My name is Brandon Castro and I am a neurosurgery resident at the University of Chicago and I am also a postdoctoral research fellow in the Lesniak and Li Chang lab at Northwestern University. Today I'm going to be talking about a B-cell based vaccine, which is a therapy that was developed in our lab, and describe how it is an effective immunotherapy in preclinical models of glioblastoma. I do not have any financial disclosures. I will first introduce our novel B-cell based vaccine, which I will refer to as B-vax from now on. I will then describe the role of cellular immunity in the therapeutic effects of B-vax and finally I will talk about the role of humoral immunity in the therapeutic effects of B-vax. Glioblastoma, as we all know, is a very aggressive brain tumor with a poor prognosis. There has been little therapeutic advancement since 2005 when the stoop protocol was first described. Therapy has been a promising therapeutic field within glioblastoma research primarily because these tumors are immunologically cold tumors. We have identified a subset of B-cells characterized by 4-1-BBL. This subset of B-cells defines antigen-experienced B-cells and we have previously shown that these cells have anti-tumor activity. 4-1-BBL positive B-cells serve as the foundation of our B-cell based vaccine. This is a schema for how we generate B-vax. We start with isolating secondary lymphoid organs from tumor bearing mice or peripheral blood from GBM patients. We then isolate the 4-1-BBL positive B-cell population. These cells are activated in culture initially with BAF, a survival factor, and anti-CD40 antibody for the first 24 hours followed by interferon gamma for another 24 hours. So the entire activation process takes 48 hours and at the end we have created our B-vax which can then be used for a cellular therapy. After finalizing the protocol for how to generate B-vax, we then started using it in our murine glioblastoma models. So we injected mice with CD2A tumor cells intracranially and then we treated the mice with B-vax or the appropriate controls and B-vax treated mice did show a survival benefit compared to the controls. Knowing that B-vax provides a survival benefit in preclinical murine models was extraordinarily exciting and our next step was to try to figure out why this is the case. We are starting by investigating the role of cellular immunity and humoral immunity. Cellular immunity is the ability of B-vax to serve as an antigen-presenting cell, thus activating CD8 T-cells which then subsequently kill tumor cells. Humoral immunity is the ability of B-vax to transition into plasma blasts, thus secreting antibodies which can travel to the tumor and kill the tumor via antibody-dependent cellular cytotoxicity. So our main question is, does B-vax improve survival through cellular immunity, humoral immunity, or both? These are some additional survival studies showing that B-vax and B-vax plus CD8 T-cells showed a survival benefit. On the right, you see a comparison with DC-vax. So DCs are the traditional antigen-presenting cells of the body and DC-vax is a cellular-based vaccine therapy that was previously developed, and we compared our B-vax side-by-side with DC-vax plus CD8 T-cells, and we showed a survival benefit of B-vax compared to DC-vax. Next we tested B-vax in conjunction with the current standard of care, which is the STOOP protocol, which involves a combination of radiation therapy and temozolomide. So when we added B-vax and CD8 T-cells to radiation therapy, it showed a survival benefit, and we saw the same thing when adding these therapies in combination with temozolomide. We further investigated the role of cellular immunity in the efficacy of B-vax by performing cytotoxicity assays. We used primary GBM cells from both primary and recurrent GBM patients and co-cultured them with CD8 T-cells, either naive CD8 T-cells or B-vax-activated CD8 T-cells, and we showed increased tumor killing in CD8 T-cells that were activated by B-vax, specifically those that were grown in spheres. Our next question was, does B-vax differentiate into plasmablasts? When we isolated B-vax from tumor-bearing mice that were treated with B-vax or controls three days after injection, we found that about half of those B-cells expressed CD138, which is a marker of plasmablast formation, and this was significantly higher than the two controls, B-naive and B-activation. When we looked at the antibodies that were generated from these plasmablasts, we found that the majority of immunoglobulins isolated from B-vax-treated mice were of the IgG isotype, whereas the B-naive and B-activation, our controls, had more of an IgM isotype. We then took these antibodies generated from B-vax and B-naive and treated tumor-bearing mice with the immunoglobulins, and we found a survival benefit in the B-vax IgG-treated mice. Now these mice did not receive the parent cell, they only received the antibodies, suggesting a role of humoral immunity in the efficacy of B-vax. To further answer the question of how antibodies improve survival, we first wanted to find out what these antibodies specifically bind to. We took sections from tumor-bearing mice. These mice were B-cell knockout mice, which means that they do not have any of their own B-cells or antibodies, because B-cells are the only antibody-producing cells in the body. We then incubated them with B-vax-produced antibodies and the respective controls, and we found that the antibodies preferentially bind to the tumor over the normal surrounding brain, and in addition, when we looked at the actual cells that these antibodies were binding to, they bind to the tumor cells specifically as opposed to other cells. When translating this research to GVM patients, we are faced with the challenge of being unable to generate antibodies in vivo. So what we have developed is a protocol for activating B-vax ex vivo, transitioning these cells to plasma blasts, and we then collect the supernatant every three days and are able to isolate the antibodies from the supernatant. We have done this so far with a variety of patient samples successfully, and we have shown that B-vax does, in fact, proliferate, which is not something typical of B-cells grown in culture. In addition, we see that on the twelfth day of activation, we have a more plasma blast-type state, and this is more so the case with B-vax compared to B-naive controls, although there is some variability between patients, as we would expect. So in summary, B-vax conveys a survival benefit in preclinical murine models of GVM. CD8 T-cells activated by B-vax induces tumor cell killing, suggesting a role for cellular immunity. B-vax differentiates into plasma blasts in vivo, and antibodies derived from B-vax lead to a survival benefit and tend to bind preferentially to tumor cells. And we are currently working on translating all of this to GVM patients and hope to bring this to a clinical trial in the near future. I'd like to thank Dr. Lesniak and Dr. Li Cheng for letting me do this work in their lab, as well as our funding sources and all of my lab mates for helping with this research.

B-vax

immunotherapy

glioblastoma

cellular immunity

humoral immunity