Personalized Peptide Vaccines & Therapies - TIDES eBook Series
This ebook explores the milestones and challenges encountered by today’s leading researchers in producing personalized vaccines and therapies.
Personalized Peptide Vaccines & Therapies
Personalized Peptide Vaccines & Therapies
Introduction
Arguably the most exciting emerging treatment in immunotherapy, personalized medicine has provided increasing hope and understanding in targeting different cancers by incorporating genetic and data-driven approaches.
In this ebook, we look at the milestones and challenges encountered by today’s leading researchers in the journey of producing personalized vaccines and therapies, including antigen selection and synthesis, machine-learning and regulatory considerations.
The next pages include whitepapers and presentation overviews from the latest TIDES events, with insights from Gyros Protein Technologies, Dana-Farber Cancer Institute, BioNTech and much more.
Jump to any article using the contents on the following page, or at any time using the Contents menu in the top left. There you can also download this eBook as a PDF.
2. Building Better Personal Cancer Vaccines
We explore how effective anti-tumor immunity in humans has been associated with the presence of T-cells directed at cancer neoantigens, as presented by Dr. Catherine J. Wu, Professor of Medicine at Dana-Farber Cancer Institute.
The Future of Peptide Vaccines and Personalized Peptide Therapies
By David Orchard-Webb
The Future of Peptide Vaccines and Personalized Peptide Therapies
Synthetic peptide vaccines are a nascent but highly promising therapeutic class.
The field unites cancer biology and infectious disease treatment principles. There is, however, a key difference, a cancer vaccine is not prophylactic. Even though infectious disease prophylactic vaccines, such as against HPV, can prevent cancer, personalized cancer vaccines are an active treatment of an existing tumor.
This whitepaper focuses on peptide cancer vaccines, although infectious disease vaccines are also touched upon. Several considerations are important such as the peptide selection, and strategies to overcome loss of MHC. Personalization opens up regulatory challenges and these are also discussed.
SYNTHETIC PEPTIDE VACCINES
Synthetic vaccines are short or long synthetic peptides derived from viruses and bacteria, fungi, cancer, as well as innovative T cell and innate immune stimulating immunogens and fusion proteins. They fall into two broad categories, cancer vaccines which can be personalized and peptide vaccines for infectious diseases.
These vaccines could be comprised of peptides as the main immunomodulatory agent or the peptides could be an adjuvant to a standard vaccine.
Peptide vaccines can be synthesized as peptides, or encoded in a vector, usually of viral origin. The argument for vectorized peptides is enhanced immunogenicity, however adjuvant and excipient technologies are capable of increasing the immunogenicity of stand-alone peptides, with more specificity than vectorized peptides, in some formulations.
PERSONALIZED CANCER VACCINES
There are a number of vaccination strategies available against cancers that utilize peptides, namely, cancer-overexpressed peptides, cancer neoantigens (nAgs), tumour tagging with exogenous pathogen-associated molecular patterns (PAMPs) peptides via viral transduction or DNA transfection, and adjuvants and excipients that activate the innate immune system.
“Tumor tagging” is a promising strategy when the tumor has employed immune evasion via down-regulation of the MHC receptors, which would normally nullify adaptive immune stimulating vaccine strategies.
More recently the discovery of an evolutionarily ancient “fitness fingerprint”, consisting of combinations of Flower membrane proteins, presents an opportunity to recognize tumors irrespective of MHC. Proteins that indicate reduced fitness are called Flower-Lose, because they are expressed in cells marked to be eliminated. [Madan] Human Flower isoforms hFWE1 and hFWE3 behave as Flower-Lose proteins, whereas hFWE2 and hFWE4 behave as Flower-Win proteins. Cancer cells have increased Win isoform expression and proliferate in the presence of Lose-expressing stroma, which confers a competitive growth advantage on the cancer cells. Tagging tumor cells with Flower-Lose isoforms via viral transduction is one possible strategy.
The best tumor associated antigens are not normally expressed in somatic cells, such as testis antigen NY-ESO-1. NY-ESO-1 antigen is commonly expressed in 10-50% of melanoma, lung, liver, esophageal, breast, prostate, bladder, thyroid and ovarian cancer cases, 60% of multiple myeloma cases, and 70-80% of synovial cell sarcoma. There are currently over 50 active clinical trials targeting NY-ESO-1. However, even this excellent overexpressed protein vaccination target may carry a fertility risk in males.
TUMOR NEOANTIGEN VACCINES
Unique to tumor cells, nAgs are expressed as a result of mutations in coding genes, generating a non-self peptide, without pre-existing immune tolerance. Unlike overexpressed tumor antigens that may also be expressed at lower levels in normal cells, targeting nAgs carries a relatively low risk of generating autoimmunity.
The potential for combination of nAg vaccines with immune checkpoint inhibitors is highlighted by the correlation between tumor mutational burden (number of nAgs) and improved response to the treatment. Interestingly in patients with pancreatic cancer tumors rich in neoantigens derived from MUC16 and peptides that mimicked viral peptides there was an association with long term survival. [Balachandran]
Individual nAgs can be concatenated, which can sometimes increase immunogenicity and increase the breadth of immune response. A scoring method has been developed whereby candidate nAgs are selected from personal next generation sequencing of the patient’s tumor based on: (i) MHC-I and II predicted binding affinity, (ii) mutation allele frequency in the tumor DNA, and (iii) mRNA expression. [D’Alise] This system can be used to select the top 30 neoantigens for incorporation into a vaccine in an automated fashion.
Successful personalized nAg vaccination strategies are dependent upon accurate prediction of high affinity binding to MHC. This can be accomplished with machine learning and deep neural networks. An interesting approach called MHCSeqNet deployed neural network architectures developed for natural language processing to model the amino acid sequences of MHC alleles and nAg peptides as sentences with amino acids as individual words. This innovation allows MHCSeqNet to accept new MHC alleles as well as peptides of any length. More generally bioinformatic services such as ElliPro can predict epitope sites from a given structure, allowing rationale design of peptides for use in vaccines against infectious disease.
COMPANIES DEVELOPING CANCER PEPTIDE VACCINES
Several companies are developing personalized cancer neoantigen vaccines including, but not limited to, Neon Therapeutics, an immuno-oncology company developing personalized neoantigen vaccines using massively parallel sequencing for detection of all coding mutations within tumours, and machine learning approaches. [Ott]
Neon has an agreement with Elicio Therapeutics. Elicio Therapeutics (formerly Vedantra Pharmaceuticals, Inc.) is developing an amphiphile platform that enables precise targeting and delivery of peptide antigens directly to the lymphatic system by interacting with albumin. [Moynihan]
Gritstone Oncology is developing personized cancer vaccines in a similar vein to Neon Therapeutics; however, they are delivering their neoantigens in a prime boost regimen derived directly from infectious disease vaccine principles. They have created the EDGE machine learning model of antigen presentation for neoantigen prediction, which they believe could enable an improved ability to develop neoantigen-targeted immunotherapies for cancer patients. [Bulik-Sullivan]
Agenus Inc. is developing personalized cancer peptide vaccines and targeting the peptides to the immune system by conjugating them with heat shock proteins. Some methods include autologous peptide extraction from patient biopsies. [Bloch]
Flow Pharma Inc.’s FlowVax platform is optimized to simultaneously deliver multiple nAg peptide targets. Each of the chemical components used in the FlowVax platform are currently part of an FDA approved vaccine or pharmaceutical, simplifying the regulatory pathway. Immuneoprofiler™ software harneses the power of artificial intelligence (AI) to identify clinically relevant and immunogenic neoantigens from next generation sequencing data. [Clancy]
A handful of companies are developing mutant RAS vaccines. The NantWorks family of companies headed by Patrick Soon-Shiong are making use of peptide vaccines including mutant RAS neoantigens, which were developed and manufactured by GlobeImmune, in combination clinical trials. [Cohn] Targovax holds IP for mutant RAS vaccines, and positive clinical data, but has chosen not to pursue development further at this time. [Birkeli]
Several companies are developing cell penetrating or lytic peptides that activate the immune system. AMAL Therapeutics SA, which was recently acquired by Boehringer Ingelheim, is developing a self-adjuvanting (TRL activating) peptide platform, with a multiantigenic domain with room for four antigens, and a cell penetrating peptide domain for efficient antigen delivery to immune cells such as dendritic cells. [Belnoue]
GV1001 is a 16-amino acid fragment of the human telomerase reverse transcriptase catalytic subunit (hTERT) developed as a cancer vaccine by KAEL-GemVax and also found to be a cell penetrating peptide through interaction with heat shock proteins. [Kim] Lytix Biopharma develops proprietary oncolytic peptides. Lytix's lead candidate, LTX-315, is developed for intratumoral treatment of solid tumors. [Sveinbjørnsson]
Peptide vaccines can make use of autologous cells. ERYTECH Pharma has developed the ERYMMUNE platform that uses artificially aged red blood cells and targets the majority of them to the spleen, where they undergo erythrophagocytosis and present their encapsulated peptides to Antigen Presenting Cells (APC), thus activating CD4/CD8 T-cells. [Banz]
The platform is being further developed by SQZ Biotech. Marker Therapeutics is developing a MultiTAA peptide therapy that is designed to educate T cells grown from stem cells. [Marker] LEAPS (Ligand Epitope Antigen Presentation System) is a CEL-SCI patented platform technology. J‐LEAPS vaccines promote the maturation of precursor cells into a unique type of dendritic cell that produce interleukin 12, but not IL‐1 or tumour necrosis factor, and presents the antigenic peptide to T cells.
IMPROVING FORMULATION CONSISTENCY OF INDIVIDUALIZED, PEPTIDE-BASED THERAPIES
The formulation of a vaccine can have a major impact on its immunogenicity. One of the major considerations in peptide vaccination strategies is how to get the peptides to the lymph nodes, where they can be presented to the immune system.
In cancer diagnosis, dye labelled albumin is crucial for identifying sentinel lymph nodes. The most abundant protein in the blood, albumin, naturally accumulates at tumours and sites of inflammation, and preferentially accumulates in the lymph nodes. This property can be used to direct peptide vaccines to the lymph nodes. This could be accomplished via fusion proteins or taking advantage albumin’s lipid binding properties.
Adjuvant technology such as lipid-based adjuvants, oil-in-water adjuvants, and plant-derived adjuvants may help target peptide vaccines to the lymph nodes. Lipid moieties may confer binding to albumin as suggested by the multiple lipid binding sites present in human albumin. [Kawai] Novel lipopeptides, LP1 and LP2, which mimic the terminal structure of the native Helicobacter pylori HpaA activate TLR2 and protect against bacterial colonization in mice.
As albumin’s apparent preference for the lymph nodes may be based on little more than its molecular weight, suitably sized non-albumin nanoparticles can also deliver peptides to the lymph nodes for antigen presentation. Virus-Like Particles (VLPs) are biological nanoparticles having a size of around 20-100 nm. Viral capsid proteins have tendency to self-assemble independently of genetic material and can incorporate suitably designed peptides. These VLPs are fully non-replicative.
REGULATORY VIEW OF PERSONALIZED MEDICINE CONCEPTS FOR THERAPEUTIC PEPTIDES
From a regulatory perspective the need to ensure the quality safety and efficacy of peptide vaccines remains paramount before marketing authorisation is granted. This regulatory consideration is designed to ensure that the risk:benefit ratio is favorable to the patients receiving the treatments.
Pharmacovigilance and continued clinical safety data collection post-authorisation, which also may include registry data or additional real-world-evidence, is important to ensure that the risk:benefit balance does not become less favorable once licensed, for example with the appearance of autoimmune disease. Pharmacovigilance allows the identification of adverse drug reactions which were not evident in the initial clinical data. This consideration could prove extremely important for cancer vaccines based on overexpressed proteins that are present in some normal tissues.
The regulatory requirements for peptide vaccines as a class of product do not differ from other forms of biologics, however developers of nAg vaccines may require close discussions with the FDA in order to ensure that the personalized nature of nAg peptides can be appropriately processed by the FDA regulatory structure.
The regulatory agencies for Europe, the EMA, and the United States, the FDA, regularly release guidelines for a range of quality, non-clinical and clinical topics. The FDA Guidance for Industry on clinical considerations for therapeutic cancer vaccines provides a detailed discussion including the development of companion diagnostics for patient selection in clinical trials.
This is an evolving space and newer personalized vaccination strategies that use patient specific nAgs may not require companion diagnositics per se, as the tumors are routinely next generation sequenced as part of the vaccine development protocol.
However, and similar to EU guidelines, patient population selection and appropriate selection of a feasible endpoint remain important considertations. Surrogates such as monitoring of the immune response is considered exploratory by the FDA and the utility of such measurements are seen as useful in proof of concept, dose finding and possible correlation with clinical efficacy.
The development of exploratory biomarkers for proof of concept is supported by the FDA. Some guidance on adjuvants and multi-antigen therapy is also available. Given that peptide vaccines are still a nascent field, there are a wide range of approaches being undertaken, which are impossible to fully account for in any guideline document. It is therefore recommended that the main clinical endpoints are clinically relevant and discussed with the FDA.
Breakthrough Therapy designation is a process designed to expedite the development and review of drugs that are intended to treat a serious condition and preliminary clinical evidence indicates that the drug may demonstrate substantial improvement over available therapy on a clinically significant endpoint.
Developers of novel peptide vaccines may seek this designation in order to have a closer working relationship with the FDA and expedite the approval process, to the extent that is regulatorily possible.
Personalized peptide vaccines for cancer exemplify a paradigm shift in cancer therapy from a one size fits all approach utilizing very large numbers of participants in clinical trials to achieve a statistically significant overall improvement in survival, which in many individual cases equates to no improvement, to a more tailored approach that in some instances is n of one.
This approach entails the need for automated selection of appropriate antigens and the development of ever better antigen efficacy prediction algorithms. Adjuvants and excipients that improve immunogenicity are crucial, especially ensuring targeting of the lymph nodes, where efficient antigen presentation can occur. The field is evolving and so are the regulatory considerations.
Moving towards something approaching n of one clinical trial requires close consultation with the FDA. Achieving therapeutic results warranting a breakthrough therapy designation can provide the access to the FDA that is essential in this endeavour.
- Balachandran VP., et al. Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer. 2017. Nature. https://doi.org/10.1038/nature24462
- Banz A., et al. Tumor growth control using red blood cells as the antigen delivery system and poly(I:C). 2012. J Immunother. https://doi.org/10.1097/CJI.0b013e3182594352
- Belnoue E., et al. Targeting self- and neoepitopes with a modular self-adjuvanting cancer vaccine. 2019. https://doi.org/10.1172/jci.insight.127305
- Birkeli R. Targovax to fully focus on ONCOS oncolytic virus program. 2019. https://www.targovax.com/en/targovax-to-fully-focus-on-oncos-oncolytic-virus-program/
- Bloch O., et al. Autologous Heat Shock Protein Peptide Vaccination for Newly Diagnosed Glioblastoma: Impact of Peripheral PD-L1 Expression on Response to Therapy. 2017. Clin Cancer Res. https://doi.org/10.1158/1078-0432.CCR-16-1369
- Bulik-Sullivan B., et al. Deep learning using tumor HLA peptide mass spectrometry datasets improves neoantigen identification. 2018. Nat Biotechnol. https://doi.org/10.1038/nbt.4313
- Clancy T. Flow Pharma, Inc. and OncoImmunity AS partner for the Development of FlowVax™ Personalized Cancer Vaccines. 2019. https://www.businesswire.com/news/home/20190312005768/en/Flow-Pharma-OncoImmunity-partner-Development-FlowVaxTM-Personalized
- Cohn A., et al. Whole Recombinant Saccharomyces cerevisiae Yeast Expressing Ras Mutations as Treatment for Patients With Solid Tumors Bearing Ras Mutations: Results From a Phase 1 Trial. 2018. J Immunother. https://doi.org/10.1097/CJI.0000000000000219
- D’Alise AM., et al. Adenoviral vaccine targeting multiple neoantigens as strategy to eradicate large tumors combined with checkpoint blockade. 2019. Nat Commun. https://doi.org/10.1038/s41467-019-10594-2
- Kawai A., et al. Crystal structure analysis of human serum albumin complexed with sodium 4-phenylbutyrate. 2018. Biochem Biophys Rep. https://doi.org/10.1016/j.bbrep.2018.01.006
- Kim H., et al. The Telomerase-Derived Anticancer Peptide Vaccine GV1001 as an Extracellular Heat Shock Protein-Mediated Cell-Penetrating Peptide. 2016. Int J Mol Sci. https://doi.org/10.3390/ijms17122054
- Madan E., et al. Flower isoforms promote competitive growth in cancer. 2019. Nature. https://doi.org/10.1038/s41586-019-1429-3
- Marker Therapeutics, Inc. Multipronged Cell Therapies For A Multifaceted Disease. https://www.markertherapeutics.com/multitaa-technology/
- Moynihan KD., et al. Enhancement of Peptide Vaccine Immunogenicity by Increasing Lymphatic Drainage and Boosting Serum Stability. 2018. Cancer Immunol Res. https://doi.org/10.1158/2326-6066.CIR-17-0607
- Ott PA., et al. An immunogenic personal neoantigen vaccine for patients with melanoma. 2017. Nature. https://doi.org/10.1038/nature22991
- Sveinbjørnsson B., et al. LTX-315: a first-in-class oncolytic peptide that reprograms the tumor microenvironment. 2017. Future Med Chem. https://doi.org/10.4155/fmc-2017-0088
Building Better Personal Cancer Vaccines
with Dr. Catherine J. Wu, Professor of Medicine at Dana-Farber Cancer Institute
Building Better Personal Cancer Vaccines
Presenting at the virtual TIDES: Oligonucleotide and Peptide Therapeutics conference in September 2020, Dr. Catherine J. Wu from the Dana-Farber Cancer Institute Boston, MA, showed how effective anti-tumour immunity in humans has been associated with the presence of T-cells directed at cancer neoantigens, a class of HLA-bound peptides that arise from tumour-specific mutations. These are highly immunogenic, because they are not present in normal tissues; and therefore, bypass central thymic tolerance.
Although neoantigens were long-envisioned as optimal targets for an anti-tumour immune response, their systematic discovery and evaluation only became feasible with the recent availability of massively parallel sequencing for detection of all coding mutations within tumours, and of machine learning approaches to reliably predict those mutated peptides with high-affinity binding of autologous human leukocyte antigen (HLA) molecules.
Vaccination with neoantigens can both expand pre-existing neoantigen-specific T-cell populations and induce a broader repertoire of new T-cell specificities in cancer patients, tipping the intra-tumoral balance in favour of enhanced tumour control. Dr. Catherine J. Wu studies demonstrated the feasibility, safety, and immunogenicity of a vaccine that targets up to 20 predicted personal tumour neoantigens (Melanoma Neovax anti-PD1). Vaccine-induced polyfunctional CD4+ and CD8+ T cells targeted unique neoantigens used across patients; with these T cells discriminating mutated from wild-type antigens, and in some cases directly recognizing autologous tumour.
More recently, Dr. Catherine J. Wu lab researchers went back to these previously treated patients and grasped how persistent these memory T cell responses are, by seeing a continuous response 5-7-years later, with a second-wave of immune response that shows epitope spreading from the initial epitope.
The question now is if doctors and researchers can better predict the antigen presentation given the ambiguity that arises from the co-expression of multiple HLA alleles. With that in mind, Dr. Wu’s lab used cell lines expressing a single HLA allele, optimizing immune-purifications, and developing an application-specific spectral search algorithm, identifying thousands of peptides bound to 16 different HLA class I alleles. These data enabled the discovery of subdominant binding motifs and an integrative analysis quantifying the contribution of factors critical to epitope presentation, such as protein cleavage and gene expression.
Next, with the help of mass spectrometry the profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines were identified; together with canonical peptide motifs per HLA allele, unique and shared binding sub-motifs across alleles, and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, Dr. Wu’s lab developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumour cell lines.
Another important question in Dr. Wu’s lab is whether they can find new classes of targets in splice variants, gene fusions, or unannotated open reading frames that are translated; and, whether they can render the tumours more immunogenic. Also, by trying to understand how cell states are involved in natural disease progression, Dr. Wu’s lab was able to realize how CD8+ cells are terminally exhausted in metastatic disease of renal cell carcinoma; and, their interaction with tumour macrophages can actually return a prognostic value.
Find out more about the 2020 TIDES: Oligonucleotide and Peptide Therapeutics virtual event in our full overview here.
Peptide Neoantigen Cancer Vaccines and T cell Therapy
with Dr. Daniel deOliveira, Sr. Director, Peptide Development, Tech. Ops. & Manufacturing, Genocea Biosciences
Peptide Neoantigen Cancer Vaccines and T cell Therapy
At the virtual TIDES: Oligonucleotide and Peptide Therapeutics conference in September 2020, Dr. Daniel deOliveira from Genocea Biosciences explained how his company moved towards differentiated immunotherapies through precision antigen selection with their personalized immune response profiling platform ATLAS, which stands for AnTigen Lead Acquisition System.
This platform mainly comprises two programs: GEN-009, a neoantigen vaccine for which a Phase 1/2a clinical trial is ongoing; and GEN-011, a neoantigen-specific cell therapy using T cells derived from peripheral blood. There is also in the Genocea Biosciences discovery pipeline the GEN-010 platform, which is a follow-on neoantigen cancer vaccine, still in Pre-IND phase; and, shared antigen cancer vaccines and vaccines for cancers of viral origin (e.g., Epstein-Barr virus).
The focus of the ATLAS profiling platform is neoantigen selection, that is the identification of mutations that allow the cancer cells to propagate and invade tissues in a thoroughly biological sense. ATLAS empirically selects the relevant neoantigens of tumour-specific T cell responses in patient and tumour-relevant conditions. Tumour biopsies together with saliva samples from patient donors are sent for Next-Generation Sequencing (NGS) analysis.
From that inquiry, plasmids are then selected for every candidate neoantigen and are inserted in a e-coli vector. At the same time, from a blood sample of the patient, a selection of dendritic cells is made, together with CD4+ and CD8+ T-cells.
Afterwards, the dendritic cells sampled are treated with the neoantigen bacterial vectors, and the ones with positive immune responses to the patient CD8+ T-cells are then selected for autologous treatment. From this personalized experimental analysis, two read-outs can be made: (1) is it a true antigen or not; and, (2) is it stimulatory or inhibitory?
As such, personalized tumour mutations presented to dendritic cells are digested and offered through MHC bound peptides that are recognized by T cells. If, from this presentation, an anti-tumour CD8+ T-cell reaction based on a normalized cytokine concentration (IFN-gamma) response is made, then a new stimulatory neoantigen has been positively selected. If, on the other hand, a negative normalized cytokine concentration (IFN-gamma) response is obtained in CD4+ T-cells, then an inhibitory antigen (Inhibigens’) is confidently obtained.
What this means is that this specific inhibigen is removed from the patient vaccine neoantigen pool, because it inhibits the immune response against the cancer.
This feature of the ATLAS platform, of having the ability to select inhibitory antigens that suppress the anti-tumour T cell response, makes the ATLAS neoantigen readout complete, since only neoantigens with a positive immune-response against the cancer cells are selected for treatment, avoiding inhibigen-specific T cells that may be responsible for hyper-progression after checkpoint blockade therapies.
In the specific case of the GEN-009 vaccine, it actually consists of 4-20 stimulatory Synthetic Long Peptides (SLPs) divided into 4 pools, with each pool containing 1-5 SLPs, administered subcutaneously and presented by circulating antigen-presenting cells directly to T cells in the lymph nodes. The expanded T cells specific to GEN-009 neoantigen then go into the circulation, look for tumour cells that show that “harmful” peptide and kill it.
The Part A GEN-009 Phase 1/2a Clinical trial showcased the vaccine safety and immunogenicity in terms of the adjuvant. The preliminary results of Part B of the ongoing trial of GEN-009 in combination with a PD-1 inhibitors in advanced solid cancers, confirmed tumour reduction or stable outcomes for all patients treated; suggesting that GEN-009 vaccination could be used in conjunction with standard-of-care checkpoint inhibitor-based regimens (CPI) to augment their effects.
In the case of GEN-011, the idea is to use already expanded T cells specific to previously selected neoantigens and give those to the patients. The selection of neoantigens is done through ATLAS as before; and, the neoantigen-specific T cell expansion is done with the PLANET platform. PLANET stands for Proliferation of Lymphocytes Activated by Neoantigens Endogenous in Tumours.
This is a new category of neoantigen T cell therapy with peripheral blood-derived ATLAS neoantigens, that are specific for 89% of all intended neoantigen targets. The IND has been filled and preliminary clinical data is expected in first half of 2021.
Find out more about the 2020 TIDES: Oligonucleotide and Peptide Therapeutics virtual event in our full overview here.
High throughput GMP neoantigen peptide synthesis brings individualized cancer immunotherapy to a new level
Gyros Protein Technologies
High throughput GMP neoantigen peptide synthesis brings individualized cancer immunotherapy to a new level
The development of therapies that effectively destroy tumors while sparing healthy tissue is the Holy Grail in clinical oncology. Efforts to improve on traditional chemotherapy have led to, for example, Nobel-prize winning developments in immunotherapy, and antibody-based therapies.
A new approach in immunotherapy that involves vaccines based on peptide neoantigens promises to bring therapeutic precision to the level of individual tumors in individual patients. Achieving this demands a combination of rapid data acquisition and analysis followed by efficient GMP-compliant parallel peptide synthesis.
Figure 1: The principle of immunotherapy based on neoantigens
Cancer is characterized by a high frequency of genetic mutations that result in mutated proteins, uncontrolled division, and proliferation of abnormally functioning cells. These mutated proteins can be processed into neoantigen peptides that are presented as immune signals on the surface of cancer cells (Figure 1).
The T cells can recognize and target these neoantigens as foreign, leading to the death of the cancer cell. Neoantigens are specific to the tumor in the individual patient making them a highly promising target for personalized, or rather individualized immunotherapy using cancer vaccines (1, 2). Certain cancers, such as melanoma, result in more mutations than others, making the production of neoantigens more likely.
Cancer therapy based on neoantigens involves generating a vaccine designed to target the individual cancer cells in a particular patient. The first step in this “needle-to-needle” process is to pinpoint missense mutations in tumor-expressed proteins by sequencing gene exons in cancer biopsies and normal tissue from the patient. Transcriptome data is also included to indicate antigen abundance.
The most critical step is the use of algorithms to identify those mutated proteins that are processed into 8- to 11-residue peptides by the proteasome for later recognition by CD8+ T cells. Once the neoantigens have been identified, they are synthesized using conventional peptide synthesis.
Cocktails of the neoantigens are incorporated into vaccines designed to stimulate the immune system to attack the specific cancer cells in the individual patient that express just these neoantigens. The result is truly individualized immunotherapy, which can be combined with other therapies such as checkpoint modulators and monoclonal antibodies.
How are neoantigens used in therapy?
Figure 2. The “needle-to-needle” process from biopsy to injection of individualized neoantigen vaccine involves rapid data acquisition, analysis and peptide synthesis.
Rapid data acquisition, analysis and peptide synthesis
Speed is key in treatment with neoantigens, since the dynamics of the mutation spectrum of individual tumors means that neoantigen-based therapy can be compared to firing a rifle bullet (‘the vaccine’) at a predator (‘the tumor’) that can move in any direction at any moment – maximizing your chances of success is based on hitting the target before it has moved too far away from its original position.
This means that the genomic snapshot of the tumor must be quickly converted into a functioning vaccine, with vaccine manufacturers aiming at a turnaround time of just a few weeks.
Neoantigen vaccines have been made possible by major technological advances, including next generation sequencing that enables the timely and cost-effective sequencing of individual genomes.
Powerful computer algorithms help identify the best neoantigens to include in a vaccine, although further development is needed to reduce the number of candidates to those that specifically trigger antitumor responses. In addition, advances in manufacturing enable rapid production of small quantities of diverse molecules for individual vaccines.
Tough demands on GMP peptide synthesis
Generating the pool of neoantigens needed requires parallel synthesis of peptides with high purity and yield. Neoantigens may be further altered through posttranslational modifications (PTMs) that occur in malignant but not healthy cells and are therefore an additional source of unique antigens that are specific to the individual patient.
Such modified neoantigens have been isolated from a number of blood cancers and research clearly shows that PTM-neoantigens make promising targets for immunotherapy (3). Mass spectrometry (MS) analysis has helped in the discovery of a large range of attractive target antigen candidates, such as phosphopeptides (4), that may be used for immunotherapy.
The synthesis of neoantigens may also present synthesis challenges involving modifications and problematic sequences. Generating phosphorylated peptides can be quite straightforward but requires specialized amino acids such as pTyr, pSer, or pThr.
However, synthesis with modified amino acids still needs much work. Currently, there are only mimics of pHist and introducing Lys(Me)2 or Lys(Me)3 can be difficult and may reduce the overall quality of peptide synthesis (5). Heating during a deprotection step can result in dephosphorylating the amino acids during synthesis and pSer and pThr can require extra base and longer coupling times (5).
Neoantigen peptide synthesis on PurePep® Chorus
PurePep Chorus peptide synthesizer was evaluated for neoantigen peptide synthesis by synthesizing candidate binding peptides for mutated kinesin family member 2C (KIF2C) identified by Lu and coworkers for targeting metastatic melanoma using tumor infiltrating lymphocytes (TILs; 6). A fast protocol at an elevated temperature of 75 °C involved the following chemistry:
- Deprotection - 2 x 2 min
- Coupling - 3 min @ 75°C
- Coupling reagents – 6-fold excess HCTU with DIPEA
The synthesis of this set of neoantigen peptides was completed in very high purity after the first synthesis run (Figure 3). This right-first-time approach is critical when producing peptides for neoantigen applications.
Figure 3. Neoantigen peptides were rapidly synthesized to a high purity using PurePep Chorus.
Case Study: How to manufacture 20 GMP peptides in 3 weeks
In a webinar from TIDES Digital Week, Alastair Hay, Account Manager (Peptides) with Almac Group Ltd. presented the challenges of high throughput GMP synthesis of neoantigen peptides and how this is being pushed to new levels of throughput.
About Almac
Almac as a peptide CMO has used parallel synthesizers for more than 15 years and has recently applied automated parallel synthesizers in a GMP setting for the manufacture of clinical grade NeoPeptide™ neoantigens for use in vaccine products.
NeoPeptide experts at Almac have been associated with the individualized cancer vaccine field for several years and the MHRA registered facility and systems have been established to enable fully GMP compliant manufacture and release of 20–30 GMP peptides within less than three weeks. A bespoke Pharmaceutical Quality System has been designed and implemented to enable high speed, fully compliant GMP manufacture. Each peptide manufacture is unique; manufacture occurs once, and the product is administered to only one patient.
A good reputation together with positive regulatory reviews received from Europe and US authorities has resulted in an unparalleled service to help enhance the lives of cancer patients.
Neoantigen developments at Almac were founded by a request from a sponsor in 2012 who asked, “Can you manufacture 20 GMP peptides in a month?”.
This presented a number of challenges compared to the manufacture of conventional peptides (Table 1) but led to the development of a plan that was favorably viewed by the sponsor and the U.S. Food and Drug Administration (FDA), and the manufacture of a 20-peptide set in 2015.
|
Conventional GMP Peptide
|
NeoPeptide
|
|
Generally, a single peptide
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Multiple peptides (10–20)
|
|
Multi-patient trial
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Single patient – fully personalised
|
|
Multi-gram quantity (100 g)
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Low quantity (10–30 mg)
|
|
Product specific process development
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Robust generic manufacturing process
|
|
Product specific analytical development
|
Robust generic analytical methods
|
|
Fully GMP compliant
|
Fully GMP compliant
|
Table 1: Conventional GMP peptide vs NeoPeptide™
Conventional GMP peptide manufacture can be a lengthy process (Fig. 4), which meant that meeting the needs of neoantigen peptide manufacture involved an 80-fold increase in manufacturing rate. This can be compared with the original goal for neoantigen manufacture turnaround of 8 weeks, with 12 weeks being more common.
The need for speed in neoantigen peptide manufacture precludes sequence-specific process development, which means that generic methods must be used. The peptide synthesizer must therefore be able to cope with a wide range of sequence motifs, and the choice of chemistry, coupling cycles and deprotection cycles must give the best chance of success in rapid synthesis.
These challenges result in an accepted attrition rate, but poor quality should only be due to the inherent nature of the sequence, or issues with solubility, rather than limitations in the chemistry or instrument. The instrument itself must be GMP compliant, qualified and calibrated and backed up with manufacture logs and maintenance procedures, together with engineering support, training and training records.
Almac met their goals using Symphony® X peptide synthesizer, which enables synthesis of up to 24 peptides at the required scale. The CMO is currently able to synthesize 20 GMP peptides in 3 weeks, with a future target for an expanded facility of 20 GMP peptides per day in a “patient a day” suite.
Figure 4. Meeting the goals for neoantigen manufacture demanded a considerable increase in manufacturing capability – from 6 months for a single peptide to 1 month for 20 peptides.
Peptide synthesizers for GMP-compliant peptide synthesis
Peptide synthesizers from Gyros Protein Technologies are used in a large number of cGMP facilities to produce peptides required in clinical studies, neoantigen trials, and cosmetic formulations. The peptide synthesizers include a number of features that support GMP-compliant manufacture:
Hardware
- Resin integrity – The resin for each synthesis is contained in a single reaction vessel (RV) and is not moved or transferred to any part of the system. This feature eliminates any risk of product cross contamination.
- Valve block – The Ultra PurePep Pathway on these instruments ensures a dedicated reagent delivery line for each RV, with no reagent cross-over and no dead volumes.
- Line clearances – Lines are automatically flushed with primary solvent and nitrogen after each delivery to ensure fluid channels are rinsed and dried before the next addition.
Software
Designed for 21CFR part 11 compliance provided by:
- User Management
- Audit Trail
- Data Integrity
- Electronic Signatures
IQ/OQ/PQ
Installation qualification (IQ) and operational qualification (OQ) support is available together with performance qualification (PQ) guidance to support work in regulated environments.
Neoantigen vaccines are a very promising approach to the treatment of cancer at the level of the individual patient. Success depends on the availability of reliable peptide synthesizers that can support robust chemistry for the rapid GMP-compliant manufacture of many peptides in parallel. Instrument reliability and technical advances have helped push the boundaries of peptide synthesis and promise an exciting future for cancer immunotherapy.
DIPEA, N, N-Diisopropylethylamine; HCTU, 2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate.
- Progress in neoantigen targeted cancer immunotherapies. Han X-J et al. Front Cell Dev Biol. 2020; 8: 728. doi: 10.3389/fcell.2020.00728
- Advances in the development of personalized neoantigen-based therapeutic cancer vaccines. Blass E & Ott PA, Nat Rev Clin Oncol. 2021 Jan 20;1-15. doi: 10.1038/s41571-020-00460-2
- Identification of glycopeptides as posttranslationally modified neoantigens in leukemia. Malaker SA et al, Cancer Immunol Res. 2017 May;5(5):376-384. doi: 10.1158/2326-6066.CIR-16-0280. Epub 2017 Mar 17.
- Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry. Bassani-Sternberg M et al, Nat. Commun. 2016 Nov 21;7:13404. doi: 10.1038/ncomms13404
- Peptide microarrays to interrogate the “Histone Code”. Rothbart, SB. Methods Enzymol. 2012; 512: 107–135. doi:10.1016/B978-0-12-391940-3.00006-8.
- Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions. Lu Y-C et al. Clin Cancer Res. 2014 Jul 1;20(13):3401-10. doi: 10.1158/1078-0432.CCR-14-0433.
Symphony X peptide synthesizer
Manufacturing neoantigen vaccines and developing therapeutics requires a lot from a peptide synthesizer.
Symphony X peptide synthesizer, with the proprietary PurePep Pathway, has the ability to run 12 independent reaction vessels and 24 reaction vessels overall, which is often desirable when dealing with peptide libraries.
- Speed – able to synthesize 24 neoantigen peptides in 12 hours (15–20 mers).
- Purity is ensured due to the focus on ‘right first time’ methodology, with no reagent or resin cross-contamination, and minimizes the risk of re-synthesis.
- Unique in providing multi-channel synthesis under inert atmosphere and with no cross contamination
- Supports GMP manufacture, designed for 21 CFR part 11 compliance, together with IQ/OQ support and PQ guidance are available to support work in regulated environments
PurePep Chorus peptide synthesizer
PurePep Chorus meets many of the demands made in the synthesis of neoantigen peptides and complex peptides for therapeutics. The proprietary PurePep Pathway comprises fluidics that minimize cross-contamination, dead volumes, and reagent carryover.
This is especially crucial for the “right-first-time” synthesis of neoantigen peptides and the synthesis of long sequences for therapeutics, in which even small amounts of impurities, side products, and incomplete reactions over many cycles can drastically reduce the final purity and yield of desired peptides.
Aggregation, secondary structure, steric hindrance, and conformational effects can still pose challenges in synthesis, and PurePep Chorus features enabling technologies that aid the synthesis of complex peptides and peptidomimetic sequences.
Intellisynth™ real-time UV monitoring optimizes reaction times to ensure complete deprotection. By monitoring at 301 nm, the instrument measures the progress of the reaction – avoiding guesswork that can lead to incomplete deprotections, deletions, and side reactions. This feature is available on all reaction vessels, providing UV monitoring on up to six peptides in a single synthesis.
Other features include:
- A modular peptide synthesizer that is in-lab upgradeable, from 2 to 4 to 6 reaction vessels to meet productivity needs
- Independent induction heating, configurable to multiple vessels simultaneously, and the ability to run multiple conditions in one run to speed up method development
- Icon-driven intuitive software platform with pre- programmed methods, ability to import sequences and reagent preparation calculators
- Designed for 21 CFR part 11 compliance, together with IQ/OQ support and PQ guidance are available to support work in regulated environments
Personal Neoantigen-Based Cancer Vaccine NEO-PV-01 in the Treatment of Metastatic Cancers
Personal Neoantigen-Based Cancer Vaccine NEO-PV-01 in the Treatment of Metastatic Cancers
At the TIDES Europe and US conferences in late 2020, Dr. Jesse Dong from BioNTech US delivered a talk about the personalized neoantigen cancer vaccine NEO-PV-01, in Phase 1b development in tumour settings at the time of presenting.
The vaccine is custom-designed and manufactured for each individual patients’ tumour mutations, focused in combination with checkpoint inhibitors in metastatic disease settings. It uses Next Generation Sequencing and proprietary bioinformatics (RECON, Real-time Epitope Computation for Oncology) to select up to 20 neoantigen peptides from tumour samples.
Initial data shows that vaccine-induced immune responses were durable and safe with no adverse events in all 82 patients with melanoma, bladder, or lung cancer included in the trial. De novo neoantigen-specific CD4 +and CD8 + T cell responses were observed post-vaccination in all of the patients. The vaccine-induced T cells had a cytotoxic phenotype and were capable of trafficking to the tumour and mediating cell killing. In addition, epitope spread to neoantigens not included in the vaccine was detected post-vaccination.
In relation to secondary endpoints including objective response rates, progression-free survival, and overall survival, these were also taken into account by the researchers. The objective response rates among patients with melanoma, lung, and bladder cancer, respectively, were 59%, 29%, and 27%. The median progression-free survival for these three histology groups was 23.5, 8.5, and 5.8 months, respectively, and the one-year overall survival rates were 96%, 83%, and 67%. The median overall survival was not reached for patients with melanoma or lung cancer but was 20.7 months for patients with bladder cancer.
BioNTech US researchers also explored indicators of patients' responses specifically to the vaccine. In a post-hoc analysis, they measured the percentage of all NEO-PV-01 vaccinating peptides that elicited interferon-gamma responses in samples of patients' peripheral blood at three time points: pre-treatment, pre-vaccine, and post-vaccine. Using the IFN Gamma EliSpot Assay, they found that among patients who had sufficient samples at all three time points, significant immune responses to multiple vaccinating peptides were observed, including 52% responses to peptides in patients with melanoma, 47% in patients with lung cancer, and 52% in patients with bladder cancer. An average of 42% and 24% of vaccinating peptides generated CD4+ and CD8+ T-cell responses, respectively.
The researchers also evaluated immune responses in patients' samples 52 weeks after the start the treatment and detected persistent immune responses for 58% of neoepitopes. The data confidently demonstrates that vaccination with NEO-PV-01 and Nivolumab results in the generation of T-cell responses that are specific and durable.
The ability of the patients' CD4+ and CD8+ T-cells to migrate to the solid tumours and kill cancer cells was also evaluated by the researchers, by looking for the expression of CD107a, a marker for T-cell degranulation. Across all three cohorts of patients, the overall surface expression of CD107a was detected in the presence of 58% of the epitopes after vaccination.
In relation to the question whether the neoantigen-specific T cells could release additional epitopes to generate a broader neoantigen-specific immune response, researchers screened several patients' samples for T-cell responses against epitopes that weren't included in the vaccine, and they found peripheral immune responses against a handful of these neoepitopes and noted that these responses had not been present before the administration of the vaccine. As such, the presence of epitope spread could be a surrogate marker for tumour cell killing by neoantigen-specific T cells induced by vaccination.
What is particularly important is that this epitope spread was correlated with patients' progression-free survival across all three tumour types, which further indicated that it may only be necessary to target a subset of neoantigens expressed by the tumour to generate a broad immune response against the expressed neoantigens. As such, the conclusion is that the clinical trial data support the safety and immunogenicity of NEO-PV-01 and Nivolumab in patients with advanced solid tumours; and the data supports future randomized trials with NEO-PV-01.
Find out more about the 2020 TIDES: Oligonucleotide and Peptide Therapeutics virtual event in our full overview here.
Neoantigen Peptide Therapies and Personalised Medicine
An Interview with Samantha Zaroff Ph.D, Genscript
“Neoantigens are making a big buzz as preferred targets for personalized immunotherapies”
We spoke to Samantha Zaroff Ph.D, Product Manager of Neoantigen Peptide Services at GenScript about neoantigen peptide therapies, their uses in personalized medicine and the challenges in producing them.
"Specifically neoantigens can be difficult to synthesise because of five characteristics: length, charge, hydrophobicity, yield and purity," she said.
Click the video above to watch Zaroff explain how Genscript has designed a neoantigen peptide synthesis platform to address the issues in their production.
