Myeloid-Derived Suppressor Cells in the Tumor Microenvironment.

Myeloid-derived suppressor cells (MDSCs) represent a heterogenous population of immature myeloid cells capable of modulating immune responses. In the context of cancer, MDSCs are abnormally produced and recruited to the tumor microenvironment (TME) to aid in the establishment of an immunosuppressive TME that facilitates tumor escape. Additionally, MDSCs contribute to non-immunologic aspects of tumor biology, including tumor angiogenesis and metastasis. The clinical significance of MDSCs has recently been appreciated as numerous studies have suggested a correlation between circulating and intratumoral MDSC frequencies and tumor stage, progression, and treatment resistance. In this chapter, we review MDSC characterization, development, expansion, and mechanisms that facilitate immunosuppression and tumor progression. Furthermore, we highlight studies demonstrating the clinical significance of MDSCs in various disease states in addition to strategies that modulate various aspects of MDSC biology for therapeutic gain.

IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production.

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 µg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.

NK Cell Therapeutics for Hematologic Malignancies: from Potential to Fruition.

PURPOSE OF REVIEW: The current review focuses on the preclinical development and clinical advances of natural killer (NK) cell therapeutics for hematologic malignancies and offers perspective on the unmet challenges that will direct future discovery in the field. RECENT FINDINGS: Approaches to improve or re-direct NK cell anti-tumor functions against hematologic malignancies have included transgenic expression of chimeric antigen receptors (CARs), administration of NK cell engagers including BiKEs and TriKEs that enhance antibody-dependent cellular cytotoxicity (ADCC) by co-engaging NK cell CD16 and antigens on tumors, incorporation of a non-cleavable CD16 that results in enhanced ADCC, use of induced memory-like NK cells alone or in combination with CARs, and blockade of NK immune checkpoints to enhance NK cytotoxicity. Recently reported and ongoing clinical trials support the feasibility and safety of these approaches. NK cell-based therapeutic strategies hold great promise as cost-effective, off-the-shelf cell therapies for patients with relapsed and refractory hematologic diseases.

Selectively targeting myeloid-derived suppressor cells through TRAIL receptor 2 to enhance the efficacy of CAR T cell therapy for treatment of breast cancer.

BACKGROUND: Successful targeting of solid tumors such as breast cancer (BC) using chimeric antigen receptor (CAR) T cells has proven challenging, largely attributed to the immunosuppressive tumor microenvironment (TME). Myeloid-derived suppressor cells (MDSCs) inhibit CAR T cell function and persistence within the breast TME. To overcome this challenge, we have developed CAR T cells targeting tumor-associated mucin 1 (MUC1) with a novel chimeric costimulatory receptor that targets tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TR2) expressed on MDSCs. METHODS: The function of the TR2.41BB costimulatory receptor was assessed by exposing non-transduced (NT) and TR2.41BB transduced T cells to recombinant TR2, after which nuclear translocation of NFκB was measured by ELISA and western blot. The cytolytic activity of CAR.MUC1/TR2.41BB T cells was measured in a 5-hour cytotoxicity assay using MUC1+ tumor cells as targets in the presence or absence of MDSCs. In vivo antitumor activity was assessed using MDSC-enriched tumor-bearing mice treated with CAR T cells with or without TR2.41BB. RESULTS: Nuclear translocation of NFκB in response to recombinant TR2 was detected only in TR2.41BB T cells. The presence of MDSCs diminished the cytotoxic potential of CAR.MUC1 T cells against MUC1+ BC cell lines by 25%. However, TR2.41BB expression on CAR.MUC1 T cells induced MDSC apoptosis, thereby restoring the cytotoxic activity of CAR.MUC1 T cells against MUC1+ BC lines. The presence of MDSCs resulted in an approximately twofold increase in tumor growth due to enhanced angiogenesis and fibroblast accumulation compared with mice with tumor alone. Treatment of these MDSC-enriched tumors with CAR.MUC1.TR2.41BB T cells led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared with CAR.MUC1 (469.79±81.46 mm3) or TR2.41BB (434.86±64.25 mm3) T cells alone. CAR.MUC1.TR2.41BB T cells also demonstrated improved T cell proliferation and persistence at the tumor site, thereby preventing metastases. We observed similar results using CAR.HER2.TR2.41BB T cells in a HER2+ BC model. CONCLUSIONS: Our findings demonstrate that CAR T cells that coexpress the TR2.4-1BB receptor exhibit superior antitumor potential against breast tumors containing immunosuppressive and tumor promoting MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.

Oncolytic adeno-immunotherapy modulates the immune system enabling CAR T-cells to cure pancreatic tumors.

High expression levels of human epidermal growth factor receptor 2 (HER2) have been associated with poor prognosis in patients with pancreatic adenocarcinoma (PDAC). However, HER2-targeting immunotherapies have been unsuccessful to date. Here we increase the breadth, potency, and duration of anti-PDAC HER2-specific CAR T-cell (HER2.CART) activity with an oncolytic adeno-immunotherapy that produces cytokine, immune checkpoint blockade, and a safety switch (CAdTrio). Combination treatment with CAdTrio and HER2.CARTs cured tumors in two PDAC xenograft models and produced durable tumor responses in humanized mice. Modifications to the tumor immune microenvironment contributed to the antitumor activity of our combination immunotherapy, as intratumoral CAdTrio treatment induced chemotaxis to enable HER2.CART migration to the tumor site. Using an advanced PDAC model in humanized mice, we found that local CAdTrio treatment of primary tumor stimulated systemic host immune responses that repolarized distant tumor microenvironments, improving HER2.CART anti-tumor activity. Overall, our data demonstrate that CAdTrio and HER2.CARTs provide complementary activities to eradicate metastatic PDAC and may represent a promising co-operative therapy for PDAC patients.

Design and Implementation of NK Cell-Based Immunotherapy to Overcome the Solid Tumor Microenvironment.

Natural killer (NK) cells are innate immune effectors capable of broad cytotoxicity via germline-encoded receptors and can have conferred cytotoxic potential via the addition of chimeric antigen receptors. Combined with their reduced risk of graft-versus-host disease (GvHD) and cytokine release syndrome (CRS), NK cells are an attractive therapeutic platform. While significant progress has been made in treating hematological malignancies, challenges remain in using NK cell-based therapy to combat solid tumors due to their immunosuppressive tumor microenvironments (TMEs). The development of novel strategies enabling NK cells to resist the deleterious effects of the TME is critical to their therapeutic success against solid tumors. In this review, we discuss strategies that apply various genetic and non-genetic engineering approaches to enhance receptor-mediated NK cell cytotoxicity, improve NK cell resistance to TME effects, and enhance persistence in the TME. The successful design and application of these strategies will ultimately lead to more efficacious NK cell therapies to treat patients with solid tumors. This review outlines the mechanisms by which TME components suppress the anti-tumor activity of endogenous and adoptively transferred NK cells while also describing various approaches whose implementation in NK cells may lead to a more robust therapeutic platform against solid tumors.

Detection of response to tumor microenvironment-targeted cellular immunotherapy using nano-radiomics.

Immunotherapies, including cell-based therapies, targeting the tumor microenvironment (TME) result in variable and delayed responses. Thus, it has been difficult to gauge the efficacy of TME-directed therapies early after administration. We investigated a nano-radiomics approach (quantitative analysis of nanoparticle contrast-enhanced three-dimensional images) for detection of tumor response to cellular immunotherapy directed against myeloid-derived suppressor cells (MDSCs), a key component of TME. Animals bearing human MDSC-containing solid tumor xenografts received treatment with MDSC-targeting human natural killer (NK) cells and underwent nanoparticle contrast-enhanced computed tomography (CT) imaging. Whereas conventional CT-derived tumor metrics were unable to differentiate NK cell immunotherapy tumors from untreated tumors, nano-radiomics revealed texture-based features capable of differentiating treatment groups. Our study shows that TME-directed cellular immunotherapy causes subtle changes not effectively gauged by conventional imaging metrics but revealed by nano-radiomics. Our work provides a method for noninvasive assessment of TME-directed immunotherapy potentially applicable to numerous solid tumors.

NK Cells Expressing a Chimeric Activating Receptor Eliminate MDSCs and Rescue Impaired CAR-T Cell Activity against Solid Tumors.

Solid tumors are refractory to cellular immunotherapies in part because they contain suppressive immune effectors such as myeloid-derived suppressor cells (MDSCs) that inhibit cytotoxic lymphocytes. Strategies to reverse the suppressive tumor microenvironment (TME) should also attract and activate immune effectors with antitumor activity. To address this need, we developed gene-modified natural killer (NK) cells bearing a chimeric receptor in which the activating receptor NKG2D is fused to the cytotoxic ζ-chain of the T-cell receptor (NKG2D.ζ). NKG2D.ζ-NK cells target MDSCs, which overexpress NKG2D ligands within the TME. We examined the ability of NKG2D.ζ-NK cells to eliminate MDSCs in a xenograft TME model and improve the antitumor function of tumor-directed chimeric antigen receptor (CAR)-modified T cells. We show that NKG2D.ζ-NK cells are cytotoxic against MDSCs, but spare NKG2D ligand-expressing normal tissues. NKG2D.ζ-NK cells, but not unmodified NK cells, secrete proinflammatory cytokines and chemokines in response to MDSCs at the tumor site and improve infiltration and antitumor activity of subsequently infused CAR-T cells, even in tumors for which an immunosuppressive TME is an impediment to treatment. Unlike endogenous NKG2D, NKG2D.ζ is not susceptible to TME-mediated downmodulation and thus maintains its function even within suppressive microenvironments. As clinical confirmation, NKG2D.ζ-NK cells generated from patients with neuroblastoma killed autologous intratumoral MDSCs capable of suppressing CAR-T function. A combination therapy for solid tumors that includes both NKG2D.ζ-NK cells and CAR-T cells may improve responses over therapies based on CAR-T cells alone.

Natural killer cells produce T cell-recruiting chemokines in response to antibody-coated tumor cells.

In the current report, we have examined the ability of natural killer (NK) cells to produce T cell-recruiting chemokines following dual stimulation with interleukin (IL)-2 or IL-12 and human breast cancer cells coated with an antitumor antibody (trastuzumab). NK cells stimulated in this manner secreted an array of T cell-recruiting chemotactic factors, including IL-8, macrophage-derived chemokine, macrophage inflammatory protein 1alpha (MIP-1alpha), monocyte chemoattractant protein 1, and regulated on activation, normal T-cell expressed and secreted (RANTES), whereas stimulation of NK cells with either agent alone had minimal effect. Furthermore, these factors were functional for T-cell chemotaxis as culture supernatants derived from costimulated NK cells induced migration of both naïve and activated T cells in an in vitro chemotaxis assay. T-cell migration was significantly reduced when neutralizing antibodies to IL-8, MIP-1alpha, or RANTES were added to culture supernatants before their use in the chemotaxis assay. In addition, coadministration of trastuzumab-coated tumor cells and IL-12 to mice led to enhanced serum MIP-1alpha. As a clinical correlate, we examined the chemokine content of serum samples from breast cancer patients enrolled on a phase I trial of trastuzumab and IL-12, and found elevated levels of IL-8, RANTES, IFN-gamma inducible protein 10, monokine induced by IFN-gamma, and MIP-1alpha, specifically in those patients that experienced a clinical benefit. Sera from these patients exhibited the ability to direct T-cell migration in a chemotaxis assay, and neutralization of chemokines abrogated this effect. These data are the first to show chemokine production by NK cells, specifically in response to stimulation with antibody-coated tumor cells, and suggest a potential role for NK cell-derived chemokines in patients receiving therapeutic monoclonal antibodies.

TGF-ß1 programmed myeloid-derived suppressor cells (MDSC) acquire immune-stimulating and tumor killing activity capable of rejecting established tumors in combination with radiotherapy.

Cancer-induced myeloid-derived suppressor cells (MDSC) play an important role in tumor immune evasion. MDSC programming or polarization has been proposed as a strategy for leveraging the developmental plasticity of myeloid cells to reverse MDSC immune suppressive functions, or cause them to acquire anti-tumor activity. While MDSC derived ex vivo from murine bone marrow precursor cells with tumor-conditioned medium efficiently suppressed T cell proliferation, MDSC derived from conditioned medium in presence of TGF-ß1 (TGFß-MDSC) acquired a novel immune-stimulatory phenotype, losing the ability to inhibit T cell proliferation and acquiring enhanced antigen-presenting capability. Altered immune function was associated with SMAD-2 dependent upregulation of maturation and costimulatory molecules, and downregulation of inducible nitric oxide synthase (iNOS), an effector mechanism of immunosuppression. TGFß-MDSC also upregulated FAS-ligand expression, leading to FAS-dependent killing of murine human papillomavirus (HPV)-associated head and neck cancer cells and tumor spheroids in vitro and anti-tumor activity in vivo. Radiation upregulated FAS expression on tumor cells, and the combination of radiotherapy and intratumoral injection of TGFß-MDSC strongly enhanced class I expression on tumor cells and induction of HPV E7 tetramer-positive CD8 + T cells, leading to clearance of established tumors and long-term survival. TGFß-MDSC derived from human PBMC with tumor conditioned medium also lost immunosuppressive function and acquired tumor-killing activity. Thus, TGFß1 mediated programming of nascent MDSC leads to a potent anti-tumor phenotype potentially suitable for adoptive immunotherapy.

Chimeric Antigen Receptor Signaling Domains Differentially Regulate Proliferation and Native T Cell Receptor Function in Virus-Specific T Cells.

The efficacy of T cells expressing chimeric antigen receptors (CARs) for solid tumors has been limited by insufficient CAR T cell expansion and persistence. The use of virus-specific T cells (VSTs) as carriers for CARs may overcome this limitation since CAR-VSTs can be boosted by viral vaccines or oncolytic viruses. However, there is limited understanding of the optimal combination of endodomains and their influence on the native T cell receptor (TCR) in VSTs. We therefore compared the function of GD2.CARs expressing the TCR zeta chain (ζ) alone or combined with endodomains from CD28 and 4-1BB in varicella zoster virus-specific (VZV) T cells. VZVSTs expressing GD2-CARs recognized VZV-derived peptides and killed GD2-expressing tumor cells. However, after repeated stimulation through their native TCR, the expansion of GD2-CAR.CD28ζ-VZVSTs was 3.3-fold greater (p < 0.001) than non-transduced VZVSTs, whereas GD2-CARζ- and GD2-CAR.41BBζ inhibited VZVST expansion (p < 0.01). Compared to control VZVSTs, GD2-CAR.ζ VZVSTs showed a greater frequency of apoptotic (p < 0.01) T cells, whereas prolonged downregulation of the native αß TCR was observed in GD2-CAR.41BBζ VZVSTs (p < 0.001). We confirmed that CD28ζ can best maintain TCR function by expressing GD2.CARs in Epstein-Barr virus-specific T cells and CD19-CARs in VZVSTs. In response to CAR stimulation VSTs with CD28ζ endodomains also showed the greatest expansion (6 fold > GD2-CAR.41BBζ VZVSTs (p < 0.001), however anti-tumor efficacy was superior in GD2-CAR.41BBζ-VZVSTs. These findings demonstrate that CAR signaling domains can enhance or diminish the function of the native TCR and indicate that only CD28ζ may preserve the function of the native TCR in tonically signaling CAR-VSTs.

IL-12 enhances the natural killer cell cytokine response to Ab-coated tumor cells.

The anti-tumor activity of recombinant mAb's directed against tumor cell growth receptors has generally been considered to result from direct antiproliferative effects, the induction of apoptosis, or possibly Ab-dependent cellular cytotoxicity mediated against tumor targets. However, it remains unclear to what degree these mechanisms actually aid in the clearance of Ab-coated tumor cells in vivo. We show here that NK cells secrete a distinct profile of potent immunostimulatory cytokines in response to dual stimulation with Ab-coated tumor cells and IL-12. This response could not be duplicated by costimulation with other ILs and was significantly enhanced in the presence of monocytes. Cytokine production was dependent upon synergistic signals mediated by the activating receptor for the Fc portion of IgG (FcgammaRIII) and the IL-12 receptor expressed on NK cells. Coadministration of Ab-coated tumor cells and IL-12 to BALB/c mice resulted in enhanced circulating levels of NK cell-derived cytokines with the capacity to augment anti-tumor immunity. These findings suggest that, in addition to mediating cellular cytotoxicity and apoptosis, the anti-tumor activity of mAb's might also result from activation of a potent cytokine secretion program within immune effectors capable of recognizing mAb-coated targets.

The antitumor effects of IFN-alpha are abrogated in a STAT1-deficient mouse.

IFN-alpha activates the signal transducer and activator of transcription (STAT) family of proteins; however, it is unknown whether IFN-alpha exerts its antitumor actions primarily through a direct effect on malignant cells or by stimulating the immune system. To investigate the contribution of STAT1 signaling within the tumor, we generated a STAT1-deficient melanoma cell line, AGS-1. We reconstituted STAT1 into AGS-1 cells by retroviral gene transfer. The resulting cell line (AGS-1STAT1) showed normal regulation of IFN-alpha-stimulated genes (e.g., H2k, ISG-54) as compared with AGS-1 cells infected with the empty vector (AGS-1MSCV). However, mice challenged with the AGS-1, AGS-1STAT1, and AGS-1MSCV cell lines exhibited nearly identical survival in response to IFN-alpha treatment, indicating that restored STAT1 signaling within the tumor did not augment the antitumor activity of IFN-alpha. In contrast, STAT1-/- mice could not utilize exogenous IFN-alpha to inhibit the growth of STAT1+/+ melanoma cells in either an intraperitoneal tumor model or in the adjuvant setting. The survival of tumor-bearing STAT1-/- mice was identical regardless of treatment (IFN-alpha or PBS). Additional cell depletion studies demonstrated that NK cells mediated the antitumor effects of IFN-alpha. Thus, STAT1-mediated gene regulation within immune effectors was necessary for mediating the antitumor effects of IFN-alpha in this experimental system.

Src homology 2-containing inositol 5'-phosphatase 1 negatively regulates IFN-gamma production by natural killer cells stimulated with antibody-coated tumor cells and interleukin-12.

We have previously shown that natural killer (NK) cells secrete a distinct profile of immunomodulatory cytokines in response to dual stimulation with antibody-coated tumor cells and interleukin-12 (IL-12). This NK cell cytokine response is dependent on synergistic signals mediated by the activating receptor for the Fc portion of IgG (FcgammaRIIIa) and the IL-12 receptor (IL-12R), both constitutively expressed on NK cells. The phosphatase Src homology 2-containing inositol 5'-phosphatase 1 (SHIP1) is known to exert inhibitory effects on Fc receptor (FcR) signaling via its enzymatic activity on phosphatidylinositol 3-kinase (PI3-K) products within many cells of the immune system, most notably mast cells, B cells, and monocytes. However, its activity in the context of FcR activation on NK cells has not been fully explored. The current study focused on the regulation of FcgammaRIIIa-induced NK cell cytokine production by SHIP1. Inhibitor studies showed that NK cell IFN-gamma production following FcR stimulation in the presence of IL-12 depended, in part, on the downstream products of PI3-K. Overexpression of wild-type (WT) SHIP1, but not a catalytic-deficient mutant, via retroviral transfection of primary human NK cells, resulted in a >70% reduction of NK cell IFN-gamma production in response to costimulation. In addition, NK cells from SHIP1-/- mice produced 10-fold greater amounts of IFN-gamma following culture with antibody-coated tumor cells plus IL-12 compared with NK cells from WT mice. Further, activation of the mitogen-activated protein kinase (MAPK) family member extracellular signal-regulated kinase (Erk; a downstream target of PI3-K) was significantly enhanced within SHIP1-/- NK cells compared with WT NK cells following costimulation. Pharmacologic inhibition of Erk activity, but not Jnk MAPK activity, led to significantly decreased IFN-gamma production from both SHIP1-/- and WT NK cells under these conditions. These results are the first to show a physiologic role for SHIP1 in the regulation of NK cell cytokine production and implicate PI3-K in the induction of MAPK signal transduction following costimulation of NK cells via the FcR and the IL-12R.

Constitutive Signaling from an Engineered IL7 Receptor Promotes Durable Tumor Elimination by Tumor-Redirected T Cells.

Successful adoptive T-cell immunotherapy of solid tumors will require improved expansion and cytotoxicity of tumor-directed T cells within tumors. Providing recombinant or transgenic cytokines may produce the desired benefits but is associated with significant toxicities, constraining clinical use. To circumvent this limitation, we constructed a constitutively signaling cytokine receptor, C7R, which potently triggers the IL7 signaling axis but is unresponsive to extracellular cytokine. This strategy augments modified T-cell function following antigen exposure, but avoids stimulating bystander lymphocytes. Coexpressing the C7R with a tumor-directed chimeric antigen receptor (CAR) increased T-cell proliferation, survival, and antitumor activity during repeated exposure to tumor cells, without T-cell dysfunction or autonomous T-cell growth. Furthermore, C7R-coexpressing CAR T cells were active against metastatic neuroblastoma and orthotopic glioblastoma xenograft models even at cell doses that had been ineffective without C7R support. C7R may thus be able to enhance antigen-specific T-cell therapies against cancer.Significance: The constitutively signaling C7R system developed here delivers potent IL7 stimulation to CAR T cells, increasing their persistence and antitumor activity against multiple preclinical tumor models, supporting its clinical development. Cancer Discov; 7(11); 1238-47. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.

MICA-Expressing Monocytes Enhance Natural Killer Cell Fc Receptor-Mediated Antitumor Functions.

Natural killer (NK) cells are large granular lymphocytes that promote the antitumor response via communication with other cell types in the tumor microenvironment. Previously, we have shown that NK cells secrete a profile of immune stimulatory factors (e.g., IFNγ, MIP-1α, and TNFα) in response to dual stimulation with the combination of antibody (Ab)-coated tumor cells and cytokines, such as IL12. We now demonstrate that this response is enhanced in the presence of autologous monocytes. Monocyte enhancement of NK cell activity was dependent on cell-to-cell contact as determined by a Transwell assay. It was hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of MICA on monocytes to NK cell NKG2D receptors. Strategies to block MICA-NKG2D interactions resulted in reductions in IFNγ production. Depletion of monocytes in vivo resulted in decreased IFNγ production by murine NK cells upon exposure to Ab-coated tumor cells. In mice receiving trastuzumab and IL12 therapy, monocyte depletion resulted in significantly greater tumor growth in comparison to mock-depleted controls (P < 0.05). These data suggest that NK cell-monocyte interactions enhance NK cell antitumor activity in the setting of monoclonal Ab therapy for cancer. Cancer Immunol Res; 5(9); 778-89. ©2017 AACR.

Phase I study of the sequential combination of interleukin-12 and interferon alfa-2b in advanced cancer: evidence for modulation of interferon signaling pathways by interleukin-12.

PURPOSE: To evaluate the safety of sequentially administered recombinant (r) human (h) interleukin-12 (IL-12) and interferon alfa-2b (IFN-alpha-2b) in patients with advanced cancer and to determine the effects of endogenously produced IFN-gamma on Janus kinase-signal transducer and activator of transcription (Jak-STAT) signal transduction in patient peripheral-blood mononuclear cells (PBMCs). PATIENTS AND METHODS: Forty-nine patients with metastatic cancer received rhIL-12 on day 1 and IFN-alpha-2b on days 2 to 6 of either a 14-day (n = 43) or a 7-day treatment cycle (n = 6). rhIL-12 was initially administered subcutaneously at a dose of 100 ng/kg, whereas IFN-alpha-2b was escalated from 1 to 10 million units (MU) in cohorts of three patients (1, 3, 5, 7, or 10 MU). rhIL-12 was subsequently administered intravenously (IV) in escalating doses (100 to 500 ng/kg) to achieve greater IFN-gamma production. Peripheral blood was drawn for measurement of plasma IFN-gamma and the induction of Jak-STAT signal transduction in PBMCs. RESULTS: No IL-12-or IFN-alpha-related dose-limiting toxicities were observed. There were no responses in 41 assessable patients. Five patients exhibited stable disease lasting 6 months or longer while on therapy. Optimal induction of IFN-gamma by IL-12 occurred after an IV dose of 250 ng/kg. Patient PBMCs exhibited increased levels of STAT1 after IL-12 administration. The peak level of IFN-gamma achieved with IL-12 therapy correlated with the peak level of intracellular STAT1 in patient PBMCs (r = 0.38, P = .021). CONCLUSION: The combination of rhIL-12 and IFN-alpha-2b can be administered sequentially with minimal toxicity. IV administration of rhIL-12 modulates IFN-alpha-induced Jak-STAT signal transduction in patient PBMCs.

Prolonged culture of vaccine-primed lymphocytes results in decreased antitumor killing and change in cytokine secretion.

Adoptive transfer of effector T cells has been used successfully to eliminate metastases in animal models. Because antitumor activity depends on the number of effector cells transferred, some human trials have used in vitro-repetitive activation and expansion techniques to increase cell number. We hypothesized that the prolonged culture period might contribute to the lack of human trial success by decreasing the potency of the effector T cells. Lymph nodes draining a progressively growing murine melanoma tumor transduced to secrete granulocyte/macrophage colony-stimulating factor were harvested and activated in vitro with anti-CD3 monoclonal antibody followed by expansion in IL-2 for a total of 5 days in culture. Some lymphocytes were reactivated and further expanded for a total of 9 days in culture. In vivo activity of the effector T cells was measured by the reduction in lung metastases and is shown to be dose dependent. The prolonged culture period resulted in nearly 3-fold more T cells but at least 8-fold less antitumor activity. This was accompanied by decreased secretion of the proinflammatory cytokine, IFN-gamma, and increased secretion of the anti-inflammatory cytokine, IL-10. Thus, although increased cell number is important to maximize the effectiveness of adoptive immunotherapy, some culture conditions may actually be counterproductive in that decreases in cell potency can outweigh the benefits of increased cell numbers. The T-cell cytokine secretion pattern predicts decreased effector cell function and may explain the decreased antitumor effect.