Gyros Protein Technologies
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.
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.
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).
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:
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.
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
Multiple peptides (10–20)
Multi-patient trial
Single patient – fully personalised
Multi-gram quantity (100 g)
Low quantity (10–30 mg)
Product specific process development
Robust generic manufacturing process
Product specific analytical development
Robust generic analytical methods
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 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
Software
Designed for 21CFR part 11 compliance provided by:
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.
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.
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: