Advances in plasma proteomics are expanding the understanding of disease mechanisms and have the potential to transform clinical practice by enabling early disease detection and personalized treatment.

High-resolution mass spectrometry (MS) has greatly improved the sensitivity and accuracy of using plasma samples for proteomics analysis by enabling the identification and quantification of low-abundance proteins and post-translational modifications (PTMs). Over the past two decades, new methodologies combined with technological advancements have helped the field of plasma proteomics evolve as an extremely powerful tool for protein scientists, biologists, and clinical researchers (1). Research has shown the capabilities of a rapid and highly reproducible proteomic workflow to deliver a systemic-view proteomic portrait of a person’s health state from a single drop of blood (2).

Yet performing protein analysis directly from plasma poses some analytical challenges. Protein expression is not only influenced by an individual's genetic background, but also by various factors such as time, location, and physiological responses to external stimuli such as stress, disease, aging, and physical activity. Additionally, a single protein or gene expression product can exist in multiple proteoforms due to alternative splicing, point mutations, PTMs, and endogenous proteolysis, each with distinct biological activities (3,4).

The use of orthogonal approaches in plasma proteomics can help address this complexity, leading to more accurate and comprehensive data that can advance our understanding of disease mechanisms and aid in the development of new treatments. Technological advances open new possibilities for improvements in early disease detection and the development of personalized treatments.

Using prm-PASEF for advanced proteomics analysis

Parallel accumulation serial fragmentation (PASEF) is a technique used in MS-based proteomics for peptide sequencing and identification. A recently developed parallel reaction monitoring-PASEF (prm-PASEF) approach allows the addition of ion mobility as a new dimension to liquid chromatography–mass spectrometry (LC–MS) proteomics and increases proteome coverage at a reduced analysis time. The combination of trapped ion mobility spectrometry (TIMS) with a rapid time-of-flight (TOF) mass spectrometer improves the sensitivity for proteomics, allowing detection of the amounts found in single cells.

As a result, prm-PASEF can take advantage of a dual ion mobility trap, enabling highly multiplexed targeted acquisition and thus requiring a shorter chromatographic separation without sacrificing the number of targeted peptides (5). Additionally, the TIMS design allows researchers to reproducibly measure the collisional cross section (CCS) values for all detected ions, to further increase the system’s selectivity, enabling more reliable relative quantitation information from complex samples and short gradient analyses (

Four-dimensional (4D) proteomics seeks to analyze proteins by combining the traditional three dimensions of proteomics (protein identity, abundance, and PTMs) with a fourth dimension, which is the localization of proteins within their cellular and tissue contexts. By analyzing proteins in all four dimensions, 4D proteomics aims to provide a more comprehensive and detailed understanding of protein function and regulation. This can be particularly valuable in the development of personalized medicine by helping to identify disease-specific biomarkers and potential targets for drug development.

Using a TIMS mass spectrometer with PASEF and high-throughput LC separation, research has shown 4D proteomics using a match between runs (MBR) approach can extract intensity information for all peptides to be quantified using narrow m/z and retention time windows for biomarker discovery in large sample cohorts of human blood plasma. 4D-MBR adds an extra CCS filter to the first m/z and retention time ones, allowing for much greater specificity (

Unlike traditional PASEF, which uses a data-dependent acquisition (DDA) approach where precursor ions are selected based on their intensity, dia-PASEF uses a data-independent acquisition (DIA) approach. In DIA, all precursor ions are fragmented, allowing for the detection and quantification of a larger number of peptides and proteins in a sample. Dia-PASEF combines DIA with the high-resolution ion mobility separation provided by TIMS, enabling the rapid and sensitive identification and quantification of peptides in complex mixtures (

). The technique has the potential to provide higher coverage and accuracy in the identification and quantification of peptides compared to traditional DIA methods. 

Dia-PASEF has been used in a variety of proteomics applications, including the identification and quantification of proteins in clinical samples, the analysis of PTMs, and the characterization of protein-protein interactions. Research has shown dia-PASEF delivers extremely reproducible identification and quantification information over a concentration of five orders of magnitude making the approach perfectly suited for the challenges of quantitative proteomics (8).

Early detection of diseases is a significant challenge in human health, and there is a pressing need for more precise biomarkers, improved patient stratification, and methods for predicting treatment response. Advancements in unbiased and comprehensive 4D plasma proteomics offer unique advantages to provide solutions to these challenges through exceptional sensitivity and selectivity. Together, TIMS and PASEF allow for high-throughput analysis of complex protein samples, resulting in improved sensitivity, dynamic range, and throughput compared to traditional proteomics methods. These technological developments have the potential to provide new insights into disease mechanisms and potential therapeutic targets.

1. Geyer, P. E.; Holdt, L. M.; Teupser, D.; Mann, M. Revisiting Biomarker Discovery by Plasma Proteomics. 

 (9), 942. DOI: 10.15252/msb.20156297
2, Geyer, P. E.; Kulak, N. A.; Pichler, G.; et al. Plasma Proteome Profiling to Assess Human Health and Disease. 

(3), 185–195. DOI: 10.1016/j.cels.2016.02.015
3. Ignjatovic, V.; Geyer, P. E.; Palaniappan, K. K.; et al. Mass Spectrometry-Based Plasma Proteomics: Considerations from Sample Collection to Achieving Translational Data. 

 (12), 4085–4097. DOI: 10.1021/acs.jproteome.9b00503
4. Gegner, H. M.; Naake, T.; Dugourd, A.; et al. Pre-Analytical Processing of Plasma and Serum Samples for Combined Proteome and Metabolome Analysis. 

, 961448. DOI: 10.3389/fmolb.2022.961448
5. Brzhozovskiy, A.; Kononikhin, A.; Bugrova, A. E.; et al. The Parallel Reaction Monitoring-Parallel Accumulation–Serial Fragmentation (prm-PASEF) Approach for Multiplexed Absolute Quantitation of Proteins in Human Plasma. 

, 4, 2016–2022. DOI: 10.1021/acs.analchem.1c03782
6. Meier, F.; Köhler, N. D.; Brunner, A. D.; et al. Deep Learning the Collisional Cross Sections if the Peptide Universe from a Million Experimental Values. 

, 1185. DOI: 10.1038/s41467-021-21352-8
7. Kosinski, T.; Heilig, R.; Bensaddek, D.; et al. Plasma Proteomics Goes High Throughput—timsTOF Pro with PASEF and 4D Feature Alignment to Quantify 500 Plasma Proteins in 11.5 Min. Bruker Daltonics (March 2019).
8. Kaspar-Schoenefeld, S.; Marx, K.; Gandhi, T.; et al. diaPASEF: Label-Free Quantification of Highly Complex Proteomes. Bruker Daltonics (September 2019).

Shourjo Ghose is proteomics business development manager—North America, and Andreas Schmidt is application development scientist; both at Bruker Daltonics.

Articles

Features

A single protein or gene expression product can exist in multiple proteoforms due to alternative splicing, point mutations, post translational modifications and endogenous proteolysis. 

New Developments in Plasma Proteomics

Antibody-drug conjugate (ADC) safety and effectiveness relates directly to the functioning of the linker connecting the cytotoxic payload to the targeting antibody. The chemistries used to link the two crucial components of ADCs have evolved over time, leading to improved performance. Selection of the optimal linker chemistry is paramount to successful development of ADCs, however. The choice depends on the attributes of the antibody and the payload, as well as the nature of the target.

Since the first ADC (Mylotarg, gemtuzumab ozogamicin, Pfizer) was introduced in 2000 (and withdrawn in 2010 for causing severe liver toxicity due to its unstable N-acylhydrazone linker, redesigned, and then reapproved in 2017), linker chemistries have evolved significantly to address issues with heterogeneity and instability. “First-generation ADCs struggled with heterogenous drug-to-antibody ratios (DAR) and unstable linkers, making them less effective and potentially more toxic,” says Campbell Bunce, chief scientific officer at Abzena. “Some of the early linkers used for ADCs were found to present a number of issues, including non-specific payload release in non-tumor tissues, leading to off-target toxicity and a limited therapeutic window,” agrees Sara Jenkins, ADC commercial manager with Sterling Pharmaceutical Services.

Specific examples include maleimide and lysine conjugation to antibodies. The former allows in vivo detachment of the linker with payload followed by reattachment to free cysteines, which can reduce ADC efficacy and impact the safety profile. The latter suffers from indiscriminate conjugation across the whole antibody, which can also interfere with binding to the cancer antigen or the Fc receptor functions. Both, according to Bunce, also suffer from variable DARs, making development of a robust manufacturing process challenging.

Much work has been undertaken to investigate the linker terminus to stabilize this reaction, find alternative methods of attaching the antibody via site-specific cysteines, or through use of enzymatic chemistry, Jenkins observes. Other efforts have been focused on ensuring that cleavable linkers cleave only in the target tumor tissue and at an appropriate rate, which can also be an issue for payload release, she adds. Increasing the number of accessible sites where linkers can be attached to payloads is another challenge currently being tackled by researchers who are, Jenkins says, exploring various functional groups on payloads and effector molecules.

Significant progress has been made on many fronts. In second-generation ADCs, advances in site-specific conjugation and cleavable linkers enable improved stability, homogeneity, and more controlled release, ensuring more targeted delivery to cancer cells and fewer off-target effects, Bunce notes. The third- and latest-generation of ADCs are further improved by using hydrophobicity-masking and solubility-enhancing elements for the payloads. “The new chemistries allow for improved PK properties; tandem or dual cleavage linkers, making payload release even more specific to the target cells; and multi-loaded linkers for increased therapeutic potency and a broader therapeutic index,” Bunce explains.

Linkers can be classified in two broad categories: cleavable and non-cleavable. Cleavable linkers, according to Nicolas Camper, senior director of chemistry with Abzena, release their payload when exposed to certain conditions in the target cell, such as low pH or high levels of a specific enzyme. Non-cleavable linkers, on the other hand, release the drug only upon catabolic degradation of the ADC.

There is still a great deal of interest in the classic approved peptide linkers such as Val-Cit-PAB (valine-citrulline-p-aminobenzylalcohol), which is used in Adcetris (brentuximab vedotin, Seagen) and more recently the GGFG (glycine-glycine-phenylanine-glycine) linker found in Enhertu (trastuzumab deruxtecan, Daiichi Sankyo), according to Jenkins.

Enzyme cleavable linkers have become increasingly popular, however, Bunce comments, as they tend to offer an optimal balance between systemic stability and efficient payload release inside cells. Jenkins points to glucuronidase triggered systems such as β-glucuronide as an example. “These linkers have a number of advantages, including the fact that lysosomal enzyme β-glucuronidase is abundant in lysosomes and over-expressed in some tumors, resulting in stable ADCs; and β-glucuronide is highly hydrophilic, leading to reduced ADC aggregation,” she says.

“The ultimate choice depends on the drug’s intended action and the biological context, although different payload release moieties deserve evaluation as part of the ADC lead-selection process,” Camper contends.

The conjugation chemistry used to connect the linker to the antibody is one of the important aspects of the linker that must be optimized. As the number of available technologies has increased, the trend has been to preferably leverage site-specific conjugation rather than stochastic (unpredictable or random) conjugation approaches, according to Camper.

Some site-specific conjugation technologies are compatible with native antibodies, Camper explains, while others require antibody engineering to introduce enzymatic conjugation tags or rare or non-natural amino acids into the antibody sequence. Not surprisingly, each approach presents its own challenges for manufacturing depending on the complexity of the process. All of these strategies, though, observes Camper, give better defined and more easily characterized ADCs while also allowing evaluation of different linker-payload conjugation locations on the antibody, which he notes can have a significant impact on biological activity.

Abzena offers its site-specific conjugation technology ThioBridge, which exploits the natural interchain disulfide bonds in antibodies, thus avoiding the need for extensive antibody reengineering. “Compared to older maleimide conjugation, ThioBridge produces uniform ADC constructs with homogenous drug loading. Its 3-carbon bridge attachment also enhances stability and improves pharmacokinetics,” Camper states. In addition to site-specific attachment, he says the technology is compatible with different linker designs and payload classes, has the potential to improve toxicity profiles, and is sufficiently flexible for DAR and hydrophilicity optimization.

New payloads require different chemistries

As research into ADCs has progressed, the number of types/classes of compounds that can serve as cytotoxic payloads has expanded. That has created the need for different attachment chemistries for these linkers other than those that must react with amines and alcohols, according to Jenkins.

Several chemistries have been reported in the literature and some have been put in practice. “Prominent among them,” says Jenkins, “is the addition of hydrophilicity to linkers using hydrophilic triggers or by adding pendant hydrophilic groups.” She also highlights different triggers under investigation by various groups, including stabilized maleimides that limit the retro-Michael elimination reaction, enzyme-independent triggers such as pH-dependent linkers that remain stable at physiological pH but unstable at the pH of the tumor microenvironment, and disulfide-based designs that take advantage of glutathione gradients to trigger release.

Determination of the optimal linker chemistry for a given antibody and payload combination depends on numerous factors. Specific considerations noted by Jenkins include stability at physiological pH and in serum; the ability to release the payload where desired, whether in the cell or the tumor microenvironment; sufficient hydrophilicity to counteract payload hydrophobicity while still reducing ADC aggregation; and a reasonable and practical manufacturing process.

Efficacy and safety are paramount, emphasizes Bunce. “The linker must ensure targeted payload delivery to maximize therapeutic impact and minimize off-target effects and toxicity. That is because from a design perspective, the choice of linker can affect ADC stability, DAR, and pharmacokinetics,” he elucidates. Manufacturability cannot be ignored, however, agrees Bunce. “The linker chemistry must align with scalable production methods to control costs and meet development timeframes,” he says.

Evaluation requires multifaceted approach

One of the main hurdles in optimizing linkers for ADCs is the number of parameters which can potentially be investigated. They include, says Camper, the antibody conjugation site and chemistry, the payload release mechanism, solubility enhancing elements, and architectures allowing accommodation of multiple copies of the payload, and positioning of these different elements in the linker. Adding to the challenge is the fact that there is a profuse offering of technologies to improve all the different elements of ADC linkers, all with their own advantages and drawbacks. “The number of possibilities to optimize an ADC can easily look overwhelming,” he concludes.

Scale up of ADC production processes can also present issues. “Advanced technical solutions working well at bench scale and providing ADCs with attractive pre-clinical therapeutic profiles may prove extremely complex to manufacture or turn into regulatory challenges due to the density of innovation introduced into the new ADC drug,” Camper observes. The result: complex decision-making for ADC linker optimization. Consequently, evaluating the performance of ADC linkers requires a multifaceted approach that combines physical experiments with analytical and predictive techniques, according to Camper.

Specific enzyme assays, such as cathepsin-B and β-glucuronidase, can be used to determine whether the payload is released at an appropriate rate and as expected, Jenkins observes. A linker’s stability in plasma can, meanwhile, be a key factor in establishing how resilient an ADC can be to unexpected cleavages or premature loss of payload when exposed to bio-fluids, she adds. In addition, cell and liver fractions such as lysosomes and S9 fractions can be utilized to see how linker-payloads, and even the toxins themselves, react to biological mixtures that are closer in nature to those found in tumors. Mass spectrometry, Jenkins continues, is a useful technique for these experiments because of its specificity and sensitivity in complex samples.

Specific methods highlighted by Camper include Incucyte FabFluor (Sartorius) labeling for confirmation of ADC internalization by the target cells and efficient intracellular tracking alongside the endo-lysosomal pathway, flow cytometry to assess on-target and off-target toxicity, EpiScreen and Cytokine Screen (Abzena) immunogenicity assays and in silico assays for safety, in vitro stability tests to ensure that the ADC remains stable in circulation and doesn’t release its payload prematurely, and pharmacokinetic and pharmacodynamic analyses to gain in vivo performance data that inform adjustments for optimal outcomes and allow selection of the best lead ADC design for further development and manufacture based on a favorable therapeutic index.

Bunce stresses, however, that as ADCs become more intricate—carrying multiple payloads or designed for controlled release mechanisms—these conventional analytical techniques may reach their limits. “Mass spectrometry, next-generation sequencing, and chemical modeling, or even novel technologies could fill this gap, providing a more comprehensive view of ADC characteristics,” he remarks. Bunce goes on to state, “these innovative techniques may well be the linchpin for overcoming the technical challenges ahead, offering a refined toolkit for the next generation of ADC linker optimizations.”

There is also potential for application of advanced digital tools in ADC linker selection and optimization. “As an industry, we’re delving deeper into the complexities of ADC linker chemistry, trying to get ahead of the next wave of optimizations that will hopefully deliver more effective therapies. One area often overlooked is the role of computational biology in predicting linker behavior,” Bunce contends.

Advanced simulations, Bunce adds, can provide insights into the dynamic interactions between the linker and the antibody, thereby reducing the need for exhaustive empirical tests. “If we can integrate machine learning algorithms, there is the possibility of bringing a greater degree of automation to the process of data analysis, making it easier for ADC experts to identify subtle but crucial trends in ADC performance,” he says.

Cytotoxic payloads used in ADCs, like many other novel small-molecule APIs, often are hydrophobic compounds with poor water solubility and thus low bioavailability. As a result, chemists in the ADC field must often identify a solution to hydrophobicity problems associated with highly lipophilic payloads, according to Stepan Chuprakov, director of chemistry at Catalent. “Depending on the chosen bioconjugation method (site-specific, stochastic) and the desired DAR, the resulting conjugate may demonstrate poor biophysical properties, including high levels of aggregation and poor pharmacokinetics (rapid plasma clearance),” he explains.

Typically, some structural elements that mask the payload’s hydrophobicity are added, including discrete polyethylene glycol (PEG) chains, polyols, and charged groups, Chuprakov notes. As examples, Jenkins suggests that the cleavage trigger can be switched from Val-Cit to Val-Ala or a more hydrophilic β-glucuronide that can lower the logP of the molecule and increase the therapeutic index. “The approach works well if β-glucuronidase is abundant in the tumor cell to facilitate cleavage, but is not as beneficial if the trigger cleavage is dependent upon peptidase enzymes,” she notes.

With respect to the insertion of a long PEG unit into the linear chain to increase the hydrophilicity of the linker payload, Jenkins comments that it has become evident that, in some cases, adding long PEG units can actually have a detrimental effect on the molecule. Instead, she says that adding pendant hydrophilic units such as PEG or polysarcosine (pSar) in the correct positions has been shown to have a masking effect on the hydrophobic payload.

On the positive side, Chuprakov observes that the available potential chemical space for linker design is vast, and chemists have a variety of synthetic tools to incorporate those additional elements. “However, synthetic chemists may want to avoid the temptation of going too far beyond what nature dictates, as some exotic structural elements may come with their own concerns, for example, immunogenicity,” points out Chuprakov.

The best strategy for assuring selection of the optimal linker for any ADC is the use of performance of a rigorous pre-clinical developability assessment encompassing all the different components of an ADC, both taken individually and as a whole, Camper insists.

“Start from the selection of antibody clones optimized for target antigen binding specificity and affinity, internalization and trafficking properties, as a well as favorable immunogenicity and stability profiles. Then, coupling the lead antibody clones to a panel of payloads and linkers to determine the best combination of linker-payloads and antibodies to carry forward into in vivo evaluation on the basis of in vitro cell cytotoxicity and serum stability profiles, the therapeutic potential of lead candidate ADCs can be assessed from efficacy, PK, and toxicology studies,” Camper says.

Using this approach, liabilities can be identified and addressed, possibly by getting help from advanced linker technologies for optimization. “Early evaluation of conjugation efficiencies and biophysical stability also provides invaluable information on ADC manufacturability and is factored into the selection of the ADC candidate to take into clinical trials,” Camper observes.

“In addition to thoroughly reviewing how safe and effective an ADC is and how easily it can be manufactured, our team also uses ongoing data to make real-time adjustments to a drug’s design. By doing so, issues that could cause problems later on in the development process can be fixed or minimized in advance, making the drug more reliable and easier to produce. It’s all about starting smart so you can progress fast while balancing efficacy, safety, and ease of production,” says Bunce.

Along with a multifaceted approach, successful linker optimization requires collaboration of many people with expertise in a wide range of fields. “The ADC field is complicated, and chemistry and biology teams need to work in parallel to create an efficacious drug. It is common for situations to occur when a chemistry problem is resolved, only to cause a new, unexpected biological problem,” comments Xiao Cai, senior scientist in chemistry at Catalent. He stresses that scientists need to bear in mind that an ADC has highly cross-functional composition, so timely and effective communication between chemists and biologists is imperative for their successful design.

While much attention goes to the scientific and technical aspects of optimizing ADC linker chemistries, Bunce believes that ethical and environmental concerns also deserve a spotlight. “Not only the materials used in creating linkers could pose environmental risks if not managed properly. Some of the manufacturing processes are energy-intensive and may warrant exploration of more sustainable alternatives,” he suggests. On the ethical front, Bunce says it is critical to consider the accessibility and affordability of advanced ADC technologies, especially in low-resource settings.

“These aspects often go under the radar but could play a significant role in the broader acceptance and deployment of new ADC technologies,” Bunce contends. “A truly integrated approach that goes beyond just the science could be instrumental in shaping the future of ADC development,” he concludes.

One of the most important points about linker optimization that these experts all stress is the fact that there is no one solution that fits all ADCs. “Linkers will differ from case to case, and there is no one universal solution to their design, so an application-driven approach is needed,” states Chuprakov.

That is largely because, Chuprakov explains, a linker is not only a connector, but also has embedded engineered functionality, such as a desired level of stability during circulation, and an optimal rate of cleavage by intramolecular machinery after ADC internalization has released the drug. Once a linker’s functionality has been established, then additional design elements can be added, particularly those that address biophysical properties of the resulting conjugate, such as hydrophilicity, while simultaneously factoring in CMC considerations.

Jenkins agrees, stressing there is also no such thing as “the perfect linker.” “Both the antibody and payload will dictate what linker is ultimately chosen, while subsequent testing is likely to determine what further optimization needs to be undertaken,” she says.

Cynthia A. Challener, PhD, is a contributing editor to 

A look at how linker technology for antibody drug conjugates has evolved.

Exploring the Optimization of Linker Chemistries for ADCs

On Dec. 5, 2023, AbbVie and BigHat Biosciences, a US-based biotech firm, announced that they have entered into a research collaboration to discover and develop next-generation therapeutic antibodies in oncology and neuroscience. The collaboration will utilize BigHat’s Milliner platform, which is a suite of machine learning (ML) technologies integrated with a high-speed wet lab, to guide the design and selection of high-quality antibodies.

Under the agreement, BigHat will receive an upfront payment of $30 million and may be eligible for up to approximately $325 million in R&D milestones. The company also has the potential to further earn commercial milestone payments as well as tiered royalties on net sales.

"This collaboration further demonstrates our commitment to integrate AI [artificial intelligence]/ML-based approaches in drug discovery and development, as we strive to accelerate our oncology and neuroscience pipeline, and deliver better medicines faster," said Jonathon Sedgwick, vice-president and global head of discovery research at AbbVie, in a company press release. "We look forward to working with BigHat's team to design novel antibody-based therapies that may help address persistent unmet needs faced by patients."

"We are thrilled to collaborate with AbbVie, a global leader in driving innovation in drug development, and look forward to employing our Milliner platform to help design next-generation therapeutic antibodies," said Mark DePristo, CEO, BigHat, in the release. "Milliner allows rapid optimization of multiple key parameters, including functionality and developability, to help deliver complex antibodies with improved characteristics faster."

This collaboration follows AbbVie’s earlier move to 

News

This collaboration will combine BigHat Biosciences’ AI/ML-guided Milliner platform with AbbVie's expertise in oncology and neuroscience to develop next-generation antibodies.

AbbVie Collaborates with BigHat Biosciences on Next-Gen Therapeutic Antibodies

Absci, a generative artificial intelligence (AI) drug creation company, announced on Dec. 4, 2023 that it is collaborating with AstraZeneca to deliver an antibody to treat cancer that is designed with AI. AstraZeneca will use Absci’s Integrated Drug Creation platform to accelerate the discovery of potential cancer treatments. The agreement includes an upfront commitment, R&D funding and milestone payments, and royalties on product sales.

The Integrated Drug Creation platform uses generative AI and scalable wet-lab technologies to create proprietary data by measuring millions of protein–protein interactions, according to Absci. The data are used to train the AI models to validate antibodies using the 

 AI models. Drug discovery may be accelerated by data collection, AI-driven design, and wet-lab validation, and potentially optimize multiple drug attributes concurrently.

“AstraZeneca is a leader in developing novel treatments in oncology, and we are excited to collaborate with them to design a therapeutic candidate antibody with the potential to improve the lives of cancer patients,” said Sean McClain, Absci founder and CEO, in a press release. “This agreement advances Absci’s goal of creating a new generation of life-changing and transformative therapeutics using its AI platform.”

“This collaboration is an exciting opportunity to utilize Absci’s 

 AI antibody creation platform to design a potential new antibody therapy in oncology,” said Puja Sapra, senior vice-president of Biologics Engineering & Oncology Targeted Delivery, AstraZeneca, in the release.

The Absci/AstraZeneca agreement follows the publication of Absci’s design and validation of 

 antibodies using the generative AI model, as well as previous collaborations with other organizations.

AstraZeneca will utilize Absci’s AI antibody drug creation platform to create an AI-designed antibody.  

Absci and AstraZeneca Collaborate on AI-Driven Oncology Candidate

In sickle cell anemia mishappen and sticky hemoglobin cells cannot carry oxygen properly and frequently clump, clogging blood vessels and sometimes causing searing pain termed a vaso-occlusive crisis. It is debilitating. β-thalassemia isn’t etiologically closely related, however, similar to sickle cell disease (SCD), genetic defects are passed on from both parents, which leads to low hemoglobin production. This creates fatigue, jaundice, shortness of breath and irregular heartbeats. A fresh (Nobel prize winning) tool to redress both mutational situations was recently given Medicines and Healthcare products Regulatory Agency (MHRA) approval, in a world-first approval for complicated next-generation therapeutics (

). Clustered regularly interspaced short palindromic repeats (CRISPR) was successfully applied to both disease states, in a therapeutic called Casgevy (exagamglogene autotemcel, or exa-cel).

CRISPR is so new that this MHRA approval caused quite a stir in both the medical and pharmaceutical worlds. Even at the end of November 2023 the American society of Hematology didn’t appear to convey much about this news, nor the attendant questions and issues that must still be addressed. These include potential immunogenicity because of biology outside of the CRISPR itself, and also access alongside cost and reimbursement.

Casgevy galvanizesproduction of a form of hemoglobin normally made only in developing fetuses. “Production of this fetal hemoglobin is typically shut off soon after birth by a gene called 

, allowing fetal-hemoglobin production to resume. This provides some hemoglobin that is not misshapen and dampens the effects of the abnormal form. Vertex and CRISPR Therapeutics reported that, at nine months after treatment, 31 of 32 participants in their clinical trial had not had a single vaso occlusive crisis. Before treatment, they’d had an average of about four each year” (2).

CRISPR remains somewhat controversial for potential off-target effects. On top of this, after it makes its cut in the DNA of the 

 gene, CRISPR relies on the cell’s natural DNA repair machinery to stitch the strands back together. The step can introduce new errors. All of this is well known and becoming better understood; however, in its advisory committee briefing, Vertex stated: “There is no known or hypothesized mechanism whereby a DNA edit would revert to reduce the reactivation offetal hemoglobin (HbF) after exa-cel treatment. The long-term engraftment of edited cells also demonstrates the successful editing of long-term hematopoietic stem cell (HSCs) that are known to persist for a person’s lifetime” (3).

What is potentially equally worrisome for Casgevy are new findings from Wellcome Sanger Institute, University of York, and Boston Children’s Hospitalscientists outlining an observation that “cell competition” following gene therapy, may increase leukemia incidence. “The cause is likely not the gene therapy treatment itself, say researchers, but the therapy process, which involves gene editing outside the body and re-transplantation of stem cells back into the patient.” The authors go on to say “Instead, the process of genetically modifying these stem cells outside the body and re-transplanting them back into the patient makes blood stem cells that already have these mutations more prominent, thereby increasing their influence on the blood and immune systems.” The authors hypothesize younger patients might be an easier cohort to manage these therapies through, as their stem cells inherent have fewer mutations to “select” as triggering undesirable outcomes, and that “Continuously tracking these mutations and gaining a deeper understanding of these processes will profoundly impact the future well-being of sickle cell patients around the world.” (4)

While they are approximately 100,000 SCD patients in the United States, the disease seems related to malaria, and hence the vast majority of sufferers live in resource-poor nations. Vertex Pharmaceuticals said it had not yet established a price for the treatment in Britain and was working with health authorities “to secure reimbursement and access for eligible patients as quickly as possible. In the US, Vertex has not released a potential price for the therapy, but a report by the nonprofit Institute for Clinical and Economic Review said prices up to around $2 million would be cost-effective. By comparison, research earlier this year, showed medical expenses for current sickle cell treatments, from birth to age 65, add up to about $1.6 million for women and $1.7 million for men.” (5)

Not only cost but access to the “manufacture” of this treatment is also a barrier to widespread implementation. “In the US, there are approximately 200 specialized centers for bone marrow transplants. There are only three such centers for all of sub-Saharan Africa—in Nigeria, Tanzania, and South Africa. Making these types of CRISPR-based therapies available throughout the continent will require massive investment in clinical infrastructure. The road from now, having gene editing that is possible and implemented where most people live, it’s not that close,” said Ambroise Wonkam, a geneticist at Johns Hopkins School of Medicine and the University of Cape Town who also serves as president of the African Society of Human Genetics. “We have to put that into perspective” (6).

Casgevy was used here as synecdochic for the surging tidal wave of new modalities. The choice might well have fallen to ARCT-154, a self-amplifying messenger RNA (sa-mRNA) COVID-19 vaccine for initial vaccination and booster for adults 18 years and older, which was even more recently given approval by Japan's Ministry of Health, Labor and Welfare (MHLW). While ARCT-154 was tested on 16,000 patients compared to Casgevy in 40 patients only, and. while the cost and access is no barrier compared with Casgevy, both therapeutics herald new ways of thinking, new types of testing and validation, and new types of manufacture and end-point monitoring. Following on from the revolutionary COVID-19 vaccines, what comes next is more complicated, more difficult to price for reimbursement, and more effective in terms of cure rates. The toothpaste is out of the tube, and the industry must learn to adapt to take best advantage of what is becoming available, no matter how messy the process.

1. MHRA. MHRA Authorizes World-First Gene Therapy that Aims to Cure Sickle-Cell Disease and Transfusion-Dependent β-thalassemia. Press Release, Nov. 16, 2023
2. Ledford, H. Is CRISPR Safe? Genome Editing Gets Its First FDA Scrutiny. 

 (7986), 234–235. DOI: 10.1038/d41586-023-03317-7
3. FDA. 

Cellular, Tissue, and Gene Therapies Advisory Committee October 31, 2023 Meeting Announcement

, Oct. 31, 2023.
4. Wellcome Sanger Institute. New Research Sheds Light on Cancer Risk in Gene Therapies. 

, Nov. 16, 2023.
5. Cheng, M. The World’s First Gene Therapy for Sickle Cell Disease Has Been Approved in Britain. 

, Nov. 16, 2023.
6. Molteni, M. With CRISPR Cures on Horizon, Sickle Cell Patients Ask Hard Questions about Who Can Access Them

Chris Spivey is the editorial director for 

Exagamglogene autotemcel seen as synechdoche.

New Medicines, Markets, and Manufacture: CRISPR for Sickle Cell Disease and β-thalassemia

TAU Systems, a US-based producer of compact laser-driven plasma accelerators and light sources, announced on Nov. 21, 2023 that it has signed a lease for a state-of-the-art building in Carlsbad, Calif., in which it will establish TAU Labs. The facility will be the first of the company’s light source applications and R&D centers, which will give users easier access to the latest particle and imaging systems. The first phase of construction is expected to be complete in 2024, according to a company press release.

The company is developing compact particle accelerators and specialized X-ray sources that combine the capabilities of large accelerators with a small footprint. This design is meant to provide easy and affordable beam-time access for any company or research institute, and, by doing so, the facility democratizes accelerator access, allowing for the progress of semiconductors, space-bound electronics, battery technology, biotechnology, and nuclear energy, among other industries, according to the press release.

TAU Systems' particle and light sources comprise the electromagnetic spectrum—from ultraviolet (UV) to gamma wavelengths—and will be installed in a 22,300-ft

 building located at the Carlsbad Research Center. The lab space will house several laser-driven particle and light sources as well as broad technical expertise. Industrial users will have wide access to extreme UV and X-ray light sources as well as million electron volt electrons and other particle beams.

"Laser-driven particle accelerators are incredibly powerful tools that enable unprecedented advances in a wide range of fields, from next-generation semiconductors and batteries, and custom-designed medicines to the advancement of fundamental physics. However, getting beam time on the very few facilities available globally is stifling progress,” said Bjorn Manuel Hegelich, CEO, TAU Systems, in the press release. “TAU's application centers will democratize access to these exciting machines and expedite crucial technological breakthroughs."

"Our high-repetition-rate laser driver also makes TAU Labs an ideal location to test the resistance to space radiation of satellite electronics. It will help make space systems more robust and prolong the life of satellites," said Jerome Paye, chief operating officer, TAU Systems, in the press release.

TAU Systems plans to establish TAU Labs, a new next-generation, laser-driven plasma accelerator laboratory, in Carlsbad, Calif.

TAU Systems to Establish TAU Labs, a New Laser Plasma Accelerator Application Center

On Nov. 21, 2023, Genentech, a member of the Roche Group, announced that it has entered into a multi-year strategic research collaboration with NVIDIA, a US-based computing company. The companies plan to combine Genentech’s artificial intelligence (AI) capabilities, extensive biological and molecular datasets, and research expertise with NVIDIA’s accelerated computing capabilities and AI to speed up drug discovery and development.

The collaboration is meant to significantly enhance Genentech’s advanced AI research programs by turning the company’s generative AI models and algorithms into a next-generation AI platform, which would be designed to expedite the discovery and delivery of novel therapies. Under the agreement, the companies aim to accelerate and optimize Genentech’s proprietary machine learning algorithms and models on the NVIDIA DGX Cloud. The cloud provides a training-as-a-service platform that has been built on dedicated NVIDIA AI supercomputing and software, including NVIDIA BioNemo for generative AI applications in drug discovery.

Also under the agreement, NVIDIA will share its computing expertise with Genentech’s teams of computational scientists, under which Genentech’s models will be optimized and scaled. This process may also improve or enhance NVIDIA’s platforms.

The collaboration will also help accelerate Genentech’s “lab in a loop”, in which extensive experimental data will feed computational models to uncover patterns and make new, experimentally testable predictions. Scientists can then quickly assess these predictions in the lab, and the results are fed back into the models to improve the underlying computational model. This loop is expected to allow for the iterative development of better therapies, according to a company press release.

“By harnessing the power of AI models and algorithms with our unique data and experiments, we're unlocking scientific discoveries with incredible speed and generating insights at an unprecedented scale,” said Aviv Regev, executive vice-president and head of Genentech Research and Early Development (gRED), in the press release. “Bringing science and technology together has always been a foundation of biomedical breakthroughs at Genentech. We are thrilled to join forces with NVIDIA to further optimize our drug discovery and development to deliver treatments that transform people’s lives.”

“The greatest impact of generative AI is to revolutionize the life science and healthcare industry,” said Jensen Huang, founder and CEO of NVIDIA, in the release. “Our collaboration to create Genentech’s next-generation AI platform will dramatically accelerate the pace of drug discovery and development.”

Under this multi-year collaboration, Genentech and NVIDIA will use AI to optimize and accelerate each company’s platforms to discover and develop innovative therapeutics.

Genentech and NVIDIA Form AI Research Collaboration for Drug Discovery and Development

MilliporeSigma, the US and Canada Life Science business of Merck KGaA, Darmstadt, Germany, announced on Nov. 21, 2023 that it has opened the second phase of its new €29 million (US$43 million) Biologics Testing Center in Shanghai, China, which opened in 2022. The expansion includes an added 1500 m2 of lab space, which are the first biosafety laboratories for the company in the Chinese market. This expansion enables the company to serve local clients, according to a company press release.

The new lab space offers testing services for cell line characterization and lot release from pre-clinical development to commercialization. MilliporeSigma can now offer biosafety testing services for monoclonal antibodies, other recombinant proteins, and cell and gene therapies to the local market.

The first phase of the new testing center allowed the company to provide testing services for viral clearance studies, while the second phase adds capabilities for cell line characterization to help ensure the safety, purity, and identity of cell banks. The testing center also offers current good manufacturing practice-compliant lot release testing for unprocessed and purified bulk harvest for meeting the requirements for biologics that are entering preclinical and clinical studies and for licensed biologics.

“The opening of the facility deepens our partnership with our Chinese clients who are at the forefront of shaping modern medicine,” said Dirk Lange, head of Life Science Services, Life Science business sector of Merck KGaA, Darmstadt, Germany, in the press release. “The Biologics Testing Center in Shanghai now provides critical local services backed by our more than 75 years of global experience in the testing market.”

MilliporeSigma has expanded its biosafety testing laboratories in Shanghai, China, with the completion of new lab space at its new €29 million (US$43 million) Biologics Testing Center.

MilliporeSigma Expands Biologics Testing Center in China

Twist Bioscience, a synthetic biology and genomics company, announced on Nov. 14, 2023 that it has launched Express Genes, a new gene synthesis service with a quick turnaround time of five to seven business days. The service, housed in the company’s Wilsonville, Ore., manufacturing facility allows for the rapid production of genes.

Twist’s technology involves the miniaturization of the chemical reaction that creates DNA. The company does this using its silicon-based DNA synthesis platform coupled with expertise, software, honed processes, and an expanded layout at its Wilsonville facility. The technology allows the company to deliver clonal genes and gene fragments at scale and with rapid turnaround times.

“Twist Express Genes is our first new offering enabled by the increased capacity and streamlined processes in our Wilsonville facility. We are now able to deliver the same NGS [next-generation sequencing]-verified clonally perfect genes that our customers have come to expect from Twist in about half the time, at an unprecedented scale. We offer the rapid turnaround that our customers need in multiple formats and at the same speed for one or thousands of genes. With this new offering, we will focus initially on our current customers who we know are making some of their own genes,” said Emily M. Leproust, CEO and co-founder of Twist Bioscience, in a company press release. “We look forward to enabling the ‘Maker’s Market’ to continue to push the boundaries of what’s possible, working to uncover new therapeutic modalities for devastating diseases, ushering in sustainable applications for enzyme engineering and chemical development, and much more. They clone their own genes because otherwise they would have to delay their research while waiting for genes they ordered, and now they won’t have to make that compromise.”

“We surveyed several hundred gene users and found that in addition to those who make their own genes entirely, a large portion of respondents both purchase DNA from a supplier and in parallel make their own,” said Anne St. Louis, director at Percepta Associates, in the press release. “These data suggest that there is a market to capture from researchers in industry and academia who currently use vendors for only part of their total gene production.”

"As a gene therapy startup developing programmable RNA medicines, we routinely generate diverse and customized DNA sequences to advance our technologies and enable application to multiple disease targets. Being able to order sequence-verified large synthetic gene constructs in just one week expands what we can screen and allows faster iterations of the design-build-test cycle," said Susan Byrne, senior principal scientist, Shape Therapeutics, in the press release.

New Technology

Twist Bioscience now offers its Express Genes rapid gene synthesis service at its Wilsonville, Ore., manufacturing facility.

Twist Bioscience Launches Gene Synthesis Service

On Nov. 14, 2023, Lonza announced the launch of its new cell line, GS Effex, which is intended for the development of therapeutic antibodies with enhanced potency. Derived from the company’s GS Xceed cell line, GS Effex was developed to meet the development needs for more sophisticated therapeutic antibodies. The new cell line combines the characteristics of good cell growth with the ability to produce therapeutic antibodies with increased potency.

GS Effex is a stable, scalable, and productive cell line that produces Fc domains free of fucose, which, according to Lonza, increases the antibody-dependent cellular cytotoxicity (ADCC) function and therapeutic potency of the final antibody. GS Effex is available under license or as a complete service offering.

"Lonza is committed to providing our customers with the right tools to develop their novel therapies. Leveraging more than 35 years of protein expression expertise, more than 75 molecules have been manufactured and brought to the clinic using our expression platforms,” said Ulrich Osswald, vice-president, Licensing, Lonza, in a company press release. “The new GS Effex cell line represents an enhanced solution especially relevant for the oncology field since improved ADCC represents a vital enhancement of the therapeutic success of an antibody product."

“At Lonza, we develop and invest in novel, disruptive technologies with potential to transform the development, analytics, and manufacturing of established and novel biotherapeutics,” said Atul Mohindra, head of Biologics R&D, Lonza, in the press release. “The GS Effex cell line provides a reliable protein expression platform with the needs of our customers in mind. This cell line enables the efficient production of monoclonal antibodies with increased ADCC, while also delivering the required product quality and efficacy profile.”

Lonza’s new GS Effex cell line can help overcome the challenges of developing therapeutic antibodies with increased potency.

BioPharm International's BP Elements, December 2023