Effector Cells In Mice Research Articles

Abstract P2-09-10: Epigenetic modulation—unlocking the potential of checkpoint inhibition in breast cancer

Abstract Immune checkpoint inhibition (ICI) has revolutionized treatment in cancers that are naturally immunogenic by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. Tumors possessing complex immunosuppressive TME's such as breast cancer present unique therapeutic obstacles as response rates to ICI remain low. Such tumors often recruit myeloid-derived suppressor cells (MDSCs) whose functioning prohibits both T cell activation and infiltration. To date, most studies focus on use of ICI in triple negative disease. Our work aims to uncover the efficacy of ICI in both early and advanced HER2 positive (HER2+) disease and to advance our understanding of how to improve response rates to these new promising therapies. We are using a HER-2/neu transgenic mouse model with tumor challenge of syngeneic cell lines to test the efficacy of different combinations of an epigenetic agent, the histone deacetylase inhibitor entinostat (ENT), checkpoint inhibitors anti-PD-1 and anti- CTLA-4, on primary and metastatic disease. We are examining treatment effects on primary tumor growth, metastatic burden, and survival. Characterization of tumor infiltrating lymphocytes and their functional capabilities are being investigated using fluorescence-activated cell sorting, gene expression profiling, and ex vivo suppression assays. Western blots, qPCR and other in vitro assays will be performed on MDSCs to investigate mechanisms behind response. In the HER2+ mouse model of early stage disease, we show that combining ENT, with ICis significantly improves survival and delays tumor growth. Preliminary data in models of advanced disease, show only ENT + a-PD-1 improves survival and metastatic burden. Conversely, in the metastatic model, ENT + a-CTLA-4 negatively effects survival and metastatic burden. In primary tumors, ENT + ICIs leads to significantly decreased suppression by granulocytic-MDSCs. However, MDSC infiltration and function is not affected in lungs containing macrometastatic disease. Interestingly, we found an increase in activated granzyme-B-producing CD8+ T effector cells in mice treated with combination therapy in both primary and metastatic tumors. Finally, gene expression profiling of MDSCs from primary tumors identified significant changes in immune-related pathways, and identified a common downstream regulator –STAT3. Studies are ongoing to evaluate the mechanistic role of STAT3 in the response observed in primary tumors and to determine if STAT3 is involved in response in the metastatic setting. In summary, addition of ENT to ICIs significantly affects overall survival in early stage models of HER2+ breast cancer however, only addition of a-PD-1 to ENT is beneficial in models of advanced disease. Additionally, the mechanism of action in early stage disease involves altered infiltration and function of MDSCs, allowing for a more robust adaptive immune response. However, a different mechanism of action is likely responsible for the responses seen in advanced stage disease. These novel findings provide a rationale for combination therapy in patients with HER2+ breast cancer and suggest responses to this combination therapy are linked to stage of disease likely due to different mechanisms of action. Citation Format: Roussos Torres ET, Rafie C, Armstrong T, Jaffee EM. Epigenetic modulation—unlocking the potential of checkpoint inhibition in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-10.

Entinostat Converts Immune-Resistant Breast and Pancreatic Cancers into Checkpoint-Responsive Tumors by Reprogramming Tumor-Infiltrating MDSCs.

Immune-checkpoint inhibition (ICI) has revolutionized treatment in cancers that are naturally immunogenic by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. Tumors possessing complex immunosuppressive TMEs such as breast and pancreatic cancers present unique therapeutic obstacles as response rates to ICI remain low. Such tumors often recruit myeloid-derived suppressor cells (MDSCs), whose functioning prohibits both T-cell activation and infiltration. We attempted to sensitize these tumors to ICI using epigenetic modulation to target MDSC trafficking and function to foster a less immunosuppressive TME. We showed that combining a histone deacetylase inhibitor, entinostat (ENT), with anti-PD-1, anti-CTLA-4, or both significantly improved tumor-free survival in both the HER2/neu transgenic breast cancer and the Panc02 metastatic pancreatic cancer mouse models. Using flow cytometry, gene-expression profiling, and ex vivo functional assays, we characterized populations of tumor-infiltrating lymphocytes (TILs) and MDSCs, as well as their functional capabilities. We showed that addition of ENT to checkpoint inhibition led to significantly decreased suppression by granulocytic MDSCs in the TME of both tumor types. We also demonstrated an increase in activated granzyme-B-producing CD8+ T effector cells in mice treated with combination therapy. Gene-expression profiling of both MDSCs and TILs identified significant changes in immune-related pathways. In summary, addition of ENT to ICI significantly altered infiltration and function of innate immune cells, allowing for a more robust adaptive immune response. These findings provide a rationale for combination therapy in patients with immune-resistant tumors, including breast and pancreatic cancers.

Abstract 4965: Entinostat transforms the suppressive tumor microenvironment of breast cancer and promotes survival and anti-responses when combined with checkpoint inhibition

Abstract Checkpoint inhibition has revolutionized treatment in cancers that are naturally immunogenic by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. While this strategy has shown some efficacy in metastatic breast cancer, most breast cancers lack immunogenic potential due to a strictly sustained immunosuppressive microenvironment and a lack of tumor antigen expression and recognition. One strategy to transform the breast TME is to use epigenetic modulation to affect activation and trafficking of myeloid derived suppressor cells (MDSCs), known to alter the immunogenicity of the TME and sensitize tumors to checkpoint modulation. We show that combinations of an epigenetic agent, the histone deacetylase inhibitor entinostat (ENT), checkpoint inhibitors anti -programmed cell death protein (a-PD-1) and anti-cytotoxic T-lymphocyte-associated protein 4 (a-CTLA-4) antibodies, with and without anti-HER2 antibodies significantly improves survival and anti-tumor effects in HER-2/neu transgenic mouse models. Using fluorescence-activated cell sorting, nanostring gene expression profiling, and other functional assays, we have characterized the various types of tumor infiltrating lymphocytes and myeloid cells and their functional capabilities within primary tumors. We show that addition of ENT to checkpoint inhibition leads to significantly decreased suppressive ability of granulocytic-MDSCs into the TME. We also demonstrate an increase in CD8+ T effector cells in mice treated with combination therapy. Flow cytometric evaluation of markers of T cell activation, exhaustion, and MDSC function demonstrate significantly increased T cell activation, exhaustion, and decreased myeloid function. Nanostring gene expression profiling of both MDSCs and lymphocytes infiltrating tumors determined significant changes in immune related pathways that likely lead to our observed outcomes. In summary, addition of ENT to checkpoint inhibition significantly increases infiltration of innate and adaptive immune cells into the highly tolerant neu-N breast tumors and leads to improved survival and decreased tumor burden. Functional assays demonstrate that this drug combination promotes a less suppressive TME and allows a more robust anti-tumor effect of infiltrating cells. It is our hope that these novel findings will provide further rationale for combination therapy and improve the response rate of these immune therapies in patients with breast cancer. Citation Format: Evanthia T. Roussos Torres, Christine Rafie, Hayley Ma, Brian Christmas, Todd Armstrong, Elizabeth M. Jaffee. Entinostat transforms the suppressive tumor microenvironment of breast cancer and promotes survival and anti-responses when combined with checkpoint inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4965.

Abstract P1-08-01: Combination checkpoint inhibition and epigenetic modulation promotes tumor suppression and improves survival in Her2+ models of breast cancer

Abstract Background: Checkpoint inhibition is a very successful treatment strategy in cancers that are naturally immunogenic by attracting T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. While this strategy has shown some efficacy in metastatic breast cancer, most breast cancers are not highly immunogenic likely due to an immunosuppressive microenvironment and a lack of tumor antigen expression and recognition. One strategy to transform the breast TME is to use epigenetic modulation to affect activation and trafficking of myeloid derived suppressor cells (MDSCs), known to alter the immunogenicity of the TME and sensitize tumors to checkpoint modulation. We hypothesize that combinatorial therapy primes the TME by altering infiltration and function of MDSCs leading to a more robust T cell response. Methods: We are using a HER-2/neu transgenic mouse model with tumor challenge of syngeneic cell lines to test the efficacy of different combinations of an epigenetic agent, the histone deacetylase inhibitor entinostat (ENT), checkpoint inhibitors anti -programmed cell death protein (a-PD-1) and anti-cytotoxic T-lymphocyte-associated protein 4 (a-CTLA-4) antibodies, with and without anti-HER2 antibodies. We will examine treatment effects on tumor growth, and hope to identify co-stimulatory and inhibitory factors regulating T cell and MDSC responses. Characterization of tumor infiltrating lymphocytes and their functional capabilities are being investigated in primary tumors using fluorescence-activated cell sorting, nanostring gene expression profiling, and immunohistochemistry. Results: We found significant improvement in survival and delay in tumor growth in mice treated with ENT in combination with a-PD-1 and/or a-CTLA-4. Addition of anti-HER2 therapy to ENT and a-CTLA4 or a-PD1 also significantly improves survival and delay in tumor growth. We also found addition of ENT to checkpoint inhibition leads to significantly increased infiltration of granulocytic-MDSCs into the TME. We demonstrate an increase in CD8+ T effector cells in mice treated with combination therapy. Flow cytometric evaluation of markers of T cell activation, exhaustion, and MDSC function demonstrate significantly increased T cell activation, exhaustion, and myeloid function however it is unclear how this directly effects the phenotype we have observed in these mice. Gene expression profiling of both MDSCs and lymphocytes infiltrating tumors is underway to help determine significant changes in immune related pathways that lead to our observed outcomes. Conclusions: Addition of ENT to checkpoint inhibition significantly increases infiltration of innate and adaptive immune cells into the highly tolerant neu-N breast tumors and leads to improved survival and decreased tumor burden. Functional assays are underway and future studies will further delineate changes in immune infiltration as well as genetic alterations responsible for these observations. It is our hope that these novel findings will provide further rationale for combination therapy and improve the response rate of these immune therapies in patients with breast cancer. Citation Format: Roussos Torres ET, Ma H, Christmas B, Armstrong T, Jaffee EM. Combination checkpoint inhibition and epigenetic modulation promotes tumor suppression and improves survival in Her2+ models of breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-08-01.

Identifying the Clonal Origins of Gvhd-Causing T Cells

In graft-versus-host disease (GVHD) donor αβ T cells in the allograft recognize host tissues as non-self and cause multi-organ damage through direct and indirect mechanisms. This multi-organ disease is mediated by T cells acquiring diverse phenotypes, including expression of homing receptors, adhesion molecules, cytokines and other effector molecules. How this diversity is generated at a clonal level is not understood. In the one extreme, each type of effector could be derived from a single T cell expressing a unique antigen receptor that may even target a unique antigen. Alternatively, a single T cell targeting a single antigen may be able to differentiate into a variety of effectors (Fig. 1). A second related question is whether GVHD is maintained within affected organs. That is, what is the importance of the continued recruitment of new blood-derived alloreactive effectors in maintaining GVHD.To answer these questions, we needed 1) a system in which GVHD-inducing T cells could be unambiguously tracked, as in polyclonal systems it is difficult if not impossible to know which T cells are disease causing; and 2) a way to distinguish GVHD-inducing T cells at the clonal level. To address the first point, we used a CD4 T cell receptor (TCR) transgenic (Tg) model of GVHD wherein GVHD is induced by the transfer of <1000 BALB/c RAG2-/- TS1 TCR Tg T cells, which recognize the S1 peptide derived from HA, into BALB/c RAG2-/- HA104 Tg recipients that ubiquitously express HA. GVHD is manifest by weight loss, death, and TS1-infiltrative pathology of the skin, liver, small intestine and colon. In order to determine the clonal contributions, we crossed TS1 Tg mice to combinations of CD45.1, CD45.2, Thy1.1, Thy1.2 and GFP backgrounds to create a matrix of up to 9 distinguishable TS1 cells.Using this model, we first characterized the variety of TS1 effector phenotypes being generated across tissues at day 14 and 21 post-transplant and will be presenting data detailing these TS1 phenotypes, including the expression of CCR9, α4β7 integrin, and cutaneous lymphocyte antigen (CLA).To confirm that different matrix cells perform similarly, we assessed the development of total TS1 effector cells in mice that had received an equal number of 5 distinct TS1 cell combinations. We found that within individual mice (n=5), there is comparable distribution of GVHD-causing TS1 effector cells (% of total TS1 cells) from all 5 TS1cell matrices across tissues 14-21 days post-transplant (Table 1).We next tested our ability to detect total effector cells derived from transferring low numbers of TS1 cells (5-500 cells) of a given matrix combination. We found that within individual mice (n=9), the transfer of 5 and 10 TS1 clones of a given matrix results in disparate distribution of TS1 effectors (% of total TS1 cells) across tissues 14-21 days post-transplant. As an example for one mouse at day 14, the fraction of TS1effectors derived from 10 TS1clones of one matrix was 0.8% (spleen), 1.9% (mLN), 1.4% (BM), 0.6% (colon), 4.4% (SI IEL), 1.1% (SI LP), 1.2% (skin) and 1.5% (liver). In that same mouse, the fraction of TS1effectors derived from 5 TS1clones of a different matrix was 0.7% (spleen), 0.9% (mLN), 0.4% (BM), 1.1% (colon), 1.1% (SI IEL), 0.6% (SI LP), 0.6% (skin) and 2.7% (liver). We made similar observations in mice at day 21, where the percentage of TS1 effectors derived from 5 TS1 clones in one mouse was 2.5% (spleen), 2.0% (BM), 2.8% (mLN), 0.2% (SI IEL), 2.3% (SI LP), 1.1% (skin) and 1.6% (liver). When focusing on specific TS1 phenotypes derived from 5 and 10 TS1clones, we also found unequal distributions of these cells across different tissues. For example, the distribution of CD69+CD103+ TS1 cells (% of total TS1 cells) derived from 5 TS1clones was 2.9% (colon), 1.5% (SI IEL) and 1.9% (SI LP) in one mouse at day 14. Similarly, TS1 cells expressing α4β7 integrin also varied in distribution across tissues, with one mouse having 2.2% (spleen), 0.5% (mLN) and 0% (BM) α4β7+ TS1 cells. These data suggest that tissue GVHD effectors are not simply in equilibrium with blood, consistent with some local GVHD maintenance. Furthermore, the presence of 5 TS1 clones in more than one effector type suggests that single cells can differentiate into multiple effectors. We are now directly addressing these possibilities by analyzing progeny derived from many sorted single cells. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract A02: The art of omission: Low fucose antibodies enhance tumor cell clearance in vivo

Abstract The use of monoclonal antibodies (mAbs) in treating cancer patients has become an important addition to chemo- and radiotherapy. Depending on the type of antibody, these mAbs induce a variety of different effector functions, including induction of apoptosis and growth inhibition that can lead to eradication of the tumor. Additionally, tumor targeting mAbs can activate the immune system resulting in induction of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP) of tumor cells by natural killer cells or myeloid cells (monocyte/macrophages and neutrophils). Currently most clinically used mAbs are primarily chimeric or humanized Immunoglobulin G1 (IgG1) mAbs, which bind to IgG Fc receptors (FcγRs). Efficient binding to FcγRs requires the IgG-Fc-glycan moieties at position 297. In humans, core-fucosylation of IgG is particularly important for binding to human FcγRIIIa and FcγRIIIb, resulting in enhanced uptake of opsonized particles and enhanced ADCC. These properties are increasingly exploited in the next generation of therapeutic anti-tumor IgG. In this study we investigated whether removal of the core fucose also enhanced killing of tumor cells by mouse effector cells, and whether this prevented tumor outgrowth in vivo. Therefore a chimeric IgG1 (TA99), targeting gp75 on B16F10 mouse melanoma cells, was generated with- and without core-fucose, and their effect was assessed in both in vitro and in vivo models. In vitro mouse immune cells were incubated with B16F10-gp75 cells in the presence or absence of wildtype or low fucose TA99 and tumor killing was analyzed. In vivo the effect of the TA99 IgG1 low fucose on tumor outgrowth was assessed by injecting both B16F10-gp75 cells and either TA99 IgG1 wt or low fucose in the peritoneal cavity of mice. Fourteen days post injection tumor outgrowth was analyzed. Mice treated with TA99 low fucose had significantly less tumors in comparison to the wildtype TA99 IgG1, showing that changing the fucosylation of the Fc domain has a remarkable effect on the effectiveness of an antibody therapy. These results emphasize that when the core fucose of therapeutic IgG antibodies is removed, efficacy of monoclonal antibody therapies will be enhanced significantly. Additionally, the effect works cross species, suggesting mouse models are also suitable to study this phenomenon and to screen antibodies in vivo for enhanced effector functions. Ultimately, this may lead to improved clinical outcome of cancer patients. Citation Format: Rens Braster, Remco Visser, Gestur Vidarsson, Marjolein van Egmond. The art of omission: Low fucose antibodies enhance tumor cell clearance in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A02.

Effect on T cell subsets and secretion of IFN-γ and IL-17 after exposure to ⁶⁰Co lethally total body irradiation in mice

To investigate the effects of letherally total body irradiation (TBI) on distribution of T-lymphocyte subtypes and their cytokine expression. The BALB/c mice were divided randomly into ⁶⁰Co gamma rays TBI group and control group. Mice were sacrificed 7 days after irradiation. The lymphocytes in spleens, mesenteric lymphonodes, livers and bone marrow were collected and counted. Changes of CD4(+) T and CD8(+) T cell subsets as well as the expressions of IFN-γ and IL-17 were analyzed by flow cytometry. (1)Compared with control group, the total number of lymphocytes in marrow, spleen, lymph node and liver distinctively decreased in TBI group [(5.34±1.14)×10⁵ vs (3.08±1.13)×10⁷, (2.10±0.54)×10⁵ vs (2.71±0.83)×10⁷, (5.89±1.07)×10⁵ vs (7.92±1.15)×10⁷ and (3.45±1.01)×10⁵ vs (7.44±0.79)×10⁶, respectively, and the significant differences were observed between two groups in each organ (P<0.05)]. (2)The level of IFN-γ produced by CD4(+) T in spleen, lymph node and liver elevated in TBI group compared to control group, which were (20.77±2.03)% vs (3.69±3.13)%, (6.28±0.46)% vs (1.11±0.17)%, (27.24±5.79)% vs (9.01±1.24)% respectively, the differences between two groups in each organ were significant (P<0.05). (3)Percentages of IFN-γ(+)CD8(+) T in spleen, lymph node and liver in TBI group increased compared to control group [(52.40±9.26)% vs (43.06±1.04)%, (33.56±5.02)% vs (21.83±4.22)%, and (44.27±8.97)% vs (19.32±3.11)%, respectively, and the differences between two groups in each tissue were significant (P<0.05)]. (4)However, IL-17A expressions in CD4(+) T and CD8(+) T cells from spleen and liver were lower than those in control group. TBI induced the reduction of lymphocytes and the expansion of IFN-γ producing Th1 and Tc1 effector cells in mice.

Epicutaneous immunization with hapten-conjugated protein antigen alleviates contact sensitivity mediated by three different types of effector cells

BackgroundAllergic contact dermatitis (ACD) is a common clinical condition in industrialized countries and often causes occupational diseases. Animal model of contact sensitivity (CS) is commonly used to study ACD in mice and can be induced by skin application of haptens. It has been previously shown that CS is mediated by CD4+ or CD8+ T effector cells. More recently it was found that also liver NK cells can play a role of CS effector cells in mice. MethodsThe aim of the present study was to test whether skin-induced suppression could inhibit CS response in vivo. ResultsHere we show that EC immunization of normal mice with hapten conjugated protein antigen prior to hapten sensitization suppresses Th1, Tc1 and NK mediated CS responses. ConclusionThese data strongly suggest that maneuver of EC immunization may have important implications for designing therapeutic schemes aimed at modulating unwanted immune responses in contact hypersensitivity.

Early Induction of CCL7 Downstream of TLR9 Signaling Promotes the Development of Robust Immunity to Cryptococcal Infection

We investigated mechanisms by which TLR9 signaling promoted the development of the protective response to Cryptococcus neoformans in mice with cryptococcal pneumonia. The afferent (week 1) and efferent (week 3) phase immune parameters were analyzed in the infected wild-type (TLR9(+/+)) and TLR-deficient (TLR9(-/-)) mice. TLR9 deletion diminished 1) accumulation and activation of CD11b(+) dendritic cells (DCs), 2) the induction of IFN-γ and CCR2 chemokines CCL7, CCL12, but not CCL2, at week 1, and 3) pulmonary accumulation and activation of the major effector cells CD4(+) and CD8(+) T cells, CD11b(+) lung DCs, and exudate macrophages at week 3. The significance of CCL7 induction downstream of TLR9 signaling was investigated by determining whether CCL7 reconstitution would improve immunological parameters in C. neoformans-infected TLR9(-/-) mice. Early reconstitution with CCL7 1) improved accumulation and activation of CD11b(+) DCs at week 1, 2) restored early IFN-γ production in the lungs, and 3) restored the accumulation of major effector cell subsets. CCL7 administration abolished the difference in lung fungal burdens between TLR9(+/+) and TLR9(-/-) mice at week 3; however, significant reduction of fungal burdens between PBS- and CCL7-treated mice has not been observed, suggesting that additional mechanism(s) apart from early CCL7 induction contribute to optimal fungal clearance in TLR9(+/+) mice. Collectively, we show that TLR9 signaling during the afferent phase contributes to the development of protective immunity by promoting the early induction of CCL7 and IFN-γ and the subsequent early recruitment and activation of DCs and additional effector cells in mice with cryptococcal pneumonia.

Chimeric, divalent and tetravalent anti‐CD19 monoclonal antibodies with potent in vitro and in vivo antitumor activity against human B‐cell lymphoma and pre‐B acute lymphoblastic leukemia cell lines

CD19 is an attractive therapeutic target for treating human B-cell tumors. In our study, chimeric (c) divalent (cHD37) and tetravalent (cHD37-DcVV) anti-CD19 monoclonal antibodies (MAbs) were constructed, expressed and evaluated for their binding to human 19-positive (CD19(+)) tumor cell lines. They were also tested for proapoptotic activity and the ability to mediate effector functions. The antitumor activity of these MAbs was further tested in mice xenografted with the CD19(+) Burkitt's lymphoma cell line, Daudi or the pre-B acute lymphoblastic leukemia (ALL) cell line, NALM-6. The cHD37 and cHD37-DcVV MAbs exhibited specific binding and comparable proapoptotic activity on CD19(+) tumor cell lines in vitro. In addition, the cHD37 and cHD37-DcVV MAbs were similar in their ability to mediate antibody-dependent cell-mediated phagocytosis (ADCP). However, the tetravalent cHD37-DcVV MAb bound more avidly, had a slower dissociation rate, and did not internalize as well. It also had enhanced antibody-dependent cellular cytotoxicity (ADCC) with human but not murine effector cells. The cHD37 and cHD37-DcVV MAbs exhibited comparable affinity for the human neonatal Fc receptor (FcRn) and similar pharmacokinetics (PKs) in mice. Moreover, all the HD37 constructs were similar in extending the survival of mice xenografted with Daudi or NALM-6 tumor cells. Therefore, the cHD37 and cHD37-DcVV MAbs have potent antitumor activity and should be further developed for use in humans. Although not evident in mice, due to its increased ability to mediate ADCC with human but not mouse effector cells, the cHD37-DcVV MAb should have superior therapeutic efficacy in humans.

5-Azacytidine enhances the anti-leukemic activity of lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia

Despite therapeutic advances, the poor prognoses for acute myeloid leukemia (AML) and intermediate and high-risk myelodysplastic syndromes (MDS) point to the need for better treatment options. AML and MDS cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, induced meaningful responses in a Phase 1 clinical trial and demonstrated anti-leukemic activity in preclinical models. Recently, it was reported that 5-azacytidine (Vidaza™) prolonged the overall survival of a group of high risk MDS and AML patients. To determine whether the combination of lintuzumab and 5-azacytidine would be beneficial, a mouse xenograft model of disseminated AML was used to evaluate the combination. There was a significant reduction in tumor burden and an increase in overall survival in mice treated with lintuzumab and 5-azacytidine. The effects were greater than that obtained with either agent alone. As the in vivo anti-leukemic activity of lintuzumab was dependent upon the presence of mouse effector cells including macrophages and neutrophils, in vitro effector function assays were used to assess the impact of 5-azacytidine on lintuzumab activity. The results show that 5-azacytidine significantly enhanced the ability of lintuzumab to promote tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytic (ADCP) activities. These results suggest that lintuzumab and 5-azacytidine act in concert to promote tumor cell killing. Additionally, these findings provide the rationale to evaluate this combination in the clinic.

Interferon‐α enhances CD317 expression and the antitumor activity of anti‐CD317 monoclonal antibody in renal cell carcinoma xenograft models

A murine (mAHM) and a humanized (AHM) monoclonal antibody against CD317 (also called tetherin, BST2, or HM1.24 antigen), expressed preferentially in neoplastic B cells such as multiple myeloma, exhibited antitumor effects as a result of antibody-dependent cellular cytotoxicity (ADCC). The putative interferon (IFN) response elements IRF-1/2 and ISGF3 are present in the promoter of the CD317 gene, and IFN has been used for the treatment of not only myeloproliferative diseases but also solid tumors such as renal cell carcinoma (RCC) and melanoma. Therefore, we examined the effects of IFN on the expression of CD317 and on the antitumor activity of AHM and mAHM in RCC and melanoma. Flow cytometry and in vitro ADCC assays with human or mouse effector cells demonstrated that IFN-alpha markedly increased the amount of cell surface CD317 and augmented the ADCC activity of mAHM and AHM in RCC cells and to a lesser extent in melanoma cells. Administration of IFN-alpha to mice bearing RCC xenografts also increased the expression of CD317 in tumor cells. When coadministered with IFN-alpha, mAHM exhibited more profound antitumor activity in both IFN-alpha-sensitive and -insensitive RCC xenograft models. Thus, AHM in combination with IFN-alpha may be an effective therapy for the treatment of RCC.

Studying Human Regulatory T Cells In vivo

In this issue of Clinical Cancer Research , Mutis et al. ([1][1]) use a model of xenogeneic graft-versus-host disease to assess the suppressive function of human regulatory T cells in vivo . This model is based on the transfer of human peripheral blood cells into RAG-2−/−γc−/− mice that are

A liposomal peptide vaccine inducing CD8 + T cells in HLA-A2.1 transgenic mice, which recognise human cells encoding hepatitis C virus (HCV) proteins

Virus specific T cell responses play an important role in resolving acute hepatitis C virus (HCV) infections. Using the HLA-A2.1 transgenic mouse model we investigated the potential of a liposomal peptide vaccine to prime a CD8 + T cell response against 10 different HCV epitopes, relevant for human applications. We were able to demonstrate the induction of strong cytotoxic T cell responses and high numbers of IFN-γ-secreting cells, which persisted at high levels for at least 3 months. Co-integrating CpG oligonucleotides into liposomes further increased the number of IFN-γ-secreting cells by 2–10-fold for most epitopes tested. The frequency of specific cells was further analysed with chimeric A2 tetramers bearing the NS31073-1081 epitope and was estimated at 2–23% of the CD8 + T cell population. Importantly, mouse effector cells, specific for this epitope, were also capable of lysing a human target cell line expressing HCV proteins. This finding and the specific protection observed in challenge experiments with recombinant vaccinia virus expressing HCV sequences emphasise the biological relevance of the vaccine-induced immune response. In conclusion, such liposome formulations represent a safe and promising strategy to stimulate the CD8 + T cell against HCV.

Identification of effector cells for TNFα-mediated cytotoxicity against WEHI164S cells

WEHI164S cells were found to be very sensitive targets for in vitro killing in a 6-h culture when liver or splenic lymphocytes were used as effector cells in mice. Of particular interest, a limiting cell-dilution analysis showed that effector cells were present in the liver with a high frequency (1/4300). In contrast to YAC-1 cells as NK targets, perforin-based cytotoxicity was not highly associated with WEHI164S killing. The major killer mechanism for WEHI164S targets was TNFα-mediated cytotoxicity. By cell sorting experiments, both NK cells and intermediate T cells (i.e., TCR int cells) were found to contain effector cells against WEHI164S cells. However, the killer mechanisms underlying these effector cells were different. Namely, NK cells killed WEHI164S cells by perforin-based cytotoxicity, TNFα-mediated cytotoxicity, Fas ligand cytotoxicity, and other mechanisms, whereas intermediate T cells did so mainly by TNFα-mediated cytotoxicity. These results suggest that TNFα-mediated cytotoxicity mediated by so-called natural cytotoxic (NC) cells comprised events which were performed by both NK and intermediate T cells using somewhat different killer mechanisms. Intermediate T cells which were present in the liver were able to produce TNFα if there was appropriate stimulation.

Priming of CD8 + CTL effector cells in mice by immunization with a stress protein–influenza virus nucleoprotein fusion molecule

Literature is accumulating which suggests the potential for stress proteins to form the basis of a novel vaccine technology. Immunization with mammalian tumor-derived stress proteins and their associated peptides promote anti-tumor immunity. Vaccination with HIV-1 p24 antigen fused to mycobacterial heat shock protein (Hsp) Hsp71 enhances p24-specific immunity, as measured by p24-specific antibody production and in vitro cell proliferation and cytokine induction. An ovalbumin-Hsp71 fusion protein primes ovalbumin-specific CTL activity and resistance to challenge with an ovalbumin-expressing tumor. We have extended these observations by using a mycobacterial Hsp65 fusion molecule to prime CTL specific for a viral antigen. Gene fusion constructs were generated from DNA encoding Mycobacterium bovis strain BCG Hsp65 and individual fragments of influenza virus nucleoprotein (NP) encompassing H-2K d-and H-2D b-restricted CTL epitopes. The ability of these purified recombinant fusion proteins to prime NP-specific CTL was assessed in mice of appropriate H-2 haplotypes. We observed that adjuvant-free immunization with either fusion protein elicited significant CTL activity when administered at doses of 10–100 μg per mouse. An NP fusion protein made with glutathione- S-transferase failed to elicit NP-specific CTL, indicating that the phenomenon requires Hsp65 sequences. A single immunization with the Hsp65-NP fusion protein elicited CTL activity which persisted for a minimum of 4 months post-immunization, at which time it could be boosted by a second immunization. To our knowledge, this is the first report of a member of the Hsp60 family priming for antigen-specific CTL activity when employed as a fusion protein partner.

Adoptive transfer: the role of perforin in mouse cytotoxic T lymphocyte rejection of human tumor xenografts in vivo.

The popliteal lymph node cells of immunocompetent mice generated a strong in vitro cytotoxic response to footpad injection of several human tumor cell lines and the resulting mouse effector cells predominantly used a perforin-mediated cytotoxic mechanism. A relatively minor FasL-dependent cytotoxic response to CEM-CCRF and Jurkat leukemias, but not colon carcinoma COLO 205 cells, was also detected in immunized perforin-deficient mice. In vitro depletion of CD3+ CD8+ T cells, but not CD4+ T or NK1.1+ cells, completely inhibited lysis of human tumor cells, suggesting that CD3+ CD8+ T cells were effectors of perforin-mediated xenospecific cytotoxicity. Xenospecific cytotoxic T cells from wild-type mice were extremely efficient at rejecting tumor when adoptively transferred into scid mice bearing established COLO 205, CEM-CCRF, or Jurkat tumor xenografts. By contrast, cytotoxic T lymphocytes of perforin-deficient mice had no effect on the growth of established tumor xenografts. These data indicate that perforin, and hence direct cytotoxicity, plays a key role in the ability of adoptively transferred CD8+ cytotoxic T lymphocytes to eradicate established xenografts.

Theiler's virus infection of beta 2-microglobulin-deficient mice

Theiler's virus, a murine picornavirus, persists in the central nervous systems of susceptible mice and induces a chronic demyelinating disease. Susceptibility or resistance to this disease is controlled in part by the H2-D locus of the major histocompatibility complex (MHC). For this reason, it has been proposed that CD8+ class I-restricted cytotoxic T cells play a main role in the pathogenesis of this viral infection. We recently reported the existence of anti-virus CD8+ cytotoxic T cells in the course of Theiler's virus infection. In the present study, we examined the role of these effector cells in mice in which the beta 2-microglobulin gene had been disrupted. These mice fail to express class I MHC molecules and therefore lack CD8+ T cells. The mice are derived from a C57BL/6 x 129/Ola cross and are H-2b, a haplotype associated with resistance to Theiler's virus infection. beta 2-Microglobulin-deficient mice (beta 2m-/-mice) failed to clear the virus, developed demyelination, and, interestingly, did not succumb to early infection. These results demonstrate that CD8+ T cells are required to clear Theiler's virus infection. In contrast with a current hypothesis, they also demonstrate that CD8+ T cells are not major mediators of the demyelinating disease.

Role of oxygen-dependent mechanisms in monoclonal-antibody-induced lysis of normal T cells by phagocytes: II.—Murine phagocytes

In the first part of this study, we reported that antibody-dependent cellular cytotoxicity (ADCC) mediated by human polymorphonuclear (PMN) and mononuclear (MN) phagocytes against anti-T-cell monoclonal-antibody-(mAb)-coated T lymphocytes, is mainly dependent upon the generation of reactive oxygen intermediates (ROI) for PMN, whereas MN-mediated ADCC depends on both oxidative and non-oxidative cytolytic events. Using mouse effector cells from various organs and at various maturation or activation states, the present report shows that in this ADCC model against mAb-coated normal T cells, resident spleen (SPC) and peritoneal exudate (PEC) cells selectively bound and developed oxidative responses to these mAb-coated normal target cells but remained ineffective in ADCC. Similar data were obtained with inflammatory recruited (thioglycollate-elicited or Biogel granuloma-induced) macrophages, whereas immunologically activated macrophages (from BCG-treated mice) mediated both strong oxidative responses and potent ADCC reactions. In contrast, and as in the human system, both resident (from bone marrow) and inflammatory PMN phagocytes exerted significant lysis of these mAb-sensitized normal lymphoid cells. These findings, which are similar to those reported in ADCC against tumour target cells, strongly suggest that the nature (normal or tumoral) of the target cell does not influence the lytic

Increased susceptibility of periodate-treated tumour cells to human but not to mouse or rat natural killer cells.

Treatment of intact cells in the cold with low concentrations (1 mM) of sodium meta periodate (PI) selectively oxidizes the surface-exposed sialic acid residues to the corresponding aldehydes. Such treated tumour cells show greatly enhanced sensitivity to lysis by fresh human NK cells but not to mouse or rat NK cells. Reduction of the PI-treated cells with sodium borohydride (NaBH4) reduced their NK sensitivity to that of untreated cells. In target conjugate formation assays PI-treated tumour cells displayed a higher binding capacity than control cells or PI+NaBH4-treated cells to both mouse and human effector cells. Neuraminidase treatment of K562 and Molt-4 increased target susceptibility to human NK cells but not to mouse, whereas the susceptibility of Yac-1 cells was left unchanged using both human and mouse effector cells. The same pattern of reactivity is shown in the target binding assay. These findings indicate that subtle molecular changes in the surface-exposed carbohydrates of target cells might have a fundamental impact on their sensitivity to lysis by NK cells from certain species, and that in cross species effector-target combinations a higher binding capacity is not sufficient for increased lysis to occur.