Generation and Characterization of Microvesicles after Daratumumab Interaction with Myeloma Cells

Abstract Daratumumab (DARA) is an anti-CD38 human mAb in phase III clinical trials in myeloma patients. DARA binding induces killing of tumor cells via complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and apoptosis. This work reports on the results obtained while dissecting the events following in vivo CD38 ligation by DARA. Treatment of myeloma cells with DARA + anti-human IgG at 37 °C influenced myeloma cytoskeleton, with redistribution of the CD38 molecules and a formation of distinct polar aggregates visualized by confocal microscopy. DARA effects are different from those observed using 8 different murine anti-human CD38 mAbs, which tended to internalize. The findings observed after DARA ligation were confirmed by exposing myeloma cells to DARA immobilized on CHO cells modified to express 4 distinct human FcRs. CHO cells adhere to plastic and mimics the events that take place in the myeloma niche. First, the interaction between Dara and the different FcRs was determined according to a biosensor-based approach on an IAsys Plus equipment (Affinity Sensors). Results show that DARA possesses the highest avidity for CD64, due to the higher kinetic stability of the complex. Conversely, the differences in the FcR-DARA recognition phase (kass) had only minor effects on final stability of the complex. The results also indicated that NK cells and monocytes are the blood populations with higher kdiss for DARA. The effects observed at 37 °C on myeloma cells in the presence of immobilized DARA were amplified when compared to those with soluble mAb. Results indicate that DARA induces CD38 target to aggregate, polarize and to release microvesicles (MV) from extrusions of the myeloma membrane. MV in culture supernatants and in bone marrow plasma of myeloma patients were characterized for concentration (particles/ml) and size by means of Malvern NanoSight NS300 equipment. DARA treatment was followed by high amounts of MV of different sizes released from myeloma cells. Same experiments repeated using DARA + anti-human IgG labeled with Alexa 488 analyzed by Malvern equipment highlighted the presence of DARA on the surface of MV. The induction of MV may be relevant for in vivo therapy: MV are outward buds of the membrane, which host molecules clustered in microdomains. The presence of CD38 has been confirmed. MV phenotype was analyzed looking for the ectoenzymes that join CD38 in the regulation of adenosine in the myeloma niche. MV phenotype included not only the presence of the expected CD38, but also of CD203a/PC-1, CD39 and CD73, the ectoenzymatic pathway leading to ADO production. A first conclusion is that DARA treatment is followed in vivo by a marked release of MV at the tumor site. The fate of the MV bearing DARA on their surface is multiple: on one side, MV may interact locally with different cells and populations of the niche. Another possibility is that MV are released into the blood stream and interact with cell populations therein. The lipid bilayer of MV consents passive movements even through tissues. MV appear as minicellular signals delivering instructions at a distance from their place of origin. Further, the ectoenzymes analyzed are also involved in cell migration or in interaction with countereceptors (e.g., CD31) expressed by endothelial cells. This issue, was investigated by testing FITC-conjugated DARA on a Laboratory-established human myeloma line. MV-DARA-FITC were then exposed to PBMC preparations obtained from normal donors. The results from a cytofluorimetric analysis highlighted the tendency of the labeled MV to cluster around CD16+ (NK cells) and CD14+ subsets (monocytes). At the moment, it is only possible to conclude that MV are associated with cell membranes, a binding likely mediated by FcRs. Not known yet whether MV interact with other cell types (e.g., macrophages, dendritic cells or lymphocytes). DARA shows a high affinity to FcRs of immune cell types (NK cells, monocytes, B cells). Given clinical data that indicate a robust increase in T cell counts, activation and clonality following DARA treatment should be expected. MV containing DARA and portions of myeloma cell membranes could help drive antigen presentation and T cell response in some patients. This is being investigated further. Disclosures Mark: Bioinvent International: Consultancy, Research Funding. Giuliani:Janssen Pharmaceutica: Research Funding; Celgene Italy: Research Funding. Sasser:Janssen Pharmaceuticals: Employment. Malavasi:Janssen: Honoraria, Research Funding.

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IMMU-08. MICROENVIRONMENT MODULATION BY TIM-3 BLOCKADE IMPROVES THE OUTCOME OF PRECLINICAL DIPG MODELS

Neuro-Oncology ◽

10.1093/neuonc/noab090.116 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i28-i28

Author(s):

Iker Ausejo-Mauleon ◽

Sara Labiano ◽

Virginia Laspidea ◽

Marc Garcia-Moure ◽

Daniel de la Nava ◽

...

Keyword(s):

T Cell ◽

Tumor Microenvironment ◽

Nk Cells ◽

Immune Cell ◽

Cell Types ◽

Pediatric Brain Tumors ◽

Immune Memory ◽

Cd8 Cells ◽

Functional Studies

Abstract Diffuse Midline Gliomas (DMGs), encompassing Diffuse Intrinsic Pontine Gliomas (DIPGs), are the most aggressive pediatric brain tumors. Their meagre survival has not changed despite the combination of radiotherapy with targeted therapies emphasizing the urgent need for effective treatments. Recent research suggested that the DIPG tumor microenvironment is neither highly immunosuppressive nor inflammatory. These analyses showed the lack of infiltrating lymphocytes and the abundance of CD11b+ cells. TIM-3 (HAVCR2) is a member of the T-cell immunoglobulin and mucin domain protein family which is expressed on multiple immune cell types including T cells, Tregs, NK cells, monocytes, dendritic cells and microglia, where it potently regulates not only adaptive immunity but also innate immunity. Therefore, the central hypothesis of this study is that TIM-3 inhibitors could stimulate a cytotoxic immune effect and challenge several components in the tumor microenvironment including microglia, thereby providing a potential effective treatment for DMGs. In silico assessment of TIM-3 expression in a DIPG datasets showed a robust expression of this gene. Moreover, single-cell sequencing analyses of DIPG biopsies uncover its expression on tumor cells, especially in the OPCs compartment. In vivo efficacy studies showed that treatment with anti-TIM-3 antibody significantly increase the overall survival in two DIPG immunocompetent orthotopic animal models (doubling the median), lead to long-term survivors (50%) and showed immune memory. Analyses of CD45+ populations in the tumor microenvironment showed a significant increase in B, NK and CD8+ cells corresponding with a T-cell activate phenotype in treated-mice. The potential therapeutic involvement of NK cells was certified using nude mice and functional studies. Involvement of microglia in currently being analysed. In summary, these data underscore TIM-3 as a potential target DIPGs and uncover the potential involvement of NKs and other immune mechanisms in the efficacy of anti-TIM-3 therapy.

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Cord Blood Derived Natural Killer Cells Loaded with a Tetravalent Bispecific Antibody Construct (AFM13) As Off-the-Shelf Cell Therapy for CD30+ Malignancies

Blood ◽

10.1182/blood-2018-99-114954 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 341-341

Author(s):

Lucila Kerbauy ◽

Mecit Kaplan ◽

Pinaki P Banerjee ◽

Francesca Lorraine Wei Inng Lim ◽

Ana Karen Nunes Cortes ◽

...

Keyword(s):

T Cell ◽

Cord Blood ◽

Nk Cells ◽

Natural Killer ◽

Research Funding ◽

Bispecific Antibody ◽

Nk Cell ◽

Ex Vivo

Abstract Chimeric antigen receptors to redirect T cell specificity against tumor antigens have shown remarkable clinical responses against CD19+ malignancies. However, the manufacture of an engineered autologous T cell product is expensive and cumbersome. Natural killer (NK) cells provide an alternative source of immune effectors for the treatment of cancer. NK cell cytolytic function can be directed towards specific targets by exploiting their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) through the NK cell Fc receptor, CD16 (FcγRIIIa). AFM13 is a tetravalent bispecific antibody construct based on Affimed's ROCK™ platform. AFM13 is bispecific for CD30 and CD16A, designed for the treatment of CD30 expressing malignancies. It binds CD16A on the surface of NK cells, thus activating and recruiting them to CD30 expressing tumor cells and mediating subsequent tumor cell killing. Since autologous NK effector function is impaired in many patients with malignancies, we propose to overcome this by the use of allogeneic NK cells in combination with AFM13. Cord blood (CB) is a readily available ("off-the-shelf") source of allogeneic NK cells that can be expanded to large, highly functional therapeutic doses. The feasibility and safety of therapy with allogeneic ex vivo expanded CB-derived NK cells have been shown by our group and others. In this study, we hypothesized that we can redirect the specificity of NK cells against CD30+ malignancies by preloading ex vivo activated and expanded CB-derived NK cells with AFM13 prior to adoptive infusion. Briefly, mononuclear cells were isolated from fresh or frozen CB units by ficoll density gradient centrifugation. CD56+ NK cells were cultured with rhIL-12, rhIL-18 and rhIL-15 for 16 hrs, followed by ex vivo expansion with rhIL-2 and irradiated (100 Gy) K562-based feeder cells expressing membrane-bound IL-21 and CD137-ligand (2:1 feeder cell:NK ratio). After 14 days, NK cells were loaded with serial dilutions of AFM13 (0.1, 1, 10 and 100 mg/ml). After washing twice with PBS, we tested the effector function of AFM13-loaded NK-cells (AFM13-NK) compared to expanded CB-NK cells without AFM13 against Karpas-299 (CD30 positive) and Daudi (CD30 negative) lymphoma cell lines by 51Cr release and intracellular cytokine production assays. AFM13-NK cells killed Karpas-299 cells more effectively at all effector:target ratios tested than unloaded NK cells (Figure 1) and produced statistically more INFγ and CD107a (P=0.0034; P=0.0031 respectively, n=4). In contrast, AFM13-NK cells and unloaded NK cells exerted similar cytotoxicity against Daudi cells. Next, we established the optimal concentration of AFM13 for loading (determined to be 100 μg/ml) and the optimal incubation time to obtain maximal activity (1 h) in a series of in vitro experiments. We also confirmed that the activity of AFM13-NK cells against Karpas-299 cells remains stable for at least 72h post-wash (Figure 2). Additionally, we characterized the phenotype of AFM13-NK vs. unloaded NK cells by flow cytometry using monoclonal antibodies against 22 markers, including markers of activation, inhibitory receptors, exhaustion markers and transcription factors. Compared to unloaded NK cells, AFM13-NK cells expressed higher levels of CD25, CD69, TRAIL, NKp44, granzyme B and CD57, consistent with an activated phenotype. We next tested the in vivo anti-tumor efficacy of AFM13-NK cells in an immunodeficient mouse model of FFluc-Karpas-299. Briefly, six groups of NOD/SCID/IL2Rγc null mice (n=5 per group) were transplanted by tail-vein injection with 1 x 10e5 FFluc-transduced Karpas cells. Group 1 and 6 received tumor alone or tumor + AFM13 and served as a control. Groups 2-4 receive Karpas FFLuc with either expanded NK cells or AFM13-NK cells (NK cells loaded with AFM13) or expanded NK cells and AFM13 injected separately. Group 5 received AFM13-NK cells without tumor. Initial studies confirm the antitumor activity of AFM13-NK cells. In summary, we have developed a novel premixed product, comprised of expanded CB-NK cells loaded with AFM13 to 'redirect' their specificity against CD30+ malignancies. The encouraging in vitro and in vivo data observed in this study, provide a strong rationale for a clinical trial to test the strategy of an off-the-shelf adoptive immunotherapy with AFM13-loaded CB-NK cells in patients with relapsed/refractory CD30+ malignancies. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

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Activation of T and NK Cells Following Lenalidomide Therapy in Patients with Follicular Lymphoma.

Blood ◽

10.1182/blood.v120.21.2766.2766 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2766-2766

Author(s):

Seema Rawal ◽

Nathan Fowler ◽

Min Zhang ◽

Zhiqiang Wang ◽

Tariq Muzzafar ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Nk Cells ◽

Research Funding ◽

Memory T Cells ◽

T Cell Subsets ◽

Central Memory T Cells

Abstract Abstract 2766 Background: Lenalidomide plus rituximab therapy is a highly effective and well-tolerated therapy in patients (pts) with follicular lymphoma (FL). In a Phase II trial, this combination induced a complete remission rate of 87% in pts with advanced stage untreated FL (Fowler et al, Ann Oncol, 2011; 22; suppl 4:137). A randomized Phase III trial was recently initiated to compare this combination with current standard of care therapies in pts with FL. Although lenalidomide is known to be an immunomodulatory drug with effects on a variety of immune cells in vitro, its effects have not been well studied in vivo in humans. Understanding the in vivo effects of lenalidomide could lead to novel combination strategies to enhance the efficacy and improve clinical outcome in FL and other malignancies. Methods: Pts received lenalidomide 20 mg/day on days 1–21 of each 28-day cycle and rituximab was given at 375 mg/m2on day 1 of each cycle. Peripheral blood mononuclear cells (PBMC) were phenotyped by multiparametric flow cytometry at baseline, on cycle 2 day 15 (C2D15), and at the end of cycle 6. In addition, peripheral blood (PB) samples were collected in PAXgene Blood RNA tubes at baseline and on C2D15 for whole genome gene expression profiling (GEP). Results: Immunophenotyping of baseline and end of cycle 6 PBMC (n=17) showed that the percentages and absolute numbers of CD3+, CD4+, CD8+, TCRgd, and Foxp3+ regulatory T cells; and NK, NKT, and myeloid dendritic cells were not significantly different between the two time points. However, a significant increase in CD4+CD45RO+ (p<0.01) and CD8+CD45RO+ (p=0.04) memory T cells was observed post-therapy. Further characterization of CD4+ T cells showed a significant increase in central memory T cells (p<0.001) and a decrease in naïve (p<0.01) and terminally differentiated (p<0.01) T cells, but no change in effector memory T cells. The increase in CD8+ central memory T cells was marginally significant (p=0.06). Plasmacytoid dendritic cells (PDC) were also significantly increased (p=0.02). In contrast, no such changes in T cell subsets or PDC were observed in FL pts (n=9) treated with 6 cycles of R-CHOP chemotherapy that received equal number of rituximab doses and analyzed at similar time points (baseline and end of cycle 6). To understand lenalidomide-induced changes on a molecular level, we compared GEP data at C2D15 vs. baseline for 7 pairs of PB samples. The paired significance analysis of microarrays method, based on Student's t test, identified 1,748 differentially expressed genes (DEG; 713 up, 1035 down), without a fold-change threshold, in C2D15 samples vs. baseline. Results were influenced by rituximab-induced depletion of B cells in C2D15 samples, but there were many changes that suggested altered PBMC physiology. Noteworthy up-regulated genes (>1.5 fold) included genes associated with T and NK cell activation including BATF, CCR2, CD1B, CD2, CD160, CTLA4, CXCR3, ICOS, and LAG3; and CD163 and CD209, phagocytic receptors expressed on monocytes/macrophages. Down-regulated genes (>1.5 fold) included CXCR5, which mediates B cell migration into follicles; and IL1B and TNFSF13B (BAFF), which are produced by activated macrophages and induce B cell proliferation. Gene set enrichment analysis of all GEP results, and Ingenuity Pathway Analysis of DEGs, indicated up regulation of multiple pathways and processes including ribosomal and mitochondrial components involved in translation and oxidative phosphorylation, CTLA4 signaling in cytotoxic T cells, and differentiation and signaling by ICOS and CD28 in T helper cells. We confirmed up regulation of CTLA4, ICOS, and LAG3 at the protein level in C2D15 PBMC by flow cytometry. Furthermore, treatment of PBMC derived from untreated FL pts with lenalidomide in vitro resulted in up regulation of these molecules in T and/or NK cells consistent with our in vivo results. Conclusions: In FL pts, lenalidomide induced multiple changes in the immune system including increases in PDC and memory T cell subsets, activation of T and NK cells, and down-regulation of certain genes mediating B cell migration and proliferation. These results provide insights into the mechanism of action of lenalidomide and suggest that it can be combined with other immunostimulatory agents such as therapeutic vaccines, adoptive T cell therapy strategies, and immune checkpoint inhibitors to further enhance its efficacy in FL and other malignancies. Disclosures: Fowler: Celgene: Research Funding. Heise:Celgene Corporation: Employment, Equity Ownership. Lacerte:Celgene: Honoraria. Samaniego:Celgene: Research Funding. Neelapu:Celgene Corporation: Research Funding.

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Exploring the Functional Relevance of BTK Beyond Chronic Lymphocytic Leukemia (CLL) Cells: BTK Expression in Non-Malignant Immune Cells of the Microenvironment Mediates CLL Development and Progression In Vivo

Blood ◽

10.1182/blood.v128.22.352.352 ◽

2016 ◽

Vol 128 (22) ◽

pp. 352-352

Author(s):

Fabienne McClanahan ◽

Sean D. Reiff ◽

Daphne Guinn ◽

Minh Tran ◽

Larry Beaver ◽

...

Keyword(s):

T Cell ◽

Nk Cells ◽

Cell Lines ◽

Immune Cells ◽

Research Funding ◽

Myeloid Cells ◽

Surrogate Marker ◽

Suspension Cells ◽

And Function

Abstract Background: Bruton's tyrosine kinase (BTK) is a critical component of the BCR pathway, but BTK and/or BTK-related enzymes such as ITK, Tec, BMX and RLK are also expressed in T, NK and myeloid cells, all of which are important sources of CLL-associated immune dysfunction. Whether BTK and/or BTK-related enzymes within non-malignant immune cells have any effects on CLL progression and immune function is not known. This is highly relevant translationally, as newer generations of BTK inhibitors selectively target BTK while sparing BTK-related enzymes in other cells. As the effects of constitutively active BCR and BTK targeting can be adequately modelled in the TCL-1 and TCL-1xXID mouse models (Woyach et al. Blood 2014), we used these preclinical tools to investigate if BTK expression in non-malignant immune cells modulates CLL progression in vivo. Our aims were: (1) to characterize differences in phenotype and function of T, NK and myeloid cells in leukemia-free and leukemic WT and BTK-inactive mice; (2) to assess BTK-related PD-L1 expression; and (3) to evaluate the in vivo relevance of BTK in non-malignant immune cells to CLL progression. Methods: Single-cell suspension cells were obtained from spleens from CLL-free matched WT B6 and BTK-inactive XID mice (n=11 each) and leukemic TCL-1xXID and TCL-1 mice (n=10 each). Flow cytometry was used to characterize the phenotype of B, T, NK and myeloid cells and myeloid-derived suppressor cells (MDSCs), PD-L1 expression, and T-cell degranulation. For survival experiments, WT B6 (n=29) and XID (n=27) mice were injected with 5x106 splenocytes from leukemic TCL1 donors and monitored daily. For in vivo depletion of monocytes/ macrophages, WT and XID (both n=12) mice received clodronate i.p. every 3 days from day 1 after injection of 3x107 splenocytes from leukemic TCL1 donors, and were euthanized after 36 days of treatment. RT qPCR was conducted on BTK-deficient XLA cell lines. Results: To explore whether BTK expression in non-malignant immune cells affects CLL development in vivo, we first conducted adoptive transfer experiments into WT and BTK-inactive XID mice. While WT mice had a median survival of 52 days after CLL development, survival was almost twice as long (90 days) in XID mice, indicating that BTK inactivation in non-leukemic cells prolongs survival. To identify whether this could be explained by differences in immune cell populations, we next characterized phenotype and function of T, NK and myeloid cells in leukemia-free and leukemic BTK functional and BTK-inactive mice. This comparison revealed that CLL-induced T-cell changes were broadly recapitulated in BTK-inactive mice, while significantly more immature (p=0.0206) and mature (p=0.008) NK cells were maintained. In the myeloid compartment, BTK activity did not affect the overall quantity of CD11b+ myeloid cells, but led to significant qualitative CLL-associated changes: while mice with functional BTK exhibited a significant CLL-driven loss of neutrophils (p=0.0002) and expansion of F4/80+CD11b+ macrophages (p=0.0001), leukemic BTK-inactive mice largely maintained these populations, suggesting anti-tumor properties of these cells. We next depleted myeloid cells with presumed BTK-associated anti-tumor properties in CLL-engrafted mice using clodronate. After 36 days of treatment, differences in spleen weights, CLL load in spleen and blood, and percentage of proliferating CLL cells were lost between WT and XID mice (all p>0.05), indicating that monocytes/ macrophages in XID mice indeed have in vivo anti-tumor functions. Neutrophils were expanded in both WT (median 44.7% of CD11b+ cells) and XID mice (43.7% vs. 6.1% in non-depleted mice). Interestingly, PD-L1 expression as a surrogate marker for tumor-associated immune suppression was generally significantly increased on antigen-presenting cells from BTK-inactive leukemic mice (CLL cells p=0.0002; inflammatory monocytes p=0.0014; patrolling monocytes p=0.001; MDSCs p=0.0201). RT qPCR experiments using XLA cell lines with modified BTK expression revealed that absence of BTK was associated with significantly higher PD-L1 RNA expression (p=0.0075), indicating a direct mechanistic link between BTK and PD-L1. Conclusions: Our data indicate that BTK in myeloid and NK cells directly mediates CLL development and progression in vivo. Targeting BTK in those cells in addition to CLL cells might provide substantial clinical benefits. Disclosures Woyach: Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding.

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iPSC-Derived NK Cells Synergize with T Cells and Anti-PD-1 Antibody to Mediate Durable Anti-Tumor Responses In Vivo

Blood ◽

10.1182/blood-2019-124961 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1933-1933

Author(s):

Jeffrey S. Miller ◽

Ryan Bjordahl ◽

Svetlana Gaidarova ◽

Sajid Mahmood ◽

Paul Rogers ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Tumor Control ◽

Tumor Spheroid ◽

Advisory Committees ◽

Tumor Reduction

Cancer immunotherapies have resulted in a paradigm shift in therapy. One of the most successful approaches has been administration of antibodies targeting immune checkpoint inhibitors (ICI), such as programmed death-1 (PD-1), that can reinvigorate functionally exhausted T cells. Unfortunately, durable tumor regression is limited to a minority of patients, and relapse remains a significant concern. Combining novel immunotherapies with ICI s a promising strategy to bolster antitumor responses and response rates. Natural killer (NK) cells mediate direct tumor cell lysis, effectively target MHC low or null transformed cells and are key regulators of T cell responses through the production of inflammatory cytokines and chemokines. In many cancers, NK cell numbers are low, and their functional responses are sub-optimal. The use of allogeneic peripheral blood NK cells for immunotherapy has shown significant clinical promise for the treatment of various cancers. However, sourcing NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs) that can be combined with ICI antibodies for multiple tumor types. To interrogate the ability of iPSC-derived NK cells to synergize with ICI therapy, we developed an in vitro 3D tumor spheroid system to model the combinatorial effects of activated CD3+ T cells, iPSC-derived NK (iNK) cells and ICI blockade for anti-tumor function in a more physiological context than the standard 2D cultures and in real time. Using SKOV-3 (an ovarian cancer line) spheroids as targets in a 160-hour killing assay, we found that iNK cells could mediate significant, but not complete destruction of tumor spheroids (46% tumor reduction). Twice as many activated CD3+ T cells by themselves also induced significant but incomplete tumor spheroid destruction (58% tumor reduction). Combined iNK and activated CD3+ T cells led to robust target cell destruction (71% tumor reduction). Importantly, adding anti-PD-1 antibody (mAb) to activated CD3+ T cells and iNK cells led to near complete elimination of tumor spheroid targets, with >99% tumor reduction. Cytokine and chemokine secretion analyses in co-cultures of activated CD3+ T cells and iNK cells revealed synergistic production of CCL3 and CCL4 for T cell recruitment, and TNF and IFN-γ to augment anti-tumor responses. To determine whether these striking 3D tumor spheroid results simulate the in vivo setting, we injected luciferase-expressing OVCAR8 (a human ovarian cancer line) cells into the peritoneal cavities of immunodeficient NSG mice. Following sublethal irradiation, we treated groups of mice with either anti-PD-1 mAb, activated CD3+ T cells or iNK cells alone or in various combinations. IL-2 was injected i.p. twice weekly for two weeks into all mice except those in the tumor alone group (Figure 1A). In mice that received iNK cells alone, significant tumor control was observed over the first 21 days but was not sustained. Tumor control was similar between groups of mice that received iNK cells and mice that received iNK cells and anti-PD-1 mAb. Mice treated with either anti-PD-1 mAb or activated CD3+ T cells alone exhibited similar rates of tumor growth relative to the untreated group. Modest tumor control was observed in the group of mice that received combined activated CD3+ T cells and anti-PD-1 mAb, though the effect did not reach statistical significance. Durable tumor control past day 21 was observed in groups of mice that received either combined activated CD3+ T cells and iNK cells or activated CD3+ T cells + iNK cells with anti-PD-1 mAb (Figure 1B). Importantly, 50% of mice in the activated CD3+ T cells + iNK cells + anti-PD-1 mAb group exhibited tumor bioluminescence readings well below other treatment group at day 35 (Figure 1C, D). Collectively, these data demonstrate that iNK cells can serve as an off-the-shelf source of high-quality NK cells and synergize with anti-PD-1 ICI therapy to enhance anti-tumor T cell responses in vitro and in vivo, providing a novel immunotherapeutic platform for tumors in which iNK cells, activated CD3+ T cells or anti-PD-1 mAb therapy alone is not sufficiently effective. The current program is under clinical investigation and can be found at clinicaltrials.gov NCT03841110. Disclosures Miller: Moderna: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bjordahl:Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Rogers:Fate Therapeutics, Inc: Employment. Moyar:Fate Therapeutics, Inc: Employment. Blazar:Abbvie Inc: Research Funding; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. OffLabel Disclosure: NK cells

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283 TIM-3 blockade modulates the tumor microenvironment improving the outcome of preclinical pediatric diffuse midline glioma models

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.283 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A307-A307

Author(s):

Iker Ausejo-Mauleon ◽

Sara Labiano ◽

Virginia Laspidea ◽

Marc Garcia-Moure ◽

Daniel de la Nava ◽

...

Keyword(s):

T Cell ◽

Tumor Microenvironment ◽

Nk Cells ◽

Immune Cell ◽

Animal Experiments ◽

Cell Types ◽

Immune Memory ◽

List Type ◽

Treg Population

BackgroundDiffuse Midline Gliomas (DMGs), encompassing Diffuse Intrinsic Pontine Gliomas (DIPGs), are the most aggressive pediatric brain tumors. Their meager survival has not changed despite the combination of radiotherapy with targeted therapies emphasizing the urgent need for effective treatments. Recent research suggested that the DIPG tumor microenvironment is neither highly immunosuppressive nor inflammatory.1 These analyses showed the lack of infiltrating lymphocytes and the abundance of CD11b+ cells. TIM-3 is a member of the T-cell immunoglobulin and mucin domain protein family expressed on multiple immune cell types, including T cells, Treg, NK cells, monocytes, dendritic cells, and microglia, where it potently regulates not only adaptive immunity but also innate immunity.2–3 Therefore, TIM-3 inhibitors could challenge several components in the tumor microenvironment, thereby providing potentially effective treatment for DMGs.MethodsNP53 and XFM murine DIPG cell lines were used for animal experiments in immunocompetent orthotopic models. The tumors were processed by mechanical and enzymatic digestion and immune populations were analyzed by a flow cytometry panel. Antibodies against NK cells (NK1.1), CD4 (GK1.5), CD8 (CD8β) were used for animal depletion experiments alone or in combination.ResultsIn silico assessment of TIM-3 expression in DIPG datasets showed a robust expression of this gene. Moreover, single-cell sequencing analyses of DIPG biopsies uncover its expression in the myeloid compartment (especially in microglia). In vivo efficacy studies showed that treatment with anti-TIM-3 antibody significantly increased the overall survival in two DIPG immunocompetent orthotopic animal models (doubling the median), lead to long-term survivors free of disease (50%) and showed immune memory. Analyses of CD45+ populations in the tumor microenvironment showed a significant increase in microglia, granulocytes, NK and CD8+ cells corresponding with a NK and T-cell activate phenotypes in treated-mice. In addition, we have a substantial decrease in the Treg population, which causes an increase in the CD8/Treg ratio. CD4 and CD8 T-cell depletion led to a significant but not total loss of treatment efficacy. NK cells depletion also reduced the effectiveness of this therapy, albeit to a lesser extent than CD4-CD8 depletion. We are currently investigating the role of microglia in the outcome of the treatment.ConclusionsOur data uncovered TIM-3 as a potential target for the treatment of DIPG tumors. Inhibition of this molecule led to a potent antitumor effect mediated by a profound tumor microenvironment remodelling.ReferencesLieberman NAP, DeGolier K, Kovar HM, et al. Characterization of the immune microenvironment of diffuse intrinsic pontine glioma: implications for development of immunotherapy. Neuro Oncol 2019;21(1):83–94. doi:10.1093/neuonc/noy145.Acharya N, Sabatos-Peyton C, Anderson AC. Tim-3 finds its place in the cancer immunotherapy landscape. J Immunother Cancer 2020;8(1):e000911. doi:10.1136/jitc-2020-000911.Wolf Y, Anderson AC, Kuchroo VK. TIM3 comes of age as an inhibitory receptor. Nat Rev Immunol 2020;20(3):173–185. doi:10.1038/s41577-019-0224-6

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Anti-tumor effects of RTX-240: an engineered red blood cell expressing 4-1BB ligand and interleukin-15

Cancer Immunology Immunotherapy ◽

10.1007/s00262-021-03001-7 ◽

2021 ◽

Author(s):

Shannon L. McArdel ◽

Anne-Sophie Dugast ◽

Maegan E. Hoover ◽

Arjun Bollampalli ◽

Enping Hong ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Blood Cell ◽

Nk Cells ◽

Red Blood Cell ◽

Safety Profile ◽

Nk Cell ◽

Cell Activity ◽

In Vivo Studies

AbstractRecombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial.

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DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

Journal of Experimental Medicine ◽

10.1084/jem.20081752 ◽

2008 ◽

Vol 205 (13) ◽

pp. 2965-2973 ◽

Cited By ~ 195

Author(s):

Susan Gilfillan ◽

Christopher J. Chan ◽

Marina Cella ◽

Nicole M. Haynes ◽

Aaron S. Rapaport ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Adhesion Molecules ◽

Cd8 T Cells ◽

Nk Cell ◽

Cd8 T Cell ◽

Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

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Phenotype of recovering lymphoid cell populations after marrow transplantation.

Journal of Experimental Medicine ◽

10.1084/jem.161.6.1483 ◽

1985 ◽

Vol 161 (6) ◽

pp. 1483-1502 ◽

Cited By ~ 139

Author(s):

K A Ault ◽

J H Antin ◽

D Ginsburg ◽

S H Orkin ◽

J M Rappeport ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Nk Cells ◽

Cell Types ◽

Lymphoid Cells ◽

The Other ◽

Hla Dr ◽

T Cell Antigens ◽

Leu 7

Four patients who received bone marrow transplants were studied sequentially during the posttransplant period to define the pattern of recovering lymphoid cell types. Three patients received T cell-depleted, HLA-matched marrow, and one received untreated marrow from an identical twin. Blood lymphoid cells were labeled with 25 different pairs of monoclonal antibodies. In each sample, one antibody was conjugated to fluorescein and one to phycoerythrin, thus allowing simultaneous assessment of the expression of the two markers using the fluorescence activated cell sorter. A total of 14 antibodies were used, routinely including HLE, Leu-M3, Leu-4, Leu-1, Leu-5, Leu-9, Leu-6, Leu-2, Leu-3, HLA-DR, Leu-7, Leu-11, Leu-15, and Leu-12. Other antibodies were used to further define some populations. This study has allowed us to define six distinct cell types that have appeared in all four patients by day 90 posttransplantation, and which account for 90-100% of all circulating lymphoid cells. These cell types are (a) T helper cells expressing Leu-1, Leu-4, Leu-9, Leu-5, Leu-3, and variable amounts of HLA-DR; (b) T suppressor cells expressing Leu-1, Leu-4, Leu-9, Leu-5, Leu-2, and variable amounts of HLA-DR; (c) B cells expressing Leu-12, B1, HLA-DR, IgD, and IgM, but none of the T cell antigens; (d) an unusual B cell phenotype (Leu-1 B) expressing all of the B cell markers, and also having low amounts of Leu-1, but none of the other T cell antigens; (e) natural killer (NK) cells expressing Leu-11, Leu-15, Leu-5 but none of the other T cell or B cell markers; (f) NK cells expressing Leu-11, Leu-15, Leu-5, and low levels of Leu-2. Both NK types also express Leu-7 on some, but not all cells. The relative frequencies of these cell types varied among the patients and with time, but the striking findings were the presence of relatively few mature T cells, large numbers of NK cells, and the preponderance of the unusual Leu-1 B cell over conventional B cells in all three patients who developed B cells. Sorting experiments confirmed the NK activity of the major NK cell phenotypes, and DNA analysis confirmed that all of the cells studied were of donor origin. In addition, analysis of Ig genes in one patient showed that the Leu-1 B cells were not clonally rearranged.(ABSTRACT TRUNCATED AT 400 WORDS)

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High Dimensional Tissue-Based Spatial Analysis of the Tumor Microenvironment of Follicular Lymphoma Reveals Unique Immune Niches inside Malignant Follicles

Blood ◽

10.1182/blood-2020-134941 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 17-18

Author(s):

Jose C Villasboas ◽

Patrizia Mondello ◽

Angelo Fama ◽

Melissa C. Larson ◽

Andrew L. Feldman ◽

...

Keyword(s):

T Cells ◽

Spatial Distribution ◽

T Cell ◽

B Cells ◽

Research Funding ◽

Memory T Cells ◽

Cell Types ◽

T Cell Subsets ◽

Cell Contact ◽

Private Company

Background The importance of the immune system in modulating the trajectory of lymphoma outcomes has been increasingly recognized. We recently showed that CD4+ cells are associated with clinical outcomes in a prospective cohort of almost 500 patients with follicular lymphoma (FL). Specifically, we showed that the absence of CD4+ cells inside follicles was independently associated with increased risk of early clinical failure. These data suggest that the composition, as well as the spatial distribution of immune cells within the tumor microenvironment (TME), play an important role in FL. To further define the architecture of the TME in FL we analyzed a FL tumor section using the Co-Detection by Indexing (CODEX) multiplex immunofluorescence system. Methods An 8-micron section from a formalin-fixed paraffin-embedded block containing a lymph node specimen from a patient with FL was stained with a cocktail of 15 CODEX antibodies. Five regions of interest (ROIs) were imaged using a 20X air objective. Images underwent single-cell segmentation using a Unet neural network, trained on manually segmented cells (Fig 1A). Cell type assignment was done after scaling marker expression and clustering using Phenograph. Each ROI was manually masked to indicate areas inside follicles (IF) and outside follicles (OF). Relative and absolute frequencies of cell types were calculated for each region. Cellular contacts were measured as number and types of cell-cell contacts within two cellular diameters. To identify proximity communities, we clustered cells based on number and type of neighboring masks using Phenograph. The number of cell types and cellular communities were calculated inside and outside follicles after adjustment for total IF and OF areas. The significance of cell contact was measured using a random permutation test. Results We identified 13 unique cell subsets (11 immune, 1 endothelial, 1 unclassified) in the TME of our FL section (Fig. 1A). The unique phenotype of each subset was confirmed using a dimensionality reduction tool (t-SNE). The global composition of the TME varied minimally across ROIs and consisted primarily of B cells, T cells, and macrophages subsets - in decreasing order of frequency. Higher spatial heterogeneity across ROIs was observed in the frequency of T cell subsets in comparison to B cells subsets. Inspecting the spatial distribution of T cell subsets (Fig. 1B), we observed that cytotoxic T cells were primarily located in OF areas, whereas CD4+ T cells were found in both IF and OF areas. Notably, the majority of CD4+ T cells inside the follicles expressed CD45RO (memory phenotype), while most of the CD4+ T cells outside the follicles did not. Statistical analysis of the spatial distribution of CD4+ memory T cell subsets confirmed a significant increase in their frequency inside follicles compared to outside (20.4% vs 11.2%, p < 0.001; Fig. 1D). Cell-cell contact analysis (Fig 1C) showed increased homotypic contact for all cell types. We also found a higher frequency of heterotypic contact between Ki-67+CD4+ memory T cells and Ki-67+ B cells. Pairwise analysis showed these findings were statistically significant, indicating these cells are organized in niches rather than randomly distributed across image. Analysis of cellular communities (Fig. 1C) identified 13 niches, named according to the most frequent type of cell-cell contact. All CD4+ memory T cell subsets were found to belong to the same neighborhood (CD4 Memory community). Analysis of the spatial distribution of this community confirmed that these niches were more frequently located inside follicles rather than outside (26.3±4% vs 0.004%, p < 0.001, Fig. 1D). Conclusions Analysis of the TME using CODEX provides insights on the complex composition and unique architecture of this FL case. Cells were organized in a pattern characterized by (1) high degree of homotypic contact and (2) increased heterotypic interaction between activated B cells and activated CD4+ memory T cells. Spatial analysis of both individual cell subsets and cellular neighborhoods demonstrate a statistically significant increase in CD4+ memory T cells inside malignant follicles. This emerging knowledge about the specific immune-architecture of FL adds mechanistic details to our initial observation around the prognostic value of the TME in this disease. These data support future studies using modulation of the TME as a therapeutic target in FL. Figure 1 Disclosures Galkin: BostonGene: Current Employment, Patents & Royalties. Svekolkin:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Postovalova:BostonGene: Current Employment, Current equity holder in private company. Bagaev:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Ovcharov:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Varlamova:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Novak:Celgene/BMS: Research Funding. Witzig:AbbVie: Consultancy; MorphSys: Consultancy; Incyte: Consultancy; Acerta: Research Funding; Karyopharm Therapeutics: Research Funding; Immune Design: Research Funding; Spectrum: Consultancy; Celgene: Consultancy, Research Funding. Nowakowski:Nanostrings: Research Funding; Seattle Genetics: Consultancy; Curis: Consultancy; Ryvu: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other; Kymera: Consultancy; Denovo: Consultancy; Kite: Consultancy; Celgene/BMS: Consultancy, Research Funding; Roche: Consultancy, Research Funding; MorphoSys: Consultancy, Research Funding. Cerhan:BMS/Celgene: Research Funding; NanoString: Research Funding. Ansell:Trillium: Research Funding; Takeda: Research Funding; Regeneron: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squibb: Research Funding; AI Therapeutics: Research Funding; ADC Therapeutics: Research Funding.

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