INTEGRATION OF PRODUCT AND PROTOCOL

INTEGRATION OF PRODUCT AND PROTOCOL

Our protocols are designed to elicit the same immunological cascade of events as occur after non-myeloablative allogeneic stem cell transplant procedures.  We utilize Mirror EffectTM technology to reverse the flow of immunological events so that they emanate from host rather than the graft. Our bioengineered product, AlloStim®, contains intentionally mis-matched, activated Th1 memory cells that express high density CD40L and large amounts of inflammatory cytokines, such as IFN-gamma, TNF-alpha and GM-CSF. The route of delivery elicits different immunological responses.

Our protocols are designed with four fundamental steps: (i) priming; (ii) in-situ vaccination; (iii) immune activation; and (iv) immune boost.  The rationale for each step and an explanation of the proposed mechanism that each step was expected to elicit are described below:

A. The Priming Step

Anti-tumor immune responses are effectuated by Th1-dominated (type I) immunity. However, metastatic cancer patients usually present with Th2-dominated (type II) immune systems. Therefore, the priming step was incorporated in the protocol as an immunomodulatory mechanism designed to mediate a shift in the immune system balance from Th2- to Th1-dominance. The priming step takes advantage of the bioengineered components of AlloStim®, including the alloantigens (potent immunogen), inflammatory cytokines (such as interferon-gamma, tumor necrosis factor-alpha and GM-CSF) and CD40L expression. The intradermal administration of AlloStim® was predicted to prime Th1 allo-specific immunity and produce high titers of allo-specific Th1 and CTL T-cells as a means to modulate the systemic Th1/Th2 balance. 

The intradermal injection of intentionally mismatched AlloStim® was predicted to attract a rejection response. The rejection of the allogeneic cells releases chaperoned alloantigens and endogenous danger signals into the local microenvironment.  Langerhan’s cells (LC) of the skin that traffic to the injection site, will engulf and process the released alloantigens. The conditioning of the microenvironment with inflammatory cytokines and endogenous danger signals as a result of both the production from the activated AlloStim® cells and as a result of the by-stander activation of host innate immune cells was predicted to help steer the maturation of the LC processing to produce IL-12. The mature IL-12+ LC expressing alloantigens on MHCI and MHCII and co-stimulatory molecules trafficking to lymph nodes create an immune environment conducive for priming of allo-specific Th1/CTL (type I) immunity. 

Subsequent intradermal injections of AlloStim® were predicted to drive maturation of an allospecific memory Th1/CTL response which would trigger a delayed-type hypersensitivity (DTH) response at the injection site. The presence of CD40L+ allo-specific memory cells activated either directly by exposure to the alloantigens or indirectly through activation by the inflammatory cytokines produced by both the AlloStim® and activated host allospecific Th1/CTL responding memory cells provides a positive feedback loop to further drive the development of allo-specific type I immunity. This in turn creating high titers of allo-specific Th1/CTL memory cells, ultimately resulting in a modulation of the resident immune balance from Th2-dominance to Th1-dominance.

B. In-Situ Vaccination Step

The in-situ vaccination step was incorporated into the protocol to elicit high titers of tumor-specific Th1/CTL immune cells. In-situ vaccination involved the cryoablation of a selected metastatic tumor lesion to release internally chaperoned tumor antigens into the microenvironment and the intralesional injection of AlloStimTM into the ablated tumor as an adjuvant to drive tumor-specific Th1/CTL immunity customized to the patient’s own tumor. 

In therapeutic vaccine design, the selection of the target antigen and the adjuvant is of utmost importance to the eventual success of the vaccine.  There are many considerations for selection of TAA to incorporate into a therapeutic vaccine platform. Generally, necrotic cell death or autophagy of stressed cells has been shown to be more immunogeneic than apoptotic cell death [1, 2]. Thus, the disruption of the tumor cell membrane and release of chaperoned tumor antigens into the microenvironment for processing by professional antigen presenting cells (APC) is a desired vaccine design platform. 

Tumor death by necrosis releases chaperone proteins which carry TAA, such as hsp70 [3], into the microenvironment.  Percutaneous cryoablation is a method for causing tumor necrosis and release of these chaperone proteins into the tumor microenvironment.  The freeze-thaw cycles of the procedure result in the disruption of the tumor cell membranes and release of endogenous chaperone proteins, as well as endogenous danger signals into  the microenvironment [4, 5]. Heat shock proteins also act as adjuvants to drive maturation of APC to a IL-12+ phenotype [6], a necessary step in the process to promote type I Th1/CTL immunity. 

The injection of allogeneic AlloStimTM cells into the ablated lesion was designed to serve as an additional adjuvant to steer the immune response to the chaperoned tumor antigens to Th1/CTL immunity.  The adjuvant effect of intralesional AlloStim® was predicted to be amplified due to the prior priming step, which would attract a rigorous Th1/CTL rejection response.  The presence of inflammatory cytokines and the surface expression of CD40L on the AlloStim® and the responding host allo-specific memory cells, as well as the release of endogenous danger signals from the rejected AlloStim®, was expected to create the highly inflammatory conditions. 

It was predicted that the highly immunogeneic alloantigens engulfed by DC cells responding to the tissue damage processed together with the chaperoned tumor antigens in a microenvironment conditioned by a combination of inflammatory cytokines, endogenous danger signals released from the necrotic cell death of both tumor cells and the rejected allogeneic cells, and the upregulated CD40L expression on AlloStim® and activated host allo-specific T-cells, together with non-specifically activated by-stander memory T cells would create the conditions necessary for maturation of the immature DC responding to the tissue damage would mature IL-12+ DC. These mature DC that express both tumor-specific and allo-specific antigens on MHC molecules could then can traffic to the draining lymph node to prime tumor-specific Th1/CTL immunity.

These DC would be processing tumor antigen under conditions very different than the conditions that were present when the tumor was first exposed to the immune system. The key concept of the in-situ vaccination step is the conditioning of the local microenvironment for the re-presentation of TAA. The intended result is a de-novo systemic tumor-specific Th1/CTL response imprinted over the previously elicited non-protective Th2-dominated immune response.

C. Activation Step

The activation step involves the intravenous infusion of AlloStim® cells. This step is designed to cause the activation, expansion and extravasation of memory effector cells into the tumor lesions and to condition the systemic circulation and tumor microenvironments to promote immune-mediated tumor lysis.  In addition, this step is designed to build upon the in-situ vaccination step to further promote development and expansion of tumor-specific Th1/CTL memory cells.

In Allo-HCT procedures, an inflammatory cytokine storm occurs that promotes both GVT and GVHD effects [7, 8].  The presence of inflammatory type I cytokines in circulation and in the tumor microenvironment is responsible, in part, for the increase in tumor immunogenicity and the concurrent down-regulation of immune suppressor cell circuits that occurs after allo-HCT procedures enabling the linked GVT and GVHD effects.  In addition, type I cytokines are able to promote development of CTL memory cells. A similar type I cytokine storm is expected to be induced upon intravenous AlloStim® infusion permitting the proposed linked HVT/HVG Mirror Effects.

Intravenous AlloStim® infusion can elicit a type I inflammatory cytokine storm by various mechanisms, including: direct production of inflammatory cytokines from the AlloStim® cells; activation of memory Th1/CTL in circulation through interaction of the systemic inflammatory cytokines and CD40L expressed on AlloStim® cells with CD40 on circulating memory cells ; by-stander activation of circulating T-cells through interaction with the CD3/CD28-conjugated microbeads on the AlloStim® cells; and, release of endogenous danger signals caused by the rejection of the intentionally mis-matched AlloStim® cells.   

The induced type I cytokine storm is capable of driving an increase in the frequency of CTL with a heightened sensitivity to the chaperoned tumor antigens encountered in the in-situ vaccination step. Type I cytokines have been shown to be required for the generation of CTL memory [9]. In addition, CTL memory can be driven by CD40-CD40L interaction whereby activated host Th1 cells and/or AlloStim® cells expressing CD40L can in turn activate APC through CD40 signaling and these “licensed” APC can then drive memory CTL responses [10].  

Activated memory cells will down-regulate CD62L expression changing their circulation pattern from the lymphoid organs to the periphery [11]. Thus the activation of allo-specific and tumor-specific Th1/CTL in circulation by intravenous AlloStim® infusion would be expected to cause these cells to extravasate and congregate in sites of inflammation, such as in the tumor beds.

In this manner, intravenous AlloStim® could create highly inflammatory environments both systemically in circulation and within the local tumor microenvironments. This type of cytokine storm has been shown to impair tumor-induced regulatory T cell (Treg) immunosuppressive functions and also inhibit the generation of Tregs from naive T cells [12].

Multiple exposures to antigens are required for memory cells to develop. In the priming step, multiple intradermal injections of AlloStim® was designed to provide increased titers of allo-specific Th1/CTL memory cells. However, it is also desired to develop high titers of tumor-specific Th1/CTL memory cells. The in-situ vaccination step provides a single exposure of chaperoned tumor antigens to the host immune system.  However, multiple in-situ vaccinations was not considered feasible.

The inflammatory cytokine storm expected after intravenous AlloStim® infusion has potential to not only activate and cause extravasation of memory T-cells, but also to potentially activate NK cells.  Since activated NK cells can cause tumor cell lysis in an MHC I independent manner, this mechanism could release additional chaperoned tumor antigens into a highly inflammatory environment. These conditions, providing for multiple exposures of tumor antigens in the context of danger signals, could help drive the differentiation of tumor-specific memory Th1/CTL.

D. The Booster Step

The booster step is designed to maintain a pool of tumor-specific memory cells and a sustained inflammatory cytokine storm in order to dys-regulate tumor mediated immunosuppressive and immunoavoidance mechanisms. This is proposed to be accomplished by repeating the intravenous infusion of AlloStim® to re-initiate the mechanisms of the activation step. 

 

REFERENCES

  1. Zitvogel L, Casares N, Pequignot MO, Chaput N, Albert ML, Kroemer G: Immune response against dying tumor cells. Adv Immunol 2004, 84:131-179.
  2. Kepp O, Tesniere A, Schlemmer F, Michaud M, Senovilla L, Zitvogel L, Kroemer G: Immunogenic cell death modalities and their impact on cancer treatment. Apoptosis 2009, 14:364-375.
  3. Zhang H, Huang W: Fusion proteins of Hsp70 with tumor-associated antigen acting as a potent tumor vaccine and the C-terminal peptide-binding domain of Hsp70 being essential in inducing antigen-independent anti-tumor response in vivo. Cell Stress Chaperones 2006, 11:216-226.
  4. Kim HK, Pyun JH, Cho S, Kang SG, Lee JG, Kim JJ, Cheon J, Park HS, Kang SH: Tumor-specific immunity induced by cryoablation in a murine renal cell carcinoma model. Korean J Urol 2014, 55:834-840.
  5. Waitz R, Solomon SB, Petre EN, Trumble AE, Fasso M, Norton L, Allison JP: Potent induction of tumor immunity by combining tumor cryoablation with anti-CTLA-4 therapy. Cancer Res 2012, 72:430-439.
  6. Zeng Y, Graner MW, Katsanis E: Chaperone-rich cell lysates, immune activation and tumor vaccination. Cancer Immunol Immunother 2006, 55:329-338.
  7. Yang YG, Wang H, Asavaroengchai W, Dey BR: Role of Interferon-gamma in GVHD and GVL. Cell Mol Immunol 2005, 2:323-329.
  8. Yang YG: The role of interleukin-12 and interferon-gamma in GVHD and GVL. Cytokines Cell Mol Ther 2000, 6:41-46.
  9. Sun JC, Bevan MJ: Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 2003, 300:339-342.
  10. Schoenberger SP, Toes RE, van der Voort EI, Offringa R, Melief CJ: T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 1998, 393:480-483.
  11. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A: Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999, 401:708-712.
  12. Janikashvili N, LaCasse CJ, Larmonier C, Trad M, Herrell A, Bustamante S, Bonnotte B, Har-Noy M, Larmonier N, Katsanis E: Allogeneic effector/memory Th-1 cells impair FoxP3+ regulatory T lymphocytes and synergize with chaperone-rich cell lysate vaccine to treat leukemia. Blood 2011, 117:1555-1564.

 

SPECIALITY INFORMATION

patients


Treatment strategy designed to use the power of the human immune system to kill tumors and prevent their recurrence.
No requirement for a matched donor or chemotherapy/radiation conditioning prior to treatment.
Innovative technology – proven and non-toxic.
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Healthcare professionals


Therapeutic anti-tumor vaccine developed from core break-through technology called the "Mirror Effect™“ which opens a pathway to treating patients with metastatic cancer that have failed all available therapy options.
Elicits a GVT-like mechanism without the GVHD toxicity.
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Investors


Privately-held Israeli biopharmaceutical company spin out from Hadassah-Hebrew University Medical Center with headquarters in Jerusalem.

Over 200 individual private shareholders and grant support from the Israel Office of the Chief Scientist.
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