Chromium Release Assay Research Articles

Analyses of a Pair of Concordant Twins with Infant ALL and Discordant Clinical Outcome Reveals Immunoescape As a Mechanism of Disease Persistence in MLL-Rearranged Leukemia

MLL-fusion is the most common genetic abnormality in acute lymphoblastic leukemia (ALL) of infancy and occurs in approximately 80% of the cases. Infant ALL represents a biologically distinctive entity with a highly immature pro-B immunophenotype associated with a particularly unfavorable prognosis due to a high proportion of early relapses. This may be due to the survival of dormant residual disease protected by the bone marrow niche.We have characterized a pair of monozygotic twin sisters diagnosed with ALL in early infancy. Tumor cells from both children carried the t(11;19) translocation (MLL-ENL fusion) and both patients were treated according to the ALL-BFM 2000 protocol. Despite good initial treatment responses in both twins, one sister developed very early relapse (twin A) and succumbed to the disease. The other went into continuous complete remission (twin B) and, as of today, is alive after 9 years. The clinical data of the 2 patients are presented in Table 1.Diagnostic bone marrow (BM) aspirates of both sisters were injected into the femoral bones of NOD SCID gamma (NSG) mice. Survival of xenografted mice bearing twin A was significantly longer due to a lower proliferative capacity of twin A as compared to twin B cells. In vivo Bromodeoxyuridine (BrdU) assays revealed that twin B cells were proliferating equally fast in BM and spleen, while in twin A cells, the BM markedly suppressed entry into S-phase. Flow cytometry from xenograft BM identified a large CD34+ population in twin A cells that was almost absent in twin B cells. Furthermore, BM of twin A xenografts showed a CD34+/CD38-/CD19- stem cell like population undetectable in twin B animals. Most interestingly, injection of minimal residual disease (MRD) negative remission bone marrow (PCR for Ig-gene rearrangements and the MLL-fusion gene) of both sisters into NSG mice resulted in a full-blown xenograft leukemia in animals bearing twin A but not twin B. Taken together, these data suggest that fatal relapse in twin A may be due to a quiescent stem cell like population kept in check by unknown mechanisms.Next, we performed gene expression analyses on xenografted leukemias from twin A (initial, leukemia amplified from remission BM, relapse) and from twin B (initial). STRING analysis of gene expression differences revealed that, amongst other findings related to cell cycle regulation and DNA-repair, twin A cells downregulated genes indicating a reduced interferon pathway activity (CXCL10, IFI30, TRAIL, STAT1, OAS1, MX1) and several genes connected to TYRO protein kinase binding protein (TYROBP) shown previously to regulate the activity of natural killer (NK) cells. This suggests that twin A cells may evade immunosurveillance as a mechanism of disease persistence. 51Chromium-release assays with IL-2 stimulated allogeneic NK cells from two healthy donors and the xenografted twin cells showed that twin A cells were significantly less sensitive to NK cell mediated lysis as compared to twin B cells. Whether the tumor cells also display differential susceptibility to antibody-dependent cell-mediated cytotoxicity is currently investigated.While the extensive genomic characterization of the twin leukemias is under way, we show evidence from xenograft experiments, gene expression profiles and functional in vitro experiments that dormant residual cells in MLL-rearranged ALL can evade immunosurveillance. These findings serve as a rationale to employ immunotherapeutic approaches in infant ALL patients in order to improve their dismal prognosis. Finally, we report the first monozygotic twin pair with MLL-rearranged ALL and discordant clinical outcomes. This provides a unique opportunity and a model to gain insight into the clonal composition of infant leukemia and the origins of leukemia relapse.Table 1:Patient characteristics of the MLL-ENL positive twin pair. WBC, white blood cells; BM, bone marrow; CNS, central nervous system; MRD, minimal residual disease.ParameterTwin ATwin BAge at diagnosis (days)98147WBC (initial)/µl334.000103.000Blasts (Peripheral Blood) %9791Blasts (BM) %9897CNS involvementNot determinedNegativeImmunophenotypePro-BPro-BCytogeneticst(11;19)t(11;19)Prednisone-ResponsePoorGoodBlasts (BM), day 15 %321MRD day 33NegativeNegativeMRD day 78Negative<10-4 *Event-free survival227 days>9 yearsTime to relapse227 daysn/aOverall survival390 days>9 years*low positive, not quantifiable DisclosuresNo relevant conflicts of interest to declare.

Switching from IgG1 to IgG3 Isotype to Boost Complement-Activating Capacities of Therapeutic Antibodies – Impact of CD55 Expression on the Mode of Action

Introduction: Complement-dependent cytotoxicity (CDC) against tumor cells is supposed to contribute to clinical efficacy of therapeutic CD20 antibodies of IgG1 isotype. However, some tumor-targeting antibodies such as cetuximab do not trigger CDC. Due to its prominent molecular flexibility and its longer hinge region human IgG3 gains advantage over human IgG1 in promoting activation of the complement system. Here, we investigated the cytotoxic activity of IgG3 isotype switch variants of the therapeutic antibodies rituximab (C2B8) and cetuximab (C225).Methods: CDC against tumor cells was assessed by 3 hours 51chromium release assays utilizing 25% v/v human serum. Antibody mediated deposition of C1q, C4b, C3b, factor Bb or C5b-9 on tumor cells as well as the release of anaphylatoxins C3a, C4a and C5a were analyzed by flow cytometry.Results: Both IgG1 and IgG3 variants of rituximab or cetuximab demonstrated similar efficiency in CD20 or EGFR binding, respectively. In the case of CD20 antibodies, C2B8-IgG3 induced stronger CDC against CD20 low-expressing cells, especially against freshly isolated CLL cells, than C2B8-IgG1, while no difference between both isotypes was detected against CD20 high-expressing cells. Importantly, CDC was also significantly triggered in an autologous setting against freshly isolated CLL cells by the IgG3 but not by the IgG1 version of rituximab. Furthermore, in the case of EGFR-targeting antibodies, only anti-EGFR-IgG3 but not anti-EGFR-IgG1 triggered significant CDC against some, but not all tested cell lines. CDC triggered by anti-EGFR-IgG3 negatively correlated with expression levels of the membrane-bound complement-regulatory proteins (mCRPs) CD55 and CD59, but not CD46. Notably, anti-EGFR-IgG3 promoted strong C1q and C3b but relatively low C4b and C5b-9 deposition on target cells. Furthermore, anti-EGFR-IgG3 triggered C4a release on all tested cell lines, but failed to induce C3a and C5a release on CD55/CD59 highly expressing cell lines. RNAi-induced knock-down or over-expression of mCRPs revealed CD55 expression to be a pivotal determinant of anti-EGFR-IgG3 triggered CDC and to force a switch from classical complement pathway activation to C1q-dependent alternative pathway amplification.Conclusions: In conclusion, an isotype switch from human IgG1 to human IgG3 is accompanied by improvement of complement activating capacities of CD20 as well as EGFR targeting antibodies. However, despite of its strong C1q fixing capacity, anti-EGFR-IgG3 lacks the ability to efficiently deposit C4b on targets’ cell surfaces and thus to initiate the complement cascade on CD55 high-expressing tumor cells. These novel findings revealed human IgG3 directed against CD20 or EGFR to encompass improved cytotoxic potential with low CD55 expression on tumor cells potentially being a promising biomarker for this novel approach. DisclosuresNo relevant conflicts of interest to declare.

Targeting AML Using an Fc-Engineered BST1/CD157 Monoclonal Antibody

Antibody-based immunotherapy represents a promising strategy to target and eliminate acute myeloid leukemia (AML) tumor cells. We evaluated BST1/CD157, a novel, slow internalizing surface antigen, for its suitability as immunotherapeutic target in AML. An Fc-optimized antibody against BST1/CD157 was developed (MEN1112/OBT357NF), which binds to the Fc-receptor on natural killer (NK) cells with enhanced affinity. The target antigen expression profile of BST1/CD157 on AML bulk cells as well as on leukemic stem cells (LSC) was analyzed with flow cytometry using a commercially available antibody. BST1/CD157 expression was detected in > 97% of AML patients at primary diagnosis (n=81) and at time of relapse (n=20) using specific fluorescence intensity (SFI, positivity: SFI > 1. 5). Importantly, BST1/CD157 was also expressed on CD34+/CD38-cells (n=20), the compartment with a high percentage of LSCs.Functional titration experiments of MEN1112/OBT357NF showed antibody-mediated lysis of U937 cells in the low picomolar range (EC50 = 30–140 pM) and demonstrated superior ADCC activity compared to its non Fc-engineered parental analogue. To evaluate the additive effect of the complement cascade, healthy donor (HD) monocytes were co-incubated with autologous NK cells and MEN1112/OBT357NF in the presence or absence of 5% donor serum. After 20 hours of incubation, a significantly enhanced lysis efficacy by MEN1112/OBT357NF through the addition of serum compared to control cultures without serum (p=0.02) was demonstrated.For further functional characterization of MEN1112/OBT357NF standard chromium release assays using AML cell lines and NK cells from HDs were performed. In this setting, efficient target cell lysis (30–50%) at various effector cell:target (E:T) ratios (50:1–6:1) was observed. Compared to Rituximab-mediated lysis of RAJI cells, MEN1112/OBT357NF triggered more efficient target cell lysis at much lower effector cell concentrations (33% lysis of OCI-AML3 cells vs. 11% lysis of RAJI cells at E:T 6:1).Furthermore, the cytotoxic potential of NK cells from AML patients after intense double induction chemotherapy on AML cell lines was tested. The results were highly variable with efficient lysis in about one third of samples tested (n=10). As a positive control NK cells from HDs were used which showed superior lysis as compared to patient-derived NK cells. In an autologous set-up using NK cells from either primary diagnosis or at time of remission, we could show efficient lysis of primary AML cells in one of two patient samples (patient in complete remission) in the presence of MEN1112/OBT357NF.In summary, our analysis demonstrates that BST1/CD157 is a promising target antigen for antibody-based therapy in AML. The effective in-vitro ADCC activity supports further development of MEN1112/OBT35NF as an immunotherapy for patients with AML. DisclosuresKrupka:Oxford BioTherapeutics Ltd: Research Funding. Jansen:Oxford BioTherapeutics Ltd: Research Funding. Aud:Oxford BioTherapeutics Ltd: Employment. Dusek:Oxford BioTherapeutics Ltd: Employment. Bisht:Oxford BioTherapeutics Ltd: Employment. Pombo-Villar:Oxford BioTherapeutics Ltd: Employment, Equity Ownership. Rohlff:Oxford BioTherapeutics Ltd: Employment. Subklewe:Oxford BioTherapeutics Ltd: Research Funding.

Defining cell-surface antigenic markers for mouse T and B cells.

Defining cell-surface antigenic markers for mouse T and B cells.

Imaging-based, non-radioactive measurements of natural killer cell activity provides comparable results as classic chromium release assays but with fewer cells and less labor

Natural Killer Cell activity has traditionally been assessed by detecting the lysis of tumor cells in The Chromium Release assay. This classic assay relies on radioactive labels, is laborious, and requires substantial quantities of patient blood. Moreover the Chromium Release assay provides low signal to noise ratios. We have developed an assay that can visualize individual target cells to detect cytolytic activity within a high signal to noise range, without involving radioactivity, via high-throughput imaging. We also developed a miniaturized version of this assay to measure NK activity with one tenth of the blood needed for a classic Chromium Release assay, and with less labor.

Immunomodulatory activity of Dioscorea membranacea Pierre rhizomes and of its main active constituent Dioscorealide B

BackgroundThe rhizomes of Dioscorea membranacea Pierre, also called Hua-Khao-Yen by Thai name, are used as ingredients in many Thai traditional medicines for the alternative or complementary treatment of cancer and AIDs. Preliminary in vitro studies have indicated that D. membranacea extracts exhibited high cytotoxic activity with several cancer cell lines, but the underlining mechanisms are far from clear. The aims of this study were to investigate the effects of ethanolic and aqueous crude extracts from D. membranacea Pierre, and pure compound from D. membranacea Pierre, Dioscorealide B, on natural killer cells activity and on lymphocyte proliferation.MethodsImmunomodulatory activity was investigated using PBMCs from healthy donors. NK cells activity was performed by the chromium release assay using PBMCs as effector cells, and K562 cells line labelled with chromium as target cells. Lymphocyte proliferation was determined by 3H-thymidine uptake. The degree of activation was expressed as the stimulation index.ResultsThe crude ethanolic extracts of D. membranacea Pierre significantly stimulated NK cells activity against K562 cells line at lower concentrations of 10 and 100 ng/ml, but not at higher concentrations. The ethanolic extracts showed no observable effect on lymphocyte proliferation. The crude water extracts significantly increased NK cell activity at concentrations of 10 ng/ml, 100 ng/ml, 1 μg/ml, 10 μg/ml and 100 μg/ml, and also activated lymphocyte proliferation at concentration of 1 ng/ml, 10 ng/ml, 100 ng/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml and 100 μg/ml. However, Dioscorealide B had no significant effect at lower concentrations (0–1 μg/ml and 0–0.1 μg/ml, respectively) on NK cell activity and lymphocyte proliferation. In fact higher concentrations (>10 μg/ml and >0.5 μg/ml) of Dioscorealide B cause a significant decrease in NK cell activity and lymphocyte proliferation.ConclusionsD. membranacea Pierre stimulated NK cells activity and lymphocyte proliferation, but Dioscorealide B either had no effect, and at higher concentrations decreased NK cell activity and lymphocyte proliferation. Our results suggest that both extracts of D. membranacea Pierre significantly increases immune function, but the underlining mechanism is not clearly understood.

Immunogenicity evaluation of a rationally designed polytope construct encoding HLA-A*0201 restricted epitopes derived from Leishmania major related proteins in HLA-A2/DR1 transgenic mice: steps toward polytope vaccine.

BackgroundThere are several reports demonstrating the role of CD8 T cells against Leishmania species. Therefore peptide vaccine might represent an effective approach to control the infection. We developed a rational polytope-DNA construct encoding immunogenic HLA-A2 restricted peptides and validated the processing and presentation of encoded epitopes in a preclinical mouse model humanized for the MHC-class-I and II.Methods and FindingsHLA-A*0201 restricted epitopes from LPG-3, LmSTI-1, CPB and CPC along with H-2Kd restricted peptides, were lined-up together as a polytope string in a DNA construct. Polytope string was rationally designed by harnessing advantages of ubiquitin, spacers and HLA-DR restricted Th1 epitope. Endotoxin free pcDNA plasmid expressing the polytope was inoculated into humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice intramuscularly 4 days after Cardiotoxin priming followed by 2 boosters at one week interval. Mice were sacrificed 10 days after the last booster, and splenocytes were subjected to ex-vivo and in-vitro evaluation of specific IFN-γ production and in-vitro cytotoxicity against individual peptides by ELISpot and standard chromium-51(51Cr) release assay respectively. 4 H-2Kd and 5 HLA-A*0201 restricted peptides were able to induce specific CD8 T cell responses in BALB/C and HLA-A2/DR1 mice respectively. IFN-γ and cytolytic activity together discriminated LPG-3-P1 as dominant, LmSTI-1-P3 and LmSTI-1-P6 as subdominant with both cytolytic activity and IFN-γ production, LmSTI-1-P4 and LPG-3-P5 as subdominant with only IFN-γ production potential.ConclusionsHere we described a new DNA-polytope construct for Leishmania vaccination encompassing immunogenic HLA-A2 restricted peptides. Immunogenicity evaluation in HLA-transgenic model confirmed CD8 T cell induction with expected affinities and avidities showing almost efficient processing and presentation of the peptides in relevant preclinical model. Further evaluation will determine the efficacy of this polytope construct protecting against infectious challenge of Leishmania. Fortunately HLA transgenic mice are promising preclinical models helping to speed up immunogenicity analysis in a human related mouse model.

Solitomab, an epithelial cell adhesion molecule/CD3 bispecific antibody (BiTE), is highly active against primary chemotherapy-resistant ovarian cancer cell lines in vitro and fresh tumor cells ex vivo.

Solitomab is a novel, bispecific, single-chain antibody that targets epithelial cell adhesion molecule (EpCAM) on tumor cells and also contains a cluster of differentiation 3 (CD3) (T-cell coreceptor) binding region. The authors evaluated the in vitro activity of solitomab against primary chemotherapy-resistant epithelial ovarian carcinoma cell lines as well as malignant cells in ascites. EpCAM expression was evaluated by flow cytometry in 5 primary ovarian cancer cell lines and in 42 fresh ovarian tumor cell cultures in ascites from patients with mainly advanced or recurrent, chemotherapy-resistant disease. The potential activity of solitomab against EpCAM-positive tumor cells was evaluated by flow cytometry, proliferation, and 4-hour chromium-release, cell-mediated cytotoxicity assays. EpCAM expression was detected by flow cytometry in approximately 80% of the fresh ovarian tumors and primary ovarian tumor cell lines tested. EpCAM-positive, chemotherapy-resistant cell lines were identified as resistant to natural killer cell-mediated or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean±standard error of the mean, 3.6%±0.7% of cells killed after incubation of EpCAM-positive cell lines with control bispecific antibody). In contrast, after incubation with solitomab, EpCAM-positive, chemotherapy-resistant cells became highly sensitive to T-cell cytotoxicity (mean±standard error of the mean, 28.2%±2.05% of cells killed; P<.0001) after exposure to peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with EpCAM-expressing malignant cells in ascites with solitomab resulted in a significant increase in T-cell activation markers and a reduction in the number of viable ovarian tumor cells in ascites (P<.001). Solitomab may represent a novel, potentially effective agent for the treatment of chemotherapy-resistant ovarian cancers that overexpress EpCAM.

Evaluation of natural killer cells activity by chromium release assay in cyclophosphamide treated CD-1 mice

Evaluation of natural killer cells activity by chromium release assay in cyclophosphamide treated CD-1 mice

Flow cytometry-based assay to study HIV-1 gp120 specific antibody-dependent cellular cytotoxicity responses

Increased attention on the role of Fc-mediated effector functions against HIV-1 has led to renewed interest into the role that antibody-dependent cellular cytotoxicity (ADCC) could play in controlling viral transmission and/or the rate of disease progression. While 51Chromium release assays have traditionally been used to study ADCC responses against HIV-1, a number of alternative flow-cytometry-based assays were recently developed. In this study, an alternative flow-cytometry-based assay was established to allow non-radioactive measurement of ADCC-mediated elimination of HIV-1 gp120 envelope glycoprotein (Env)-coated target cells. This assay relies on staining target and effector cells with different dyes, which allows precise gating and permits the calculation of the number of surviving target cells by normalization to flow-cytometry particles. By using small concentrations of recombinant gp120 Env, suitable targets cells that recapitulate the ADCC response mediated against HIV-1-infected cells were generated. Finally, this method was applied successfully to screen human sera for ADCC activity directed against HIV-1 gp120 Env.

Decreased expression of NKG2D, NKp46, DNAM-1 receptors, and intracellular perforin and STAT-1 effector molecules in NK cells and their dim and bright subsets in metastatic melanoma patients.

Although natural killer (NK) cells play an important antitumor role, melanoma cells may affect their effector functions. In this study, we analyzed the expression of various receptors and effector molecules in NK cells and their subsets in metastatic melanoma (MM) patients compared with healthy controls (HCs). In HC and MM patients, we analyzed NK cell activity using a chromium release assay and the expression of CD107a degranulation marker, activating NKG2D, NKp46, DNAM-1, and inhibitory CD158a and CD158b receptors, IL-12R beta 1, IL-12R beta 2, intracellular interferon (IFN)-γ, perforin, and STAT-1 in CD3-CD56+ NK cells, and cytotoxic CD3-CD56 and immunoregulatory CD3-CD56 subsets by flow cytometry. MM patients compared with HC not only had significantly decreased NK cell activity, lower expression of CD107a, and impaired IFN-γ production but also had decreased expression of activating NKG2D, NKp46, and DNAM-1 receptors, which was followed by lower expression of perforin, STAT-1, and both IL-12R subunits in NK cells. In MM patients only, there was a positive correlation between NKG2D expression and degranulation capacity, as well as IFN-γ production in NK cells. Analysis of the expression of various parameters of NK cell effector functions between MM patients with different localization of distant metastases showed that patients in the unfavorable M1c subclass had decreased expression of NKG2D and NKp46 on NK cells compared with patients in the M1a+b group. Downregulated NKG2D, NKp46, and DNAM-1 receptors associated with impaired NK cell effector function are important biomarkers of advanced disease with a poor prognosis in melanoma patients.

Redirection of CD4+ and CD8+ T lymphocytes via a novel antibody-based modular targeting system triggers efficient killing of PSCA+ prostate tumor cells.

There is still a need for new therapeutic options against prostate cancer. Conventional single-chain bispecific antibodies (bsAbs), that directly cross-link T cells and tumor cells, hold great potential for efficient tumor treatment. However, rapid development of novel bsAbs is hampered by laborious optimization to improve their efficacy and reduce potential side effects. To accelerate the development of a novel antibody tool for the redirection of T cells to different tumor-associated antigens, we recently introduced a modular targeting system. We here describe a novel modular system for treatment of prostate cancer by retargeting of T cells to the prostate stem cell antigen (PSCA). Functionality of the novel PSCA-specific modular system was investigated in vitro by T cell activation and chromium release assays as well as in immunodeficient mice. Similar to a conventional bsAb CD3-PSCA, the novel PSCA-specific modular system induces activation of both CD4+ and CD8+ T cells leading to secretion of pro-inflammatory cytokines and highly efficient target-specific tumor cell lysis. The novel TM was ready-to-use from the time point of construction and functional at low E:T ratios and picomolar concentrations without further optimization. In addition, the PSCA-specific modular system delays outgrowth of s.c. tumors in mice comparable to bsAb CD3-PSCA. We have developed a novel PSCA-specific modular system which triggers an efficient T cell-mediated killing of PSCA+ tumor cells in vitro and in vivo. The new Ab-based targeting strategy can functionally replace conventional bsAbs and allows a flexible redirection of T cells to different tumor-associated antigens.

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting.

An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and natural killer cells. The addition of Adeno-CD40L further improved the median survival of animals to 80 ± 15 days and this effect was abolished only when using anti-CD8 antibodies. Chromium-51 (51Cr) release assay showed cytotoxic T lymphocyte (CTL) activation, suggesting efficient dendritic cell (DC) activation with CTL activation after the treatment. Furthermore, when surviving mice were rechallenged with BNL-ETK cells on the foot pad, RT + HSV-TK/GCV + Flt3L + CD40L-treated mice developed a small tumor on day 56 but the tumor eventually disappeared after 105 days. Mice treated with RT + HSV-TK/GCV + Flt3L showed a slowed tumor growth curve compared with untreated mice. Therefore, combination therapy using Flt3L to induce DC proliferation and CD40L to enhance DC maturation holds great promise for immunomodulation of radiation therapy to enhance HCC tumor control and prevent progression of disease in patients with diffuse HCC.

Embryonic stem cell‐derived haematopoietic progenitor cells down‐regulate the CD3 ξ chain on T cells, abrogating alloreactive T cells

Murine embryonic stem (ES) cell-derived haematopoietic progenitor cells (HPCs) engraft and populate lymphoid organs. In vivo, HPCs engraft across MHC barriers protecting donor-type allografts from rejection. However, the underlying phenomenon remains elusive. Here, we sought to determine the mechanism by which ES cell-derived HPCs regulate alloreactive T cells. We used the 2C mouse, which expresses a transgenic T-cell receptor against H2-L(d) to determine whether HPCs are deleted by cytotoxic T lymphocytes (CTLs). Previously, we reported that HPCs express MHC class I antigens poorly and do not express class II antigens. In vitro stimulated 2C CTLs failed to lyse H2-L(d) HPCs in a standard 4-hr (51) chromium release assay. Similarly, when the HPCs were tested in an ELISPOT assay measuring the release of interferon-γ by CTLs, HPCs failed to induce CTL degranulation. In addition, mice that were injected with HPCs showed a marked decrease in T-cell responses to alloantigen and CD3 stimulation, but showed a normal response to PMA/ionomycin, suggesting that HPCs impaired T-cell signalling through the T-cell receptor/CD3 complex. Here, we show that HPCs secrete arginase, an enzyme that scavenges l-arginine, leading to metabolites that down-regulate CD3 ζ chain. Indeed an arginase inhibitor partially restored expression of the CD3 ζ chain, implicating arginase 1 in the down-regulation of T cells. This previously unrecognized property of ES cell-derived HPCs could positively enhance the engraftment of ES cell-derived HPCs across MHC barriers by preventing rejection.

T-DM1, a novel antibody-drug conjugate, is highly effective against primary HER2 overexpressing uterine serous carcinoma in vitro and in vivo.

Amplification of c-erbB2 has been reported in over 30% of uterine serous carcinoma (USC) and found to confer poor survival because of high proliferation and increased resistance to therapy. In this study, we evaluated for the first time Trastuzumab emtansine (T-DM1), a novel antibody–drug conjugate, against multiple epidermal growth factor receptor-2 (HER2)-positive USC cells in vitro followed by developing a supportive in vivo model. Fifteen primary USC cell lines were assessed by immunohistochemistry (IHC) and flow cytometry for HER2 protein expression. C-erbB2 gene amplification was evaluated using fluorescent in situ hybridization. Sensitivity to T-DM1 and trastuzumab (T)-induced antibody-dependent cell-mediated cytotoxicity was evaluated in 5-h chromium release assays. T-DM1 and T cytostatic and apoptotic activities were evaluated using flow-cytometry-based proliferation assays. In vivo activity of T-DM1 versus T in USC xenografts in SCID mice was also evaluated. High levels of HER2 protein overexpression and HER2 gene amplification were detected in 33% of USC cell lines. T-DM1 was considerably more effective than trastuzumab in inhibiting cell proliferation and in causing apoptosis (P = 0.004) of USC showing HER2 overexpression. Importantly, T-DM1 was highly active at reducing tumor formation in vivo in USC xenografts overexpressing HER2 (P = 0.04) and mice treated with TDM-1 had significantly longer survival when compared to T-treated mice and control mice (P ≤ 0.0001). T-DM1 shows promising antitumor effect in HER2-positive USC cell lines and USC xenografts and its activity is significantly higher when compared to T. T-DM1 may represent a novel treatment option for HER2-positive USC patients with disease refractory to trastuzumab and traditional chemotherapy.

Construction and evaluation of a novel humanized HER2-specific chimeric receptor.

IntroductionThe human epidermal growth factor receptor 2 (HER2) represents one of the most studied tumor-associated antigens (TAAs) for cancer immunotherapy. The monoclonal antibody (mAb) trastuzumab has improved the outcomes of patients with HER2+ breast cancer. However, a large number of HER2+ tumors are not responsive to, or become resistant to, trastuzumab-based therapy, and thus more effective therapies targeting HER2 are needed.MethodsHER2-specific T cells were generated by the transfer of genes that encode chimeric antigen receptor (CAR). Using a multistep overlap extension PCR method, we constructed a novel, humanized HER2 CAR-containing, chA21 single-chain variable fragment (scFv) region of antigen-specific mAb and T-cell intracellular signaling chains made up of CD28 and CD3ζ. An interferon γ and interleukin 2 enzyme-linked immunosorbent assay and a chromium-51 release assay were used to evaluate the antitumor immune response of CAR T cells in coculture with tumor cells. Furthermore, SKBR3 tumor–bearing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were treated with HER2 CAR T cells to evaluate antitumor activity. Human CD3+ T cell accumulation in tumor xenograft was detected by immunohistochemistry.ResultschA21-28z CAR was successfully constructed, and both CD4+ and CD8+ T cells were transduced. The expanded HER2 CAR T cells expressed a central memory phenotype and specifically reacted against HER2+ tumor cell lines. Furthermore, the SKBR3 tumor xenograft model revealed that HER2 CAR T cells significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed robust accumulation of human CD3+ T cells in regressing SKBR3 lesions.ConclusionsThe results of this study show that novel chA21 scFv-based, HER2-specific CAR T cells not only recognized and killed HER2+ breast and ovarian cancer cells ex vivo but also induced regression of experimental breast cancer in vivo. Our data support further exploration of the HER2 CAR T-cell therapy for HER2-expressing cancers.

5-Fluorouracil and interleukin-2 immunochemotherapy enhances immunogenicity of non-small cell lung cancer A549 cells through upregulation of NKG2D ligands.

The aim of this study was to investigate the anti-cancer effects and mechanisms of immunochemotherapy of 5-fluorouracil (5-FU) and interleukin-2 (IL-2) on non-small cell lung cancer (NSCLC) A549 cells. In order to detect whether 5-FU+IL-2 could effectively inhibit tumor growth in vivo, we established an A549-bearing nude mouse model. The cytotoxicity of natural killer (NK) cells was evaluated using a standard chromium release assay. To evaluate the relevance of NK cells in 5-FU+IL-2- mediated tumor inhibitory effects, we depleted NK cells in A549-bearing mice by injecting anti-asialo-GM-1 antibodies. Effects of 5-FU+IL-2 on the expression and promoter activity of NKG2D ligands (MICA/MICB) in A549 cells in vitro were also assessed. In A549-bearing nude mice, combination therapy significantly inhibited tumor growth in comparison with monotherapy with 5-FU or IL-2 and enhanced the recognition and lysis of tumor cells by NK cells. Further study of mechanisms showed that NK cells played a vital role in the anticancer immune response of 5-FU+IL-2 immunochemotherapy. In addition, the combination therapy synergistically stimulated the expression and promoter activity of MICA/MICB. 5-FU and IL-2 immunochemotherapy significantly inhibited tumor growth and activated NK cytotoxicity in vivo, and these effects were partly impaired after depleting NK cells in tumor-bearing mice. Combination treatment of 5-FU and IL-2 upregulated the expression and the promoter activity of MICA/MICB in A549 cells, which enhanced the recognition of A549 cells by NK cells. All of the data indicated that immunochemotherapy of 5-FU and IL-2 may provide a new treatment option for patients with lung cancer.

Production of high expressing A20/DAF‐ transgenic pigs on a GGTA1‐KO background

BackgroundPig‐to‐human xenotransplantation is promising to ameliorate the worldwide shortage of suitable human donor organs. The transplantation of xenografts evokes severe immunological rejection responses. Different approaches led to considerable progress in this respect: transgenic expression of human complement regulatory proteins such as human decay‐accelerating factor (hCD55), membrane co‐factor protein (hCD46, MCP) or hCD59 and/or knockout of the porcine α1,3‐galactosyltransferase gene (GGTA1‐KO) prevent the hyperacute rejection response (HAR) [1, 2]. Moreover, human anticoagulant and/or anti‐apoptotic and anti‐inflammatory transgenes, such as human A20 (hA20) and human heme oxygenase‐1 (hHO‐1), could protect porcine xenografts from being rejected by the acute vascular rejection response [3, 4].Recently, we produced pigs transgenic for hA20 which provides cytoprotective properties in porcine aortic endothelial cells in vitro [5]. However, hA20 was expressed only at low levels in heart, skeletal muscle and porcine aortic endothelial cells. Here, we generated a new tri‐cistronic hA20/hCD55/Puro vector based on the Sleeping Beauty system (kindly provided by Dr. Zoltán Ivics) and transfected it into porcine GGTA1‐KO cells that served as donor cells for somatic cell nuclear transfer (SCNT).MethodsWe co‐transfected GGTA1‐KO [6] porcine fibroblasts with a tri‐cistronic cDNA expression vector coding for hA20/hCD55/Puro and the SB transposase 100X plasmid. The advantage of this vector is a stable genomic integration without any plasmid‐based vector backbone and the combined integration of all transgenes at a single genomic locus which avoids transgene segregation after mating. Following puromycin selection (5 μg/ml), cells were screened by PCR for genomic integration of the vector and used for SCNT.ResultsIn total, 1,028 embryos were transferred to 13 recipients of which five remained pregnant. Two pregnancies were in too early stages for ultrasound diagnosis at the time of writing. One pregnancy was terminated on day 25 of gestation and eight fetuses were obtained. The hA20 mRNA levels of these fetuses were increased 21‐ to 44‐fold compared to another line (hA20/hHO‐1/GGTA1‐KO) and even higher than in the first generation hA20 transgenic pigs. Preliminary immunofluorescence analysis data showed delta mean fluorescence intensities (MFI) for hCD55 (hCD55 MFI minus control antibody MFI) of 1,016 in average in transgenic fibroblasts in comparison to delta MFI 299 in HUVECs and 114 in PBMCs. The susceptibility of the cells to NK cell‐ and T cell‐mediated cell death compared to wild‐type fibroblasts will be determined in a 51chromium release assay. Three gilts are expected to deliver piglets in November of this year. The expression and organ distribution of hA20 and hCD55 will be characterised subsequently.ConclusionsOur goal is the production of multi‐transgenic pigs with an increased expression of xeno‐relevant transgenes. The combination of ubiquitous, high expression of anti‐apoptotic and‐inflammatory transgenes such as hA20 together with a complement regulatory protein like hCD55 on a GGTA1‐KO background has the potential to prolong the survival of porcine grafts after pig‐to‐primate xenotransplantation.

Measuring Cytotoxicity by Bioluminescence Imaging Outperforms the Standard Chromium-51 Release Assay

The chromium-release assay developed in 1968 is still the most commonly used method to measure cytotoxicity by T cells and by natural killer cells. Target cells are loaded in vitro with radioactive chromium and lysis is determined by measuring chromium in the supernatant released by dying cells. Since then, alternative methods have been developed using different markers of target cell viability that do not involve radioactivity. Here, we compared and contrasted a bioluminescence (BLI)-based cytotoxicity assay to the standard radioactive chromium-release assay using an identical set of effector cells and tumor target cells. For this, we stably transduced several human and murine tumor cell lines to express luciferase. When co-cultured with cytotoxic effector cells, highly reproducible decreases in BLI were seen in an effector to target cell dose-dependent manner. When compared to results obtained from the chromium release assay, the performance of the BLI-based assay was superior, because of its robustness, increased signal-to-noise ratio, and faster kinetics. The reduced/delayed detection of cytotoxicity by the chromium release method was attributable to the association of chromium with structural components of the cell, which are released quickly by detergent solubilization but not by hypotonic lysis. We conclude that the (BLI)-based measurement of cytotoxicity offers a superior non-radioactive alternative to the chromium-release assay that is more robust and quicker to perform.