Leonid Gorelik

Cutting Edge: BAFF Regulates CD21/35 and CD23 Expression Independent of Its B Cell Survival Function

Herein we demonstrate that B cell-activating factor of the TNF family (BAFF), a B cell survival factor, also regulates CD21/35 and CD23 expression. BAFF blockade in wild-type mice down-modulates CD21/35 and CD23 on B cells while survival remains intact, and BAFF exposure causes elevated CD21/35 and CD23 expression. Similar down-modulation is observed in bcl-2-transgenic mice treated with a BAFF inhibitor. This is the first evidence that BAFF has a function independent of B cell survival. Reports using CD21/35 and CD23 expression to assess splenic B cell subsets in BAFF-null mice concluded a lack of B cells beyond the immature stage. Since CD21/35 and CD23 are inadequate for delineating B cell subpopulations in BAFF-null mice, we used expression of BAFF-R and several B cell markers to identify more mature splenic B cells in these mice. These data broaden our understanding of BAFF function and correct the view that BAFF-null mice lack mature B cells.

Low-dose melphalan-induced shift in the production of a Th2-type cytokine to a Th1-type cytokine in mice bearing a large MOPC-315 tumor

The current studies demonstrate that MOPC-315 tumor cells secrete large amounts of interleukin-10 (IL-10), which contributes to the inhibitory activity of MOPC-315 culture supernatants for the in vitro generation of antitumor cytotoxicity by MOPC-315-“immune” spleen cells. Moreover, addition of neutralizing monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of cells from the tumor infiltrated spleens of mice bearing a large MOPC-315 tumor resulted in the generation of enhanced anti-MOPC-315 cytotoxicity. In contrast, addition of monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of splenic cells from mice that are in the final stages of immune-mediated tumor eradication as a consequence of low-dose melphalan (l-phenylalanine mustard; L-PAM) therapy (and whose spleens no longer contain metastatic tumor cells) did not lead to enhancement in the in vitro generation of antitumor cytotoxicity. The cessation of IL-10 secretion as a consequence of low-dose L-PAM therapy of MOPC-315 tumor bearers was found to be accompanied by the acquisition of the ability to secrete interferon γ (IFNγ) by the splenic cells. In addition, by day 2 after low-dose L-PAM therapy a drastic decrease in the amount of IL-10 secreted by the s.c. tumor nodules was noted, which preceded the accumulation of tumor-infiltrating lymphocytes capable of secreting IFNγ. Thus, low-dose L-PAM therapy of mice bearing a large MOPC-315 tumor leads to a shift in cytokine production from a Th2-type cytokine to a Th1-type cytokine, and it is conceivable that this shift in cytokine production plays an important role in the low-dose L-PAM-induced acquisition of antitumor immunity by hitherto immunosuppressed mice bearing a large MOPC-315 tumor.

Importance of IL-10 for CTLA-4-Mediated Inhibition of Tumor-Eradicating Immunity

In this study, we show that engagement of CTLA-4 on tumor-infiltrating lymphocytes from low-dose melphalan (l-phenylalanine mustard (l-PAM))-treated MOPC-315 tumor bearers led to IL-10 secretion. In addition, the inhibitory activity of CTLA-4 ligation for IFN-γ secretion following stimulation with anti-CD3 plus anti-CD28 mAb depended on IL-10 production. Consistent with the importance of IL-10 for CTLA-4-mediated inhibition, administration of neutralizing anti-IL-10 mAb to low-dose l-PAM-treated MOPC-315 tumor bearers (administration of blocking anti-CTLA-4 mAb) resulted in enhanced tumor-infiltrating lymphocyte-mediated anti-MOPC-315 cytotoxicity and led to complete tumor eradication in a higher percentage of mice than that observed with low-dose l-PAM alone. The percentage of MOPC-315 tumor-bearing mice cured following administration of neutralizing anti-IL-10 mAb to low-dose l-PAM-treated MOPC-315 tumor bearers was comparable to that observed following administration of blocking anti-CTLA-4 mAb. Moreover, IL-10 neutralization together with CTLA-4 blockade did not provide added therapeutic benefits to low-dose l-PAM-treated MOPC-315 tumor bearers. Taken together, these results indicate that CTLA-4 blockade improves the therapeutic outcome of low-dose l-PAM for MOPC-315 tumor bearers by inhibiting IL-10 secretion as a consequence of blocking CTLA-4 ligation.

Low-dose-melphalan-induced up-regulation of type-1 cytokine expression in the s.c. tumor nodule of MOPC-315 tumor bearers and the role of interferon γ in the therapeutic outcome

We have previously shown the importance of endogenous tumor necrosis factor (TNF) production for the curative effectiveness of low-dose melphalan (L-phenylalanine mustard) for mice bearing a large MOPC-315 tumor. In the current study we demonstrate that low-dose melphalan is actually associated with enhanced expression of mRNA for TNFα in the s.c. tumor nodule. Moreover, the expression of mRNA for interferon γ (IFNγ) and interleukin-12 (IL-12; p40) is also elevated at the tumor site. However, while elevation in the expression of mRNA for TNFα and IFNγ is evident within 24 h after the chemotherapy, elevation in the expression of mRNA for IL-12(p40) is first evident 72 h after the chemotherapy. Moreover, neutralizing anti-IFNγ mAb, like neutralizing anti-TNF mAb but not neutralizing anti-IL-12 mAb, reduced the curative effectiveness of low-dose melphalan for MOPC-315 tumor bearers. Studies into the mechanism through which IFNγ mediates its antitumor effect in low-dose-melphalan-treated MOPC-315 tumor-bearing mice revealed that MOPC-315 tumor cells, which are not sensitive to the direct antitumor effects of TNF, display some sensitivity to the antiproliferative activity of high concentrations of IFNγ. However, unlike TNFα, IFNγ is unable to promote the generation of anti-MOPC-315 cytotoxic T lymphocyte activity and, in fact, exerts an inhibitory activity on CTL generation. Taken together, our studies illustrate that low-dose melphalan therapy of MOPC-315 tumor bearers is associated with the rapid elevation in the expression of mRNA for IFNγ and TNF, two cytokines which are important for the curative effectiveness of low-dose melphalan, and which mediate their antitumor effect, in part, through distinct mechanisms.

Abstract 4606: Preclinical characterization of a novel fully human IgG1 anti-PD-L1 mAb CK-301

Antibodies targeting Programmed Death-1 (PD-1), or its ligand, PD-L1, have demonstrated remarkable efficacy in subsets of cancer patients, with inhibition of the interaction between PD-1 on T-cells and PD-L1 on tumor cells leading to the recovery of anti-tumor immune response and immune-mediated eradication of tumors. However, not all patients respond to existing PD-1 and PD-L1 targeting agents and relapses to therapy still occur. Therefore, there exists a need to identify additional therapeutics and approaches to engage the immune system to enhance the efficacy of current anticancer therapies. Using phage and yeast display approaches, we have discovered and optimized a novel, fully human, PD-L1 specific IgG1 antibody, CK-301, which exhibits subnanomolar binding affinity for PD-L1. CK-301 blocks binding of PD-L1 to both PD-1 and B7-1 in enzyme-linked immunosorbent assays (ELISA) and cell-based competition assays. Using an assay measuring inhibition of a nuclear factor of activated T-cells (NFAT) reporter caused by PD-L1 binding to PD-1, we demonstrate that CK-301 completely reverses reporter inhibition at concentration of less than 1 µg/ml, IC50 of the dose response curve is 80ng/ml. CK-301 enhances IFN-gamma secretion in allogeneic mixed lymphocyte reaction (MLR) using primary human T-cells and immature dendritic cells. CK-301 can also trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) mediated killing of PD-L1+ cell lines, including lymphoma cells. CK-301 has similar subnanomolar affinity for cynomolgus monkey PD-L1 as for human PD-L1, hence we chose Macaca fascicularis for pre-clinical toxicology and safety pharmacology studies. Single-dose administration of CK-301 to monkeys up to the highest tested dose of 100 mg/kg was shown to be safe and demonstrated linear dose-dependent pharmacokinetic (PK) properties over the dose range from 1 to 100 mg/kg with a half-life of 15 days at 100 mg/kg. A first-in-human Phase 1 study of CK-301 is planned to commence in mid-2017. Citation Format: Leonid Gorelik, George Avgerinos, Yune Kunes, Wayne A. Marasco. Preclinical characterization of a novel fully human IgG1 anti-PD-L1 mAb CK-301 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4606. doi:10.1158/1538-7445.AM2017-4606