Industrializing AI-powered drug discovery: lessons learned from the Patrimony computing platform.

INTRODUCTION: As a mid-size international pharmaceutical company, we initiated 4 years ago the launch of a dedicated high-throughput computing platform supporting drug discovery. The platform named 'Patrimony' was built up on the initial predicate to capitalize on our proprietary data while leveraging public data sources in order to foster a Computational Precision Medicine approach with the power of artificial intelligence. AREAS COVERED: Specifically, Patrimony is designed to identify novel therapeutic target candidates. With several successful use cases in immuno-inflammatory diseases, and current ongoing extension to applications to oncology and neurology, we document how this industrial computational platform has had a transformational impact on our R&D, making it more competitive, as well time and cost effective through a model-based educated selection of therapeutic targets and drug candidates. EXPERT OPINION: We report our achievements, but also our challenges in implementing data access and governance processes, building up hardware and user interfaces, and acculturing scientists to use predictive models to inform decisions.

Proteomics in support of immunotherapy: contribution to model-based precision medicine.

INTRODUCTION: Proteomics encompasses a wide and expanding range of methods to identify, characterize, and quantify thousands of proteins from a variety of biological samples, including blood samples, tumors, and tissues. Such methods are supportive of various forms of immunotherapy applied to chronic conditions such as allergies, autoimmune diseases, cancers, and infectious diseases. AREAS COVERED: In support of immunotherapy, proteomics based on mass spectrometry has multiple specific applications related to (i) disease modeling and patient stratification, (ii) antigen/ autoantigen/neoantigen/ allergen identification, (iii) characterization of proteins and monoclonal antibodies used for immunotherapeutic or diagnostic purposes, (iv) identification of biomarkers and companion diagnostics and (v) monitoring by immunoproteomics of immune responses elicited in the course of the disease or following immunotherapy. EXPERT OPINION: Proteomics contributes as an enabling technology to an evolution of immunotherapy toward a precision medicine approach aiming to better tailor treatments to patients' specificities in multiple disease areas. This trend is favored by a better understanding through multi-omics profiling of both the patient's characteristics, his/her immune status as well as of the features of the immunotherapeutic drug.

A dynamic COVID-19 immune signature includes associations with poor prognosis.

Improved understanding and management of COVID-19, a potentially life-threatening disease, could greatly reduce the threat posed by its etiologic agent, SARS-CoV-2. Toward this end, we have identified a core peripheral blood immune signature across 63 hospital-treated patients with COVID-19 who were otherwise highly heterogeneous. The signature includes discrete changes in B and myelomonocytic cell composition, profoundly altered T cell phenotypes, selective cytokine/chemokine upregulation and SARS-CoV-2-specific antibodies. Some signature traits identify links with other settings of immunoprotection and immunopathology; others, including basophil and plasmacytoid dendritic cell depletion, correlate strongly with disease severity; while a third set of traits, including a triad of IP-10, interleukin-10 and interleukin-6, anticipate subsequent clinical progression. Hence, contingent upon independent validation in other COVID-19 cohorts, individual traits within this signature may collectively and individually guide treatment options; offer insights into COVID-19 pathogenesis; and aid early, risk-based patient stratification that is particularly beneficial in phasic diseases such as COVID-19.

Structural and Functional Characterization of the Major Allergen Amb a 11 from Short Ragweed Pollen.

Allergy to the short ragweed (Ambrosia artemisiifolia) pollen is a major health problem. The ragweed allergen repertoire has been recently expanded with the identification of Amb a 11, a new major allergen belonging to the cysteine protease family. To better characterize Amb a 11, a recombinant proform of the molecule with a preserved active site was produced in Escherichia coli, refolded, and processed in vitro into a mature enzyme. The enzymatic activity is revealed by maturation following an autocatalytic processing resulting in the cleavage of both N- and C-terminal propeptides. The 2.05-Å resolution crystal structure of pro-Amb a 11 shows an overall typical C1A cysteine protease fold with a network of molecular interactions between the N-terminal propeptide and the catalytic triad of the enzyme. The allergenicity of Amb a 11 was confirmed in a murine sensitization model, resulting in airway inflammation, production of serum IgEs, and induction of Th2 immune responses. Of note, inflammatory responses were higher with the mature form, demonstrating that the cysteine protease activity critically contributes to the allergenicity of the molecule. Collectively, our results clearly demonstrate that Amb a 11 is a bona fide cysteine protease exhibiting a strong allergenicity. As such, it should be considered as an important molecule for diagnosis and immunotherapy of ragweed pollen allergy.

Identification of the cysteine protease Amb a 11 as a novel major allergen from short ragweed.

BACKGROUND: Allergy to pollen from short ragweed (Ambrosia artemisiifolia) is a serious and expanding health problem in the United States and in Europe. OBJECTIVE: We sought to investigate the presence of undescribed allergens in ragweed pollen. METHODS: Ragweed pollen proteins were submitted to high-resolution gel electrophoresis and tested for IgE reactivity by using sera from 92 American or European donors with ragweed allergy. Pollen transcriptome sequencing, mass spectrometry (MS), and recombinant DNA technologies were applied to characterize new IgE-binding proteins. RESULTS: High-resolution IgE immunoblotting experiments revealed that 50 (54%) of 92 patients with ragweed allergy were sensitized to a 37-kDa allergen distinct from Amb a 1. The full-length cDNA sequence for this molecule was obtained by means of PCR cloning after MS sequencing of the protein combined with ragweed pollen RNA sequencing. The purified allergen, termed Amb a 11, was fully characterized by MS and confirmed to react with IgEs from 66% of patients. This molecule is a 262-amino-acid thiol protease of the papain family expressed as a combination of isoforms and glycoforms after proteolytic removal of N- and C-terminal propeptides from a proform. Three-dimensional modeling revealed a high structural homology with known cysteine proteases, including the mite Der p 1 allergen. The protease activity of Amb a 11, as well as its capacity to activate basophils from patients with ragweed allergy, were confirmed. The production of a nonglycosylated recombinant form of Amb a 11 in Escherichia coli established that glycosylation is not required for IgE binding. CONCLUSION: We identified the cysteine protease Amb a 11 as a new major allergen from ragweed pollen. Given the similar physicochemical properties shared by the 2 major allergens, we hypothesize that part of the allergenic activity previously ascribed to Amb a 1 is rather borne by Amb a 11.

Expression and characterization of natural-like recombinant Der p 2 for sublingual immunotherapy.

BACKGROUND: Recombinant allergens with a native conformation represent an alternative to natural extracts for immunotherapy and diagnostic purposes. METHODS: We produced the Der p 2 mite allergen in Pichia pastoris and Escherichia coli. After purification by cation exchange chromatography, recombinant molecules were compared to their natural counterpart based upon structural (disulfide bonds, secondary structure, thermal stability) and immunological properties (antibody reactivity, basophil and T cell activation, tolerance induction in a murine sublingual immunotherapy model). RESULTS: The Der p 2.0101 isoform was confirmed to be prevalent in Dermatophagoides pteronyssinus extracts. It was then produced as a secreted molecule in P. pastoris or refolded from E. coli inclusion bodies. The yeast-expressed rDer p 2 molecule exhibits a natural-like disulfide bridge distribution and secondary structure, whereas the E. coli-derived rDer p 2 presents some heterogeneity in cysteine bonds and a lower stability following thermal stress. The two recombinant as well as natural Der p 2 molecules exhibit comparable IgE recognition and activate basophil and CD4+ T cells. Sublingual immunotherapy of nDer p 2- sensitized mice using either one of the rDer p 2 molecules efficiently decreases airway hyperresponsiveness as well as Th2 responses. CONCLUSIONS: Natural and recombinant Der p 2 molecules produced in P. pastoris and E. coli exhibit comparable immunological properties despite distinct structural features. Natural-like cysteine pairing is a critical parameter to identify stable, well-folded and homogenous proteins appropriate for immunotherapy and diagnostic purposes.

Identification of a new phenotype of tolerogenic human dendritic cells induced by fungal proteases from Aspergillus oryzae.

We characterized a new pathway to induce tolerogenic dendritic cells (DCs) following treatment of human monocyte-derived DCs with proteases from the fungus Aspergillus oryzae (ASP). ASP-treated DCs (ASP-DCs) exhibit a CD80(-)CD83(-)CD86(-)Ig-like transcript (ILT)2(-)ILT3(-)ILT4(+) phenotype, do not secrete cytokines or chemokines, and express tolerogenic markers such as glucocorticoid-induced leucine zipper, NO synthetase-2, retinaldehyde dehydrogenase-1 or retinaldehyde dehydrogenase-2. When cocultured with naive CD4(+) T cells, ASP-DCs induce an anergic state that can be reversed by IL-2. Generated T cells mediate a suppressive activity in third-party experiments that is not mediated by soluble factors. A comparison between dexamethasone-treated DCs used as a reference for regulatory T cell-inducing DCs and ASP-DCs reveals two distinct phenotypes. In contrast to dexamethasone, ASP treatment induces glucocorticoid-induced leucine zipper independently of glucocorticoid receptor engagement and leads to NF-κB p65 degradation. Abrogation of protease activities in ASP using specific inhibitors reveals that aspartic acid-containing proteases are key inducers of regulatory genes, whereas serine, cysteine, and metalloproteases contribute to NF-κB p65 degradation. Collectively, those features correspond to a previously unreported anergizing phenotype for human DCs. Such regulatory mechanisms may allow fungi to downregulate host immune responses and provide clues for new approaches to treat proinflammatory disorders.

Recombinant fusion proteins assembling Der p 1 and Der p 2 allergens from Dermatophagoides pteronyssinus.

BACKGROUND: Fusion proteins assembling multiple allergens can be engineered by recombinant DNA technologies in order to produce tools for diagnostic and immunotherapeutic purposes. Herein, we developed and characterized chimeras assembling Der p 1 and Der p 2 allergens as potential candidate vaccines against house dust mite allergy. METHODS: Fusion proteins encompassing Der p 2 with either mature or proDer p 1 were expressed in Escherichia coli or Pichia pastoris. Forms with mutation in Der p 1 catalytic site were also engineered. Purified chimeras were characterized by immunoblotting, circular dichroism, disulfide bond mapping, basophil and T lymphocyte stimulation assays. RESULTS: Four fusion proteins were expressed in E. coli as inclusion bodies, whereas only chimeras comprising proDer p 1 were obtained in yeast. All such hybrids formed polymers and aggregates, and yeast-expressed chimeras were unstable. Circular dichroism analysis performed after refolding of bacteria expressed chimeras encompassing mature Der p 1 confirmed partial folding, consistent with the occurrence of both correct and inappropriate intramolecular disulfide bonds. All fusion molecules were recognized by Der p 1- and Der p 2-specific human IgEs, monoclonal and polyclonal antibodies. Fusion proteins activate basophils from mite-allergic patients and trigger the proliferation of specific CD4+ T cells, albeit to a lower level when compared to individual allergens. CONCLUSIONS: Production of multiple Der p 1-Der p 2 fusion proteins exhibiting partial folding and proper antigenic properties has been achieved. Nonetheless, significant solubility and stability issues currently limit the application of such chimeras for immunotherapy or diagnostic.

Single cell assessment of allergen-specific T cell responses with MHC class II peptide tetramers: methodological aspects.

BACKGROUND: We report herein critical methodological principles for assessing, at a single cell level, allergen-specific T cell responses using MHC class II peptide tetramers. METHODS: We developed MHC class II peptide tetramers to monitor T cell responses against the immunodominant Bet v 1(141-155) peptide in individuals with either an HLA-DRB1*0101, DRB1*0401 or DRB1*1501 background. In vitro stimulation was performed with serially truncated versions of the Bet v 1(141-155) epitope chemically conjugated to the Ii-Key peptide. RESULTS: Identification of Bet v 1(141-155) as a high-affinity epitope for multiple HLA-DRB1 allotypes led to the development of corresponding tetramers detecting Bet v 1(141-155)-specific T cells with a high specificity and sensitivity. Stimulation with Bet v 1(141-155) Ii-Key conjugate peptides is the most efficient procedure to expand Bet v 1(141-155)-specific CD4+ T cells, allowing to detect such cells in both allergic and healthy individuals. MHC class II Bet v 1(141-155) tetramer-positive T cells produce IFN-gamma and IL-10 in healthy individuals, and IL-5 in allergic patients. Frequencies of Bet v 1-specific CD4+ T cells circulating in the blood of allergic or nonallergic individuals range from approximately 10(-5) to 10(-3) CD4+ T cells, outside or within the pollen season, respectively. CONCLUSIONS: MHC class II peptide tetramers are valuable tools to assess allergen-specific T cell responses, both qualitatively and quantitatively. Selection of a high-affinity T cell epitope, as well as optimization of in vitro stimulation conditions to expand rare T cell progenitors are critical success factors in those analyses.

Suspension-cultured BY-2 tobacco cells produce and mature immunologically active house dust mite allergens.

The replacement of crude allergen extracts by selected allergens currently represents a major goal for the improvement of allergy diagnosis and immunotherapy. Indeed, the development of molecularly defined vaccines would facilitate both standardization and enhance batch-to-batch reproducibility as well as treatment specificity. In this study, we have investigated the potential of tobacco plant cells to produce biologically active forms of the two major allergens from the house dust mite. A detailed characterization of these plant-made allergens has shown similar proteolytic maturation and folding as well as comparable immunoreactivity to their natural counterparts. Altogether, our results exemplify that suspension-cultured BY-2 tobacco cells represent a low cost and environmentally safe expression system suitable to produce recombinant allergens from Dermatophagoides pteronyssinus under a form appropriate for diagnostic and therapeutic purposes.

Production and proteomic characterization of pharmaceutical-grade Dermatophagoides pteronyssinus and Dermatophagoides farinae extracts for allergy vaccines.

BACKGROUND: House dust mites (HDM) such as Dermatophagoides pteronyssinus and Dermatophagoides farinae represent a major cause of type 1 allergies worldwide. Hence large quantities of well-characterized HDM extracts are needed to prepare pharmaceutical-grade allergy vaccines. To this aim, the present study was undertaken to define optimal conditions for large-scale cultures. METHODS: D. pteronyssinus and D. farinae were grown on different media combining various proportions of wheat germ, yeast and synthetic amino acids (the latter resembling the composition of the human stratum corneum). Extracts thus obtained were analyzed for their total allergenic activity, as well as major allergen and protein contents, using immunosorbent assays, HPLC, immunoblotting, two-dimensional electrophoresis and peptide mass fingerprinting. RESULTS: An optimal culture medium (Stalmite APF) based on wheat germ, yeast and amino acids in defined proportion (42, 42 and 15% w/w, respectively) was selected to grow various HDM species with high yields. A detailed proteomic analysis revealed that D. pteronyssinus extracts generated under such conditions did not contain allergens originating from culture medium components and that major prevalent HDM allergens (i.e. groups 1, 2, 7, 10, 13 and 20) are found among the most abundant proteins in the D. pteronyssinus extract. Semiquantitative dot-blot assays confirmed the presence of Der p 3-10 as well as Der p 13 and 14 allergens within the extracts. CONCLUSIONS: We developed a well-defined medium allowing to grow various HDM species at an industrial scale in a highly reproducible manner. Extracts from mites produced under such pharmaceutical conditions contain all the relevant allergens for desensitization purposes and in vivo diagnosis.

Tumoral and immunologic response after vaccination of melanoma patients with an ALVAC virus encoding MAGE antigens recognized by T cells.

PURPOSE: To evaluate the toxicity, antitumoral effectiveness, and immunogenicity of repeated vaccinations with ALVAC miniMAGE-1/3, a recombinant canarypox virus containing a minigene encoding antigenic peptides MAGE-3(168-176) and MAGE-1(161-169), which are presented by HLA-A1 and B35 on tumor cells and can be recognized by cytolytic T lymphocytes (CTLs). MATERIALS AND METHODS: The vaccination schedule comprised four sequential injections of the recombinant virus, followed by three booster vaccinations with the MAGE-3(168-176) and MAGE-1(161-169) peptides. The vaccines were administered, both intradermally and subcutaneously, at 3-week intervals. RESULTS: Forty patients with advanced cancer were treated, including 37 melanoma patients. The vaccines were generally well tolerated with moderate adverse events, consisting mainly of transient inflammatory reactions at the virus injection sites. Among the 30 melanoma patients assessable for tumor response, a partial response was observed in one patient, and disease stabilization in two others. The remaining patients had progressive disease. Among the patients with stable or progressive disease, five showed evidence of tumor regression. A CTL response against the MAGE-3 vaccine antigen was detected in three of four patients with tumor regression, and in only one of 11 patients without regression. CONCLUSION: Repeated vaccination with ALVAC miniMAGE-1/3 is associated with tumor regression and with a detectable CTL response in a minority of melanoma patients. There is a significant correlation between tumor regression and CTL response. The contribution of vaccine-induced CTL in the tumor regression process is discussed in view of the immunologic events that could be analyzed in detail in one patient.

Enhanced multiepitope-based vaccines elicit CD8+ cytotoxic T cells against both immunodominant and cryptic epitopes.

A frequent issue in vaccinology is to elicit balanced T cell responses against both immunodominant and cryptic T cell epitopes, from one or several antigens presented at the same time to the immune system. Using HLA-A2.1.1 restricted epitopes from the Melan A/MART-1 or gp 100 melanoma-associated antigens as a model, we engineered a series of constructs in the ALVAC canarypox vector system: T cell epitopes were expressed either as linear polyepitopes (with or without spacers), or as minigenes encoding a single epitope. The latter were found to allow the best processing and presentation of most T cell epitopes, following infection by ALVAC recombinants of the HLA A2+ bladder carcinoma cell line and stimulation of epitope-specific human TIL lines. These various constructs were also used to immunize HLA-A2.1.1 HHD transgenic mice to compare their capacity to elicit T cells responses. Polyepitopes but also minigenes encoding wild-type epitopes could not elicit in a reliable manner balanced CTL responses against all target epitopes from gp100. We could rescue T cells responses against poorly immunogenic epitopes after introducing appropriate point mutations to enhance their interaction with MHC Class I molecules. Epitope enhancement within either polyepitope, multiepitopes (i.e. minigenes expressed under the control of separate promoters) or full length immunogens should be systematically considered when designing vaccines containing both cryptic and immunodominant target epitopes.

Immunization of colorectal carcinoma patients with a recombinant canarypox virus expressing the tumor antigen Ep-CAM/KSA (ALVAC-KSA) and granulocyte macrophage colony- stimulating factor induced a tumor-specific cellular immune response.

PURPOSE: Colorectal carcinoma cells express the tumor-associated antigen epithelial cellular adhesion molecule (Ep-CAM)/KSA. Passive immunotherapy with monoclonal antibodies using this antigen has shown promising results. Ep-CAM might also be a target for active specific immunotherapy. Expression of the tumor antigen in a viral vector may facilitate appropriate antigen presentation. The feasibility of an Ep-CAM/KSA-specific therapeutic vaccination was investigated in cancer patients. EXPERIMENTAL DESIGN: The full-length Ep-CAM gene was inserted into the avipox virus ALVAC (ALVAC-KSA). Twelve radically operated colorectal carcinoma patients without evidence of remaining macroscopic disease (stages I, II, and III) entered the study. The first 6 patients were immunized with three injections of ALVAC-KSA (10(7.09) CCID(50) per immunization) alone in weeks 0, 3, and 6. The subsequent 6 patients received the same schedule of ALVAC-KSA together with the adjuvant cytokine granulocyte macrophage colony-stimulating factor (GM-CSF; 75 micro g/day for 4 consecutive days). RESULTS: The adverse reactions to the vaccinations were mild except for local skin reactions. In the ALVAC-KSA group a weak T-cell response was induced in 2 of 6 patients. In the ALVAC-KSA/GM-CSF group a marked IFN-gamma response (enzyme-linked immunospot) was induced in 5 of 6 patients. The T-cell response appeared late, 1 month after the last immunization, with a peak at 4-5 months after immunization. No IgG antibodies against Ep-CAM were detected. Before vaccination the majority of patients had a type 1 T-cell response (IFN-gamma) against the vector, which was noted in healthy donors as well. All of the patients developed high titers of IgG antibodies against the vector, and the T-cell response was vigorously boosted. CONCLUSIONS: ALVAC-KSA, in combination with low dose local administration of GM-CSF may induce a strong, IFN-gamma T-cell response (type 1). ALVAC-KSA seems to be an interesting candidate as a cancer vaccine for future clinical development.

Safety of intravenous administration of a canarypox virus encoding the human wild-type p53 gene in colorectal cancer patients.

Overexpression of p53 occurs in more than 50% of colorectal cancers. Therefore, p53 represents an attractive target antigen for immunotherapy. We assessed the safety of a canarypox virus encoding the human wild-type p53 gene given intravenously to end-stage colorectal cancer patients in a three-step dose escalation study aimed at inducing p53 immune responses. Patients with metastatic disease of p53-overexpressing colorectal cancers were vaccinated three times at 3-week intervals, each time with 10(6.5) CCID(50) (CCID(50)=cell culture infectious dose 50%; group 1, n=5), 10(7.0) CCID(50) (group 2, n=5) or 10(7.5) CCID(50) (group 3, n=6). Vital signs and the occurrence of adverse events were monitored and blood was analyzed for biochemical and hematological parameters as well as signs of auto-immune safety. In all, 16 patients were enrolled and 15 patients completed three vaccinations. No anaphylactic reaction or unwanted auto-immune reactions were observed. A total of 16 serious adverse events (SAEs) occurred: 10 in group 1, three in group 2 and three in group 3. All SAEs were tumor-related complications. There was no difference in the frequency of adverse events between the three groups, except for fever. Fever was the only vaccination-related adverse event consistently observed and was most frequent and outspoken in the group 3 patients. The majority was a grade 1 or 2 fever (93%) and grade 3 fever (7%) was observed in three patients of group 3. Some patients showed humoral and cellular responses against p53, following vaccinations. After having completed his initial treatment cycle, one patient (group 2) received a second treatment cycle of three doses of 10(7.5) CCID(50) and subsequently showed stable disease. All other patients showed progressive disease. We conclude that ALVAC-p53 can be administered intravenously to colorectal cancer patients without serious toxicity or pathological autoimmunity and can induce immune responses against p53.

Induction of p53-specific immune responses in colorectal cancer patients receiving a recombinant ALVAC-p53 candidate vaccine.

PURPOSE: The tumor-associated auto-antigen p53 is commonly overexpressed in various types of human cancer, including colorectal cancer. Experiments in preclinical models have shown that it can serve as a target for T-cell-mediated tumor-eradication. The feasibility of a p53-specific therapeutic vaccination was investigated in cancer patients. EXPERIMENTAL DESIGN: A Phase I/II dose-escalation study was performed that evaluated the effect of a recombinant canarypoxvirus (ALVAC) vaccine encoding wild-type human p53 in 15 patients with advanced colorectal cancer. Each group of five patients received three i.v. doses of one-tenth of a dose, one-third of a dose, or 1 dose of the vaccine [1 dose = 1 x 10(7.5) cell culture infectious dosis (CCID)50]. RESULTS: Potent T-cell and IgG antibody responses against the vector component of the ALVAC vaccine were induced in the majority of the patients. Enzyme-linked immunosorbent-spot assay (ELISPOT) analysis of vaccine-induced immunity revealed the presence of IFN-gamma-secreting T cells against both ALVAC and p53, whereas no significant interleukin-4 responses were detected. Vaccine-mediated enhancement of p53-specific T-cell immunity was found in two patients in the highest-vaccine-dose group. CONCLUSIONS: This study demonstrated the feasibility, even in patients with advanced cancer, to elicit immune responses against the ubiquitously expressed tumor-associated auto-antigen p53. Our results form the basis for additional studies that will explore the antitumor capacity of p53 containing multivalent vaccines in cancer patients with limited tumor burden.