Liposomal bisphosphonates as vaccine adjuvants

In experiments designed to assess the antigen presenting cell response to vaccination, we observed that mice vaccinated with a protein antigen vaccine mixed with a liposome-TLR adjvuant and administered s.c. induced a large influx of myeloid cells into the nearest vaccine draining lymph nodes. We termed these cells vaccine-elicited myeloid cells (VEMC) and noted that they appeared in the bloodstream and lymph nodes within hours of vaccination and reached very high levels by 24h after vaccination. Using multicolor flow cytometry, we also found that VEMC were comprised almost entirely of CD11b+CCR2+ cells, which are defined as inflammatory monocytes.

Thus, VEMC were comprised primarily of inflammatory monocytes, which was confirmed by cell sorting experiments (data not shown). Moreover, the influx of VEMC into vaccine draining lymph nodes was triggered by a number of different vaccine adjuvants (not shown).Previously, it has been assumed that inflammatory monocytes differentiated into DC within lymph nodes and contributed to enhancing vaccine immunity. However, our new data suggest that instead inflammatory monocytes actually potently suppress vaccine immune responses, which represents the key intellectual advance covered in this filing.

The idea that inflammatory monocytes (VEMC) actually suppress vaccine responses is based on several lines of evidence. For one, when monocytes and macrophages are depleted at the time of vaccination using a non-specific monocyte/macrophage depleting agent (liposomal clodronate; LC), vaccine responses are markedly enhanced. For example, antibody responses were increased by 2-3 logs in mice that were vaccinated and treated concurrently with LC. This amplification of vaccine immunity was also observed with a variety of different vaccine adjuvants, and with cancer vaccines. Not only were antibody responses enhanced by LC treatment, but cellular immune responses were also significantly increased.

To show that VMEC were immune suppressive, we examined the effects of LC administration on VEMC numbers in vaccine draining lymph nodes. We found that administration of LC at the time of vaccination markedly blocked the increase in VEMC in vaccine draining lymph nodes, which coincided with increased vaccine immunity. Thus, these data suggests that VEMC were the cells that mediated vaccine suppression.

Since the immune suppressive cell population was comprised primarily of CCR2+ monocytes, we hypothesized that treatment with a specific CCR2 antagonist could also be used to enhance vaccine immunity. To test this hypothesis, mice were treated with a small molecule specific CCR2 antagonist, RS102895, a spiropiperidine drug. The drug was administered once daily (either s.c. or i.p.) at -24h, time 0, and +24h after immunization. We observed a marked enhancement of antibody titers and cellular immune responses (data not shown) in mice that were treated with RS102895 and vaccinated. We also observed that a second CCR2 antagonist also induced a significant enhancement of vaccine immunity, as did a small molecule antagonist of the CSF-1 receptor (data not shown). Finally, we also found that treatment with LC or with RS102895 also efficiently blocked the recruitment of immune suppressive VEMC into vaccine draining lymph nodes.

We therefore concluded that drugs that either depleted monocytes, or blocked their mobilization from the bone marrow or their recruitment into lymph nodes, ould be used to markedly enhance vaccine immunity in response to a variety of different adjuvants with different mechanisms of action. The mobilization of VEMC appears to be mediated primarily in responses to MCP-1, such that chemokine receptor antagonists that block monocyte migration appear to be particularly effective. We have termed the new approach adjuvant-adjuvants, since administration of chemokine receptor antagonists functions to enhance to activity of conventional vaccine adjuvants.

The ability of orally administered small molecule chemokine receptor antagonists to mediate this vaccine amplifying effect suggests that the simple administration of a tablet immediately before and after vaccination could provide a simple means of improving overall vaccine efficacy. Such an approach could be used for both prophylactic and therapeutic vaccines. Indeed, we have found that administration of a CCR2 antagonist can significantly improve the effectiveness of therapeutic cancer vaccines in several different mouse cancer vaccine models (data not shown). Moreover, such an approach could be used to significantly improve vaccine efficacy in hard-to-immunize populations such as young children and the elderly.

Inventor Information:

Steven Dow

Angie Duffy

Leah Mitchell