AlloStim™ can be a potentially powerful inflammatory agent.  The use of an inflammatory agent as an adjuvant for active immunotherapy has previously been shown to be a viable strategy.  Using autologous tumor lysates as a source of tumor antigen and Bacillus Calmette-Guérin (BCG) as adjuvant, for example, reduced the overall recurrence rate after resection of the primary in Stage II colorectal cancer, but not Stage III presumably due to the lower tumor burden in the former [48].  

BCG is the most effective intravesical agent for the treatment of superficial bladder cancer and prophylaxis against recurrent disease. Among the many immunological events associated with BCG stimulation, induction of an array of cytokines appears to be a major effect. The Type 1 cytokines IL-1, IL-2, IL-12, TNF- , GM-CSF, and IFN-  have been documented to be readily detectable in responding bladder cancer patients.  IFN-  is the most predominant Th1 cytokine observed in clinical responders undergoing BCG therapy [49].

The active ingredient in AlloStim™ is ex-vivo differentiated Th1 memory cells derived from CD4+ naïve precursor cells purified from normal blood donors.  AlloStim™ is intentionally mis-matched to the patient.  Intratumoral AlloStim™ injection is expected to provide a potent adjuvant effect for the development of Type 1 anti-tumor immunity and the down regulation of tumor immunoavoidance mechanisms.  The adjuvant effect of AlloStim™  is based upon three main features of the experimental drug: (1) the ability to produce large amounts of Type 1 cytokines; (2) the surface expression of CD40L; and (3) the allogeneic nature of the cells.  That the alloantigens expressed by AlloStim™ will have an adjuvant effect is supported by observations that immune responses against weak tumor antigens can be augmented by co-immunization against potent antigens such as xeno-, allo- or viral antigens [50, 51].  

AlloStim™ produces large amounts of the Type 1 cytokines: IFN-γ, TNF-α and GM-CSF.  IFN-γ  is a pivotal Type 1 cytokine necessary to promote Type 1 anti-tumor immunity.  It has been shown that IFN-γ can mediate anti-tumor effects by directly inhibiting tumor cell growth and inducing T cell-mediated anti-tumor responses [52, 53]. IFN-  secretion independently contributes to the NK cell response [54] and enhances the NK cell response activated by IL-12 [55].

The importance of TNF-α is demonstrated by evidence that infusion of this cytokine alone is sufficient to cure certain established animal tumors [56, 57].  TNF-α is part of a family of Type 1 cytokines and ligands that can effectively destroy cancer cells by inducing apoptosis .[58, 59].  IFN-γ and TNF-α not only have an adjuvant effect on anti-tumor effector cells, but can also directly induce apoptosis of tumors [60, 61].

GM-CSF production by AlloStim™ also provides a powerful adjuvant effect.  GM-CSF induces production of Type 1 cytokines by human PBMC, T lymphocytes, and APC and down-regulates Type 2 cytokine expression, promotes differentiation of monocytes into DC with a preferential expansion of DC1 (IL-12-producing DC) and activation of NK activity [62]. GM-CSF autologous tumor vaccination was effective for the treatment of hepatic colorectal metastases [63].

Ex-vivo studies have demonstrated that the cytokines produced by AlloStim™  can cause DC to mature and produce IL-12.  IL-12 is known as a primary initiator of Type 1 immune response and acts as an upstream positive regulator for IFN-  production from NK and Th1 cells.  IL-12 activates cytotoxic T cells [64, 65] causes CD4+ lymphocytes to differentiate to Th1 phenotype [66, 67] and tilts the balance between Type 1 and Type 2 immune responses in favor of Type 1 [67, 68].  IL-12 is known to have a strong adjuvant effect in promoting Type 1 immunity. Transduction of vaccine cells with IL-12 genes can substantially improve their immunogenicity [69].