ALBERT

Description: Lymphocyte-based immunotherapy has emerged as a breakthrough in cancer therapy for both hematologic and solid malignancies. In a subpopulation of cancer patients, this powerful therapeutic modality converts malignancy to clinically manageable disease. However, the T cell- and chimeric antigen receptor T (CAR-T) cell-mediated antimetastatic activity, especially their impacts on microscopic metastatic lesions, has not yet been investigated. Here we report a living zebrafish model that allows us to visualize the metastatic cancer cell killing effect by tumor- infiltrating lymphocytes (TILs) and CAR-T cells in vivo at the single-cell level. In a freshly isolated primary human melanoma, specific TILs effectively eliminated metastatic cancer cells in the living body. This potent metastasis-eradicating effect was validated using a human lymphoma model with CAR-T cells. Furthermore, cancer-associated fibroblasts protected metastatic cancer cells from T cell-mediated killing. Our data provide an in vivo platform to validate antimetastatic effects by human T cell-mediated immunotherapy. This unique technology may serve as a precision medicine platform for assessing anticancer effects of cellular immunotherapy in vivo before administration to human cancer patients.

Description: Natural killer (NK) cell killer immunoglobulin-like receptor (KIR) interactions with self MHC class I molecules can regulate NK cell function; such interactions typically inactivate NK cells potentially providing a dominant mechanism through which malignant cells evade host NK cell-mediated immunity. Recently we found that the proteasome inhibitor bortezomib up-regulated surface expression of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) on a variety of different human malignant cells rendering them susceptible to NK cell-mediated apoptosis in vitro; this effect appears to override KIR ligand-mediated NK cell inactivation, overcoming tumor resistance to both allogeneic KIR ligand-matched and autologous NK cell cytotoxicity. We also found that murine tumors were sensitized by bortezomib to the cytotoxic effects of syngeneic NK cells; the killing of RENCA and LLC1 tumors in vitro by syngeneic BALB/c and C57BL/6 NK cells respectively was enhanced when tumors were exposed to 10nM of bortezomib for 18h. Here, we show that the combined treatment of bortezomib followed by syngeneic NK cell infusions significantly delays tumor growth in tumor bearing animals. While treatment with bortezomib or interleukin-2 activated syngeneic NK cells alone had little effect on tumor growth, the combined treatment significantly delayed growth of RENCA tumors in BALB/c mice and LLC1 in C57BL/6 mice (p

Description: Previous studies have shown that granulocyte colony stimulating factor (G-CSF) mobilization skews T-cells toward a type 2 cytokine profile, potentially impacting GVHD and other immune mediated events that occur after allogeneic hematopoietic stem cell transplantation (HCT). AMD3100, a selective antagonist of CXCR4, rapidly mobilizes hematopoietic progenitor cells into the circulation, has a synergistic effect on CD34+ cell mobilization when combined with G-CSF, and is currently being evaluated as a single agent to mobilize allografts. Apheresis collections mobilized with a single injection of AMD3100 contain a similar number of T-cells as those collected following 5 daily doses of G-CSF. We investigated whether T-cells mobilized with AMD3100 undergo changes in cytokine polarization status as described to occur with G-CSF mobilization. Using real time PCR, we investigated the expression of 84 genes associated with TH1, TH2, and TH3 T-cell pathways at baseline and following mobilization with a single injection of AMD3100 (dosed at 240 or 320 mcg/kg; n=12 subjects) or following 5 daily doses of G-CSF(n=5 subjects). RNA was extracted from CD3+ T-cells isolated using immunomagnetic beads (〉95% purity) from PBMCs collected immediately before mobilization and 6 hours after AMD3100 administration or 5 days after G-CSF mobilization. The RT2 Profiler ™ PCR Array was used which contains pathway specific cytokine genes associated with TH1, TH2, and TH3 cells. Expression levels of 16 genes changed significantly (false discovery rate=0.10) from baseline following G-CSF mobilization; 9 genes were up-regulated and 7 genes were down-regulated from baseline. Five up-regulated and 4 down-regulated genes had greater than a 2-fold change in expression (Figure). In contrast, none of the 84 genes examined, including the 16 altered with G-CSF, changed significantly following AMD3100 administration. Our results are concordant with current literature that shows the expression of several genes effecting T-cell cytokine polarization are altered in G-CSF mobilized T-cells. It has been suggested that the TH2 polarization in G-CSF mobilized products contributes to the comparable incidence of acute GVHD and the higher incidence of chronic GVHD compared to bone marrow allografts. In contrast, T-cells mobilized with AMD3100 appear similar to non-mobilized T-cells, and do not undergo a change in TH1- and TH2-related gene expression. Whether the differences in cytokine polarization of T-lymphocytes mobilized with AMD3100 compared to G-CSF will impact immune reconstitution or other immune sequela (i.e. GVHD, graft-vs.-tumor) associated with HCT is currently being assessed in a pilot allogeneic transplantation trial in humans using AMD3100 to mobilize donors. Figure: Heat map showing expression levels of 16 genes in CD3+ T-cells that changed significantly from baseline following G-CSF mobilization in 5 healthy donors. Samples were analyzed with a two-sample paired t-test, and the corresponding p-values were evaluated based on the permutation technique at a 10% false discovery rate. All samples were normalized to the center of the mean of the pre G-CSF samples with black denoting up-regulated expression and white denoting down-regulated expression. Figure:. Heat map showing expression levels of 16 genes in CD3+ T-cells that changed significantly from baseline following G-CSF mobilization in 5 healthy donors. Samples were analyzed with a two-sample paired t-test, and the corresponding p-values were evaluated based on the permutation technique at a 10% false discovery rate. All samples were normalized to the center of the mean of the pre G-CSF samples with black denoting up-regulated expression and white denoting down-regulated expression.

Description: Ligation of inhibitory receptors renders natural killer (NK) cells inactive against autologous tumors. Recently, the proteasome inhibitor bortezomib was shown to sensitize tumors to autologous NK-cell cytotoxicity in vitro. Here, we show bortezomib augments the antitumor effects of syngeneic NK-cell infusions in tumor-bearing animals; this effect is further enhanced in regulatory T cell (Treg cell)–depleted hosts. In vitro, bortezomib-treated tumors had higher tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and perforin/granzyme-mediated caspase-8 activity, which enhanced their susceptibility to NK-cell lysis. Bioluminescence imaging of mice with established tumors showed treatment with bortezomib and syngeneic NK cells reduced tumor growth and prolonged survival compared with controls receiving bortezomib or NK cells alone. In contrast, tumor progression was not delayed when animals received bortezomib and perforin-deficient NK cells, showing drug-induced augmentation in NK-cell cytotoxicity was mediated through perforin/granzyme. Furthermore, tumor growth was slower in bortezomib-treated recipients when host Treg cells were eradicated with anti-CD25 antibody before infusing NK cells compared with mice without Treg-cell ablation (tumor doubling time, 16.7 vs 4.9 days, respectively; P = .02). These findings suggest that depletion of Treg cells followed by bortezomib-induced tumor sensitization to autologous NK cells could be used as a novel strategy to treat cancer.

Description: The activation of NK cell inhibitory receptors may limit the antitumor efficacy of adoptive autologous and allogeneic NK cell infusions. Recently, we found that exposing malignant cells to the proteosome inhibitor bortezomib upregulated surface expression of death receptors for TRAIL, resulting in significant enhancement of autologous NK cell tumor cytotoxicity in vitro and in vivo. Here we show that NK cells expanded in vitro in the presence of IL-2 and EBV-LCL feeder cells upregulate surface expression of TRAIL which significantly augments bortezomib-induced tumor sensitization to NK cell killing. CD56+/CD3– NK cells were isolated from normal donors by immuno-magnetic bead selection and were co-cultured with irradiated EBV-LCL feeder cells in X-VIVO 20, 10% human AB serum, and 500 IU/ml hrIL-2 for up to 21 days. Depending on culture conditions, a 300 to 10,000 fold increase in NK cell numbers was achieved. Non-expanded and expanded NK cells were analyzed by flow cytometry for the expression of CD56, CD16, TRAIL, FasL, NKG2D, LFA-1, perforin, and granzymes A and B at baseline and ≥ 10 days following in vitro expansion. Chromium release assays were performed to assess fresh vs. expanded NK cell cytotoxicity of renal cell carcinoma (RCC) tumor targets treated with 10 nM bortezomib for 18 hr vs. untreated RCC controls. Freshly-isolated NK cells did not express TRAIL or FasL; in contrast NKG2D, LFA-1, perforin and granzymes A and B were constitutively expressed in fresh NK cells. After expansion, there was a dramatic increase in surface expression of TRAIL and NKG2D; on fresh vs. expanded NK cells from 3 different donors, TRAIL expression increased from 0% to 80.8±15.4% (mean fluorescence intensity [MFI] of TRAIL increased from 6.0±5.1 to 37.9±3.2). The MFI of NKG2D surface expression also increased following NK cell expansion (432.0±70.9 from 48.3±16.3). Expression of LFA-1 and perforin did not change, although there was a small increase in surface and intracellular expression of FasL and granzymes A and B respectively. At a 1:1 effector to target ratio, fresh NK cells lysed 3.4± 2.1% and 5.0± 2.7% of untreated and bortezomib-treated RCC tumor cells respectively. In contrast, there was a dramatic increase in bortezomib-treated tumor susceptibility to killing by expanded NK cells; NK cells expanded for 12-18 days killed 27.6± 9.3% and 55.8± 8.3% of untreated vs. bortezomib-treated RCC tumor cells respectively. Conclusion: In vitro-expanded NK cells are phenotypically and functionally different from non-expanded NK cells. Expanded cells have increased NKG2D and TRAIL expression and greatly enhanced TRAIL-mediated tumor cytotoxicity compared to non-expanded NK cells. Based on these findings, a phase I trial investigating the safety and anti-tumor effects of escalating doses of adoptively-infused ex vivo-expanded autologous NK cells following bortezomib treatment in patients with advanced metastatic tumors and hematological malignancies has recently been initiated. Figure: Freshly isolated and expanded Nk cell lysis of renal cell carcinoma cell line with and without treatment of tumor cells with bortezomib Figure:. Freshly isolated and expanded Nk cell lysis of renal cell carcinoma cell line with and without treatment of tumor cells with bortezomib

Description: The proteasome inhibitor bortezomib was recently found to render tumor cells susceptible to natural killer (NK) cell-mediated apoptosis in vitro and in vivo. This sensitization appears to occur as a consequence of this agent up-regulating surface expression of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) on human malignant cells rendering them susceptible to TRAIL-mediated NK cell cytotoxicity. We hypothesized that bortezomib would likewise sensitize tumors to the cytotoxic effects of antigen specific T-cells through similar apoptotic pathways, thereby providing an incentive to use bortezomib as a universal immune-sensitizing agent. The HLA-A2+, gp100+, MART-1+ melanoma cell lines 526 and 624 were treated with 10nM bortezomib for 18 hrs then were analyzed by FACS for expression the cell surface markers (HLA-ABC, MIC-A/B, TRAIL-R1/2 and Fas) and Cr51 cytotoxicity assay for susceptibility to CD8+/HLA-A2+ restricted gp100 and MART-1 specific CTL-mediated lysis. As observed previously, NK cell-mediated apoptosis was significantly higher in tumor cells treated with bortezomib compared to untreated tumor cells. In contrast, an unanticipated and significant reduction in CTL-mediated cytotoxicity was observed in tumors treated with bortezomib compared to untreated tumors; at an effector:target ratio of 3:1, NK cell cytotoxicity increased from 43±2% to 70±2% (p

Description: Key Points Cytokine-activated NK cells display distinct gene expression programs in response to cytokine withdrawal. IL-15 sustains antitumor functions of NK cells through mTOR-governed metabolic processes.

Description: Graft-vs-tumor (GVT) effects following NMHCT induce disease regression in a subset of patients (pts) with advanced metastatic kidney cancer. At present, little is known about the antigens serving as tumor targets in responding pts. In an effort to characterize GVT effectors and their tumor antigens, we generated RCC cell lines to use as targets in cytotoxicity assays in three pts (one non-responder and two responders) undergoing a cyclophosphamide/fludarabine-based NMHCT from HLA matched siblings. Peripheral blood lymphocytes (PBL) were collected from pts at multiple time-points after transplantation and were expanded in-vitro with either irradiated patient (pre-transplant) PBL/EBV-LCL or autologous tumor cells. RCC pt #1 developed grade II skin GVHD on day 22 but did not have an objective tumor response and died from progressive tumor on day 203. In a Cr51 release assay, minor histocompatibility antigen specific (mHa) T-cell lines generated using pre-transplant pt PBL/EBV-LCL stimulators lysed 98% of pt CD40-ligand stimulated B cells (CD40L-B) but did not kill pt RCC cells. RCC pt #2 developed grade II skin GVHD on Day 51 and was noted to have regression of lung metastasis on day 183. Using PBL obtained during tumor regression, mHa- reactive T-cell lines were generated (pre-transplant pt PBL/EBV-LCL used as stimulators) that lysed 76% and 12% of pt EBV-LCL and autologous RCC tumor cells at a 20:1 E:T ratio. Following limiting dilution cloning, 6 CD8+ T cell clones were expanded that killed pt EBV-LCL (but not donor) including one MHC class-1 restricted T cell clone that lysed both pt EBV-LCL and pt tumor cells. RCC pt #3 developed grade III skin GVHD on day 120, had regression of lung metastasis on day 160, and survives more than 4 years after transplantation. PBL collected from this pt 40 months after transplant contained CD8+ T-cell populations that secreted IFN-g (0.9% by intracellular cytokine staining) when co-cultured with pt RCC cells but not after co-culture with pt CD40L-B cells. CD3+/CD8+ CTL were expanded from these PBL using pt RCC cells as stimulators; in vitro, these CTL killed the pt’s RCC cells but did not kill pt fibroblasts or pt EBV-LCL (Figure A). Following co-culture with tumor, intracellular IFN-g staining combined with TCR Vb antibodies revealed 3 tumor reactive CD8+ T-cell populations (TCR Vb7+, TCRVb5.1+, and TCRVb non-staining); 25.2% of these CTL were TCRVb7+. IFN-g secretion by TCR Vb7+ T cells was blocked when tumor cells were pre-incubated with mAbs to CD8, pan MHC class I and HLA-A11 (Figure B) consistent with recognition of an HLA-11 restricted tumor antigen. Following limiting dilution cloning, several CD8+ T-cell clones (both TCR Vb7+ and TCR Vb non-staining) with RCC-specific cytotoxicity were identified. We conclude that donor T-cells with both broad alloreactivity and tumor specificity play a role in mediating GVT effects in RCC pts having disease regression following NMHCT. Figure Figure

Description: Killer immunoglobulin-like receptor (KIR)-HLA ligand mismatches in the graft-versus host direction (i.e. the recipient lacks an inhibitory HLA ligand for one or more donor KIRs) appears to be associated with improved outcome in patients (pts) receiving HLA-matched or mismatched T cell-depleted HCT. We investigated the impact of genotypic KIR-ligand mismatches in pts undergoing T cell-replete HCT from an HLA-identical or 5/6 antigen-matched related donor following nonmyeloablative conditioning. Donor KIR genotype (KIR2DL1,KIR2DL2/3,KIR3DL1) and HLA-B and C genotypes were analyzed in 92 pts who received an unmanipulated, G-CSF mobilized peripheral blood HCT from a 6/6 (n=88) or 5/6 (n=4) HLA-matched related donor following conditioning with cyclophosphamide (120mg/m2) and fludarabine (125mg/m2). The conditioning regimen included anti-thymocyte globulin (160mg/kg) for pts at increased risk for graft rejection (heavily transfused, 5/6 HLA-matched donors, or non-sibling related donors, n=19). Indications for transplantation included metastatic renal cell cancer (RCC, n= 37), treatment refractory solid tumors (n=26), hematologic malignancies (n=9) and non-malignant hematologic disorders (n=20). Cyclosporine, alone (n=11) or in combination with either mycophenolate mofetil (n=33) or methotrexate (n=48) was used as graft versus host disease (GVHD) prophylaxis. Sixty-eight donors (73%) demonstrated genotypic evidence of at least one KIR for which the pt lacked a corresponding inhibitory HLA C or B ligand (‘missing KIR ligand’). There was a trend towards a higher incidence of grades 3–4 acute GVHD (37% vs. 17%, p=0.08) and a significantly higher incidence of steroid refractory GVHD (16% vs 0%, p=0.02) in pts lacking an HLA ligand for one or more donor KIR. The ‘missing KIR ligand’ effect on response rate was further evaluated in a subset of pts with metastatic RCC undergoing HCT. An HLA ligand for one or more donor KIR was absent in 28/38 (76%) RCC pts. A higher objective response rate (PR or CR) was seen in pts with one or more missing KIR-ligands (12/28, 43%) compared to those whose HLA genotype would predict inhibition of all donor KIRs (2/9, 22%, p=0.24). This effect was most pronounced in pts homozygous for HLA Bw6 who lacked HLA Bw4 ligands capable of inhibiting donor KIR3DL1. Compared to pts not homozygous for HLA-Bw6, Bw6 homozygous RCC pts had a higher response rate (24% vs 58%; p=0.047) and significantly prolonged survival (median 419 vs 1496 days; p=0.005) following HCT. A univariate analysis considering patient age, acute and chronic GVHD, and KIR-HLA ligand mismatches (C mismatch, B mismatch, or 〉 1 mismatch) showed that a missing HLA-Bw4 ligand and the absence of a ligand for 〉 1 donor KIR were each associated with improved survival in RCC patients; in a multivariate analysis, absence of an HLA Bw4 ligand for donor KIR3DL1 was the only factor that remained significantly associated with improved survival (p=0.015, OR 0.276, CI 0.097–0.781). Conclusion: KIR-HLA ligand mismatches appear to impact multiple transplant outcomes including the incidence and severity of acute GVHD, tumor response and survival following T-cell replete nonmyeloablative transplantation from HLA-matched related donors. Evaluation of a larger cohort of patients to confirm these findings is currently underway.