Anti-tumor activity of alectinib in the orthotopic in vivo imaging model with NCOA4-RET fusion positive tumor cells

Abstract Abstract 646 Adoptively transferred virus or tumor antigen-specific T-cells have demonstrated efficacy in initial clinical trials, but while clearance of viral infection is sustained, responses to tumor-specific T-cells are usually short-lived. Therefore, current efforts are focused on distinguishing attributes of virus-specific T-cells that contribute to their persistence and formulating strategies to sustain anti-tumor effects of T-cell (TC) therapies. We have developed an in-vivo model to compare the relative efficacy of T-cells specific for a tumor antigen (WT-1) versus T-cells specific for a viral antigen (CMVpp65) using human colon carcinoma cells that express the oncofetal protein WT-1 which were transduced to co-express CMVpp65 (WT-1[+] cocapp65) as a surrogate system. Groups of 6 NOD/Scid-IL2Rgc-KO/J mice (NSG) were each subcutaneously injected with 3 × 105 WT-1 [+] cocapp65 on the R flank. Each animal was also injected with 3 × 105 cells from a WT-1[+] ovarian carcinoma cell line (SKOV3-A2) on the L shoulder to compare the efficacy of WT-1 specific T-cells (WT1-CTLs) against different WT-1 [+] tumor cell types. Expression of WT-1 protein is lower in SKOV3-A2 than in cocapp65 cells. Both tumors are HLA A0201[+] and were transduced to express a GFP-firefly luciferase gene. T-cells were administered intravenously 5 days after tumor injection to enable vascularization and tumor growth was quantitated using bioluminescence. These experiments evaluated (1) the relative capacity of CMVpp65 specific T-cells (CMV-CTLs) versus WT-1 CTLs to eradicate WT1[+] cocapp65 cells that co-express a viral and tumor antigen (2) the relative efficacy of WT-1 CTLs against 2 different HLA A0201 [+] WT-1 expressing tumors; an ovarian carcinoma and a colon carcinoma, and (3) the contribution of IL-15/15Rα complex in augmenting the efficacy of antigen specific T-cells by using intraperitoneally (i.p) injected Baf-3 cells transduced to express human IL-15/15Rα complex. The treatment groups were as follows: (1) Control – no T-cells + IL-2 (2000 U) (2) Control – no T-cells + IL-15/IL-15Rα (5 × 106 baf-3 cells) (3) WT1 CTLs + IL-2 (2000 U) (4) CMV-CTLs + IL-2 (2000 U) (5) WT1 CTLs + IL-15/IL-15Rα (5 × 106 baf-3 cells) (6) CMV-CTLs + IL-15/IL-15Rα (5 × 106 baf-3 cells). IL-2 and irradiated baf-3 cells were administered intraperitoneally twice weekly. When the doses of antigen specific interferon gamma (IFNg) [+] T-cells were equivalent in the infused CMVpp65 and WT1 specific T-cells, the CMV-CTLs induced greater, and more sustained suppression of the growth of the WT-1[+] cocapp65 cells in-vivo than the WT-1 CTLs (Fig.1). The anti-tumor activity of the WT-1 CTLs was greater against WT-1[+] cocapp65 than against the WT-1[+] ovarian carcinoma (SKOV3-A2), potentially reflecting the higher expression of WT-1 in cocapp65. The SKOV3-A2 tumor began to re-grow by 24 days post T-cell infusion approaching the size of control tumors by day 38, while the WT-1[+] cocapp65 still demonstrated slower growth through day 38. The addition of IL-15/15Rα increased the efficacy of the transferred T-cells, the difference being more pronounced for the anti-tumor activity of WT-1 CTLs. Fig. 1 Comparative Efficacy of CMV and WT-1 CTLs against Human Tumor Targets Co-expressing CMVpp65 and WT-1 Fig. 1. Comparative Efficacy of CMV and WT-1 CTLs against Human Tumor Targets Co-expressing CMVpp65 and WT-1 These studies demonstrate that equivalent doses of IFNg[+] WT-1 CTLs can also suppress WT-1[+] cocapp65 tumor xenografts, but are less effective than CMV-CTLs, and that IL-15 supplementation augments the cytotoxic activity of the CTLs in-vitro and enhances the duration of the anti-tumor effects in-vivo. This model permits side by side comparisons of the anti-tumor activity of human T-cells directed against viral and tumor antigens expressed on the same clonogenic human tumor target. Because both responses are directed against the same cells, this model could thereby facilitate identification of the distinguishing features of T-cells specific for viral or tumor antigens as well as differences in the presentation of viral and oncofetal “self” antigens by tumor cells that contribute to disparities in their anti-tumor activity and persistence in-vivo. Disclosures: No relevant conflicts of interest to declare.

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Tissue-Specific Homing and Different Impact in Gvhd Prevention of NK Cell Subpopulations.

Blood ◽

10.1182/blood.v120.21.3004.3004 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3004-3004

Author(s):

Kathrin Meinhardt ◽

Ruth Bauer ◽

Irena Kroeger ◽

Julia Schneider ◽

Franziska Ganss ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Nk Cells ◽

Tumor Cells ◽

In Vivo Imaging ◽

Nk Cell ◽

Cell Subset ◽

Target Organs ◽

Cell Subpopulations

Abstract Abstract 3004 Clinical studies exploiting the impact of natural killer (NK) cells in allogeneic hematopoietic stem cell transplantation (HSCT) have provided promising results. It is known that NK cells are a heterogeneous population and can be divided into functionally distinct NK cell subpopulations. Murine NK cells can be separated along their expression of CD27 and CD11b and CD117 (c-kit). However, the functional relevance of distinct NK cell subsets in graft-versus-host-disease (GVHD) has not been investigated in detail so far. We have established different protocols for ex vivo isolation and expansion of murine NK cell subpopulations. These NK subsets were further analyzed in vitro and in vivo in an allogeneic murine GVHD model. Here we report on different genomic, phenotypic and functional properties of 4 NK cell subsets. Our data clearly demonstrate that CD27+ NK cells revealed the highest IFN-g production upon coculture with tumor cells and/or IL-2. Interestingly, the CD11b+ NK cells express multiple genes of cytotoxic pathways and develop the highest cytotoxic capacity towards tumor cells. We observed up to 60% tumor lysis by CD27- CD11b+ NK cells compared to 40–45% by CD27+ CD11b+, about 25% by CD27+ CD11b- and 10% by c-kit+ CD11b- NK cells at an effector-target ratio of 5:1, respectively. Furthermore, the CD11b+ NK cell subset significantly reduced T cell proliferation induced by allogeneic dendritic cells in mixed lymphocytes reactions. Next, we analyzed the migratory capacity and tissue-specific homing of FACS-sorted NK cell subsets by adoptive transfer of congeneic CD45.1+ and Luc+ NK cell subpopulations in autologous and allogeneic bone marrow transplantation. Of interest, FACS analysis and in vivo imaging showed that CD11b+ NK cells migrated to peripheral GVHD target organs, whereas CD27+ NK cells preferentially homed to the bone marrow. Finally, this study addressed for the first time the role of distinct NK cell subpopulations in the development of GVHD in a fully MHC mismatched HSCT mouse model. Importantly, we identified the CD11b+ NK cell population as the NK cell subset that significantly diminished GVHD. In vivo imaging of Luc+CD11b+ NK cells revealed that this subset migrates to the colonic tissue to prevent development of GVHD colitis as shown by colonoscopy. In summary, our comparative study outlines that only CD11b+ NK cells, migrating to the peripheral GVHD target organs and providing the most efficient cytolytic capacity directed against allogeneic dendritic cells, protect against GVHD. These new insights are highly relevant for the selection of optimal NK cell subsets in the field of cellular immunotherapy. Disclosures: No relevant conflicts of interest to declare.

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Use of an antibody-drug conjugate targeting tissue factor to induce complete tumor regression in xenograft models with heterogeneous target expression.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.3066 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 3066-3066 ◽

Cited By ~ 1

Author(s):

Esther CW Breij ◽

David Satijn ◽

Sandra Verploegen ◽

Bart de Goeij ◽

Danita Schuurhuis ◽

...

Keyword(s):

Tissue Factor ◽

Tumor Cells ◽

Tumor Regression ◽

Antibody Drug Conjugate ◽

Xenograft Models ◽

Anti Tumor Activity ◽

Complete Tumor ◽

Antibody Drug

3066 Background: Tissue factor (TF) is the main initiator of coagulation, that starts when circulating factor VII(a) (FVII(a)) binds membrane bound TF. In addition, the TF:FVIIa complex can initiate a pro-angiogenic signaling pathway by activation of PAR-2. TF is aberrantly expressed in many solid tumors, and expression has been associated with poor prognosis. TF-011-vcMMAE, an antibody-drug conjugate (ADC) under development for the treatment of solid tumors, is composed of a human TF specific antibody (TF-011), a proteaseEcleavable valine-citrulline (vc) linker and the microtubule disrupting agent monomethyl auristatin E (MMAE). Methods: TF-011 and TF-011-vcMMAE were functionally characterized using in vitro assays. In vivo anti-tumor activity of TF-011-vcMMAE was assessed in human biopsy derived xenograft models, which genetically and histologically resemble human tumors. TF expression in xenografts was assessed using immunohistochemistry. Results: TF-011 inhibited TF:FVIIa induced intracellular signaling and efficiently killed tumor cells by antibody dependent cell-mediated cytoxicity in vitro, but showed only minor inhibition of TF procoagulant activity. TF-011 was rapidly internalized and targeted to the lysosomes, a prerequisite for intracellular MMAE release and subsequent tumor cell killing by the ADC. Indeed, TF-011-vcMMAE efficiently and specifically killed TF-positive tumors in vitro and in vivo. Importantly, TF-011-vcMMAE showed excellent anti-tumor activity in human biopsyEderived xenograft models derived from bladder, lung, pancreas, prostate, ovarian and cervical cancer (n=7). TF expression in these models was heterogeneous, ranging from 25-100% of tumor cells. Complete tumor regression was observed in all models, including cervical and ovarian cancer xenografts that showed only 25-50% TF positive tumor cells. Conclusions: TF-011-vcMMAE is a promising new ADC with potent anti-tumor activity in xenograft models that represent the heterogeneity of human tumors, including heterogeneous TF expression. The functional characteristics of TF-011-vcMMAE allow efficient tumor targeting, with minimal impact on coagulation.

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3P290 Noninvasive in vivo imaging of tumor cells in a novel xenograft model(27. Bioimaging,Poster)

Seibutsu Butsuri ◽

10.2142/biophys.53.s260_1 ◽

2013 ◽

Vol 53 (supplement1-2) ◽

pp. S260

Author(s):

Sayaka Kita ◽

Hideo Higuchi

Keyword(s):

Tumor Cells ◽

In Vivo Imaging ◽

Xenograft Model

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2P297 Noninvasive in vivo imaging of tumor cells and tissue in mouse auricles(27. Bioimaging,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

Seibutsu Butsuri ◽

10.2142/biophys.54.s244_3 ◽

2014 ◽

Vol 54 (supplement1-2) ◽

pp. S244

Author(s):

Sayaka Kita

Keyword(s):

Tumor Cells ◽

Annual Meeting ◽

In Vivo Imaging ◽

Biophysical Society

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Development of Chimeric Antigen Receptor-Expressing iPSC-Derived Macrophages with Improved Anti-Tumor Activity

Blood ◽

10.1182/blood-2021-148687 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1693-1693

Author(s):

Somayeh Pouyanfard ◽

Manuel Fierro ◽

Dan S Kaufman

Keyword(s):

Ovarian Cancer ◽

Stem Cells ◽

Tumor Cells ◽

Pluripotent Stem Cells ◽

Stock Options ◽

Ovarian Cancer Cells ◽

Anti Tumor Activity ◽

Privately Held

Abstract Previous studies by our group demonstrate the ability to routinely derive hematopoietic and immune cells from human pluripotent stem cells. Here, we demonstrate the efficient derivation of macrophages from human induced pluripotent stem cells (iPSCs). These macrophages have phenotypic and genotypic characteristics similar to monocytes/macrophages isolated from human peripheral blood. We also demonstrate the ability to polarize these iPSC-derived macrophages (iPSC-Macs) to M1 and M2 populations. Specifically, M1 iPSC-Macs have pro-inflammatory characteristics including expression of CD40 and CD80 on the cell surface, produce increased amounts of TNF-a and IL-6 detected in the supernatant, as well have increased expression of inflammatory cytokines/chemokines (TNF-a, IL-6, IL-1b, IL-12, CCL2, CCL3 and TRAIL) and increased expression of matrix metalloproteases (MMPs). Function of these iPSC-Macs was initially assessed by phagocytosis of fluorescently-labeled beads. These studies demonstrated both the iPSC-M1 and M2 macrophages efficiently phagocytized these beads, and at similar amounts as their peripheral blood counterparts. Next, we tested the ability of the iPSC-Macs to phagocytize human tumor cells. Using A1847 ovarian tumor cells, we found while the iPSC-Macs alone had limited ability to phagocytize the tumor cells (9%), addition of either an anti-CD47 mAb (41%) or anti-EGFR (41%) lead to markedly increased phagocytosis, with the combination of the 2 antibodies being even better (55% phagocytosis). We then tested iPSC-Macs in vivo against luciferase (luc)-expressing A1847 ovarian cancer cells as a xenograft model in NSG-SGM3 mice that express human IL3, GM-CSF and SCF. Using bioluminescent imaging, we found that the combination of iPSC-Macs with both anti-CD47 and anti-EGFR demonstrated significantly improved anti-tumor activity, with median survival of 75 days, compared to 50-60 days for mice treated with only iPSC-Macs, only mAbs or with iPSC-Macs combined either single mAb. Next, we aimed to use the iPSC platform to produce iPSC-Macs engineered to express chimeric antigen receptors (CARs) to further improve their anti-tumor activity. Here, we developed and tested novel macrophage specific CARs that were stably expressed in undifferentiated iPSCs using transposon-mediated gene transfer, similar to our previous studies to derive iPSC-derived CAR-expressing NK cells that have now been translated into clinical trials. We used an anti-mesothelin (meso) scFv combined with 8 different CAR constructs with distinct intracellular signaling components. We found that the iPSC-Macs could express good levels of the CARs (iPSC-CarMacs). Function was again tested in vitro by phagocytosis of the Meso+ A1847 ovarian cancer cells. The iPSC-CarMacs with a Bai1 stimulatory domain consistently demonstrated the best activity in this assay system. We next tested the anti-meso-iPSC-CarMacs in vivo using the A1847 cells. Again, we demonstrate the iPSC-CarMacs combined with anti-CD47 mAb mediate significantly improved anti-tumor activity using this in vivo model compared to the non-CAR-iPSC-Macs + anti-CD47, p <0.005 (Figure). Survival studies are still ongoing. Together, these studies demonstrate that iPSCs can be used to routinely and efficiently derive macrophages with potent anti-tumor activity. Additionally, CARs that are optimized for macrophage-mediated activity can be expressed to generate iPSC-CarMacs that effectively kill tumor cells in vitro and in vivo. These iPSC-CarMacs provide another approach to provide a standardized, targeted, off-the-shelf cell therapy product that can be used to treat both hematological malignancies as well as diverse solid tumors. Figure 1 Figure 1. Disclosures Kaufman: Shoreline Biosciences: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qihan Biotech: Consultancy, Current holder of stock options in a privately-held company; VisiCELL Medical: Consultancy, Current holder of stock options in a privately-held company.

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