Development of a Novel Virus-like Particle (VLP) Vaccine for Personalized B-Cell Lymphoma and Chronic Lymphocytic Leukemia Therapy
Authors: Chan Wei , Theriault Thomas , Levy Ronald , Swartz James 
Published: October 2019

Abstract
Background
For decades since its discovery, the idiotype protein (Id) expressed on the surface of B-cell malignancies has been pursued as a tumor-specific target in therapeutic antibody and vaccine development programs. The challenge in vaccine development has been to present the Id antigen so as to stimulate an effective anti-tumor response. Advances in our understanding of the immune system have revealed the importance of innate receptors, particularly the toll-like receptor (TLR) family, in stimulating adaptive immunity. Further understanding of virus-induced immunity reveals features such as viral trafficking to lymph nodes and presentation of antigens in an ordered array that facilitate immune response generation. Our strategy combines this knowledge with advanced protein engineering in order to produce a stabilized virus-like particle (sVLP) that displays Id in the context of innate immune stimulants. Multiple studies using the 38C13 lymphoma model were performed to evaluate and maximize the efficacy of Id-VLP vaccines using the historical "gold standard" Id-KLH as a reference.
Methods
VLP vaccines were constructed by conjugating the tumor-associated Id antigen and immune stimulants, GM-CSF, CpG DNA, and flagellin, to the exterior surface of either the MS2 or Hepatitis B core (HBc) VLP. The bioconjugates were engineered to maintain biologic activity of the immune stimulants and present unperturbed heavy and light chain variable regions. A total of 14 different vaccines displaying 3 different 38C13 Id formats, IgM, F(ab')2 and scFv, with or without one or more of the three immune stimulant were produced. For example, with the scFv Id format VLP vaccines, 7 of the 8 possible combinations of immune stimulants were made.
Three in vivo experiments were performed to evaluate vaccine compositions. C3H mice were vaccinated three times within a one-month period with control or experimental vaccines. Following vaccination, the mice were challenged with 38C13 Id-bearing tumor cells administered by intraperitoneal or subcutaneous routes. Sera samples were collected prior to, during, and after vaccination for analysis of total and isotype-specific anti-Id antibody titer.
Results
The first study compared MS2- and HBc-based VLP vaccines. While both viral structures exhibited immune inhibition on their own, the HBc VLP vaccine conjugated with GM-CSF and CpG elicited a strong and protective humoral immune response as compared to experimental controls. This was confirmed in a second study where different HBc VLP vaccines explored additional combinations of immune stimulants and the different Id formats. While all HBc VLP vaccines elicited strong and protective immune responses, the VLP with scFv Id format and CpG provided better protection against tumor challenge than the Id-KLH conjugate. (70% vs 30% event-free survival). The third study further examined the scFv Id format and immune stimulants on stabilized HBc-based vaccines where disulfide bonds were introduced between dimers within the axes of symmetry to create a VLP structure in which every atom is indirectly covalently bonded to every other atom. Once again, the scFv Id-CpG-sVLP vaccine elicited a strong and protective response.
Conclusions
Collectively, these studies provide strong rationale for the selected BB-001 composition (scFv Id-CpG-HBc sVLP) as a vaccine for Id-bearing lymphomas, as well as solid evidence that HBc sVLPs are a versatile and effective platform for further vaccine development. While all HBc-based Id vaccines with immune stimulants were statistically significantly superior to placebo groups in terms of efficacy and/or immune response, the simpler VLP vaccine with the TLR9 agonist CpG outperformed the combinations with GM-CSF and/or flagellin. The subcutaneous tumor challenge was more demanding, and showed a statistically significant survival advantage for BB-001(70%) versus Id-KLH (30%). Consistent with previous 38C13 studies, humoral immune response was critical for protection from challenge. Evidence of antibody class switching in the humoral immune response was also observed.

Disclosures
Theriault: Bullet Biotechnology, Inc.: Employment, Equity Ownership. Levy:Bullet Biotechnology, Inc.: Consultancy. Swartz:Bullet Biotechnology, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.