Executive Summary
Both live and on-demand, 175+ presentations and discussions explored a diverse range of topics across the spectrum of oligonucleotide & peptide therapeutics.
Biomedical researcher and writer Dr. Catarina Carrao reviews the key themes of the event, addressed through popular keynotes and panel discussions.
The dialogue started with the importance of sequence confirmation for oligonucleotides, which continues to be a debated issue. In the point of view of Dr. Rene Thürmer, Deputy Head at BfArM Federal Inst for Drugs & Med Device, sequencing should always be part of the drug substance specification in identity testing; and, a tip given by him of a good insight on this topic is the recent publication by Capaldi and colleagues on this regard (Nucleic Acid Therapeutics, August 2020).
Dr. Claus Rentel, Executive Director, Analytical Development/QC at Ionis Pharmaceuticals, Inc. asked Dr. Thürmer about the possibility to use Next Generation Sequencing to validate oligonucleotide sequencing, which he agreed to be a possibility; even though, in relation to MS/MS-sequencing, Dr. Rentel was critic of the fact that it’s difficult to fully sequence the oligo with this technique.
When questioned about low-level impurities, Dr. Thürmer remitted to his talk that justifications based on science and ICH Q11 should always be provided; with a comprehensive data set, characterization, suitable and validated analytical methods (which are usually not required for small molecule starting materials), description of synthesis and tight specifications (i.e., justification for each attribute should guarantee consistent quality). Also, he stretched that there needs to be a high number batch of analysis to demonstrate consistency, with an elucidation and criticality assessment of the impurity profile; and genotoxic impurity assessment in line with ICH M7 requirement.
Criticality of starting materials and impurities was a point raised by Dr. Rentel, which was also supported by Dr. Thürmer, emphasizing how significant this aspect of the regulatory file is for the marketing approval.
Dr. Punit Seth, Vice President, Medicinal Chemistry at Ionis Pharmaceuticals, started by answering a question about target delivery in the muscle. His recent research has shown that attaching palmitate, tocopherol or cholesterol to phosphorothioate (PS) antisense oligonucleotides (ASOs) can influence their activity and distribution in muscle tissues, increasing the possibilities of breaching extra-hepatic delivery of oligonucleotides.
The next question to the panel related to tumor vasculature via integrin targeting, to which Dr. Seth replied that they have tried RGD peptide (Arginylglycylaspartic acid) and were disappointed with the results.
Dr. Muthiah Manoharan, Senior Vice President of Drug Discovery at Alnylam, further replied that integrins can be the right target, even though research is incomplete. He added that previous studies together with Prof. Rudy Julyano actually showed a variety of approaches to link oligonucleotides to integrins with high specificity and affinity, including direct chemical conjugation, incorporating oligonucleotides into lipoplexes, and use of various polymeric nanocarriers including dendrimers.
Next, the audience asked what were the receptors responsible for the FALCON (Fatty Acid Ligand Conjugated OligoNucleotide) conjugates, to which Dr. Charles Allerson, Vice President of Chemistry & Drug Development at DTx Pharma, replied that this specific platform offers receptor-mediated uptake through proprietary fatty acids, even though they are still not sure of which specific receptors are responsible for delivery.
As moderator, Dr. Manoharan said the audience was interested in antibody oligo-conjugates (AOCs), to which Dr. Seth responded that there is already proof-of-concept publications for this new emerging therapeutic application; and, in his opinion, the exploitation of protein binding specificity of antibodies to deliver ASOs for the silencing of cell type-specific genes, will continue to grow.
Afterwards, the enthusiastic panel members took questions from each other, with Dr. Allerson asking Dr. Seth about Ionis’ oral delivery of oligo compounds, to which he replied that they used a permeation enhancer to open the tight junctions and cellular transporters, work that has been previously published by Ionis. Dr. Seth was then interested in the lipid transporters studied at DTx, to which Dr. Allerson replied that, even though they haven’t found specific transporters yet, they are able to compete the uptake with fatty acids in an active mechanism.
Dr. Tomi K. Sawyer, Chief Drug Hunter and President at Maestro Therapeutics, gave an exciting keynote presentation about the odyssey of peptide drug discovery, and how it has evolved from highly focused efforts on specific receptors and proteases, to a plethora of targets bridging receptors to enzymes and protein–protein interactions. As he explained, penetrating into cells to modulate intracellular targets is no longer a challenge, opening the “druggable” target space.
Dr. Sawyer explained that over recent years, peptide macrocyclization has seen a rebirth as a key driving force of the third wave of peptide drug discovery. Leveraging both structure-based design and super-diverse library screening of synthetic peptides has accelerated the generation, optimization and development of these types of peptide drugs.
Macrocyclic peptide natural products (e.g., cyclosporine A or CsA), designed macrocyclic peptidomimetic inhibitors of proteases (e.g., hepatitis C virus (HCV) protease inhibitor), designed stapled α-helical peptides (e.g., dual MDM2/X antagonist), CsA-inspired mRNA display library-generated macrocyclic peptides (e.g., SIRT2 inhibitor), CsA-inspired DNA encoded library-generated macrocyclic peptides (e.g., XIAP antagonist) and cell-penetrating peptide (CPP) hybridized synthetic library-generated macrocyclic peptides (e.g., KRAS antagonist) are providing novel chemotypes with unique biophysical and pharmacological properties to tackle the so-called “undruggable” targets.
There is significant progress in the study of stapled α-helical peptides, and the application of varying synthetic chemistries (e.g., ring-closing metathesis, azide–alkyne cycloaddition and thioether). The highly potent and in vivo effective dual MDM2 and MDMX antagonist ATSP-7041 (Aileron Therapeutics) has become a benchmark stapled peptide, due to its intrinsic biophysical properties (e.g., amphipathicity, solubility, cell permeability and metabolic stability); and, has successfully progressed into the clinic.
Using the ATSP-7041 has a model system, with a combination of computational and innovative experimental and predictive screening tools, has allowed the exploration of the intracellular target space with a collection of resources of structurally diverse macrocyclic peptides along with design rules that advance the peptide-drug world. The third wave of peptide discovery is rising and it will be huge.
Moderator Dr. Trishul Shah, Head of Sales, North America at PolyPeptide Group, Director Business Development, started the discussion by asking Dr. Catherine Wu, Professor of Medicine at Dana-Farber Cancer Institute and Harvard Medical School, about the car analogy in her path towards the discovery of personalized peptide vaccines. Dr. Wu responded from an oncologist point of view, that the knowledge about checkpoint blockade is comparable to “taking the foot out of the brake pedal”, and has allowed “opening the door” of the tumour and “speeding up the way” for an efficient patient therapy.
Next, Dr. DeOliveira, Sr. Director, Peptide Development, Tech, Ops. & Manufacturing at Genocea Biosciences, talked about the proprietary ATLAS platform that enables an optimization of neoantigens for inclusion in immunotherapies, by excluding specific inhibitory antigens that suppress the patient immune system, the so-called Inhibigens. In his opinion this will be a “game-changer” in the field of personalized peptide vaccines, by allowing a blockage of the internal growth signals specific of the cancer cells.
When questioned whether Inhibigens could be a neoantigen for a different patient, Dr. DeOliveira replied that their research shows that neoantigens and Inhibigens tend to have the same features across patient profiles.
Asked how the decision is made between which patient takes Genocea’s GEN-009 or GEN-011 personalized vaccines, Dr. DeOliveira replied that even though preliminary, GEN-009 is meant for a patient that still has a viable immune system that drives the anti-tumour response.
On the contrary, since the GEN-011 is an ACT or 'adoptive T cell therapy', it involves the isolation and expansion of tumor- or neoantigen-specific T cells to create the therapeutic; as such, it is meant for a situation where the patient is already immunocompromised and needs a full battalion of engineered T-cells to fight the tumor.
Dr. Annie De Groot, Founder, CEO and CSO at Epivax, Inc., joined the panel discussion, and asked whether the oncologists were concerned with impurities in the starting materials for the personalized vaccines.
Dr. Wu replied that they usually “take a really good look” at the traces of the manufacturers, to make sure of the specificity of the product. They then complete this analysis in the lab, with a sound track of product testing (e.g., Mutant vs Wild Type) to be certain of what they will be injecting into the patient and possible side effects.
Dr. Jesse Dong, Vice President, Peptide Chemistry at BioNTech US, was asked about the induction of neoantigen-specific immunity by NEO-PV-01 with adjuvant anti-PD-1. He replied that they have seen epitope spreading of the immune response to neoantigens that were not included in the vaccine in 15 out of 22 patients, which further shows that the vaccine induced a broadened immune response and had positive effects on survival. Dr. Dong further emphasized that these results, together with the results from Dr.Wu that show that the personalized vaccine alone has epitope spreading effects (with no adjuvant added), are very promising for the future of personalized cancer treatment. In his and Dr. Wu’s opinion, this is a very exciting outlook for the field.
The moderator of this engaging panel session, Dr. Stephen Spagnol, Senior Scientist, Large Molecule Drug Product Development at Johnson & Johnson, began by asking what the panelists’ thoughts are on the major challenges for the delivery of macromolecules as therapeutics.
Dr. Krishanu Saha, Associate Professor at University of Wisconsin-Madison, explained how, in their research, they have tried traditional agents for nonviral polymeric delivery of protein-based CRISPR systems, but ended up using the protein-RNA complex in itself, taking advantage of the cell’s natural mechanisms to deliver the payload to the nucleus. In his opinion, in the genome-editing space, protein-RNA therapeutics are very promising because, for CRISPR systems, there is only the need for a short half-life of activity, since the modifications are stable over time. Longer exposure could conduct to enhanced cell effects which are not desirable.
Dr. Vadim Dudkin, Senior Director & Head, RNA & Targeted Therapeutics, Discovery Sciences at Janssen R&D, added that his research has focused not just on surface binding to the target, but in increasing the capacity for internalization and kinetics, as a way to rise the dosage of the payload to the targeted cell and its native biodistribution properties. In his opinion, understanding of intracellular trafficking and endosomal escape, both short and long-term, is of the utmost importance for an effective siRNA.
Dr. Craig Duvall, Cornelius Vanderbilt Professor of Biomedical Engineering at Vanderbilt University, then talked about how much time of his research has been dedicated to screening methods for the most endosomal-escape effective ligands. They have designed two genetically encoded split-luciferase turn-on reporter assays that can be measured rapidly in well plates on live cells using a luminometer: G8C2 system and G8G8 system.
Next, Dr. Spagnol asked Dr. Jessica Rouge, Assistant Professor of Chemistry at University of Connecticut, about the advantages of enzymatic-responsive materials, a focus of her research work. Dr. Rouge said their research seeks to repurpose enzymes to recognize peptide-modified oligonucleotide substrates for the sequence-specific assembly of RNA/DNA at the surface of micelle-like nanomaterials. The idea is to protect the native function, using mild and biocompatible attachment-cleavage chemistries naturally used by enzymes or exosomes.
Dr. Sriram Sathy, Vice President, Biology and Translational Medicine at Codiak Biosciences, “took the bait” and emphasised how Codiak has developed the engEx Platform, which engineers’ exosomes with distinct properties and direct tropism, so they can reach specific target cells and load them with various types of therapeutic molecules. They have discovered the surface glycoprotein PTGFRN as an optimal protein scaffold, that is amenable to genetic fusion with a broad class of therapeutic proteins.
Dr. Anton McCaffrey, Senior Director of Emerging Science & Innovation at TriLink BioTechnologies Inc., explained that TriLink’s proprietary co-transcriptional capping procedure generates CleanCap in an efficient reaction, producing an extremely low-immunogenic cap with a natural structure that promotes an efficient translation.
Pseudouridine-5'-triphosphate (Pseudo-UTP) is used to impart desirable mRNA characteristics such as increased nuclease stability, increased translation or altered interaction of innate immune receptors with in vitro transcribed RNA. Pseudo-UTP, along with 5-methylcytidine-5'-triphosphate (5-methyl-CTP) have shown innate immune suppression in culture and in vivo, while enhancing translation. Dr. McCaffrey emphasized that TriLink is a leading manufacturer of Pseudo-UTP and chosen by many researchers in the field.
Next, Dr. McCaffrey elucidated that a variety of mRNA purification options are also available at TriLink, including silica gel purification, liquid chromatography isolation (LC-Isolation), and Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC). According to his words, for some applications, LC-Isolation may be enough; although, the extra RP-HLC purification step is able to further reduce interferon-induction in THP-1 cells in many cases, increasing the efficiency of the mRNA.
Dr. McCaffrey then talked about self-amplifying RNAs (saRNA) and how this is an exciting new platform for rapid development of potent vaccines; where TriLink and Imperial College of London are collaborating to produce a saRNA vaccine encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle (LNP). Dr. McCaffrey ended his talk by saying that TriLink takes pride in providing GMP mRNA manufacturing suites for all clinical needs, and a collection of analytical services that allow a thorough characterization of therapeutic mRNA.
Dr. Angelika Fretzen, Technology Translation Director at Harvard’s Wyss Institute for Biologically Inspired Engineering, was the moderator of the panel session, and asked how important are the milestones before researchers can go into manufacturing.
Dr. Weston Daniel, Vice President of Translational Research at Exicure Inc., said the first thing to think about is the basic: know your drug-product; what does the research group/start-up want to achieve with manufacture runs (e.g., supporting toxicology, clinical trials, etc.), and how much material needs to be made in the long-run? The advice is that, even though expensive, hiring a consultant will probably pay off, because many issues in Regulation, Quality Control and Manufacturing are not easy to perceive at first.
Dr. Andrew Latham Director, Business Development & Licensing at Merck was then asked how he looks at the licensing discussions, to which he replied that having an experienced CRO has many advantages when negotiating with a large company like Merck; also, a second source of supply is always important. Dr. Fretzen replied that in her in experience, sometimes a smaller COO and a small biotech company have a better alignment. Dr. Daniel agreed and emphasized that the right sized partner is very important for a successful move.
All panel members agreed that having a strong relationship between partners from the beginning onwards is as an important aspect for a successful endeavour. This relationship can take quite some time to grow, and timing needs to be right for both associates. As such, it’s important to focus on defining the product-drug critical attributes; make sure that “no skeletons are hiding in the closet” of stakeholders that could hunt the pipeline in the future; and, ensure that a trust bond between partners is built upon. Also, an alignment of interests and a global momentum goes a long way, and ensures that diligence talks move speedily and effectively.
