LAMP Allergy Vaccines Theory & Proof of Concept with Der P2 Allergen in Mice New Vaccine for Japanese Red Cedar Immunomic Therapeutics, Inc. bhearl@immunomix.com www.immunomix.com 240-731-5232
www.immunomix.com
What Causes Allergies? •
Allergy happens because of an educational error of the bodies’ immune system. Normally, the body uses its immune system to defend itself against foreign invaders like bacteria and viruses through experience – by encountering, battling, and then remembering one enemy after another. The immune system is better equipped to fight off invaders that it has seen before than those it first encounters. This process is accomplished through special ‘memory’ white blood cells and unique protein molecules called antibodies.
•
Although there are many classes of antibodies, only one is involved in all true allergic reactions: IgE (also known as ‘the allergy antibody’).
•
Allergic reactions occur when the immune system misidentifies harmless foreign substances and reacts to them as if they were as harmful. These substances are called allergens. Common pollens, mold spores, house dust mites, cockroaches, and animal dander (shed skin, fur, or feathers) are among the allergens that most frequently cause problems. Allergic reactions to allergens such as insect venom, latex, and certain types of food or medications are more rare.
•
Irritants like tobacco smoke, perfumes, or strong cleaning products can make allergy symptoms worse and trigger asthma, but are not considered true allergens themselves.
www.immunomix.com
Current Allergy Treatment Options Treatment for allergies fall into three main categories: avoidance, symptom treatment and immunotherapy. None of the existing options provide a long term satisfactory solution to allergies. The basic medications for allergies are antihistamines, which counteract the histamine chemicals that cause the allergic reactions. Prescription anti-inflammatory steroid drugs, such as prednisone, may also be used for severe symptoms. In emergency situations -- when anaphylactic shock occurs -- injections of epinephrine are used to open up airway passages. Allergy shots -- also called immunotherapy or allergy desensitization therapy -- may come close to eliminating some allergies by introducing small amounts of the offending allergens in order to help the body learn to deal with them, but require up to 36 months of weekly or bi-weekly injections to become “effective.� Results from current immunotherapy vary widely and this course of treatment is not an option for those suffering from highly reactive allergies (e.g., peanut, penicillin). www.immunomix.com
The Allergic Response & LAMP Immunotherapy There is increasing recognition of the potential benefit of genetic immunization as a method for both prophylactic and therapeutic treatment of the broad spectrum of protein allergens. The underlying rationale is that allergen protein encoded as a DNA vaccine will preferentially activate allergen-specific T-helper type 1 (Th1) cellular responses with the production of interferons by antigen presenting cells (APC), natural killer cells (NK), and T cells, rather than the characteristic Th2-type responses, such as secretion of interleukin (IL) -4, -5, and -13, and the formation of immunoglobulin E (IgE) by B lymphocytes and the maturation and recruitment of eosinophils in late-phase reactions. It is believed that (a) the cellular trafficking properties of the allergen protein in transfected cells Is one of the major determinants of the immune system response to the allergen, and that (b) a design of the allergen-encoded DNA vaccine that will facilitate trafficking into the MHC II processing and presentation pathway of APCs is critical for optimal and precise expression of Th1 immune responses. Lysosomal Associated Membrane Protein or “LAMP” is a protein that localizes in antigen presenting cells (APC) to the same compartment as the Major Histocompatibility Complex Type II (MHC-II). Work in the laboratory of Dr. J. Thomas August at Johns Hopkins University showed that when the protein sequence that would otherwise be expressed in the cytoplasm of the cell was linked to the protein sequence of LAMP, the chimeric protein will migrate to the MHC-II lysosomes in APC’s. ITI’s LAMPvax vaccine formulations utilize this intra-cellular trafficking function to access the MHC-II pathway and in the case of allergy vaccines, convert the immune system response from an IgE allergen response to an IgG antigen response with the concomitant elimination of allergy symptoms. www.immunomix.com
The Allergic Response & LAMP – Directed Immune System Re-Programming The ITI approach allergy vaccines involves attacking the problem using a traditional method – converting the immune response from an IgE mediated response to allergen to an IgG mediated response.
Figure based on diagram at dynavax.com
www.immunomix.com
LAMP-vax Allergy vaccines introduce the allergen (antigen) to the immune system through the MHC-II / Th1 pathway which favors the generation of an IgG response.
A Unique Immunotherapy Allergy Solution ITI’s LAMPvax DNA vaccines are composed of 3 major elements: the luminal domain of LAMP, the antigenic protein sequence, and the Transmembrane / Cytoplasmic signaling domain of LAMP.
When this unique chimeria is synthesized in the cell, the luminal domain of LAMP and the antigenic protein are localized inside the Golgi and the cytoplasmic targeting sequence remains external to the Golgi and facilitates subcellular localization.
www.immunomix.com
LAMP Chimera Co-Localizes with MHC-II
From Arruda, et al, 2006
www.immunomix.com
The LAMP chimera protein has been shown to localize in the MHC-II compartment in many studies by confocal microscopy. In the figure to the left, cells were transfected with a LAMP/gag chimera and stained for anti-Gag (red) and anti-MHCII (green). The coincidental location of both LAMP and Gag are shown in yellow. Virtually all of the Gag protein is found coincidentally with MHC-II.
Dendritic Cells Are Key to Immune System Presentation A fundamental first step in vaccination is that the vaccine proteins must reach the internal compartment of dendritic cells and then be processed and presented to the immune system. Following injection of a plasmid DNA vaccine, the encoded gene is expressed inside the cell and then processed for MHC presentation.
Source: wikimedia, dendritic cell
www.immunomix.com
Vaccines that include the LAMP gene sequences present through the MHC-II , accessing the primary immune system presentation pathway.
LAMPvax Activation of Dendritic Cells The next few slides diagram the process of DNA vaccination and presentation to the immune system mediated by LAMP. Step 1. DNA is taken up by the DC. This can be naked DNA or DNA complexed to lipids or other carriers.
1 2
Step 2. The DNA enters the nucleus of the cell and begins to prepare mRNA. It is also possible to deliver mRNA directly.
Step 3. The mRNA directs the synthesis of the chimeric LAMP – Antigen protein which is localized first in the Golgi and then in the maturing MHC-II+ lysosomes.
www.immunomix.com
The Lysosome Compartment is Key to Immune System Activation The MHC-II molecules, essential to antigen presentation to the immune system, are found in the lysosomes. These vesicles receive imported antigens that are then processed (i.e., digested into peptides) for binding to the MHC-II complex. The lysosomes naturally contain the highly glycosylated protein, Lysosomal Associated Membrane Protein or “LAMP.� ITI’s vaccines take advantage of the natural intracellular localization of LAMP to bring the antigen synthesized by the cell to this key immune processing center. www.immunomix.com
Lysosome Compartment MHC-II LAMP – Allergen Chimera In this expanded view of the lysosome compartment from a LAMPvax transfected cell, the two of the key components of the immune response are diagramed. The LAMP – allergen chimera is anchored inside the vesicle with the allergen protein completely sequestered from the cell exterior providing a unique safety element for LAMPvax allergy applications.
www.immunomix.com
Digestion by Proteases Release Allergen Peptides
As the lysosome matures, the pH of the internal environment drops and enzymes digest the proteins inside the lysosome.
www.immunomix.com
Peptides “Load� on the MHC-II Peptides released during the digestion process then bind to the MHC-II molecule in a very specific binding cleft in the protein.
www.immunomix.com
At the Cell Surface, the Antigenic Peptide is Presented to the Immune System Following the antigen peptide digestion, the mature lysosome moves to the cell surface and presents the MHC-II to the immune system. Shown at the left is an expanded view of the MHC-II molecule containing the antigenic peptide. Thus, in an allergy application, no free allergen is ever exposed outside the cell. The only presentation of allergen is through the very controlled presentation in the binding cleft of the MHC-II molecule.
www.immunomix.com
Data with Dust Mite Allergen Preliminary results An initial study of mouse immunization with a LAMP/allergan construct has been published. Mice were immunized with 3 DNA constructs containing the Der p2 mite allergen, Der p 2 without a signal peptide (p2), with a signal peptide (p52), and with a signal peptide plus the LAMP lysosomal-targeting sequence (p52-LAMP), and followed by a Th2-skewed protein challenge. • Mice immunized with p52-LAMP showed strong and clear-cut Th1-type response, as evident by high IFN-γ production and elevated levels of Der p 2-specific IgG2a production whereas the native Der p 2 construct induced only moderate levels of Th1 response. • Mice immunized with construct p52 containing a signal sequence showed a mixed TH1/TH2 phenotype and produced substantial circulating Der p 2 protein. • Mice adoptively transferred with DCs primed by the p52 construct containing a signal sequence and presumably producing secreted Der p 2, but not by the p2 or p52-LA constructs, were sensitized to produce high levels of Der p 2-specific IgE. • It was concluded that immunization with a DNA construct encoding a signal peptide could prime TH2-skewed responses and IgE production and despite the DNA immunization that the inclusion of lysosomal-targeting sequences to such construct could improve the safety and efficacy of DNA vaccination against allergy. The data on the following slides (16-20) are re-presented from Chua, et al. (2006) Vaccine and examine the immune response in mice following immunization with dust mite allergen and a DNA vaccine coding Der P2. www.immunomix.com
Immunization with Der P Protein Induces Strong IgE response / low IFN-γ In this figure, mice were immunized with the dust mite protein and then monitored over time for the production of IgE, IgG and IFN-γ. Mice so immunized produced high levels of IgE and IL-4 and relatively low levels of IgG and IFN-γ.
See Chua, et al. (2006)
www.immunomix.com
P52-LAMP Induces IgG with Minimal Detectable IgE when challenged with Der P2 protein
Mice were immunized with DNA vectors, pCI (control), p2 (Der P2 with no signaling), p2 (Der P52 with secretory signal) and p52-LA (Der P2 with secretory signal and LAMP) and then challenged with Der P2 protein on days 35, 39 & 43. The results show strong IgG bias for the LAMP immunized mice with undetectable levels of IgE.
See Chua, et al. (2006)
www.immunomix.com
LAMP vector has low IL-4 response and Strong IFN-Îł
Mice were immunized with DNA Der P2 vectors and then examined for cytokine levels. The spleenocytes from each study group were incubated with Der P2 protein for 72 (T72) or 96 (T96) hours prior to cytokine assay. LAMP immunized mice showed the greatest interferon response and low IL-4 production. See Chua, et al. (2006)
www.immunomix.com
Adoptive transfer of DCs from DNA vaccinated mice
DC’s from mice immunized with DNA vaccines to Der P2 were transferred to naïve mice and then challenged with Der P2 protein. Mice receiving DC’s from non-LAMP mice showed a Th2 / IgE bias while mice receiving the DC’s from p52-LA favored a Th1 / IgG response. See Chua, et al. (2006)
www.immunomix.com
Addition of LAMP eliminates free circulating allergen
Mice immunized with DNA vaccines to Der P2 were evaluated for circulating Der P antigen. The addition of the LAMP targeting sequence reduced circulating antigen to non-detectable levels. See Chua, et al. (2006)
www.immunomix.com
New Vaccine for Japanese Red Cedar
IMAGES AVAILABLE UNDER CONFIDENTIALITY AGREEMENT ONLY
www.immunomix.com
ITI has developed a new immunotherapeutic approach to address allergenic response to cedar pollen, a major allergen in Japan which causes JRC pollinosis.
Cry J1/J2 Vaccine Validated CONFIDENTIAL DATA
www.immunomix.com
The LAMP Advantage • LAMP is a proven technology for activating the immune system as a vaccine. • LAMP allergen vaccines do not produce any free protein (allergen), significantly increasing safety. It may make targets such as peanut viable candidates for immunotherapy. • Allergy vaccines are designed as immune system “reprogrammers.” That is, the objective is to create a TH1 – favored, IgG response. Animal data and human clinical data indicate this result is achieved with LAMPvax DNA vaccines.
www.immunomix.com
Prospectus • Cry J1/J2 vaccine is being evaluated in a mouse model. Recent data has shown our vaccine produces a strong IgG response; no IgE antibodies have been detected. • ITI is seeking a partner to support a Phase I clinical study. • Additional formulations for important allergy targets are in development. www.immunomix.com