Multi-Drug Resistant Leukemic Cells Highly Express HLA Class I Molecules and Single-Chain Fv Diabody Specific to HLA-A Overcomes Drug Resistance in These Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2376-2376
Author(s):  
Ali Jalili ◽  
Shuji Ozaki ◽  
Naoki Kimura ◽  
Masahiro Abe ◽  
Toshio Matsumoto
Keyword(s):  
Tumor Cells ◽  
Cell Lines ◽  
Hla Class I ◽  
Resistant Cell ◽  
Class I ◽  
Single Chain ◽  
Expression Levels ◽  
Hla Class I Molecules ◽  
Single Chain Fv ◽  
Mdr 1

Abstract Many tumor cells become resistant to commonly used cytotoxic drugs due to the overexpression of ATP-binding cassette (ABC) transporters. Specifically, p-glycoprotein (MDR-1) is frequently up-regulated in chemotherapy-resistant tumor cells, which is associated with poor prognosis. On the other hand, human leukocyte antigen (HLA) class I molecules are known to be significantly down-regulated in advanced tumor cells to escape from immune surveillance. However, the relationship between MDR-1 expression and HLA expression is not fully understood. Recently, we have developed a recombinant single-chain Fv diabody specific to HLA-A and demonstrated that this agent mediates cell death in HLA-overexpressing lymphoid tumor cells but not in normal cells (Cancer Res2007; 67:1184). Here, we investigated the expression levels of HLA class I in chemo-resistant leukemic cells and evaluated the therapeutic potential of single-chain Fv diabody specific to HLA-A, C3B3-DB (Chugai Pharmaceutical Co. Ltd., Tokyo, Japan). Chemotherapy-resistant cells were established by subculturing of myeloid leukemia cell line HL60 and Burkitt’s lymphoma cell line BL-TH in increasing doses of vincristine (VCR), and named HL60/VCR and BL-TH/VCR, respectively. MDR-1 is strongly expressed in HL60/VCR and BL-TH/VCR cells both at the mRNA and protein levels, but not in the parental cells. Interestingly, expression levels of HLA class I molecules are 8 times higher in HL60/VCR and BL-TH/VCR cells than in the parental cells, suggesting that MDR-1 modulates cell surface expression of HLA by its transporter function. Next, we examined the cytotoxic activity of C3B3-DB on these chemo-resistant cell lines. C3B3-DB induced apoptosis in HL60/VCR and BL-TH/VCR cells and these chemo-resistant cell lines were more sensitive to C3B3-DB than the parent cells. Combination of C3B3-DB with chemotherapeutic agents such as VCR and daunorubicin (DNR) resulted in enhanced cytotoxicity against HL60/VCR and BL-TH/VCR cells. Importantly, pretreatment of these chemo-resistant cell lines with C3B3-DB reduced expression levels of MDR-1 and increased drug retention in these cells as detected by flow cytometry and confocal microscopy. Furthermore, combination of C3B3-DB with VCR significantly blocked the cell cycle at the G2 phase compared with VCR alone. Similar results were obtained with primary acute myeloid leukemia cells from 2 patients, resulting in up-regulation of both HLA class I and MDR-1 molecules at relapse phase compared at diagnosis. These results suggest that C3B3-DB enhances cytotoxicity of chemotherapeutic agents and provides a novel approach for overcoming drug resistance in hematological malignancies.

A Recombinant HLA Class I-Specific Single-Chain Fv Diabody Can Target Cancer Stem Cell-Like Side Population Cells in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2799-2799
Author(s):  
Akishige Ikegame ◽  
Shuji Ozaki ◽  
Daisuke Tsuji ◽  
Takeshi Harada ◽  
Shingen Nakamura ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Side Population ◽  
Hla Class I ◽  
Class I ◽  
Chemotherapeutic Drugs ◽  
Single Chain ◽  
Single Chain Fv ◽  
Rpmi 8226

Abstract Abstract 2799 Poster Board II-775 Multiple myeloma (MM) remains an incurable disease despite high overall response rates induced by combination therapy of chemotherapeutic drugs and new agents such as thalidomide, lenalidomide, and bortezomib. Recently, the existence of cancer stem cells is proposed for several tumors including MM, and such cells are considered as an important target for curative therapy. The side population (SP) cells are identified by their ability to efflux Hoechst 33342 dye, which represent a small fraction with stem cell properties. Our previous studies have demonstrated that MM cells expressed HLA class I at high levels than normal hematopoietic cells, and that antibodies against HLA class I specifically induced MM cell death by Rho-mediated actin aggregation. In this study, we characterized SP fraction in MM cell lines (RPMI 8226, U266, and MM.1S) and primary MM cells (n=3) by flow cytometry, and investigated the efficacy of chemotherapeutic drugs as well as a recombinant single-chain Fv diabody specific to HLA class I (C3B3-DB, Chugai Pharmaceutical Co., Ltd, Tokyo, Japan). MM cell lines and primary MM cells contained a distinct fraction of SP cells ranging from 0.01% to 0.6% of the gated cells, which was confirmed by disappearance after treatment with verapamil. Treatment with melphalan (10 μM, 48 hours) decreased the percentage of non-SP cells (16.7% to 3.9%) but not of SP cells (0.6% to 0.7%) in RPMI 8226. In contrast, treatment with C3B3-DB (1 μg/ml, 48 hours) caused a significant reduction in both non-SP cells (16.7% to 4.0%) and SP cells (0.6% to 0.3%) in RPMI 8226. Similar results were observed in primary MM cells enriched from patient bone marrow cells by negative selection with antibody cocktail. Next, we isolated SP cells and non-SP cells in RPMI 8226 using a cell sorter, and characterized in detail. SP cells exhibited elevated levels of ABCG2 and low levels of CD138 compared with non-SP cells, but HLA class I was expressed at high levels in both SP and non-SP cells by flow cytometry. Annexin/PI assay showed that SP cells were 1.5- and 2.0-fold more resistant to melphalan and bortezomib than non-SP cells. Whereas both SP cells and non-SP cells showed similar sensitivity to C3B3-DB. Methylcellulose colony-forming assay showed that SP cells have a higher potential for colony formation (numbers of colonies, 13.0±1.0) than non-SP cells (1.3±0.6), and the colony formation of SP cells was significantly inhibited by C3B3-DB (0.7±0.6, p<0.01). Notably, RPMI 8226 cells expressed the pluripotency-associated transcription factors including Oct3/4, Sox2, and Nanog as detected by RT-PCR, but only Sox2 mRNA expression was decreased at 6 hours after C3B3-DB treatment. Furthermore, when C3B3-DB-treated SP cells were inoculated subcutaneously in SCID mice (n=4), there was a significant decrease in a tumor volume as compared with untreated SP cells (679±148mm3 vs 3217±562 mm3, p<0.01). SP cell analysis of these tumors showed that the percentage of SP fraction of C3B3-DB-treated SP cell tumors was significantly low (0.01%) compared with that of untreated SP cell tumors (0.33%). These results indicate that C3B3-DB has a potential activity for eradicating MM cancer stem cell-like SP cells, and that the molecular targeting of such drug-resistant cells provides an important strategy for improving the efficacy of current therapies in MM. Disclosures: No relevant conflicts of interest to declare.

844 Immunomodulatory activity of epigenetic drugs combinations in mesothelioma: laying the ground for new immunotherapeutic strategies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A896-A896
Author(s):  
Sara Cannito ◽  
Health Biology ◽  
Ornella Cutaia ◽  
Carolina Fazio ◽  
Maria Fortunata Lofiego ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Hla Class I ◽  
Class I ◽  
Immunomodulatory Activity ◽  
Epigenetic Drugs ◽  
Hla Class I Antigens ◽  
Hla Class I Molecules ◽  
Mesothelioma Cell Line ◽  
Mesothelioma Cell

BackgroundGrowing evidence are demonstrating the therapeutic efficacy of immune checkpoint inhibitors (ICI) in mesothelioma; however, a limited percentage of patients benefits from this therapeutic approach. Epigenetic modifications play a relevant role in negatively regulating the cross-talk between neoplastic and immune cells, and in contributing to the highly immunosuppressive mesothelioma microenvironment. A better understanding of mesothelioma epigenetic landscape could open the path to novel and potentially more effective approaches combining ICI and epigenetic drugs. We investigated the immunomodulatory potential of epigenetic agents by comparing the activity of DNA hypomethylating agents (DHA) with histone deacetylases inhibitors (HDACi) and EZH2 inhibitors (EZH2i), alone or combined with DHA, in mesothelioma cells.MethodsFour mesothelioma cell lines were treated with the DHA guadecitabine 1μM, or with the HDACi, Valproic Acid (VPA) 1mM, or the EZH2i, EPZ-6438 1μM, alone or combined with guadecitabine. We investigated the expression of HLA class I molecules by flow-cytometry and of PD-L1, cancer testis antigens (CTA: NY-ESO, MAGE-A1), Natural Killer Group 2 member D Ligands (NKG2DLs: MIC-A, MIC-B, ULBP2) and EMT-regulating cadherins (CDH1, CDH2) by quantitative Real-Time PCR. Fold change (FC) expression for each treatment vs untreated cells was reported as mean values (FCm) among investigated cell lines. A positive modulation of the expression was considered if FCm>1.5.ResultsGuadecitabine upregulated the expression of HLA class I antigens (FCm=1.75), PD-L1 (FCm=2.38), NKG2DLs (MIC-A FCm=1.96, MIC-B FCm=2.57, and ULBP2 FCm=3.56), and upregulated/induced CTA expression. Similarly, VPA upregulated HLA class I antigens (FCm=1.67), PD-L1 (FCm=3.17), NKG2DLs (MIC-A FCm=1.78, MIC-B FCm=3.04, and ULBP2 FCm=3.75) expression; however, CTA expression was modulated only in 1 mesothelioma cell line. Conversely, EPZ-6438 up-regulated only NY-ESO-1 and MIC-B expression in 1 mesothelioma cell line.The addition of both VPA and EPZ-6483 to guadecitabine strengthened its immunomodulatory activity. Specifically, guadecitabine plus VPA or EPZ-6438 upregulated the expression of HLA class I antigens FCm=2.55 or 2.69, PD-L1 FCm=8.04 or 2.65, MIC-A FCm=3.81 or 2.26, MIC-B FCm=8.00 or 3.03, ULBP2 FCm=6.24 or 4.53, respectively. Higher levels of CTA upregulation/induction were observed with combination treatments vs guadecitabine alone.Cadherins modulation was mesothelioma histotype-related: CDH1 expression was induced in the 2 constitutively-negative sarcomatoid mesothelioma cells by guadecitabine alone or combined with VPA or EPZ-6438; CDH2 expression was upregulated by VPA alone (FCm=1.53) or plus guadecitabine (FCm=2.54).ConclusionsCombination of DHA-based immunotherapies with other classes of epigenetic drugs could be an effective strategy to be pursued in the mesothelioma clinic.

Specific PML Isoforms and SATB1 Affect HLA Expression In Classical Hodgkin Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4234-4234
Author(s):  
Yuxuan Liu ◽  
Anke Van Den Berg ◽  
Bea Rutgers ◽  
Rianne Veenstra ◽  
Debora de Jong ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mrna Level ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Expression Levels ◽  
Hla Genes ◽  
Enhanced Expression ◽  
Positive Correlations

Abstract Introduction Hodgkin Reed-Sternberg (HRS) cells are characterized by a general loss of B cell phenotype, whereas antigen presenting properties are commonly retained. HLA class I and class II are expressed in most EBV+ cHL cases, with an even enhanced expression in some cases. The mechanisms underlying enhanced or lost HLA expression are unknown. Promyelocytic leukemia protein (PML) is the main component of nuclear bodies (NBs) and it organizes the chromatin structure into loops by anchoring matrix attachment regions to the nuclear matrix. The PML transcript can be processed into seven different isoforms. Downregulation of PML isoform III or V results in a reduced HLA-A and HLA-G expression in Jurkat T cells, whereas PML enhances HLA class II expression by upregulating the class II transactivator (CIITA). Special AT-rich region binding protein 1 (SATB1) has been shown to be associated with PML-NBs in the HLA region. Downregulation of SATB1 in Jurkat cells results in an enhanced expression of HLA-A, HLA-G and HLA-H. Aim To determine if specific PML isoforms and SATB1 regulate HLA expression in cHL cell lines. Methods We stained HLA class I, HLA class II, PML-NBs and SATB1 in 54 EBV+ cHL cases and in 27 EBV- cHL cases. We analyzed HLA class I, HLA class II, PML isoforms I to V and SATB1 mRNA expression levels by qRT-PCR in cHL cell lines (n=6). Next, we inhibited SATB1 and PML expression in the cHL cell line L1236 using shRNA constructs. The effects of the shRNA constructs on HLA protein expression levels were assessed by Western blot and flow cytometry. Results We observed a normal HLA class I membrane expression in 40% of EBV+ and 20% of EBV- cases. There was a stronger than normal HLA class I protein expression in approximately 40% of EBV+ cHL and not in EBV- cHL patients. The number of PML-NBs was positively correlated to the level of HLA class I expression (p<0.01), whereas the percentage of SATB1 positive cells was inversely correlated with the level of HLA class I expression in EBV+ cHL cases (p<0.05). We observed normal HLA class II expression in 50% of EBV+ and 50% of EBV- cases. A stronger than normal HLA class II expression was observed in 4 EBV+ and 1 EBV- cHL patients. The number of PML-NBs and the percentage of SATB1 positive cells were not correlated with classical HLA class II expression in EBV+, EBV- or the total cHL patient group. We further investigate the association of PML, SATB1 and HLA class I and II in six cell lines. At the mRNA level, significant positive correlations were found between PML-III and HLA-C and between PML-III and β2M. Borderline significant positive correlations were found between PML-V and CIITA and between PML-V and HLA-DPB1. Suggestive, though not significant, correlations were observed of various PML isoforms and SATB1 for several HLA genes. The limited number of cell lines, precluded more definitive conclusions for the other comparisons. To further study the role of specific PML isoforms and SATB1 in regulation of HLA gene expression, we inhibited PML-III, PML-V or SATB1 in L1236 by shRNA-based knockdown. The PML-III shRNA induced a ∼80% reduction at the PML-III mRNA level, but did not affect membranous HLA class I expression and possibly affected HLA class II expression levels in L1236. The shRNA for PML-V was not effective and needs to be optimized and experiments with a pan-PML shRNA are ongoing. The SATB1 shRNA1 induced a ∼75% at the SATB1 mRNA level and a ∼80% reduction at the protein level, while SATB1 shRNA2 showed a ∼75% reduction and a ∼25% reduction, respectively. Inhibition of SATB1 by shRNA1 significantly upregulated HLA class II expression (P=0.02) and no significant effect on HLA class I expression. Inhibition of SATB1 by shRNA2 showed no significant effect on HLA class I and II expression. Conclusion PML and SATB1 protein levels are associated with HLA class I in HRS cells. Various negative associations of SATB1 with HLA genes at the mRNA level support a possible regulatory effect. This was indeed observed upon inhibition of SATB1 in L1236 that induced enhanced HLA-DR/DP/DQ expression. The PML regulation appears to be complex with different PML isoforms showing different associations with specific HLA genes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals HLA Class I Knockout Converts Allogeneic Primary NK Cells Into Suitable Effectors for “Off-the-Shelf” Immunotherapy

2021 ◽  
Vol 11 ◽  
Author(s):  
Keven Hoerster ◽  
Markus Uhrberg ◽  
Constanze Wiek ◽  
Peter A. Horn ◽  
Helmut Hanenberg ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Genome Editing ◽  
Nk Cell ◽  
Human Cancer ◽  
Hla Class I ◽  
Class I ◽  
Cellular Immunotherapy ◽  
Single Chain ◽  
Hla Class I Molecules

Cellular immunotherapy using chimeric antigen receptors (CARs) so far has almost exclusively used autologous peripheral blood-derived T cells as immune effector cells. However, harvesting sufficient numbers of T cells is often challenging in heavily pre-treated patients with malignancies and perturbed hematopoiesis and perturbed hematopoiesis. Also, such a CAR product will always be specific for the individual patient. In contrast, NK cell infusions can be performed in non-HLA-matched settings due to the absence of alloreactivity of these innate immune cells. Still, the infused NK cells are subject to recognition and rejection by the patient’s immune system, thereby limiting their life-span in vivo and undermining the possibility for multiple infusions. Here, we designed genome editing and advanced lentiviral transduction protocols to render primary human NK cells unsusceptible/resistant to an allogeneic response by the recipient’s CD8+ T cells. After knocking-out surface expression of HLA class I molecules by targeting the B2M gene via CRISPR/Cas9, we also co-expressed a single-chain HLA-E molecule, thereby preventing NK cell fratricide of B2M-knockout (KO) cells via “missing self”-induced lysis. Importantly, these genetically engineered NK cells were functionally indistinguishable from their unmodified counterparts with regard to their phenotype and their natural cytotoxicity towards different AML cell lines. In co-culture assays, B2M-KO NK cells neither induced immune responses of allogeneic T cells nor re-activated allogeneic T cells which had been expanded/primed using irradiated PBMNCs of the respective NK cell donor. Our study demonstrates the feasibility of genome editing in primary allogeneic NK cells to diminish their recognition and killing by mismatched T cells and is an important prerequisite for using non-HLA-matched primary human NK cells as readily available, “off-the-shelf” immune effectors for a variety of immunotherapy indications in human cancer.

Expression of the ULBP Ligands for NKG2D by B-NHL Cells Plays An Important Role in Determining Their Susceptibility to Rituximab-Induced ADCC

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 218-218
Author(s):  
Atsushi Inagaki ◽  
Takashi Ishida ◽  
Hiroki Yano ◽  
Fumiko Mori ◽  
Asahi Ito ◽  
...  
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Mhc Class I ◽  
Cell Lines ◽  
Associated Factors ◽  
Rank Correlation ◽  
Class I ◽  
51Cr Release ◽  
Expression Levels ◽  
Nkg2d Ligands

Abstract Purpose: ADCC is a major antitumor mechanism for the action of therapeutic monoclonal antibodies (mAbs) such as rituximab, trastuzumab and cetuximab. Therefore, a better understanding of ADCC will allow the development of novel, more effective treatment strategies, and may help overcome the resistance which can develop against the effects of the therapeutic mAbs. However, the tumor-associated factors which determine susceptibility to rituximab-induced ADCC have not been identified. In the present study, we focused on this issue, especially focused on the molecules expressed by the tumor cells that interact with NK cells, such as NKG2D ligands, because of the importance of NK cells for rituximab-induced ADCC. The aim of this study was to identify tumor associated factors which determine susceptibility to rituximab-induced ADCC. Experimental Design: 30 different CD20+ non-Hodgkin lymphoma (B-NHL) cell lines were phenotyped for characteristics of cell surface protein: expression levels of CD20, MHC class I, NKG2D ligands (ULBP1-3, MICA, and MICB), CD48 (2B4 ligands), HLA-G, cathepsin B, and complement inhibitors (CD46, CD55, and CD59), and the influence thereof on susceptibility to rituximab-induced ADCC was established. Result: The degree of rituximab-induced ADCC were correlate with the expression levels of CD20 and ULBP1-3, and inversely correlate with the expression levels of MHC class I among 30 different CD20+ B-NHL cell lines. The importance of the ULBPs was confirmed using antibody blockade. In the presence of blocking mAb to ULBP1, 2 or 3, a decrease of rituximab-induced ADCCs against B-NHL cell lines were observed. In addition, the present study clearly identified the key mechanism of rituximab-induced ADCC as antibody-dependent target-specific cytotoxicity mediated by highly activated NK cells. Strong NK cells activation was due to the combination of Fc„dR stimulation via the Fc portion of rituximab, together with stimulation of activating NK cell receptors via their ligands expressed on the tumor cells, particularly ULBPs, which occurred in a robustly synergistic manner. Conclusions: Tumor cell susceptibility to rituximab-induced ADCC was determined by three major tumor-associated factors: the amount of the target molecule, CD20; the amount of the ligands for inhibitory killer Ig-like receptors, MHC class I; and the amounts of some of the NKG2D ligands, especially ULBP1-3. This is the first report to show the importance of ULBPs on tumor cells for rituximab-induced ADCC. The ULBPs could be valuable diagnostic biological makers and significant targets for immunotherapy to improve efficacy not only of rituximab but also of other therapeutic mAbs. Figure. Correlations between the expression of the CD20, MHC class I, and NKG2D ligands ULBP1-3, and rituximab-induced ADCC in B-NHL cell lines. ADCC in the presence of 10μg/mL rituximab against 30 different CD20+ B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of CD20 (upper left panel), MHC class I (upper right panel), ULBP-1 (lower left panel), ULBP-2 (lower middle panel), and ULBP-3 (lower right panel). Each dot plot in each panel represents one cell line. The coefficients and P values assessed by Spearman rank correlation coefficient testing are indicated in each panel. Figure. Correlations between the expression of the CD20, MHC class I, and NKG2D ligands ULBP1-3, and rituximab-induced ADCC in B-NHL cell lines. ADCC in the presence of 10μg/mL rituximab against 30 different CD20+ B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of CD20 (upper left panel), MHC class I (upper right panel), ULBP-1 (lower left panel), ULBP-2 (lower middle panel), and ULBP-3 (lower right panel). Each dot plot in each panel represents one cell line. The coefficients and P values assessed by Spearman rank correlation coefficient testing are indicated in each panel.

Downregulation of HLA Class I Molecules in Primary Oral Squamous Cell Carcinomas and Cell Lines

2009 ◽  
Vol 40 (4) ◽  
pp. 256-263 ◽  
Author(s):  
Qiusha Tang ◽  
Jianqiong Zhang ◽  
Bing Qi ◽  
Chuanlai Shen ◽  
Wei Xie
Keyword(s):  
Cell Lines ◽  
Squamous Cell ◽  
Hla Class I ◽  
Squamous Cell Carcinomas ◽  
Class I ◽  
Oral Squamous Cell Carcinomas ◽  
Hla Class I Molecules

scholarly journals Cross-Presentation Is a Source of Tumor Antigens for Multiple Myeloma Immunotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2104-2104
Author(s):  
Alexander A. Perakis ◽  
Haley L. Peters ◽  
Jason Roszik ◽  
Mao Zhang ◽  
Anna Sergeeva ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Antigen Processing ◽  
Tumor Antigens ◽  
Hla Class I ◽  
Class I ◽  
Cross Presentation ◽  
Hla Class I Molecules

Abstract Introduction Multiple myeloma (MM) remains incurable and accounts for 20% of deaths from hematologic malignancies. Immune modulating drugs and immunotherapy have been shown to be beneficial in MM patients, highlighting the importance of the immune system in MM. The success of these interventions relies on the unique ability of the immune system to specifically distinguish between MM and normal cells. One example involves the use of cytotoxic T-lymphocytes (CTL) that eliminate cancer cells by recognizing tumor specific antigens (TSA) that are presented on human leukocyte antigen (HLA) class I molecules on the tumor cells. TSA are presented on HLA class I via the canonical antigen-processing pathway, which causes the tumor cells to become targets for killing by CTL. On the other hand, exogenous antigens are typically presented on HLA class II molecules. Through cross-presentation, exogenous antigens are taken up and presented on HLA class I molecules. We have recently identified cross-presentation as a mechanism by which tumor cells, including MM, present exogenous antigens, leading to an expanded repertoire of possible tumor antigens that can be targeted. Here we demonstrate that targeting cross-presented antigens is a feasible immunotherapeutic strategy in MM. To this end, we used the anti-PR1/HLA-A2 antibody, 8F4, which effectively eliminates PR1/HLA-A2-expressing tumor cells, and identified novel cross-presented antigens. Experimental design NSG mice were sublethally irradiated with 200 Rad. After 24 hours, 2x106 U266 cells transduced with luciferase/GFP were IV injected. Engraftment was confirmed by measuring human IgE in the mouse serum by ELISA and bioluminescent imaging (BLI). Mice were treated with 8F4 (10 mg/kg), IgG isotype control (10 mg/kg) or PBS. Mice were sacrificed on week 9, bone marrow was harvested and analyzed by flow cytometry. To identify novel cross-presented antigens, polymorphonuclear cells (PMN) were isolated from HLA-A2- healthy donors. PMN were lysed and cultured with U266 cells for 24 hours at a 2:1 PMN:U266 cell ratio. U266 cells were lysed, incubated with anti-HLA-ABC antibody and protein-A/G-sepharose beads. Immunoprecipitated proteins were recovered by mild acid elution and peptides were discovered using tandem mass spectrometry. HLA-A2 specific peptides with strong predicted in silico binding were analyzed using the HLArestrictor 1.2 algorithm. Antigen processing machinery (APM) of primary patient samples were analyzed using western blots. Results 8F4 treated mice showed a 2 fold decrease in the percent of U266 in the bone marrow compared to isotype and untreated mice (P<0.05) (Figure 1). ELISA demonstrated approximately a 3-fold decrease in human IgE ng/mL in serum of mice treated with 8F4 (P<0.001). BLI quantification showed approximately a 0.5 fold decrease in total flux (p/s) in mice treated with 8F4 compared to isotype and untreated mice. In addition to illustrating the therapeutic potential of targeting PR1, we also sought to identify other cross-presented MM peptides. Initially, over 4x103 total peptides were discovered by mass spectrometry (Figure 2). Using our reality score indexing and bioinformatics we identified approximately 5x102 unique, high confidence, cross-presented peptides. Our top candidate peptides have predicted HLA-A2 binding affinities of <18 nM, ion scores >21, retention time >30.42 minutes, and are rank 1 for their spectral matches. Since we have demonstrated the capability of MM to cross-present antigens, we investigated whether the APM was dysregulated in MM patients. Patients show dysregulation in tapasin, LMP2/7, and calnexin by western blot analysis. Conclusion Collectively, our data demonstrate that the MM antigen repertoire is much larger than previously appreciated, and that there is a new catalogue of potential immunotherapeutic targets in MM derived from exogenous antigens. Disclosures Sergeeva: Astellas Pharma: Patents & Royalties. Molldrem:Astellas Pharma: Patents & Royalties.

scholarly journals 835 Harnessing the microbiota to increase response rates to immunotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A876-A876
Author(s):  
Valentina Ferrari ◽  
Alessia Melacarne ◽  
Francesca Algieri ◽  
Maria Rescigno
Keyword(s):  
Flow Cytometry ◽  
Statistical Analysis ◽  
Tumor Cells ◽  
Cell Lines ◽  
Hla Class I ◽  
Surface Expression ◽  
Vehicle Control ◽  
Class I

BackgroundTumor cell clearance by cytotoxic T lymphocytes (CTL) requires expression of relevant antigens on HLA Class I molecules on the surface of tumor cells. Reduced levels of HLA Class I expression is a common method of immune escape, as it hampers tumor-specific CTLs’ ability to detect, recognize, and eliminate tumor cells. Recent data have shown that gut microbiota have a major impact on the clinical response to immune checkpoint inhibitors (ICIs), which could be due to a direct effect on malignant cells. Our hypothesis is that microbiota can influence the immune response by altering HLA Class I expression on tumor cells.MethodsTo investigate the ability of bacteria-based products to upregulate HLA Class I expression, we tested two different proprietary microbial derivatives (MDs) on multiple murine and human tumor primary and immortalized cell lines from various tissues, including: breast, myeloid, melanoma, and colon. We next examined if the change in HLA expression was functional by measuring activation levels and cytotoxic capacity of MART-1-specific CTLs following tumor cell treatment with MDs. Lastly, we administered MDs intra-peritoneally in 4T1-bearing Balb/c mice to sensitize 4T1 tumors to combination treatment with anti-PD-1 ICI.ResultsOur results to date show that in vitro treatment with MDs can upregulate surface HLA, albeit not uniformly across all tumor types, with breast and myeloid tumor cells showing the largest increase across the cell lines tested (figure 1). The MD-dependent HLA increase subsequently boosted CTL recognition of tumor cells without increasing background reactivity. The increased CTL degranulation correlated to the tumor cells’ increased surface HLA expression and was consistent whether the antigen was endogenous (5% increase, p<0.0001, figure 2A) or added exogenously (15%–30% increase, p<0.01 and p<0.0001 figure 2B). In combination with anti-PD-1 in vivo, MD treatment significantly abrogated tumor growth when compared to anti-PD-1 combined with the vehicle control (p<0.0001, figure 3A) and tumors harvested from MD-treated mice expressed higher levels of MHC Class I compared to the vehicle control cohort (p<0.05, figure 3B). Additionally, splenocytes from MD-treated mice showed increased recognition of 4T1 tumor cells when re-challenged in vitro (10% increase in CD8+41BB+ cells, p<0.0001, figure 3C).Abstract 835 Figure 1Class I surface expression after MD treatment. (A) breast (B) colon (C) melanoma and (D) myeloid human cancer cell lines were incubated with 5 (light bars) or 10 (dark bars) mg/mL MD#1, MD#2, and 10 mg/mL respective vehicle control (empty bars). (E) and (F) were treated with 10 mg/mL (dark bars) MD#1, MD#2, or respective vehicle controls (empty bars). After 48 hours, HLA Class I (A-D), H-2kb (E), and H-2kd (F) surface expression was measured by flow cytometry. Experiments repeated at least in duplicate. Statistical analysis by 2-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.Abstract 835 Figure 2Antigen-specific CTL activation. Tumor cells were pre-treated for 48 hours with 10 mg/mL vehicle or MD, then washed and co-cultured for 5 hours with MART-1 specific CTL. A) primary HLA-A2+ melanoma cells that are negative (Mel12) or positive (Mel13) for the MART-1 antigen, and B) Thp1 loaded or not with MART-1 peptide. CD8+CD107a+ cells measured by flow cytometry. Experiments repeated in triplicate, statistical analysis by two-way ANOVA.Abstract 835 Figure 3In vivo treatment with MD. Fifteen 6-week-old Balb/c mice were subcutaneously inoculated with 1.5 × 1054T1 tumor cells and divided into 3 treatment groups on day 3 based on equivalent tumor size. Mice were treated with 250 µg microbial derivatives (MD#1) or vehicle control (vehicle #1) in combination with anti-PD-1 (200 µg; clone 29F.1A12) starting on day 3 and continued every other day for a total of 4 injections (black arrows). (A) Tumor measurements were taken every other day using a caliper and volume calculated using the formula: tumor volume = (length x width2) ÷ 2 (B) 2 × 105 splenocytes were co-cultured 1:1 with 4T1 tumor cells in vitro and T cell activation (percent CD8+41BB+) was measured by flow cytometry. Experiment repeated in duplicate, statistical analysis by 2-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).ConclusionsOur results thus far confirm that our proprietary MDs can increase HLA expression on tumor cells, and that this can lead to increased recognition by antigen-specific CTL both in vitro and in vivo. This suggests that MDs could be explored in combination with ICIs to enhance clinical anti-cancer immune responses.

Sign in / Sign up

Export Citation Format

Share Document