Synergy between chemotherapeutic agents and CTLA-4 blockade in preclinical tumor models.

Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) binding agent, has proven to be an effective monotherapy for metastatic melanoma and has shown antitumor activity in trials when administered with other therapeutic agents. We hypothesized that the combination of ipilimumab with chemotherapeutic agents, such as ixabepilone, paclitaxel, etoposide, and gemcitabine, may produce therapeutic synergy based on distinct but complementary mechanisms of action for each drug and unique cellular targets. This concept was investigated using a mouse homolog of ipilimumab in preclinical murine tumor models, including SA1N fibrosarcoma, EMT-6 mammary carcinoma, M109 lung carcinoma, and CT-26 colon carcinoma. Results of CTLA-4 blockade in combination with one of various chemotherapeutic agents demonstrate that synergy occurs in settings where either agent alone was not effective in inducing tumor regression. Furthermore, when combined with CTLA-4 blockade, ixabepilone, etoposide, and gemcitabine elicited prolonged antitumor effects in some murine models with induction of a memory immune response. Future investigations are warranted to determine which specific chemo-immunotherapy combinations, if any, will produce synergistic antitumor effects in the clinical setting.

Interleukin-21 combined with PD-1 or CTLA-4 blockade enhances antitumor immunity in mouse tumor models.

Recent advances in cancer treatment with checkpoint blockade of receptors such as CTLA-4 and PD-1 have demonstrated that combinations of agents with complementary immunomodulatory effects have the potential to enhance antitumor activity as compared to single agents. We investigated the efficacy of immune-modulatory interleukin-21 (IL-21) combined with checkpoint blockade in several syngeneic mouse tumor models. After tumor establishment, mice were administered recombinant mouse IL-21 (mIL-21) alone or in combination with blocking monoclonal antibodies against mouse PD-1 or CTLA-4. In contrast to monotherapy, IL-21 enhanced antitumor activity of mCTLA-4 mAb in four models and anti-PD-1 mAb in two models, with evidence of synergy for one or both of the combination treatments in the EMT-6 and MC38 models. The enhanced efficacy was associated with increased intratumoral CD8+ T cell infiltrates, CD8+ T cell proliferation, and increased effector memory T cells, along with decreased frequency of central memory CD8+ T cells. In vivo depletion of CD8+ T cells abolished the antitumor activities observed for both combination and monotherapy treatments, further supporting a beneficial role for CD8+ T cells. In all studies, the combination therapies were well tolerated. These results support the hypothesis that the combination of recombinant human IL-21 with CTLA-4 or PD-1 monoclonal antibodies could lead to improved outcomes in cancer patients.

Pharmacokinetics, biodistribution, and radioimmunotherapy with monoclonal antibody 776.1 in a murine model of human ovarian cancer.

776.1 is a murine IgG1 monoclonal antibody to the human ovarian cancer antigen CA 125 that has the unique property of having a clear preference for binding to the cell-associated form of the antigen. We have examined the tumor localization properties and efficacy of 776.1 in a subcutaneous OVCAR-3 xenograft mouse model of human ovarian cancer. Biodistribution experiments using (125)I-labeled 776.1 demonstrated a peak uptake in tumors at 72 hours postinjection, with an average of 17.7% of injected dose per gram localized to the tumor. Little uptake in other organs was observed. Further experiments using CA 125-transfected syngeneic tumors, as well as an immunoprecipitation assay using human chimeric 776.1, both clearly demonstrated that 776.1 localizes to the tumor in a CA 125-dependent manner. DOTA-776.1 (1,4,7,10-tetraazacyclododecane-N,N',N",N'" tetraacetic acid-conjugated 776.1) was labeled with (90)Y and used in efficacy studies. [(90)Y-DOTA]776.1 at a single dose of 150 microCi was able to mediate efficient reduction of tumor growth, with regression observed in a subset of animals for a period ranging from 3 to 48 days, equivalent to 3 weekly administrations of cisplatin at 6 mg/kg. No significant regression was observed in groups receiving [(90)Y-DOTA]MOPC-21 control antibody at any dose. These results suggest that 776.1 may be a promising radioimmunotherapeutic agent for the treatment of human ovarian cancer.

Radiotherapy of human xenograft NSCLC tumors in nude mice with a 90Y-labeled anti-tissue factor antibody.

Tissue factor (TF) is a type I transmembrane protein and the initiator of the extrinsic blood coagulation pathway. TF plays a critical role in tumor development and its overexpression is observed in many tumors. To understand the prevalence and relative level of TF expression in non-small-cell lung cancer (NSCLC), we analyzed 50 NSCLC tumors by immunohistochemical staining and found that 88% of human NSCLC tumors overexpressed TF. We then generated a high affinity anti-TF antibody, TF278, which specifically binds TF on the surface of cells and is internalized upon binding. An 111In-labeled TF278 demonstrated favorable tumor accumulation in an SW-900 xenograft tumor model with a maximum mean percent of injected dose per gram of tissue (%ID/g) of 73.1% at 96 hours postinjection. In addition, we labeled the antibody with 90Y and tested its ability to inhibit the growth of tumors in an SW-900 xenograft tumor model in immunocompromised mice. The 90Y-TF278 slowed the growth of SW-900 tumors at a 50 microCi dose and completely regressed SW-900 tumors at a 150 microCi dose with little toxicity.

Paradoxical activation of T cells via augmented ERK signaling mediated by a RAF inhibitor.

RAF inhibitors selectively block ERK signaling in BRAF-mutant melanomas and have defined a genotype-guided approach to care for this disease. RAF inhibitors have the opposite effect in BRAF wild-type tumor cells, where they cause hyperactivation of ERK signaling. Here, we predict that RAF inhibitors can enhance T cell activation, based upon the observation that these agents paradoxically activate ERK signaling in BRAF wild-type cells. To test this hypothesis, we have evaluated the effects of the RAF inhibitor BMS908662 on T cell activation and signaling in vitro and in vivo. We observe that T cell activation is enhanced in a concentration-dependent manner and that this effect corresponds with increased ERK signaling, consistent with paradoxical activation of the pathway. Furthermore, we find that the combination of BMS908662 with CTLA-4 blockade in vivo potentiates T cell expansion, corresponding with hyperactivation of ERK signaling in T cells detectable ex vivo. Lastly, this combination demonstrates superior anti-tumor activity, compared to either agent alone, in two transplantable tumor models. This study provides clear evidence that RAF inhibitors can modulate T cell function by potentiating T cell activation in vitro and in vivo. Paradoxical activation of ERK signaling in T cells offers one mechanism to explain the enhanced antitumor activity seen when RAF inhibitors are combined with CTLA-4 blockade in preclinical models.

Synergistic effects of combined therapy using paclitaxel and [90Y-DOTA]776.1 on growth of OVCAR-3 ovarian carcinoma xenografts.

OBJECTIVE: 776.1 is a monoclonal antibody prepared against the human ovarian cancer antigen CA 125 that demonstrates preferential binding to the cell-associated form of the antigen and has shown promising results as an yttrium-90-labeled antibody in pre-clinical studies examining the effects on tumor growth in a murine xenograft model of human ovarian cancer. The purpose of the present study was to examine the effects of combined therapy with [90Y-DOTA]776.1 and paclitaxel compared with monotherapy with either agent on the growth of OVCAR-3 xenografts in nude mice. METHODS: Mice bearing OVCAR-3 xenografts were treated with paclitaxel alone, 50 microCi or 150 microCi [90Y-DOTA]776.1 alone, or a combination of both treatments. Control groups were included which consisted of a nonspecific antibody, MOPC-21, labeled to a similar degree, administered as monotherapy or in combination with paclitaxel. The effects of administration of radioimmunotherapy prior to or following chemotherapy were also examined. RESULTS: Treatment with paclitaxel and [90Y-DOTA]776.1 had a synergistic anti-tumor effect on the growth of OVCAR-3 xenografts. Synergy was only observed when a tumor-specific antibody was used in radioimmunotherapy. While no difference in tumor growth was observed with order of dosing, reduced toxicity was seen when paclitaxel was administered prior to radioimmunotherapy. CONCLUSION: The combination of radioimmunotherapy using an anti-CA 125 monoclonal antibody and chemotherapy with paclitaxel was shown to be effective in an in vivo model of ovarian cancer and may hold promise as a treatment regimen for patients with ovarian cancer.