Clay L. Efferson

Inhibition of Tumor Growth Progression by Antiandrogens and mTOR Inhibitor in a Pten-Deficient Mouse Model of Prostate Cancer

Androgen receptors have been shown to play a critical role in prostate cancer. We used ultrasound imaging techniques to track tumor response to antiandrogen and rapamycin treatment in a prostate-specific Pten-deleted mouse model of cancer. Depletion of androgens by either surgical or chemical castration significantly inhibited tumor growth progression without altering the activation of Akt and mammalian target of rapamycin (mTOR). We also showed for the first time that targeting mTOR along with antiandrogen treatment exhibited additive antitumor effects in vivo when compared with single agents. Our preclinical data suggest that combination of antiandrogens with mTOR inhibitors might be more effective in treating androgen-dependent prostate cancer patients.

Melanoma differentiation-associated gene-7 (mda-7 )/interleukin (IL)-24 induces anticancer immunity in a syngeneic murine model

Previous studies have shown that the human melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) has tumor-suppressor activity in vitro and in vivo. Additionally, in vitro studies using human peripheral blood mononuclear cells indicate that mda-7/IL-24 has TH1 cytokine-like activity. However, the individual properties of mda-7/IL-24 have been previously examined separately. Thus, there is not a single study that has examined both, antitumor and proimmune properties of mda-7/IL-24. Furthermore, the tumor suppressive activity and the cytokine activity of mda-7/IL-24 have not been previously tested in an immunocompetent setting. We therefore in the present study evaluated the antitumor and immune properties of mda-7/IL-24 in a murine syngeneic tumor model. In vitro, adenovirus-mediated mda-7 gene (Ad-mda7) transfer to murine fibrosarcoma (UV2237m; MCA16) and normal (10T1/2) cells significantly inhibited growth (P=0.001) and induced apoptosis in tumor cells but not in normal cells. In vivo, intratumoral administration of Ad-mda7 resulted in significant inhibition of tumor growth (P<0.05), with a subset of mice showing complete tumor regression. We next evaluated the immune potentiation activity of Ad-mda7 in a cancer vaccine model. UV2237m cells transfected with Ad-mda7 and injected into syngeneic immunocompetent C3H mice were unable to grow; however, they did grow in immunocompromised nude mice. These tumor-free C3H mice, when challenged with parental tumor cells experienced no tumor growth, suggesting induction of systemic immunity. Moreover, splenocytes prepared from vaccinated C3H mice demonstrated higher proliferative activity and produced elevated levels of TH1 cytokines compared with those from control mice. An in vitro subset analysis of splenocytes from vaccinated mice demonstrated a significant increase in the CD3+CD8+ but not the CD3+CD4+ cell population (P=0.019). Thus Ad-mda7 treatment of syngeneic tumors induces tumor cell death and promotes immune activation, leading to anticancer immunity.

Fine Specificity of High Molecular Weight-Melanoma-Associated Antigen-Specific Cytotoxic T Lymphocytes Elicited by Anti-Idiotypic Monoclonal Antibodies in Patients with Melanoma

HLA-A2-restricted CTLs, which lysed high molecular weight (HMW)-melanoma-associated antigen (MAA)+ melanoma cells, were induced in patients with melanoma immunized with MELIMMUNE, a combination of the murine anti-idiotypic (anti-id) monoclonal antibodies (mAb) MEL-2 and MF11–30 (MW Pride et al., Clin Cancer Res 1998;4:2363.). In the present study we investigated whether CTL epitopes are present in anti-id mAb MF-11–30 and activate T cells to recognize HMW-MAA on melanoma cells. One candidate epitope in the mAb MF11–30 VH chain, VH (3–11), was selected based on the presence of HLA-A2 anchor residues and partial homology with the HMW-MAA epitope, HMW-MAA (76–84). Lymphocytes from HLA-A2+-immunized patients proliferated to VH (3–11) peptide and to a variant HMW-MAA peptide to a significantly greater extent than autologous lymphocytes stimulated with an irrelevant peptide and lymphocytes from nonimmunized patients. No proliferative response was detected to the wild-type HMW-MAA peptide (76–84). Significant increase in IFN-γ production but not in interleukin 10 production in response to VH (3–11) and to variant HMW-MAA peptide (76–84) was observed in lymphocytes from the immunized patients. Stimulation of lymphocytes from HLA-A2+ patients with the two peptides induced CTL, which lysed HMW-MAA+/HLA-A2+ A375SM melanoma cells. This is the first report documenting the presence of immunogenic peptides in a murine anti-id mAb for a defined epitope expressed by a human melanoma-associated antigen. These results may be relevant for development of novel vaccines based on homology between anti-id mAb and tumor-associated antigen amino acid sequences.

Induction of Tumor-Reactive CTL by C-Side Chain Variants of the CTL Epitope HER-2/neu Protooncogene (369-377) Selected by Molecular Modeling of the Peptide: HLA-A2 Complex

To design side chain variants for modulation of immunogenicity, we modeled the complex of the HLA-A2 molecule with an immunodominant peptide, E75, from the HER-2/neu protooncogene protein recognized by CTL. We identified the side chain orientation of E75. We modified E75 at the central Ser5 (E75 wild-type), which points upward, by removing successively the HO (variant S5A) and the CH2-OH (variant S5G). Replacement of the OH with an aminopropyl (CH2)3-NH3 (variant S5K) maintained a similar upward orientation of the side chain. S5A and S5G were stronger stimulators while S5K was a weaker stimulator than E75 for induction of lytic function, indicating that the OH group and its extension hindered TCR activation. S5K-CTL survived longer than did CTL induced by E75 and the variants S5A and S5G, which became apoptotic after restimulation with the inducer. S5K-CTL also recognized E75 endogenously presented by the tumor by IFN-γ production and specific cytolysis. S5K-CTL expanded at stimulation with E75 or with E75 plus agonistic anti-Fas mAb. Compared with S5K-CTL that had been restimulated with the inducer S5K, S5K-CTL stimulated with wild-type E75 expressed higher levels of E75+ TCR and BCL-2. Activation of human tumor-reactive CTL by weaker agonists than the nominal Ag, followed by expansion with the nominal Ag, is a novel approach to antitumor CTL development. Fine tuning of activation of tumor-reactive CTL by weak agonists, designed by molecular modeling, may circumvent cell death or tolerization induced by tumor Ag, and thus, may provide a novel approach to the rational design of human cancer vaccines.

Activation of Tumor Antigen-Specific Cytotoxic T Lymphocytes (CTLs) by Human Dendritic Cells Infected with an Attenuated Influenza A Virus Expressing a CTL Epitope Derived from the HER-2/neu Proto-Oncogene

ABSTRACT The development of cancer vaccines requires approaches to induce expansion and functional differentiation of tumor antigen-specific cytotoxic T lymphocyte (CTL) effectors which posses cytolytic capability and produce cytokines. Efficient induction of such cells is hindered by the poor immunogenicity of tumor antigens and by the poor transduction efficiency of dendritic cells (DCs) with current nonreplicating vectors. We have investigated the use of influenza A virus, a potent viral inducer of CTLs, as a vector expressing the immunodominant HER-2 CTL epitope KIF (E75). For this purpose, an attenuated influenza A/PR8/34 virus with a truncated nonstructural (NS1) gene was generated containing the E75 epitope in its neuraminidase protein (KIF-NS virus). Stimulation of peripheral blood mononuclear cells from healthy donors and of tumor-associated lymphocytes from ovarian and breast cancer patients with DCs infected with KIF-NS virus (KIF-NS DC) induced CTLs that specifically recognized the peptide KIF and HER-2-expressing tumors in cytotoxicity assays and secreted gamma interferon (IFN-γ) and interleukin-2 at recall with peptide. Priming with KIF-NS DCs increased the number of E75+ CD45RO+ cells by more than 10-fold compared to nonstimulated cells. In addition, KIF-NS virus induced high levels of IFN-α in DCs. This is the first report demonstrating induction of human epitope-specific CTLs against a tumor-associated antigen with a live attenuated recombinant influenza virus vector. Such vectors may provide a novel approach for tumor antigen delivery, lymphocyte activation, and differentiation in human cancer vaccine development.

Prostate Tumor Cells Infected with a Recombinant Influenza Virus Expressing a Truncated NS1 Protein Activate Cytolytic CD8+ Cells To Recognize Noninfected Tumor Cells

ABSTRACT Many viral oncolytic approaches against cancer are based on the ability of specific viruses to replicate in tumors expressing components of the constitutively activated Ras/mitogen-activated protein kinase (MAPK) pathways and/or inhibited or dysregulated alpha/beta interferon (IFN-α/β) response pathways. A major issue when considering these approaches is their applicability to tumors that lack activated Ras. To identify the effector mechanisms activated by oncolytic viruses, we investigated inhibition of proliferation of the prostate cancer line LNCap by the recombinant TR-NS1 influenza A virus, a genetically attenuated influenza A/PR8/34 virus expressing a truncated nonstructural protein (NS1) of 126 amino acids. LNCap cells lack constitutively activated MAPK, extracellular signal-regulated kinase (ERK), and p38 and are resistant to death by IFN-α. Truncation of the NS1 protein of influenza viruses is known to result in viral attenuation due to a reduced ability of the NS1 to inhibit the IFN-α/β response. Infection with TR-NS1 virus rapidly activated ERK-1 more than ERK-2 in LNCap cells. Importantly, TR-NS1 virus infection transiently inhibited cell proliferation and induced apoptosis in LNCap cells. Addition of peripheral blood mononuclear cells (PBMC) and interleukin 12 (IL-12) to TR-NS1 virus-infected LNCap cells (TR-NS1-LNCap) resulted in faster elimination of TR-NS1-LNCap cells compared with LNCap cells. Moreover, TR-NS1-LNCap cells induced IFN-γ in PBMC. The levels of IFN-γ were amplified by IL-12. TR-NS1-LNCap cells also induced tumor-lytic cytotoxic T lymphocytes (CTL). These CTL lysed noninfected LNCap cells in a CD8-dependent manner. Activation of cellular immunity to tumor cells by viruses is an intriguing effector pathway, which should be especially significant for elimination of human tumors that lack activated Ras.

Taxol Increases the Amount and T Cell–Activating Ability of Self-Immune Stimulatory Multimolecular Complexes Found in Ovarian Cancer Cells

It has been proposed that chemotherapy enhances tumor antigen (TA)-specific immunity. The molecular form of TA from ovarian tumor that activates cellular immunity is unknown. We report here identification of a novel molecular form of immunogenic TA for CD8(+) cells named self-immune stimulatory multimolecular complexes (ISMMC). ISMMC consist of a molecular complex of polyosome/ribosome-bound ubiquitinated nascent HER-2 polypeptides. This complex is chaperoned by heat shock protein Gp96, which mediates ISMMC uptake by antigen-presenting cells through the scavenger receptor CD91. RNAs in ISMMC stimulate immature dendritic cells to secrete interleukin 12 and induce IFN-gamma in peripheral blood mononuclear cells. ISMMC dissociate, retrotranslocate from the lysosome to cytoplasm, and are processed to peptides by the proteasome. At subpharmacologic doses, Taxol increased the amount of ISMMC by three to four times and modified their composition by inducing the attachment of cochaperones of HSP70, such as the mitotic-phase phosphoprotein 11J. On a total protein basis, Taxol induced ISMMC, expanded more CD8(+) cells, activated more CD56(+) NKG2D(+) cells to produce IFN-gamma, and were more potent inducers of high T-cell receptor density Perforin(+) cells than native ISMMC and peptide E75. Elucidation of the composition of ISMMC and identification of adducts formed by Taxol should be important for developing molecular cancer vaccines.

Sensitivity of Undifferentiated, High-TCR Density CD8+ Cells to Methylene Groups Appended to Tumor Antigen Determines Their Differentiation or Death

CD8(+) cells expressing high numbers of TCR per cell (TCR(hi)) are considered important mediators of antitumor effects. To understand the relationship between TCR density and antigen affinity for TCR in the outcome of stimulation with antigen and differentiation of CTL recognizing tumor antigen, we analyzed perforin induction in ovarian tumor-associated lymphocytes in response to the smallest possible changes in the atomic forces of interaction between antigen and TCR. Stimulating undifferentiated, apoptosis-resistant CD8(+) cells expressing high levels of E75-TCR (TCR(hi)) with variants of the CTL epitope E75, HER-2 (369-377), induced their stepwise differentiation, first to IFN-gamma(+) Perf(-) and to TCR(hi) IFN-gamma(+) Perf(+) cells. Blocking caspase-9 activation at antigen stimulation also enhanced the generation of TCR(hi) Perf(hi) cells, demonstrating that TCR density dictated the pathway of death activated by stimulation with the same agonist. Expansion and differentiation of TCR(hi) Perf(+) CTL required an agonist of optimal CH(2) side chain length, which in this study was equal to two CH(2) groups appended to E75 at the Gly(4) position. Side chains one CH(2) shorter or longer than optimal were either less stimulatory or induced death of TCR(hi) Perf(+) cells. Differentiation of TCR(hi) CD8(+) cells can be finely tuned by synthetic amino acids in the peptide, whose side chains induce small increments in the affinity of the antigen for TCR below the affinity which induce apoptosis.

Prostate tumor cells infected with a recombinant influenza virus with truncated NS1 gene NS1 (1-126) activate cytolytic CD8+ cells which recognize non-infected tumors

Cystemustine (60 or 90 mg/m). We observed 6 complete responses (CR) of long duration, and 14 partial responses (mean duration 24 weeks).The main target is DNA, with generation of O-chloroethylguanine and cross-linking with cDNA strands (Madelmont 1994); but the suicidal DNA repair protein O6-alkylguanine-DNA alkyltransferase (MGMT) removes O6alkylguanine by accepting the alkyl group. MGMT level varies considerably in normal and tumor cells, and cells that exhibit a low MGMT level are more sensitive to CENU: they are designated Mer-, i.e. about 15–20% of humans. CR are obtained after a limited number of courses: probably tumors presenting with a low constitutive level of MGMT. Cystemustine has also been observed to interfere with cell differentiation by stimulating melanoma markers expression (Buchdahl, 1998). Cystemustinetreated B16 melanoma cells acquired a new phospholipid metabolism phenotype (Morvan, 2002; 2003). During growth inhibition phase, phosphocholine and phosphoethanolamine levels increased at a hight level, definitively acquired once tumours recovered growth: this could participate to tumour cells redifferentiation. In syngenic C57Bl6 model (Demidem, 2001), cystemustine treated tumours appeared to confer after resection a protective effect towards secondary grafted untreated tumours: phospholipid changes at the plasmatic membrane could be involved in the immune response (Sakai, 1997; Bloom 1997). These mechanisms could explain a late effect of chemotherapy: our partial response patients were obtained after several courses. To improve cystemustine efficacy, MGMT expression and the phospholipid metabolism would be strategic points. Methionine restriction, in vitro/vivo inhibits growth of human and animal tumors with: i)a decrease of MGMT ARNm and activity in tumoral cells (Kokkinakis, 1997); ii) changes in phospholipid metabolism (Demidem, 2002). The effect of a methionine-less diet on cystemustine treatment has been shown on B16 melanoma bearing mice (Morvan, AACR 2002). Hence, we initiated a phase I trial of dietary methionine restriction, in association with cystemustine for recurrent gliomas and metastatic melanomas to determine the optimal methionine-free diet duration and the feasability of this association, including MGMT evaluation.