Stanford University researchers say clinical trials of therapeutic approach are warranted.

Investigators say use of a CD137 agonist mAb significantly boosts the anticancer effects of the tumor-targeting antibody trastuzumab (Genentech’s Herceptin) and may  represent a promising strategy for increasing the effectiveness of other anticancer antibodies including rituximab and cetuximab. A Stanford University-led team showed that anticancer treatment with trastuzumab followed by the CD137 agonist mAb boosted the ability of natural killer (NK) cells to destroy trastuzumab-coated tumor cells. The cytotoxic effects of this regimen in vitro translated into marked tumor regression and increased survival in mice carrying Her2-expressing primary human breast tumors.

Ronald Levy, Ph.D., and colleagues report their findings in The Journal of Clinical Investigation in a paper titled “Stimulation of natural killer cells with a CD137-specific antibody enhances trastuzumab efficacy in xenotransplant models of breast cancer.” The authors say clinical trials of the dual antibody therapy are now warranted.

The response rates of metastatic Her2-positive breast cancer to Herceptin (trastuzumab) monotherapy are only about 10–15%, and a number of strategies have been devised to try and enhance the antitumor activity of the Her2-targeting antibody. Trastuzumab kills breast cancer cells through a number of mechanisms, and one approach to boosting its effectiveness is to bolster antibody-dependent cellular cytotoxicity (ADCC). ADCC depends primarily on natural killer (NK) cells binding via their Fc receptor (FcγRIII or CD16) to the IgG1 Fc heavy chain portion of trastuzumab. Binding activates the NK cells, which release cytotoxic granules that lyse the trastuzumab-bound breast cancer cells.

CD137 is an inducible, co-stimulatory molecule expressed on activated CD4 and CD8 T cells, and the majority of research investigating the potential therapeutic use of CD137 mAbs or ligands has focused on increasing the proliferation and survival of T cells. However, CD137 is also expressed by NK cells, monocytes, dendritic cells, and nonhematopoietic cells, the authors state. Recent work has indicated that when human NK cells interact with antibody-bound tumor cells they upregulate CD137, which improves antitumor activity.

Building on this observation, the Stanford University-led team carried out a series of studies to see whether administration of a CD137 mAb agonist would enhance breast cancer killing by trastuzumab both in vitro and in vivo. In initial assays the team confirmed that incubating NK cells from healthy human subjects with Her2-overexpressing BT474M1 breast cancer cells and trastuzumab resulted in the upregulation of CD137 expression. In contrast, CD137 expression wasn’t upregulated in the presence of a trastuzumab variant that doesn’t bind to human FcγRs or when NK cells were incubated with trastuzumab and a breast cancer cell line that doesn’t overexpress Her2 (MCF7).

The investigators then evaluated the effects of adding an anti-CD137 mAb to NK cells from healthy donors that had already been activated by trastuzumab-bound Her2-expressing breast cancer cells for 24 hours. The results confirmed that administration of CD137-agonist to the activated NK cells led to much higher levels of trastuzumab-induced IFN-γ secretion and increased the ability of activated NK cells to kill trastuzumab-coated tumor cells. “This effect was observed only against Her2-overexpressing breast cancer cell lines, confirming that the killing was due to ADCC, even though the NKs had been preactivated,” they write.

Encouragingly, the beneficial cytotoxic effects of combining already activated NK cells with the CD137 agonist mAb weren’t restricted to the same trastuzumab-coated cell type used for activation. Rather, the CD137 agonist boosted the ability of NK cells activated using trastuzumab-coated breast cancer to kill rituximab-coated lymphoma cells. Similarly, the CD137 antibody boosted the ADCC activity of NK cells activated by rituximab-coated lymphoma cells against trastuzumab-coated breast cancer cells. Notably, the dual regimen also significantly increased NK cytotoxicity against a Her2-overexpressing breast cancer cell line that is intrinsically resistant to trastuzumab.

The team then moved on to evaluate whether their in vitro findings would translate into in vivo therapeutic benefits. An initial cohort of athymic mice (which have fully competent NK cells but lack functional T cells) carrying human breast cancer (BT474M1 cell line) tumors was treated using trastuzumab alone. In these animals trastuzumab monotherapy had only a modest effect on tumor growth. The team then evaluated therapy in this mouse model using trastuzumab plus the CD137 agonist mAbs, administered at different time points. Trastuzumab was injected on the third day after tumor inoculation, and the CD137 mAb was injected on either day 2 (i.e., the day before trastuzumab therapy), on the same day (day 3), or on the day after. Treatment was then repeated in 14 days following the first therapeutic injection.

The results demonstrated the importance of administering the CD137 mAb after trastuzumab. This regimen resulted in the greatest reductions in tumor size and mortality, whereas administering the antibody a day before trastuzumab had the least beneficial effect. Treating the mice according to the trastuzumab followed by antibody regimen weekly for three weeks resulted in prolonged control of tumor growth and a significant improvement in survival. When treatment was evaluated in animals carrying breast cancer tumors from different cell lines, the benefits of CD137 mAb administration were, as expected only seen in Her2-overexpressing tumors. Promisingly, the combination of trastuzumab and the CD137 mAb treatment also significantly reduced tumor growth and prolonged survival in sublethally irradiated SCID mice carrying primary Her2-overexpressing primary breast tumors.

In a final set of studies, the researchers looked at CD137 expression in human breast cancer patients receiving trastuzumab therapy. Peripheral blood mononuclear cells (PBMCs) were collected prior to therapy and again at four hours, 24 hours, and seven days following completion of transtuzumab infusion. Across the 11-patient cohort, the mean NK cell CD137 expression level increased significantly from about four hours after the start of infusion, until it reached twofold higher than baseline at 24 hours after infusion.  The greatest magnitude of increase in CD137 expression occurred in two patients receiving their first doses of trastuzumab, the team remarks.

“This antibody strategy targeting both the tumor and the immune system may thus offer new opportunities to enhance the efficacy of therapeutic antibodies in cancer,” they conclude. “This approach is widely applicable across cancers, including to known antibodies and known antigen-expressing tumors, such as rituximab and CD20-expressing lymphomas and cetuximab and EGFR-expressing head and neck as well as colorectal tumors and to yet undeveloped antibodies to currently unknown tumor antigens.

“In vivo, we expect NK cells in contact with trastuzumab-bound tumor cells to be activated regardless of anatomic site such as within the primary tumor, tumor-involved lymph nodes, or at distant sites of metastatic breast cancer.  As efficacy of anti-CD137 mAb is partially dependent upon expression of CD137 on NK cells, the magnitude and timing of CD137 expression in the peripheral blood or at the tumor site following antibody therapy may provide a predictive biomarker of response.”

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