A prime/boost vaccine platform efficiently identifies CD27 agonism and depletion of myeloid-derived suppressor cells as therapies that rationally combine with checkpoint blockade in ovarian cancer.

Cancer immunotherapies have generated remarkable clinical responses for some patients with advanced/metastatic disease, prompting exploration of rational combination therapies to bolster anti-tumor immunity in patients with limited response or those who experience tumor progression following an initial response to immunotherapy. In contrast to other tumor indications, objective response rates to single-agent PD-1/PD-L1 blockade in ovarian cancer are limited, suggesting a need to identify combinatorial approaches that lead to tumor regression in a setting where checkpoint blockade alone is ineffective. Using a pre-clinical model of aggressive intraperitoneal ovarian cancer, we have previously reported on a heterologous prime/boost cancer vaccine that elicits robust anti-tumor immunity, prolongs survival of tumor-bearing mice, and which is further improved when combined with checkpoint blockade. As tumor control in this model is CD8 + T cell dependent, we reasoned that the prime/boost vaccine platform could be used to explore additional treatment combinations intended to bolster the effects of CD8 + T cells. Using whole tumor transcriptomic data, we identified candidate therapeutic targets anticipated to rationally combine with prime/boost vaccination. In the context of a highly effective cancer vaccine, CD27 agonism or antibody-mediated depletion of granulocytic cells each modestly increased tumor control following vaccination, with anti-PD-1 therapy further improving treatment efficacy. These findings support the use of immunotherapies with well-defined mechanisms(s) of action as a valuable platform for identifying candidate combination approaches for further therapeutic testing in ovarian cancer.

Identification, characterization, and cloning of TIP-B1, a novel protein inhibitor of tumor necrosis factor-induced lysis.

Some cancer cells evade elimination by virtue of their insensitivity to agents that induce apoptosis. Conversely, the side effects of anticancer agents could be diminished if normal cells were more resistant. To further elucidate the factors that contribute to the susceptibility of a cell to apoptosis, these investigations were designed to identify proteins isolated from cells exposed to low concentrations of tumor necrosis factor (TNF) that, when incubated with normally TNF-sensitive cells, protect these cells from TNF-induced cytotoxicity. TIP-B1, a novel protein, has been identified, purified, and characterized from cytosolic extracts of TNF-treated human fibroblasts. The approximately 27 kDa pI-4.5 TIP-B1 protein is unique based on both the sequence of three internal peptides (comprising 51 amino acids) and the nucleotide sequence of the corresponding 783-bp cDNA partial clone. Western blot analyses using polyclonal antisera raised against both the purified native TIP-B1 and the approximately 14 kDa product of the cDNA partial TIP-B1 clone, as well as Northern blot analyses using the cDNA insert as a probe, indicate that TIP-B1 may belong to a family of proteins that are expressed in a number of cell lines from diverse tissues. TNF-sensitive cells, when exposed to 4-10 microg/ml concentrations of TIP-B1 prior to the addition of TNF, are completely protected from TNF-induced lysis. Furthermore, TIP-B1 protects cells from apoptotic lysis induced by TNF. Preincubation of TIP-B1 with TNF does not affect the ability of TNF to induce lysis. Moreover, TIP-B1 does not seem to interfere with the interactions between TNF and the TNF receptors, based on a preliminary flow cytometric analysis of the cellular binding of biotinylated TNF. On the basis of these characteristics, TIP-B1 is not a soluble TNF receptor, an anti-TNF antibody, nor a protease that degrades TNF; yet TIP-B1 functions when added exogenously to cells. These characteristics, its novel sequence, and its function when added exogenously to cells indicate that TIP-B1 is unique and is not one of the other proteins reported previously to be involved in resistance to TNF. The ability of TIP-B1 to function after exogenous incubation with target cells makes TIP-B1 a likely candidate for therapeutic manipulation of TNF-induced effects.

Doxorubicin plus tumor necrosis factor alpha combination treatments in EL4-lymphoma-bearing C57BL/6 mice.

The therapeutic efficacy of a total of 42 single-agent or combination protocols involving doxorubicin (Adriamycin, ADM) and tumor necrosis factor alpha (TNFalpha) were evaluated in the syngeneic murine lymphoma model, C57BL/6-EL4. Combination treatments were the most effective and the therapeutic effects were schedule-dependent; e.g. it was generally advantageous for ADM to precede TNFalpha administration. Two protocols selected for further study were 4 mg/kg ADM i.v. on days 1 and 8 plus TNFalpha, i.v., at either 16000 U (7 microg)/injection, on days 1 and 8 or 4000 U (1.7 microg)/injection, on days 11-15. Survival of mice bearing one of four EL4 sublines having different in vitro drug sensitivities was assessed. These sublines were E10 (ADM-sensitive/TNFalpha-resistant), E16 (sensitive/sensitive), ER2 (ADM-resistant/TNFalpha-sensitive) and ER13 (resistant/resistant). Between 80% and 100% long-term survivors (i.e. tumor free on day 60) were obtained with the two treatments in mice bearing ADM-sensitive sublines, even though one of these sublines, E10, was resistant to TNFalpha in vitro. Induction of long-term survival appeared, therefore, to correlate with in vitro defined sensitivity/resistance to ADM, but not to TNFalpha Treatment-induced modulations of tumoricidal immune effector functions were also examined. Taken together, the results indicated that induction of long-term survival involved complex interactions of: (1) ADM-induced tumor modifications, including, but not limited to, tumor debulking, (2) combination-treatment-induced modifications of splenic cytolytic T cell and macrophage activities, and (3) the restoration of thymus cellularity. Finally, when long-term survivors resulting from treatment of E10- or E16-bearing mice were implanted with ER2 on day 120, the majority survived, indicating that long-term immune memory, capable of recognizing drug resistant variants, had been established.

Murine splenic macrophage tumoricidal activation by cytokines.

Interleukin-2 (IL-2), IL-1 beta, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha), each alone and in all possible combinations, were studied for their capacity to activate murine resident splenic macrophages to a tumoricidal state. Two approaches were used in these studies. The first approach was to add cytokine directly to the adherent macrophages that had been washed free of nonadherent spleen cells. The only agent effective alone was IL-2, inducing significant tumoricidal activity in macrophages obtained after culturing whole spleen cell suspensions for 4, but not 1 to 3, days. Nonadherent splenic populations were required during this 4-day macrophage "culture conditioning." Only combinations of cytokines containing IL-2 were effective, but none more than IL-2 alone. The second approach was to add cytokine to the whole spleen cell suspensions for an activation period before isolation of adherent macrophages. Again, the only agent effective alone was IL-2. Macrophage tumoricidal activity was highest when IL-2 was added to the whole spleen cell suspensions at the initiation of the 4-day activation culture. In addition, TNF-alpha, but none of the other cytokines, significantly augmented the IL-2-induced effect. The tumoricidal activity was not a consequence of lipopolysaccharide contamination or of lymphokine-activated killer cells. Based on the utilization of neutralizing antibodies, IL-1 alpha, IL-1 beta, and IFN-gamma were not involved as soluble mediators during the activation of tumoricidal splenic macrophages by IL-2 with or without TNF-alpha.

TNF-alpha potentiation of the lymphokine-activated killer response of murine thymus cells.

The role of tumor necrosis factor (TNF-alpha) on in vitro generation of lymphokine-activated killer (LAK) cells from murine thymocytes was investigated and compared to that on generation of LAK from splenocytes. TNF-alpha increased the potential of interleukin-2 (IL-2) at suboptimal concentrations to generate LAK activity in thymocytes even more than in splenocytes. In parallel, augmented [3H]thymidine uptake by thymocytes and splenocytes was seen. However, no net increase in viable cell number was observed. LAK effector cells from TNF-alpha plus IL-2 cultures responded with an increased [3H]thymidine uptake to restimulation by IL-2 alone. These results suggest that TNF-alpha + IL-2 may be inducing the expansion of a small subset of cells. NK1.1+ cells are a very minor subset of thymocytes, nevertheless phenotype analysis showed that in thymocytes, IL-2 + TNF-alpha generates NK1.1+ CD8- LAK effectors in contrast to NK1.1- CD8+ cells found with IL-2 alone. This result is consistent with the finding in the proliferation studies. The fact that thymocytes are stimulated by the TNF-alpha + IL-2 combination to proliferate as well as to develop a phenotypically distinct effector supports the role of TNF-alpha in intra- and extrathymic regulation.

Immunological responses critical to the therapeutic effects of adriamycin plus interleukin 2 in C57BL/6 mice bearing syngeneic EL4 lymphoma.

A safe and effective therapeutic combination of moderate doses of Adriamycin (doxorubicin, 4 mg/kg, IV, Days 1 and 8 or only Day 8) plus prolonged administration of moderate doses of interleukin 2 (2 micrograms, b.i.d., Days 9-40) was developed in the syngeneic EL4 (5 x 10(4) cells, IP, Day 0) lymphoma--C57B1/6 mouse model and has been reported in the companion paper. The studies described herein demonstrate that the effectiveness of this combination treatment against EL4 lymphoma growing intraperitoneally in C57B1/6 mice was dependent upon the presence of CD8+ cells. Thus, the induction of long-term survivors (60-80%) by Adriamycin plus interleukin 2 was completely ablated by pretreatment of mice with anti-CD8 monoclonal antibody (MAb), whereas pretreatment with anti-CD4 MAb only partially inhibited the therapeutic effects and anti-NK1.1 MAb had no effect. A close association between survival, an increase in phenotypically identified CD8+ cells, and an increase in specific anti-EL4 cytolytic activity was demonstrated with cells from the tumor site (peritoneum) but not consistently with cells from the spleen. No association was observed between survival and modulations in natural killer (NK), lymphokine-activated killer (LAK), or tumoricidal macrophage activity of spleen or peritoneal cells. Taken together the results indicate that, in this model, the most relevant correlate of a therapeutically effective host antitumor response is the level of specific EL4 tumor killing by cells present at the tumor site. Based on the findings reported herein, it can be predicted that weakly immunogenic tumors may be eradicated by immunologic mechanisms elicited in conjunction with properly designed therapeutic regimens.

Selective modulation by alpha-difluoromethylornithine of T-lymphocyte and antibody-mediated cytotoxic responses to mouse tumor allografts.

alpha-Difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, has been shown to be growth inhibitory in a wide variety of normal and tumor cell systems. Since cells of the host defense system are among the most rapidly proliferating cells in the body, DFMO may inhibit certain components of this system. In order to assess this possibility, four randomized groups of C57BL/6 mice were maintained either on water throughout (controls) or on 2% DFMO in drinking water for various periods of time. Mice were given DFMO from Days -4 to 0, Days 0 to +4, or Day 0 to day of assay. On Day 0 randomly selected mice from each group received P815 tumor allografts. Daily from Days +3 to +14, pools of spleen cells from three mice per group were assessed for allospecific cytolytic T-lymphocyte, antibody formation, natural killer cell, and phagocytic cell activities. While natural killer cell and phagocytic cell activities remained essentially unchanged under all conditions, both cytotoxic T-lymphocyte and antibody responses were modified. Somewhat similar effects were seen with both responses and involved to varying degrees: (a) a delay of the initiation of rapid increase in the response but not in the onset of first detectable response; (b) delay in the time of peak response; (c) increased level of maximal response; (d) two peaks of maximal response. The data indicate that DFMO treatment of whole animals, dependent upon schedule of administration and time of assay, induces very selective effects on both cytotoxic T-lymphocyte and antibody responses, without apparent modification of nonspecific host defense mechanisms, with the overall effect being a prolongation of the period of specific response.

The effect of PS-K, a protein bound polysaccharide, on immune responses against allogeneic antigens.

A single dose of PS-K administered to C57B1/6J mice after immunization augmented both the humoral and cellular allogeneic responses against P815 tumor cells. PS-K addition to primary alloantigen sensitization cultures (C57B1/6J spleen cells against X-irradiated P815 cells) resulted in augmented 3H-thymidine uptake and cell mediated cytolytic activity. Ten daily administrations of PS-K to DBA/2J mice after implantation of a mammary tumor of DBA/2HaDD origin results in increased tumor regression and prolonged survival of the mice. PS-K addition to human mixed lymphocyte cultures caused an augmentation of both cell mediated cytolytic activity and 3H thymidine uptake. The dose dependence of the PS-K effects were described by a bell shaped curve and PS-K did not appear to affect the day of peak development of the various responses. The effects were consistently greater if PS-K was administered at the same time or after antigen presentation. Thus, the immunoaugmenting effects induced by PS-K were similar in each model system tested.