Vincent G. Brichard, M.D. Ph.D.
Senior Vice President
Head of the Immunotherapeutics Business Unit
GlaxoSmithKline Biologicals
 

Live metabolically competent bacteria are significantly more complex than synthetic molecules or antibodies, and they can generate more complex and therapeutically more competent immune responses. Using highly attenuated and safe Listeria monocytogenes (Lm) to secrete antigens fused to a highly adjuvant Listeria protein listeriolysin O (LLO). Lm-LLO vaccines are sufficiently attenuated that the CDC and the ZKBS (Germany) have determined them to be non-pathogenic (BSL-1). The multiplicity of Lm-LLO mechanisms is unlike other therapies. Lm-LLO vaccines; strongly stimulate innate immunity and both arms of the adaptive immune system (they do not require an adjuvant), reduce intratumoral inhibition by decreasing the percentage of Tregs and MDSC within tumors, stimulate persistent immune memory after a brief exposure, stimulate new immune cell synthesis in the marrow and cause immature immune cells to mature into targeted effector cells, increase chemo-attractants and their receptors that attract immune cells to tumors, facilitate circulating immune cells to migrate into tumors, as well as having other effects. In phase 1 we were able to show that 2 doses of ADXS-HPV administered to advanced, metastatic cervix cancer patients who had failed prior therapy and who have a poor prognosis increased median survival from a historic 182 days to 347 days and a historic 1 year survival of 5% to 53%. There are currently 4 phase 2 trials of ADXS-HPV ongoing. We have treated >145 patients with >250 well tolerated doses. 2 new agents will enter phase 1 trials in 1Q2012. Interim clinical results will be discussed.

Benefits:
• About the emerging field of “live drugs”
• How Listeria monocytogenes is uniquely suited to bioengineering as a therapeutic agent
• The use of Lm-LLO creates a safe and highly efficacious agent beyond the abilities of Lm alone
• About Advaxis phase 1 and interim phase 2 data on its first clinical agent ADXS-HPV, and its plans to bring other agents to the clinic

The responsiveness of the immune system to counter unwanted antigens is a prominent medical parameter. This responsiveness is determined by the cell counts and ratios of different leukocyte populations, which are promising diagnostic approaches. Current standard methods - FACS in peripheral blood, Immunohistochemistry (IHC) in solid tissue - have a limited ability for exact cell quantification owing to arbitrary/subjective gating protocols for FACS and lacking precision for IHC. For regulatory T-cells (Tregs), additional problems are manifested due to lack of specificity of Treg identifying proteins (CD25, FoxP3). Both proteins are synthesized also in activated effector T-cells, leading to detection of mixed cell populations.

The discovery of cell type specific epigenetic markers allows precise quantitation of immune cells in all human samples. The tests are based on quantitative PCR targeting genomic DNA. Therefore, readout is stable and samples can be frozen and easily shipped. This allows monitoring of patients in multicenter studies, retrospective studies, comparison of results between different studies, and routine monitoring.

In addition to technical benefits, higher Treg specificity is observed using epigenetic FOXP3 gene analysis, showing epigenetic activation only in true Tregs and epigenetic suppression in all other tested cells.

Application of epigenetic assays for Tregs, overall T-cell population, NK cells and further subpopulations will be presented in cancer and immune disease patients. The results reveal the same trends as measurements with alternative methods, while showing higher precision. Furthermore, the tests can be applied on both blood and tissue allowing measurements of circulating and tissue-infiltrating immune cells.

ImmusanT, a privately held biotech company based in Cambridge, MA, is revolutionizing the diagnosis and treatment of celiac disease, which affects approximately 1% of the population and currently has no pharmacological treatment.  Celiac Disease is an inherited autoimmune disease triggered by the consumption of foods containing gluten.  The chronic inflammation of the intestine in this disease is caused by the immune response to gluten.  Untreated disease is associated with other health conditions such as osteoporosis, anemia and liver disease.  ImmusanT is harnessing the specificity of the immune reaction to gluten (protein in wheat, rye and barley) to develop diagnostics and therapeutics.  Their scientists isolated the three toxic peptides responsible for the damage to the intestinal lining and alteration of nutritional absorption.  The Company has now translated the scientific discovery of these immunodominant T-cell stimulatory gluten peptides into a diagnostic and therapeutic.   It is believed that upon repeated dose administration of Nexvax2,  ImmunsanT’s peptide based immunotherapy, the number of gluten-specific T cells capable of triggering a pro-inflammatory response will subside and regulatory suppressor T cells will increase in number to establish tolerance to dietary gluten.  The company plans to leverage the learning to other auto-immune disease and allergies.

Flow cytometry allows for the simultaneous measurement of multiple cellular components both on the cell surface and within intracellular compartments. It is also amenable to the measurement of soluble analytes such as cytokines, drug compound, or anti-drug antibodies in solution. This flexible, powerful technology can be applied to all stages the drug development process -- from target discovery and characterization, to the evaluation of clinical responses. Compared to other methodologies commonly used in drug development such as plate-based immunoassays and mass spectrometry, flow cytometric methods can be more complex to develop and validate. The increasing variety of sample types, reagents, instrumentation, and software analysis programs combined with a lack of reference material to demonstrate accuracy of measurements, contribute to unique validation challenges. This presentation will discuss the application of “Fit-for-Purpose” method validation approach to flow cytometric assays.

Benefits of the talk:
Provide an understanding of the unique contribution of flow cytometry to drug development
Provide an understanding of basic flow cytometry
Provide an understanding of the Fit-for-Purpose assay validation concept
Provide an understanding of the challenges associated with validating flow cytometric methods
Provide an understanding of implementing flow cytometric methods for global clinical trials.

By directing recombinant HLA peptide complexes to the surface of target cells via an antibody delivery system we are able to redirect the lytic activity of virus specific T cells to tumour cells or produce expansion of a specific group of T cells dependant on the cell targeted. This technology has been demonstrated to produce tumor cell killing in animal models and the expansion of oligoclonal T cell populations in vivo and in vitro when directed to an antigen presenting cell.

Whilst this technology offers the potential for use as a therapy for malignancy or as an oligoclonal vaccine for infectious diseases and some types of cancer, scaling up production of the reagents for clinical studies still holds some challenges.
The same technology can also be used to manipulate T cells in vitro and may offer applications in Immune Monitoring. Using the technology to immobilize recombinant HLA class I or class II/peptide complexes on the surface of HLA –ve cells we can produce HLA mono-specific APCs/targets that bear only a single immunological identity. These cells are highly specific, reproducible, easy to prepare and require only a single cell line to be kept in culture to allow testing of T cell functional responses against all designated HLA/peptide combinations.

Our data indicates that this system is of high specificity and similar efficacy to more complex dendritic cell based systems in producing epitope specific T cell expansion in vitro. When used for T cell functional assays the results with the HLA mono-specific cells in Elispot and intra-cellular cytokine staining are comparable to those obtained with the current standard of peptide pulsed cells.

Assays using HLA mono-specific cells with immobilized HLA-A24/peptide complexes and HLA class II DR15 complexes demonstrate that the system can be used to produce high quality results from both PBMCs and expanded T cell populations for any chosen non-HLA-A2 epitope by simply changing the identity of the HLA complex employed.

This novel system should allow both reproducible T cell expansion and the simple, accurate and economic measurement of T cell functional activity against any chosen HLA/peptide complex.

The field of targeted delivery of cytotoxic agents to tumors via antibody-drug conjugates is currently experiencing a renaissance. If a decade ago only few groups were actively pursuing this approach, presently almost every large pharmaceutical company has entered the field. This increased interest in antibody-drug conjugates can be attributed to the recently reported successes of antibody-auristatin, antibody-calicheamicin and antibody-maytansinoid conjugates in the clinics. This presentation will focus on approaches to design well tolerated antibody-maytansinoid conjugates that are highly potent against “hard-to-kill” cancers, such as solid tumors that express target antigen heterogeneously and/or are resistant to traditional chemotherapy. The strategy outlined in this presentation may be applicable more broadly, to conjugates of other cytotoxic drug.

We are developing therapeutic antibodies that are highly optimized for efficacy and a superior safety profile. Our technology is composed of recombinantly fusing cytotoxic cytokines to the antibodies to arm the antibodies with a cytotoxic payload. This approach allows us to highly improve therapeutic antibodies to selectively target disease causing cells while reducing the systemic toxicity of the cytokines. Our monoclonal antibody- IFN-alpha fusion molecules that are designed to deliver IFN-alpha activity to tumors with broad therapeutic index will be discussed.

Recent advances in cancer immunology and vaccine technology, as well as patient profiling, in the last decade have advanced the field towards translating these insights into addressing significant unmet medical needs for patients.

NeuVax (E75) is one of the most studied and tested cancer peptide vaccines. Originally eluted from human breast and ovarian tumor samples in complex with HLA-A2, E75 is a short peptide (9 amino acids) derived from the extracellular domain of HER2. It is a HER2-derived epitope that has been proven to bind to both HLA-A2 and A3, and predicted to bind other alleles as well such as HLA-A26 and A24. The E75/HLA complex displayed on APCs elicits a robust and highly specific CD8+, cytotoxic T-lymphocyte (CTL) response against HER2-expressing tumor cells.

Based on promising Ph 2 results which demonstrated >75% reduction in recurrence rates, the FDA granted a Special Protocol Assessment (SPA) to Galena for a pivotal Phase 3 trial in low to intermediate HER2 breast cancer patients in the adjuvant setting for the prevention of recurrence.

Measurements of Cell Mediated Immunity (CMI) during Immunotherapy Trials requires Monitoring Strategies for Multiple Biomarkers and Efficient PBMC Processing and Logistics

Cell Mediated Immunity plays a central role in the efficacy of Immunotherapy. An important challenge for pharmaceutical and regulatory entities has been the slow translation of advances in basic science into useful, practical tools that produce standardized, reliable, clinically relevant information. In addition large pre-clinical and clinical trials have to remain economically feasible, while providing the required data.
Objective parameters for efficacy in humans are necessary to provide guidance for development and clinical trial evaluation, without such, informed regulatory approval of more effective Immunotherapeutics and vaccines and will remain an elusive goal.

Much effort has been spent on the assessment of phenotypic complexities in combination with the often not truly functional evaluation of the size of such Lymphocyte populations. However, recent research highlights that efficacy appears to be determined rather by the functional effector characteristics of e.g. antigen-specific T cells and their clonal size frequency.

True functional testing strategies include the requirement to establish logistics for maintaining live viable specimens in sufficient quantity. Early considerations especially for the standardization of specimen processing, cryopreservation, sample management as well as for appropriate assay systems and objective, computerized evaluation equipment to be used are vital steps for the successful design and execution of pre-clinical and clinical trials.

Benefits:
• Highlights how detection of relevant responses and safety data requires a careful choice of test systems and cytokines to be monitored.
• Elaborates how highly sensitive, standardized, GLP compliant ELISPOT assays can aid the understanding and progress of Immunotherapy trials.
• Explains how standardization of specimen processing, cryopreservation and sample management are vital steps for successful immunogenicity testing strategies during multicenter clinical trials.

Panelist: Karim Dabbagh, Executive Director and Head, Pfizer Research Units, External R&D Innovation, Worldwide R&D, Pfizer