Marker Therapeutics Inc (MRKR)

[Rkmatters]

While GALE (E75) was in development Herceptin was approved so they changed their game plan. The results were okay, but it was post hoc analysis. See below attachment. I honestly think it comes down to the trial design, the recruitment of patients with low expression. And their use of placebo which is a cytokine (GM-CSF), which is known to recruit DC, and that in tail assist an immune response. Herceptin is a mAB, and so the standard of care patients would have benefited from placebo cytokine and that E75 (NeuVax) solo will have minimized the small benefit seen in comparison. Plus they were recruiting patients of low HER2 expression - which was the opposite with what a non-immune memory vaccine should do (fine for TPIV but not for GALE); remember GALE's E75 vaccine isn't a complete the successful components of the vaccine as it only has t-killer cells and no t-helper cells (needed for sustainability, immune memory (to attack recurrence) and expansion). I had thought the booster shots would have helped with to minimize the need for immune memory, but it apparently did not. Maybe the vaccine killer T was just not strong enough, that's a possibility. At the end of the day, we won't know until GALE analyzes the results and reports why it may have showed no difference between we won't know.

http://galenabiopharma.com/wp-content/uploads/2013/10/Combination-immunotherapy-with-trastuzumab-and-the-HER2-vaccine-E75-in-low-and-intermediate-HER2.pdf

https://clinicaltrials.gov/ct2/show/NCT01479244?term=NeuVax&rank=3


Article from TapImmune explains their vaccine difference.

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Immunotherapy in the Age of Vaccinomics

TapImmune’s Approach to a Her-2/Neu Therapeutic Vaccine - White paper by Mark Reddish, TapImmune

Vaccinomics - The matching of individual immune response genetics to specific target peptides in vaccines. Designs for success.
The field of immunology and vaccine design has reached a new paradigm of understanding as to how vaccines work, or don’t work, based on our understanding of immune response genetics. In principle the genetic variables, or ‘alleles’ of an individual’s Major Histocompatability Complex (MHC) determine which peptides or ‘targets’ an individual can or cannot respond to.. In addition to this level of genetic complexity, we now also understand clearly what peptide targets tumor cells will and will not present to the immune system. This antigen presentation (a process which results in peptides being recognized as self or non-self by T-cells) question is entirely separate from that of immune response genetics. Proper tumor ‘targets’ must be naturally derived via something called ‘antigen processing’. The antigen processing machinery (which includes TAP proteins playing a critical role) actually ‘digests’ a cells proteins, like Her-2/neu, into small peptide fragments or targets and transporting them to the surface of the cell. Once a protein is digested the targets are ‘transporting’ by TAP proteins to bind with MHC molecules, making them available for immune recognition on the surface of the cell. It is essential to understand that both an individual’s MHC genetics combined with the cellular function of ‘antigen processing’ that will ultimately determine if an individual can mount an effective response to a specific tumor or tumor vaccine. Thus the design of an immunotherapeutic cancer ‘vaccine’ requires an understanding of ‘immune response genetics’ of the person being vaccinated (MHC ‘types’) and the cellular biology of the tumor that processes proteins into small peptide targets. Over the last 20 years the Her-2/neu onco-protein has been thoroughly dissected into literally dozens of target peptides formulated into vaccines and these have been clinically tested and analyzed. This vaccine development history allows us now to look back and understand the immunology of Her-2/neu using the concepts of vaccinomics. Both MHC I (killer targets) and MHC II (helper targets) peptides have been mapped and tested as vaccine components, allowing for an informed design of a predictably broadly reactive vaccine that can target functional killer peptide and via T cell help to amplify and sustain these responses with an optimal vaccine formulation.
The Role of Our Immune Response Genes-The Major Histocompatibility Complex-MHC
The MHC genes that determine our individual ability to respond to vaccines (or disease) come in two types or classes, these referred to as Class I MHC and Class II MHC. In parallel, the immune system has two types of T cells that perform different and supportive functions in the overall immune response to threats like viruses or cancers. These two kinds of T cells are called ‘ T killers’ and ‘T helpers’, and these two T cell types respond to different peptide targets presented by Class I MHC (killers) or Class II MHC (helpers). The ultimate goal of an immunotherapeutic cancer vaccine is to cause the clonal expansion of MHC restricted Killer T cells (CD8 cells) and to ‘expand and support’ these killer T cells with ‘help’ as they invade tumors and deliver ‘killing’. Clonal expansion and functional activation of these killer T cell responses requires Her-2/neu specific helper T cell responses. The helper cells provide the growth factors and cytokines that support clonal expansion and differentiation of nai¨ve T cells into mature functional killer cells. In earlier Her-2/neu peptide vaccine clinical studies, killer T cell responses/function were seen to wane in the absence ‘T cell help’, with T cell responses returning to baseline within 2-5 months after the last immunization (1). Therefore, vaccine products designed without a supportive T helper cell function can potentially allow disease recurrence after active immunizations have been stopped and T cell killing functions have faded. In studies of tumor specific cytotoxic T cell therapies, it has been observed that killer cell activity was lost in just two weeks after infusion of such activated ‘killer cells’ in melanoma patients (5). A critical parameter of an effective tumor vaccine requires a vaccine formulation to include both killer T cell targets and helper T cell targets (2,3) for their supportive functions. In vaccine studies, comprised of HER-2/neu killer cell targets and Her-2/neu helper cell targets (4), substantial T cell responses were notable well over a year after the last immunization. This is in stark contrast to the results noted above (1) where killer cell activity was gone after less than 5 months. Thus the need for repetitive booster vaccinations can be obviated if a vaccine formulation includes a broadly reactive helper T cell component and the proper killer cell targets. Even short term killer cell function in the tumor microenvironment requires that helper T cells be present and activated in the tumor environment such that these helpers continuously secrete the activating cytokines (predominantly IL-2) that sustain the killer cells in their function (6,7).
One strategy that has been utilized in tumor vaccine development is to use ‘universal helper peptides’ such as ‘PADRE’ (8) or the Ii key peptide (9) as a means of generating strong ‘helper T cell responses’. These universal helper peptides react across MHC genetic restriction barriers (broadly reactive) and do provide for strong helper T cell derived growth factors to amplify killer T cell responses. However, the activating antigen for these helpers is not present after the immunization, and is never present in the tumor microenvironment, and thus those ‘universal’ helpers do not support the actual function of killer T cells, only their initial generation. Immune response analysis of vaccines utilizing this strategy reveals that the CD4 T cells primarily respond to the universal helper peptide, leaving only weak responses that are actually generated to the actual Her-2/neu target peptide. Indeed a look at the T cell response data for the AE37 vaccine (helper peptide formulation with Ii-Key fusion) shows that 75% of the ‘helper activity’ is actually specific for the Ii-key component, and only 25% is directed to the HER-2/neu peptide (9). This observation is not uncommon for ‘universal peptides’ when used as fusion protein targets, as these tend to be strongly immune-dominant leaving the actual cancer target peptide under recognized instead of enhanced.
In order to fully engage the cellular immune response in the tumor microenvironment, it is critically important that both CD4+ helper cells and CD8+ killer cells be a part of the tumor ‘infiltrate”(6,7). Both helper and killer T cells require that antigen be present in their environment for their activation and function. Helper T cells provide the growth factors and cytokines required for killer T cells to maintain their killing functions. Having helper T cells that are specific to ‘non-tumor derived antigens, such as Ii- key, does not provide for an activated helper cell in the tumor environment, thus for an optimal HER-2/neu vaccine, it must be designed to present individual and distinct peptides that will bind and presented by both the MHC Class I (killer targets) and MHC Class II pathways (helper targets), and these activating antigens should both be present in the tumor microenvironment. This is exactly the design of TapImmune’s Her-2/Neu vaccine, with the added benefits of the enhanced antigen processing and presentation provided by TAP expression technology.
Detailed Immuno-genetic Comparison of Class I targets p369-377 (E75) with the newly identified p373-382 target
Clinical studies of ‘MHC binding’ peptide based vaccines, requires that patients be genetically ‘qualified’ for inclusion into these trials. Individuals who do not have the correct or matched MHC types cannot respond and should be excluded based on their immune response genetics. The clinical utility of a genetically designed vaccine across the population as a whole is based on the number and variety of target peptides, and how many MHC types can be included as ‘responders’ to the collection of ‘mapped peptides’ in a given vaccine formulation. Design strategies that include multiple peptides that bind multiple MHC types, and or use ‘promiscuous’ peptides that bind to multiple MHC types, can provide for a vaccine that can be used more broadly in the population with greater predicted efficacy. The selected target peptides must exist in the tumor microenvironment for these CD4 and CD8 responders to function optimally, generating specific and long lasting killer cell function and lasting immunologic memory.
TapImmune has agreed to an exclusive license from the Mayo Foundation for use of the highly promiscuous and immunodominant HER-2neu peptide, p373-382 for use in TapImmune’s, HER-2/neu vaccine. This high affinity MHC I binding CTL target peptide discovered in Keith Knutson’s labs has been extensively tested and compared to the p369-377 peptide target found in the NeuVax, therapeutic vaccine. The p373-383 peptide binds to HLA A2 with an approximately log greater binding affinity compared to p369-377 (Figure 1D). In addition p373-382 ‘stabilized’ a higher maximum level of class I expression on the surface of cells using the T2 cell based MHC Class I binding assay. (Figure 1- A,B,C, found in appendix 1). This peer-reviewed work has been accepted for publication in the Journal of Immunology and will appear in electronic form in Nov/Dec. 2012 (16).
The higher affinity of binding and natural processing of the p373-382 peptide creates a better, more functional class I restricted killer cell response, either on peptide pulsed cells (Figure 2) or on MHC matched human tumor cells that express HER-2neu. In fact a high degree of specific killing is noted on a variety of HLA-A2 matched, HER-2/neu positive tumor cells irrespective of which peptide was used for priming (See figure 3). The killing activity against human tumor cell targets is 4-5 times greater when cells are primed with the high affinity p373-382 peptide compared to the lower affinity p369-377 peptide. Interestingly the killing of peptide pulsed targets is higher on p373-382 pulsed targets no matter if the priming peptide was p369-377 or p373-382. This data proves that the killing observed when p369-377 is used for priming, is actually cross-reactivity to the p373-382 natural target as opposed to representing a distinct epitope. The biochemical observation that the 369-377 target peptide is not generated by the proteasome antigen processing machinery, confirms this cross reactive explanation of the clinical results seen to date with E75.
In addition to the HLA-A2 binding affinity data, the promiscuous nature of the p373-382 peptides binding to HLA class I molecules creates a greater utility for this vaccine component in a larger percentage of the population. The p369-377 peptide is bound by both of the common class I alleles, HLA-A2 and A3, that are expressed by 45.6% and 23.8% of the Caucasian population of North America(HLA Matchmaker, http//tpis.upmc.edu/tpis/HLAMatchmaker/). Thus a vaccine that is comprised of an A2 and A3 binding peptide will be a ‘match’ for 45.6 + (23.8 X .55)= 59.5% of the Caucasian population. Percentages for Hispanic, Asian, and African American populations will be considerable lower than this based on lower A2 and A3 allele frequencies in these ethnic groups. Utility in these groups calculate asfollows:for African Americans who express A2 at 22.03% and A3 at 18.07% the potential utility is 22.07+ (.78 x 18.07)=36.13%. Utility for Asian Americans who express A2 at 18.1% and A3 at <5% the overall utility is 18.1 + (.82 X 5)=22.1%. For Hispanic Americans who express A2 at 37.1% and A3 at 14.3% the calculated utility is 37.1 +(.629 x 14.3)=46.1%. Thus the overall blended averages are approximately 50% of the North American population. In contrast the TapVax target peptide, p373- 382 is referred to as promiscuous, due to the observed high affinity of binding to several class I allelic products, these are listed in Table 1 (see Appendix 1). The calculated utility or ‘responsiveness’, based North American MHC Class I population genetics is greater than 90% of the population, while E75 predicts response rates at approximately 50% of the same population.
Detailed MHC Class II Antigens Binding Profile of TapImmune’s Her-2Neu Vaccine Candidate
In addition to the MHC genetics of the Class I target peptide, it is also required to provide a vaccine formulation that binds to a wide genetic variety of MHC Class II molecules in order to provide the required T cell ‘help’. As noted above, the vaccine called AE 37 from Antigen Express, includes a Her-2/Neu helper peptide (AE36) that is fused with the universal helper peptide Ii-key to form AE37 (9). Although the Ii-key approach does provide for helper cell activation across the genetic spectrum, this helper epitope is not found in the tumor, and thus activated T helper cells do not localize there and provide for the continuous activation derived source of T cell ‘help’. The AE36 peptide alone is a Class II binding peptide (p776-790) which is restricted by a single MHC Class II type, and thus does offer some help for this smaller subset of patients (approximately 15%). The TapImmune formulation includes a collection of 4 MHC Class II binding peptides that are described as ‘degenerate’ due to their high affinity of binding to a wide array of MHC Class II alleles or types (15). The approach taken in the discovery of these peptides was to synthesize dozens of peptide analogs based on computer predictions and to test the binding of theses helper peptides to isolated MHC-Class II molecules. From the dozens predicted, a set of 4 peptides that bound with high affinity to a wide array of Class II molecules were selected. (see Table 2). The hypothesis was then tested by evaluating human immune responses in vitro, this demonstrated that patients can indeed mount immune responses to these targets as predicted (15) and that immune tolerance was not an issue restricting immune responses. Using MHC population genetics this collection of Class II binding peptides are predicted to be reactive with approximately 84% of the North American population, similar to the broad responsiveness predicted for the Class I target peptide. Thus the TapImmune vaccine is designed with proprietary targets for both Class I and Class I MHC that are broadly reactive across a far larger percent of the population when compared to the two major competitors vaccines. A summary of competitive vaccines, their compositions, strengths and weaknesses is provided in Table 5.
The Role of Antigen Processing in tumor cells to present targets for killing
As described above, it is now possible both in the lab, and on a computer, to identify or predict which small peptide fragments will bind to individual MHC Class I or Class II molecules. However, being able to generate immune responses to isolated peptide is only half of the story, as tumor cells must actually display these same peptides on their cell surface, bound to the same MHC molecules for a killer T cell to actually kill. Some synthetic peptide vaccines can induce apparently strong killer T cell responses by‘fitting’ into Class I MHC molecules, but are still poor vaccines due to the absence of the matched natural target displayed on tumor cells. To understand this paradox we must understand antigen processing. Inside of all cells, tumor cells included, are small barrel shaped organelles called proteasomes who’s function it is to ‘process’ proteins, like HER-2/neu, into the small target sized peptide fragments. The specific fragments that they generate by a proteasome (or immunoproteasome) are a function of the protein clipping enzymes or proteases that are inside of these barrel shaped organelles, creating a predictable set of peptide fragments from any given protein. Generating immune responses tofragments that are not naturally derived via this ‘processing step’ will not generate effective killing responses when real or natural human tumor cells are the targets. Thus there are some some ‘immunogenic peptides’ that serve as target structures in cancer vaccines that generate killer cells that do not actually kill HER-2/neu positive tumors (10). The E75 antigen (p369- 377) is just such a peptide target. This 9mer target peptide is bound by HLA A2 and A3 Class I molecules, and individuals can generate killer cells that target this peptide MHC complex. These peptide specific killers do indeed kill ‘peptide pulsed target cells’ quite efficiently. However, peptide pulsed target cells do not require antigen processing, as the readymade synthetic peptide target is supplied in the experimental design, creating the illusion of killing. However, the killing of actual tumor cells that express Her-2/neu is relatively poor or absent (10). This observation was not fully understood until recently when proteasome/immunoproteasome in vitro processing assays revealed that the p369-377 peptide was not generated by either tumor cell derived proteasomes or immune- proteasomes from HER-2/neu protein precursors (11). The small amount of killing activity that has been observed in association with some vaccine trials, appears now to be a result of ‘cross reaction’ with the overlapping portion of the p373-382 peptide (overlap at residues 373-377) that was identified by the Knutson lab at the Mayo clinic. The p373-383 immuno-dominant peptide is now being incorporated in the TapImmune’s HER-2/neu vaccine as the immune-dominant killer cell target peptide. In fact the Knutson group utilized an in vitro proteasome antigen processing assay to demonstrate that the target of the NeuVax vaccine, p369-377, is not actually produced when Her- 2/neu proteins/peptides are naturally processed (see Table 4). This biochemical study of antigen processing provides for a rational understanding of clinical results and design rationale for a superior vaccine design.
The Role of TAP in cancer and TAP Based Vaccines
As noted above ‘antigen processing’ plays a critical role in the immune system’s ability to ‘perceive’ and respond to either cancer or viral infections. The MHC Class I antigen presentation pathway is best described as a QC system to examine what proteins are being made inside of a cell on any given day. The density of specific target peptides on the surface of cells, either virally infected or cancerous, is a critical parameter that determines if a T cell response will be triggered initially, and for the recognition and killing by killer cells once they are produced. The most common strategy to avoid immune recognition, by both tumors and viruses, is to turn off the antigen processing ‘machinery’ and thereby stop the display of target peptides on the surface of a cell. This is accomplished most often by down- regulating the Transporters of Antigen Processing, or TAP molecules that are the critical step that moves target peptides into the endoplasmic reticulum (ER) where they bind to MHC Class I. Across the spectrum of cancer types there is an extensive body of literature that shows failure to express TAP1 and/or TAP2 results in low/or no Class I MHC expression, and a poor overall prognosis for survival. A bibliography of such publications is provided in the appendix. TapImmune’s core technology approach is based on genetic expression systems (viral or plasmid based) that insert the genes to enhance or restore TAP1/2 levels, and thereby enhance or restore antigen presentation. In murine preclinical models of lung cancer or melanoma, the restoration of TAP1 alone using an adenovirus expression system. This provides sufficiently restored immune recognition to enhance anti-tumor T cell immunity and significantly improved survival following tumor challenge. (12,13). This result is based solely on enhanced recognition of the tumors after implant, and is not enhanced by any vaccination strategy. In preclinical studies TAP expression technologies have been shown to enhance immune recognition at both the ‘generation of a response’ stage, and at the effector level once killer T cells are generated. In TAP based vaccines, engineered viral ‘vectors’ or expression systems are designed to express (produce) the TAP proteins (transporters) and simultaneously produce the identified target peptides. By including ‘trafficking’ signals, the Class II peptides are targeted for secretion and helper cell activation, while the class I peptide is targeted for proteasome processing and killer T cell activation. As noted previously the density of Class I target peptides is critical for triggering a cytotoxic T cell response. By producing high copy number of the proprietary target peptides into the cytosol, at the same time as producing the ‘TAP transporters’ the antigen processing machinery is flooded with the high affinity target peptides, thereby exceeding the threshold of T cell recognition and generating an enhanced killer cell response. This enhanced T cell response is quite clear in preclinical models of our small pox vaccine candidate in which an engineered ‘vaccinia’ virus was used to enhance immune recognition, and resulted in a thousand fold more potent ‘protective’ immune response (14). This combination of broadly responsive unique patented antigens combined with the TAP expression platform forms the basis for this second generation Her-2/neu cancer vaccine. The approach to replace deficient TAP and to incorporate it into a vaccine strategy is applicable to a wide range of cancers including other Her-2/neu positive cancers such as colorectal and ovarian.
Summary
The 20 year history of Her-2/neu vaccine development provides a body of information allowing now for a rational understanding of the clinical results observed to date, and more importantly the aspects appropriate for an advanced vaccine design today. The TapImmune Her-2/neu vaccine incorporates an ‘array’ of target peptides that cover greater than 85% of the population. This design includes the necessary targets for both helper and killer T cells, each with broad population targeting. These patented targets are incorporated into the TAP expression platform to provide an enhanced killer cell response, which is sustained by a broadly reactive tumor specific helper response. This combination of targets and presentation strategies represent a state of the art vaccine formulation that has only now entered in clinical testing. The current phase I trial at the Mayo clinic is designed to establish the safety of the peptide targets, while TAP expression technology will be added to this format as a prime and boost vaccine with prospects for utility across a wide spectrum of genetic types.

References
* 1) Knutson, K.L., Schiffman, K. and M.A. Cheever (2002) Immunization of Cancer Patients with a HER- 2/neu, HLA-A2 Peptide, p369-377, Results in Short Lived Peptide Specific Memory. Clin. Cancer Research 8:1014-1018 ?
* 2) Widmann, C., Romero, P., Maryanski, J.L., Corradin, G., and Valmori, D., (1992) T Helper epitopes enhance the cytotoxic response of mice immunized with MHC class I restricted malaria peptides. J. Immunol. Methods 155:95-99 ?
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* 4) Knutson, K., Schiffman, K., Disis, M.L., (2001) Immunization with a HER-2/neu helper peptide vaccine generates CD8 T-cell immunity in cancer patients. J. Clin Investigation 107:477-484 ?
* 5) Dudley, M.E., Wunderlich, J., Nishimura, M.I., Yu, D., Yang, J.C., Topalian, S.L., Leitman, S.F., and Rosenberg, S.A. (2001) Adoptive transfer of cloned melanoma-reactive T lymphocytes for the treatment of patients with metastatic melanoma. J Immunotherapy 24:363-373. ?
* 6) Hung, K., Hayashi, R., Lafond-Walker, A., Lowenstein, C., Pardoll, D., and Levitsky, H., (1998) The central role of CD4+ T cells in the antitumor immune response. J Exp. Med 188:2357-2368 ?
* 7) Kalamas, S.A., and Walker, B.D. (1998) The critical need for CD4 help in maintaining effective cytotoxic T lymphocyte responses. J Exp Med 188: 2199-2204. ?
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* 9) Holmes, J.P., et al. (2008) Results of the first Phase I Clinical Trial of the Novel Ii-Key Hybrid Preventative HER-2/Neu Peptide (AE37) Vaccine. J Clin. Oncol. 26:3426-3433. ?
* 10) Zaks, T.Z., and Rosenberg, S.A. (1998) Immunization with a peptide epitope (369-377) from HER- 2/neu leads to peptide specific cytotoxic T lymphocytes that fail to recognize Her-2/neu + tumors. Cancer Research 58:4902-4908. ?
* 11) Henle, A.M., Erskine, C.L., Benson, L.M., and Knutson, K.L. (2012) Identification of HER-2/neu peptide p373-382 as a naturally processed immunodominant HLA-A2 binding epitope.Poster American Association Immunol. Boston, May 2012. ?
* 12) Yuanmei, L., et al (2005) Cancer Research Vol. 65 (17) pp.7926-7933. Restoration of the Expression of Transporters Associated with Antigen Processing in Lung Cancer Increases Tumour-Specific Immune Response and Survival. ?
* 13) Lou, Y., et al Vaccine Vol.25 (12) pp.2331-2339 Tumour immunity and T cell memory are induced by low dose inoculation with a non-replicating adenovirus encoding TAP1. ?
* 14) Vitalis, T.Z. et al (2005) PLoS Pathogens 1(4) e36. Using TAP Component of Antigen-Processing Machinery as a Molecular Adjuvant. ?
* 15) Karyampudi, L., et al (2010) Clin Cancer Res. 16:825-834 A Degenerate HLA-DR Epitope Pool of HER-2/Neu Reveals a Novel Immunodominant Epitope, HER-2/Neu 88-102. ?
* 16) Henkle, A., et al (2012) J. Immunol.(In Press). Enzymatic discovery of a HER-2/neu epitope that generates cross-reactive T cells ?http://www.tapimmune.com/wp-content/uploads/2014/12/White-Paper-Immunotherapy-in-the-Age-of-Vaccinomics-Mark-Reddish-TapImmune-Inc.pdf?