43·22±2·98 versus 20·71±1·08%; P<0·001). However, there was no significant difference in PD-1 expression of CD4+ T cells and natural killer (NK) cells between the NDMM and NC groups. Additionally, the co-culture assays revealed that BMSCs significantly suppressed CD8+ T cell function. However, the PD-L1 inhibitor effectively reversed BMSC-mediated suppression in CD8+ T cells. We also found that the combination of PD-L1 inhibitor and pomalidomide can further enhance the killing effect of CD8+ T cells on MM cells. In summary, our findings demonstrated that BMSCs in patients with MM may induce apoptosis of CD8+ T cells through the PD-1/PD-L1 axis and inhibit the release of perforin and granzyme B from CD8+ T cells to promote the immune escape of MM.

Inflammation targets CDK11 in autoimmune diabetes as a protective mechanism against beta cell apoptosis.

Abstract Autoimmune diabetes (T1D) is caused by the immune-mediated destruction of insulin-producing beta cells in the pancreas leading to hyperglycemia. Apoptosis is the main mechanism responsible for beta cell demise, including Fas-receptor engagement and Perforin release as essential death executers. However, the chronic inflammatory milieu pervading pancreatic islets prior to diabetes onset, alters beta cells function and primes beta cells for apoptosis long before the debut of the disease. To identify those genes that are targeted by inflammation and are causally related to beta cell fitness and viability we have used the microarray technology. We have identified several candidate genes being downregulated by inflammation, one of this genes is Cdk11, a cyclin-dependent kinase which is involved in transcription (CDK11p130), mitosis and apoptosis (CDK11p58). L-type cyclins are the partners for Cdk11p130, and Cyclin D3 is the partner of CDK11p58. The Cdk11 gene, when is transcribed, generates a single mRNA form, that can be translated either into CDK11p130 or CDK11p58 by a differential IRES (Internal Ribosome Entry Site) usage, being CDK11p58 only expressed during mitosis, while CDK11p130is expressed during all cell cycle phases. We have addressed the role of CDK11 in autoimmune diabetes using the T1D-prone NOD mouse model hemideficient in CDK11, since the CDK11 null mutation is embryonically lethal. First of all, we have confirmed the effect of inflammation on Cdk11 mRNA expression in islet cells by submitting NODSCID mice to adoptive transfer of increasing amounts of diabetogenic NOD splenocytes, and, observed that Cdk11 expression in islet endocrine cells is downregulated by inflammation in a dose-response dependent manner.

A Deadly Cargo: Gene Repertoire of Cytotoxic Effector Proteins in the Camelidae.

Cytotoxic T cells and natural killer cells can kill target cells based on their expression and release of perforin, granulysin, and granzymes. Genes encoding these molecules have been only poorly annotated in camelids. Based on bioinformatic analyses of genomic resources, sequences corresponding to perforin, granulysin, and granzymes were identified in genomes of camelids and related ungulate species, and annotation of the corresponding genes was performed. A phylogenetic tree was constructed to study evolutionary relationships between the species analyzed. Re-sequencing of all genes in a panel of 10 dromedaries and 10 domestic Bactrian camels allowed analyzing their individual genetic polymorphisms. The data showed that all extant Old World camelids possess functional genes for two pore-forming proteins (PRF1, GNLY) and six granzymes (GZMA, GZMB, GZMH, GZMK, GZMM, and GZMO). All these genes were represented as single copies in the genome except the GZMH gene exhibiting interspecific differences in the number of loci. High protein sequence similarities with other camelid and ungulate species were observed for GZMK and GZMM. The protein variability in dromedaries and Bactrian camels was rather low, except for GNLY and chymotrypsin-like granzymes (GZMB, GZMH).

Optimizing Ex-Vivo Expanded NK Cell- Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) Combined with NKTR-255 in Chronic Lymphocytic Leukemia (CLL), Follicular Lymphoma (FL), and Burkitt Lymphoma (BL)

Background: The CD20 molecule is universally expressed by normal B cells in all stages of development, from the pre-B cell up to the mature plasma cell as well as by most B cell malignancies including CLL, FL and BL (Chu/Cairo, BJH, 2016). Rituximab, a monoclonal chimeric anti-CD20 antibody, has been widely used as a chemoimmunotherapeutic regimen in the frontline therapy for patients with CD20+ BL and diffuse large B-cell lymphoma. The addition of rituximab to the CHOP backbone or to standard FAB/LMB therapy has greatly improved outcomes without significantly increasing toxicity in patients with B-NHL (Goldman/Cairo, Leukemia, 2013, Coiffier et al, NEJM, 2002). However, patients who relapse have a poor clinical response to rituximab retreatment. Obinutuzumab is a humanized, type II anti-CD20 monoclonal antibody glycoengineered to enhance Fc receptor affinity. It has lower complement-dependent cytotoxicity than rituximab but greater ADCC, phagocytosis and direct B-cell killing effects (Chu/Cairo, BJH, 2018). Obinutuzumab has been successfully utilized in front-line therapy in FLL (Marcus, et al, NEJM, 2017) and CLL (Goede, et al, NEJM, 2014; Moreno, et al, Lancet, 2019). Our group has successfully expanded functional and active peripheral blood NK cells PBNKwith irradiated feeder cells to target B-NHL (Chu/Cairo, et al, Can Imm Res 2015). We previously demonstrated that obinutuzumab has significantly enhanced expanded PBNK mediated cytotoxicity against BL and pre-B-ALL cell lines compared to rituximab (Tiwari/Cairo et al, BJH, 2015). NKTR-255 is an IL-15 receptor agonist designed to activate the IL-15 pathway and expand natural killer (NK) cells and promote the survival and expansion of memory CD8+ T cells without inducing suppressive regulatory T cells (Kuo/Zalevsky, Cancer Res. 2017). NKTR-255 stimulates proliferation and survival of NK, CD8+ T cells, and enhances long-term immunological memory which may lead to sustained anti-tumor immune response. Objective: To investigate the effects of NKTR-255 on the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL. Methods: NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells as previously described (Denman/Dean Lee, PLoS One, 2012). Expanded PBNK cells were isolated using Miltenyi NK cell isolation kit. NKTR-255 was generously provided by Nektar Therapeutics. In vitro cytotoxicity was examined using luminescence reporter-based assays. IFNg, granzyme B and perforin levels were examined by standard enzyme-linked immunosorbent assays as we previously described (Chu/Cairo, ASH, 2018). MEC-1 (CLL), PGA-1 (CLL), DOHH2 (FL) and Rituximab-resistant BL cells Raji-2R and Raji-4RH were used as target cells. Results: NKTR-255 significantly enhanced the in vitro cytotoxicity of expanded NK cells when combined with rituximab against MEC-1 (E:T=3:1, p&amp;lt;0.001), PGA-1 (E:T=3:1, p&amp;lt;0.001), and DOHH2 (E:T=3:1, p&amp;lt;0.001) as compared to the control groups (Fig.1A). NKTR-255 also significantly enhanced granzyme and perforin release from expanded NK cells when combined with rituximab against MEC-1 (granzyme: p&amp;lt;0.05; perforin: p&amp;lt;0.001), PGA-1(granzyme: p&amp;lt;0.05; perforin: p&amp;lt;0.05), DOHH2 (granzyme: p&amp;lt;0.05; perforin: p&amp;lt;0.001) as compared to controls. NKTR-255 significantly enhanced the in vitro cytoxicity of expanded NK cells when combined with obinutuzumab agains rituximab-resistant BL cells like Raji-2R (E:T=3:1, p &amp;lt;0.01), and Raji-4RH (E:T=3:1, p&amp;lt;0.01) as compared to the control groups (Fig.1B). NKTR-255 also significantly enhanced IFN-g, granzyme and perforin release from expanded NK cells when combined with obinutuzumab against Raji-2R (E:T=3:1, IFN-g: p&amp;lt;0.001, granzyme: p&amp;lt;0.001 and perforin: p&amp;lt;0.001) and Raji-4RH (E:T=3:1, IFN-g: p&amp;lt;0.001, granzyme: p&amp;lt;0.01 and perforin: p&amp;lt;0.01) as compared to controls. Conclusion: We found that NKTR-255 significantly enhanced the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells in vitro with enhanced IFN-g, granzyme B and perforin release. The in vivo effects of NKTR-255 with expanded NK cells and anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells using humanized NSG models are under investigation. Disclosures Lee: Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties. Madakamutil:Nektar Therapeutics: Current Employment. Marcondes:Nektar Therapeutics: Current Employment. Klein:Roche: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Cairo:Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding.

Bone Marrow-Derived Mesenchymal Stem Cells Inhibit CD8+ t Cell Immune Responses Via PD-1/PD-L1 Pathway in Multiple Myeloma

High expression of the inhibitory receptor programmed death ligand 1 (PD-L1) on tumor cells and tumor stromal cells have been found play a key role in tumor immune evasion in several human malignancies. However, the expression of PD-L1 on bone marrow mesenchymal stem cells (BMSCs) and whether the PD-1/PD-L1 signal pathway is involved in the BMSCs versus T cell immune response in Multiple Myeloma (MM) remain poorly defined. In this study, we explored the expression of PD-L1 on BMSCs from newly diagnosed MM (NDMM) patients and the role of PD-1/PD-L1 pathway in BMSCs-mediated regulation of CD8+T cells. The data showed that the expression of PD-L1 on BMSCs in NDMM patients was significantly increased than that in normal controls (NC) (18.81±1.61% vs. 2.78±0.70 %; P&amp;lt;0.001). Furthermore, the PD-1 expression on CD8+T cells with NDMM patients was significantly higher than that in normal controls (43.22±2.98% vs. 20.71±1.08%; P&amp;lt;0.001). However, there was no significant difference in PD-1 expression of CD4+ T cells and NK cells between NDMM group and NC group. Additionally, the co-culture assays revealed that BMSCs significantly promoted CD8+ T cells apoptosis and suppressed CD8+ T cells function. However, PD-L1 inhibitor effectively reversed BMSCs-mediated suppression in CD8+ T cells. We also found that the combination of PD-L1 inhibitor and pomalidomide can further enhance the killing effect of CD8+ T cells on MM cells. In summary, our findings demonstrated that BMSCs in patients with MM may induce apoptosis of CD8+T cells through the PD-1/PD-L1 axis and inhibit the release of perforin and granzyme B from CD8+ T cells so as to promote the immune escape of MM. Disclosures No relevant conflicts of interest to declare.

A Phase I Trial of Incorporating Natural Killer (K-NK) Cells for Patients with Chronic Myeloid Leukemia (CML) and Molecular Residual Disease after Tyrosine Kinase Inhibitor (TKI) Therapy

Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of patients with chronic myeloid leukemia (CML) ushering in an era where, in select patient populations, treatment planning goals have shifted towards the achievement of treatment free remission (TFR) after TKI cessation. Both duration and depth of response to TKI therapy are predictors of future success in achieving a lasting TFR and with improved outcomes independent of TKI cessation. Unfortunately, molecular residual disease (MRD) persists in many patients despite optimal therapy and predicts for worse outcomes over time and decreased ability to maintain a TFR after TKI cessation. Achievement of a major molecular response (MMR) and probability of TFR have been associated with increased numbers of NK cells, particularly mature cytolytic NK cells. Kiadis K-NK003 cells are off-the-shelf NK cells from a universal donor expanded using PM21, proprietary membrane particles modified to express membrane bound IL-21 and 4-1bb ligand. The resulting expanded K-NK003 cells have a hyperfunctional phenotype that are simultaneously highly cytotoxic with high release of perforin and Granzyme B, and potent producers of the cytokines IFN-γ, TNF-α and IL-2. This is an open label, non-randomized, prospective phase I pilot study designed to evaluate safety and to examine whether the addition of K-NK003 to ongoing TKI therapy for CML patients with persistent MRD will allow patients to achieve MRD negative status. Patients will be treated with K-NK003 on day 1 of each 14 day cycle, for a total of 6 cycles, in conjunction with their ongoing TKI therapy. The primary endpoint is safety. The efficacy objective is to estimate the rate of optimal molecular responses (negative to at least MR4.5). Secondary and exploratory endpoints include the proportion of patients with a reduction in BCR-ABL transcripts and NK cell number and function. Adult patients with chronic phase CML who have been on TKI therapy for at least 1 year prior to enrollment in the study will be eligible. Patients must have been on their most recent TKI consistently for at least 6 months prior to enrollment on study and must be expected to remain on current TKI for the duration of the study. Patients with current accelerated or blast crisis phase disease will be excluded. Disclosures Rein: Celgene: Consultancy; Blueprint Medicine: Consultancy; Novartis: Consultancy; Clinical Care Options: Consultancy, Other: Speaker. Rizzieri:Bayer: Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees; Celltrion: Membership on an entity's Board of Directors or advisory committees; abbvie: Membership on an entity's Board of Directors or advisory committees; Mustang: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Acrobiotech: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite: Honoraria, Speakers Bureau; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Using Expanded Natural Killer Cells as Therapy for Invasive Aspergillosis.

Invasive aspergillosis (IA) is a major opportunistic fungal infection in patients with haematological malignancies. Morbidity and mortality rates are high despite anti-fungal treatment, as the compromised status of immune system prevents the host from responding optimally to conventional therapy. This raises the consideration for immunotherapy as an adjunctive treatment. In this study, we evaluated the utility of expanded human NK cells as treatment against Aspergillus fumigatus infection in vitro and in vivo. The NK cells were expanded and activated by K562 cells genetically modified to express 4-1BB ligand and membrane-bound interleukin-15 (K562-41BBL-mbIL-15) as feeders. The efficacy of these cells was investigated in A. fumigatus killing assays in vitro and as adoptive cellular therapy in vivo. The expanded NK cells possessed potent killing activity at low effector-to-target ratio of 2:1. Fungicidal activity was morphotypal-dependent and most efficacious against A. fumigatus conidia. Fungicidal activity was mediated by dectin-1 receptors on the expanded NK cells leading to augmented release of perforin, resulting in enhanced direct cytolysis. In an immunocompromised mice pulmonary aspergillosis model, we showed that NK cell treatment significantly reduced fungal burden, hence demonstrating the translational potential of expanded NK cells as adjunctive therapy against IA in immunocompromised patients.

Liposomal Delivery of Mitoxantrone and a Cholesteryl Indoximod Prodrug Provides Effective Chemo-immunotherapy in Multiple Solid Tumors.

We developed a custom-designed liposome carrier for codelivery of a potent immunogenic cell death (ICD) stimulus plus an inhibitor of the indoleamine 2,3-dioxygenase (IDO-1) pathway to establish a chemo-immunotherapy approach for solid tumors in syngeneic mice. The carrier was constructed by remote import of the anthraquinone chemotherapeutic agent, mitoxantrone (MTO), into the liposomes, which were further endowed with a cholesterol-conjugated indoximod (IND) prodrug in the lipid bilayer. For proof-of-principle testing, we used IV injection of the MTO/IND liposome in a CT26 colon cancer model to demonstrate the generation of a robust immune response, characterized by the appearance of ICD markers (CRT and HMGB-1) as well as evidence of cytotoxic cancer cell death, mediated by perforin and granzyme B. Noteworthy, the cytotoxic effects involved natural killer (NK) cell, which suggests a different type of ICD response. The immunotherapy response was significantly augmented by codelivery of the IND prodrug, which induced additional CRT expression, reduced number of Foxp3+ Treg, and increased perforin release, in addition to extending animal survival beyond the effect of an MTO-only liposome. The outcome reflects the improved pharmacokinetics of MTO delivery to the cancer site by the carrier. In light of the success in the CT26 model, we also assessed the platform efficacy in further breast cancer (EMT6 and 4T1) and renal cancer (RENCA) models, which overexpress IDO-1. Encapsulated MTO delivery was highly effective for inducing chemo-immunotherapy responses, with NK participation, in all tumor models. Moreover, the growth inhibitory effect of MTO was enhanced by IND codelivery in EMT6 and 4T1 tumors. All considered, our data support the use of encapsulated MTO delivery for chemo-immunotherapy, with the possibility to boost the immune response by codelivery of an IDO-1 pathway inhibitor.

&lt;p&gt;Drugs for Multiple Sclerosis Activate Natural Killer Cells: Do They Protect Against COVID-19 Infection?&lt;/p&gt;

COVID-19 infection caused by the newly discovered coronavirus severe acute respiratory distress syndrome virus-19 (SARS-CoV-2) has become a pandemic issue across the globe. There are currently many investigations taking place to look for specific, safe and potent anti-viral agents. Upon transmission and entry into the human body, SARS-CoV-2 triggers multiple immune players to be involved in the fight against the viral infection. Amongst these immune cells are NK cells that possess robust antiviral activity, and which do not require prior sensitization. However, NK cell count and activity were found to be impaired in COVID-19 patients and hence, could become a potential therapeutic target for COVID-19. Several drugs, including glatiramer acetate (GA), vitamin D3, dimethyl fumarate (DMF), monomethyl fumarate (MMF), natalizumab, ocrelizumab, and IFN-β, among others have been previously described to increase the biological activities of NK cells especially their cytolytic potential as reported by upregulation of CD107a, and the release of perforin and granzymes. In this review, we propose that such drugs could potentially restore NK cell activity allowing individuals to be more protective against COVID-19 infection and its complications.