Kenneth F. May

Combination Therapy with Anti–CTL Antigen-4 and Anti-4-1BB Antibodies Enhances Cancer Immunity and Reduces Autoimmunity

The majority of cancer antigens identified thus far have limited expression in normal tissues. It has been suggested that autoimmune disease is a necessary price for cancer immunity. This notion is supported by a recent clinical trial involving an anti-CTL antigen-4 (CTLA-4) antibody that showed significant clinical responses but severe autoimmune diseases in melanoma patients. To selectively modulate cancer immunity and autoimmunity, we used anti-CTLA-4 and anti-4-1BB antibodies to treat mice with a preexisting cancer, MC38. The combination of the two antibodies led to CD8 T-cell-mediated rejection of large established MC38 tumors and long-lasting immunity to the same tumor cells, although the same regimen was not effective for B16 melanoma. More importantly, whereas individual antibodies induced inflammation and autoimmune manifestations, combination therapy increased cancer immunity while reducing autoimmunity. The reduction of autoimmune effects correlates with an increased function of regulatory T cells. Our results suggest a novel approach to simultaneously enhance cancer immunity and reduce autoimmunity.

Failed adoptive immunotherapy with tumor-specific T cells: reversal with low-dose interleukin 15 but not low-dose interleukin 2.

Adoptive immunotherapy with tumor-specific T cells has emerged as a valid approach for prevention or treatment of diseases, such as melanoma and EBV-associated lymphoma. As interleukin (IL) 15 promotes survival of CD8+ memory CTLs, we hypothesized that it could be used to enhance antitumor immunity in vivo through the maintenance of adoptively transferred memory CTL. To test this, we treated mice bearing P1A+ tumors with adoptively transferred T cells possessing a transgenic Vα8+ T-cell receptor specific for the P1A tumor antigen (called P1CTL). Mice were then randomized to receive daily low-dose IL-15 (0.5 μg/day) or PBS. Mice receiving the transgenic P1CTL and IL-15 experienced a significantly delayed tumor relapse or complete tumor regression (P < 0.002 compared with PBS), with a striking persistence of the CD8+ Vα8+ P1CTL compared with mice receiving the CD8+ Vα8+ P1CTL and PBS vehicle (26.3 versus 5.1% P < 10−5). Animals exhibiting complete tumor regression had a significant population of CD8+ Vα8+ P1CTL (46%) that persisted with IL-15 treatment until 140 days after adoptive transfer and successfully defended them against tumor rechallenge without IL-15. Low-dose IL-2 afforded no protection over vehicle and resulted in lower percentages of T cells with an activated memory phenotype, lower Bcl-2 expression, and lower ex vivo antitumor cytotoxicity compared with mice treated with IL-15. Collectively, the data support the notion that exogenous low-dose IL-15 therapy can enhance and even reverse the limited efficacy of adoptively transferred tumor-specific T-cell therapy and may do so in a fashion that is superior and distinct from exogenous IL-2 therapy.

B7-deficient autoreactive T cells are highly susceptible to suppression by CD4(+)CD25(+) regulatory T cells.

CD4+CD25+ regulatory T cells (Tregs) suppress immunity to infections and tumors as well as autoimmunity and graft-vs-host disease. Since Tregs constitutively express CTLA-4 and activated T cells express B7-1 and B7-2, it has been suggested that the interaction between CTLA-4 on Tregs and B7-1/2 on the effector T cells may be required for immune suppression. In this study, we report that autopathogenic T cells from B7-deficient mice cause multiorgan inflammation when adoptively transferred into syngeneic RAG-1-deficient hosts. More importantly, this inflammation is suppressed by adoptive transfer of purified wild-type (WT) CD4+CD25+ T cells. WT Tregs also inhibited lymphoproliferation and acquisition of activation markers by the B7-deficient T cells. An in vitro suppressor assay revealed that WT and B7-deficient T cells are equally susceptible to WT Treg regulation. These results demonstrate that B7-deficient T cells are highly susceptible to immune suppression by WT Tregs and refute the hypothesis that B7-CTLA-4 interaction between effector T cells and Tregs plays an essential role in Treg function.

A novel method for the expansion of cytotoxic T-cells using an artificially created cell (CHO) in the presence of MHC dimer-peptide in naive and primed cell lines

S: POSTER PRESENTATIONS P1 Assessment of the Teratogenicity of Isosuifan Blue (Lymphazurin 1%) S.B. Banks,* Q. Miller, J.G. Harre, M.C. Lilly, D.M. Rose. Keesler Medical Center, Biloxi, MS. INTRODUCTION: This study evaluated the effects of isosulfan blue (Lymphazurin 1%) in developing fetuses of pregnant female rats. Ifisosulfan blue were found to be safe for use in pregnant human patients, for sentinel lymph node biopsy, a full lymph node dissection could be avoided in selected cases of breast cancer or melanoma that occurs in association with pregnancy. METHODS: Forty-seven pregnant rats received either subcutaneous injection (1.0 ml) of isosulfan blue or normal saline on one of the following gestational days: seven, fourteen, or eighteen. Each rat underwent cesarean section either the following day (24 hours post injection), or was delayed until full-term (20 days). Amniotic fluid samples were drawn and fetuses were examined grossly while weight was recorded in full-term fetuses only. Fullterm fetuses were then fixed in either Bouins solution or 95% ethanol to allow subsequent histological examination of the soft tissues or skeletal structures for any malformations. RESULTS: A total of 689 fetuses were harvested. Only two malformations were found in the control arm. There were 521 amniotic fluid samples collected; the average absorbance of the isosulfan blue arm was 0.01563 +/0.02, while the control average was 0.01532 +/0.03. The fetal abortion/resorption rate was equivalent in both treatment and control groups at 4.34%. The overall weight ofgestational day 20 fetuses obtained by cesarean section in the isosulfan blue group was 3.56 +/1.06 grams and the overall birthweight of the control group was 3.69 +/1.00 grams. The malformation rates, amniotic fluid light absorbance averages, full-term fetal abortion rates, or full-term average weights were not statistically different between groups (p>.05). No additional abnormalities were identified following histologic examination. CONCLUSIONS: No statistically significant differences were found between the isosulfan and control groups (p>.05). Isosulfan blue dye was administered in this study of gravid rats without an increased fetal malformation risk or abortion rate. It is highly unlikely that isosulfan blue crosses the placental barrier. P2 A Novel Method for the Expansion of Cytotoxic T-cells Using an Artificially Created Cell (CHO) in the Presence of MHC DimerPeptide in Naive and Primed Cell Lines S.E Abdessalam,* K. May, E. Kocak, E. Martin, P. Zheng, Y. Liu. The Ohio State University Hospitals, Columbus, OH. OBJECTIVE: Insufficient tumor antigen presentation by either tumor cells or host antigen presenting cells can be a major cause of the absence of an immune response in a tumor bearing host. Attempts at inducing a specific immune response to malignancies have been tried with limited success. The purpose of this study was to test the development of cytotoxic T-cells (CD8+ cells) using a novel artificial antigen presenting cell system in both naive and primed lymphocyte populations. METHODS: Spleens were harvested from naive Balb/C mice or primed Balb/C mice (J558-B7 tumor cells injected subcutaneously) and crushed into single cell suspension to isolate mononuclear cells. These cells were then placed into cultures containing CHO-FcR, CHO-B7, CHO-FcR/B7, or no CHO and containing Ld dimer complexed with a respective peptide LCMV [lymphocytic choriomeningitis virus peptide], MCMV [cytomegalovirus peptide], or P1A [J558 sarcoma peptide]. Every 5-8 days the cells were re-stimulated with their respective CHO dimer-peptide combination. The cells were stained with CD8 FITC and Ld:peptide PE at each round. RESULTS: In the naive expansion experiments, starting with 0% CD8+/peptide+ cells, using dimer loaded with MCMV, LCMV, or P1A peptide and respective CHO cell, the MCMV stimulation resulted in a maximum 5.7% CD8+ cells specific for MCMV peptide, the LCMV stimulation resulted in a maximum 9.9% CD8+ cells specific for LCMV peptide, and the P1A stimulation resulted in a maximum of 11.1% CD8+ cells specific for P1A peptide. In the primed expansion experiments, starting with 0.3-1.7% CD8+ cells specific for P1A peptide, a maximum of 49.4% CDS+ cells specific for P1A peptide were identified. CONCLUSIONS: The expansion of cytotoxic T-cells specific against a variety ofpeptides (viral and tumor) is possible using an artificial antigen cell presenting system consisting of CHO cells containing Fc and B7 receptors and dimer loaded with peptide in both a naive model and a primed model. This has potential implications in both the treatment of established tumors and in the prevention of viral diseases. P3 Blockade of IGF-IR Function Reduces COX-2 Expression in Human Pancreatic Cancer Cells O. Stoeltzing, 1. E Fan, 2 W. Liu, 2 A.A. Parikh, 2 N. Reinmuth, 2 D.J. Hicklin, 3 L.M. Ellis. 2 1. Dept. of Surgery, University of Regensburg, Regensburg, Germany; 2. MD Anderson Cancer Center, Houston, TX; 3. ImClone Systems, New York, NY. INTRODUCTION: Both the insulin-like growth factor-I (IGF-I) receptor and cyclooxygenase-2 (Cox-2) are frequently overexpressed in human pancreatic cancer (PC). We hypothesized that IGF-IR may lead to induction of COX-2 expression in pancreatic caner and sought to investigate the signaling pathways mediating this effect. METHODS: Human PC ceils (L3.6pl) were treated with IGF-I to determine activated signaling intermediates. COX-2 expression in PC cells was examined by Western blot and Northern blot analyses. Signaling pathways mediating COX-2 up-regulation were identified by inhibiting MEK/Erk, PI-3K/Akt, and P38 pathways. Effects of IGF-IR inhibition on COX-2 expression were first investigated by stably transfecting L3.6pl cells with a dominant-negative receptor construct (IGF-IR DN) or empty vector (pcDNA) (control). To further investigate IGF-R function, we used a selective IGF-IR blocking antibody (A12) in vitro. Effects of A12 on IGF-IR phosphorylation, signaling pathway activation, and COX-2 expression were subsequently determined by Western blot analyses. Impact of IGFIR inhibition on cell proliferation and survival were investigated using the MTT assay, with or without A12. RESULTS: IGF-1 induced COX-2 expression via the MAPK (Erkl/2) signaling pathway. Surprisingly, inhibition of PI3K/Akt function increased constitutive and inducible COX-2 expression in cells. Constitutive COX-2 expression in IGF-IR DN cells was dramatically reduced compared to pcDNA controls. IGF-I induction of COX-2 expression was also blunted in IGF-IR DN cells. Inhibition of IGF-IR by Al2 antibody effectively blocked IGF-IR phosphorylation and decreased constitutive plus inducible Erk activation. In addition, A12 treated cells showed a 50% reduction in COX-2 expression and a blunted response to IGF-I. Treatment with A12 significantly decreased cell numbers after 48 h after treatment with IGF-I. CONCLUSION: The IGF-IR system is an important regulator of COX2 expression in human pancreatic cancer. Blockade of IGF-IR function significantly reduced COX-2 expression and cell survival, and may therefore be a promising approach for the treatment of pancreatic cancer. P4 Rapamycin Synergistically Enhances the Cytotoxicity of Paclitaxel in Breast Cancer Cells W. Mondesire,* W. Jian, H. Zhang, J. Peng, J. Dong, E Meric-Bernstam. MD Anderson Cancer Center, Houston, TX. Introduction: Rapamycin inhibits the serine-threonine kinase mTOR, inhibiting protein translation and leading to cell cycle arrest in G1. Although rapamycin and its analogues are currently in Phase II/III clinical trials, few patients have had a clinically significant response with single agent therapy.

Anti-4-1BB monoclonal antibody enhances rejection of large tumor burden by promoting survival but not clonal expansion of tumor-specific CD8+ T cells

84 SSO 57TH ANNUAL CANCER SYMPOSIUM