Characterizing clonal evolution in blood cancers like acute myeloid leukemia (AML) is critical for understanding their mutational histories and how cell populations change during disease development.

In this GEN webinar, Robert Bowman, PhD, will discuss his lab’s approaches for modeling clonal evolution in mouse models of disease. His group deploys multi-recombinase models to study the stepwise acquisition of mutations seen in AML. These approaches allow for the evaluation of how mutation order impacts disease development. They have characterized the hierarchy of cellular differentiation using flow cytometry and single cell RNA sequencing, recently integrating the ScaleBio Single Cell RNA Kit into their workflow. He will discuss a specific study focusing on FLT3-mutant AML, present data comparing genetic deletion versus chemical inhibition with FDA-approved tyrosine kinase inhibitors, and finally, his plans to further deploy models of oncogene-dependency.

A live Q&A session will follow the presentation, offering you a chance to pose questions to our expert panelist.

Webinar produced with support from:

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These so-called microproteins or noncoding ORF-derived (ncORF) peptides are small proteins expressed only by cancer cells that can be used to activate the immune system against cancer. Furthermore, these molecules are generated by genes that were once considered incapable of encoding proteins. The scientists discovered the proteins in this study by integrating and analyzing information from tumor and healthy tissue collected from over a hundred patients with hepatocellular carcinoma including RNA sequencing, immunopeptidomics, and ribosome profiling data. 

There is significant interest in cancer vaccines which rely on the immune system’s ability to recognize foreign proteins generated as a result of mutations in cancerous cells. The challenge lies with cancers with low mutation rates like liver cancers. Microproteins could be a solution in these cases. The results reported in the paper highlight the potential of using microproteins exclusively expressed in tumor cells as targets for new treatments. Specifically, the researchers identified “a subset of 33 tumor-specific long noncoding RNAs expressing novel cancer antigens shared by more than 10% of the HCC samples analyzed, which, when combined, cover a large proportion of the patients,” according to the paper.

In fact, “we have seen that some of these microproteins can stimulate the immune system, potentially generating a response against cancer cells,” said Puri Fortes, PhD, one of the paper’s authors and a researcher at CIMA as well as the Network Biomedical Research Center for Liver and Digestive Diseases (CIBERehd). According to the paper, when the team tested four ncORF-derived peptides in transgenic mice, they found that two of them could generate a significant immune response involving CD8+ T cells.  “This response can be enhanced with vaccines, similar to the coronavirus vaccines, but producing these microproteins. These vaccines could stop or reduce tumor growth,” said Fortes. 

Also, unlike other types of vaccines based on patient-specific mutations, a potential anticancer vaccine that targets ncORF peptides could be used to treat multiple people, as the same microprotein is expressed in various patients, the researchers noted. 

The post Liver Tumor Microproteins Could Be Key to Developing New Cancer Vaccines appeared first on GEN - Genetic Engineering and Biotechnology News.

In general, gene drives involve the propagation of genetic material through a population. The goal: Direct the evolution of the population to instigate the population’s collapse, all while preventing the population from developing resistance.

The gene drive approach appealed to scientists at Penn State who were frustrated by the way cancer treatment can turn into a deadly version of the “Whac-A-Mole” game. During treatment, various kinds of resistance from various cancer cell populations can pop up at any time. Rather than play the old game more vigorously, or wield additional mallets, the scientists decided to play a new game. They created a modular genetic circuit that turns cancer cells into a “Trojan horse,” causing them to self-destruct and kill nearby drug-resistant cancer cells. Tested in human cell lines and in mice as proof of concept, the circuit outsmarted a wide range of resistance.

The findings were published in Nature Biotechnology, in an article titled, “Programming tumor evolution with selection gene drives to proactively combat drug resistance.” The researchers also filed a provisional application to patent the technology described in the paper.

“We show that tumor evolution can be reproducibly redirected to engineer therapeutic opportunity, regardless of the exact ensemble of pre-existing genetic heterogeneity,” the article’s authors wrote. “We develop a selection gene drive system that is stably introduced into cancer cells and is composed of two genes, or switches, that couple an inducible fitness advantage with a shared fitness cost.

The idea, the scientists explained, is to combine selection (redirecting tumor evolution toward more benign states) with bystander function (taking advantage of the bystander-killing activity that suicide gene–carrying cells have against neighboring cells).

“Our complete dual-switch circuits demonstrate the ability to eliminate preexisting resistance, including complex genetic libraries of resistance variants within a drug target and across the genome,” the authors continued. “Finally, model-guided switch engagement demonstrates robust efficacy in vivo, highlighting the benefits of leveraging evolutionary principles rather than combating them.”

“This idea was born out of frustration,” said Justin Pritchard, PhD, a professor of biomedical engineering at Penn State and the senior author on the paper. “We’re not doing a bad job of developing new therapeutics to treat cancer, but how can we think about potential cures for more late-stage cancers?

“Selection gene drives are a powerful new paradigm for evolution-guided anticancer therapy. I love the idea that we can use a tumor’s inevitability of evolution against it.”

Newer personalized cancer medicines often fail, not because the therapeutics aren’t good but because of cancer’s inherent diversity and heterogeneity, Pritchard explained. Even if a frontline therapy is effective, resistance eventually develops and the medication stops working, allowing the cancer to return. Clinicians then find themselves back at square one, repeating the process with a new drug until resistance emerges again. The cycle escalates with each new treatment until no further options are available.

The gene drive idea, the researchers reasoned, could be used to eliminate resistance mechanisms before cancer cells had a chance to evolve and pop up unexpectedly. Also, it could be used to force a specific resistance mechanism to emerge—one that they would prefer to see, one that they would be prepared to fight.

What started as a thought experiment is proving to work. The team created a modular circuit, or dual-switch selection gene drive, to introduce into non-small lung cancer cells with an EGFR gene mutation. This mutation is a biomarker that existing drugs on the market can target.

The circuit has two genes, or switches. Switch one acts like a selection gene, allowing the researchers to turn drug resistance on and off, like a light switch. With switch one turned on, the genetically modified cells become temporarily resistant to a specific drug, in this case, to a non-small lung cancer drug. When the tumor is treated with the drug, the native drug-sensitive cancer cells are killed off, leaving behind the cells modified to resist and a small population of native cancer cells that are drug-resistant. The modified cells eventually grow and crowd out the native resistant cells, preventing them from amplifying and evolving new resistance.

The resulting tumor predominantly contains genetically modified cells. When switch one is turned off, the cells become drug-sensitive again. Switch two is the therapeutic payload. It contains a suicide gene that enables the modified cells to manufacture a diffusible toxin that’s capable of killing both modified and neighboring unmodified cells.

“It not only kills the engineered cells, but it also kills the surrounding cells, namely the native resistant population,” Pritchard said. “That’s critical. That’s the population you want to get rid of so that the tumor doesn’t grow back.”

The team first simulated the tumor cell populations and used mathematical models to test the concept. Next, they cloned each switch, packaging them separately into viral vectors and testing their functionality individually in human cancer cell lines. They then coupled the two switches together into a single circuit and tested it again. When the circuit proved to work in vitro, the team repeated the experiments in mice.

However, the team didn’t just want to know that the circuit worked; they wanted to know it could work in every way. They stress tested the system using complex genetic libraries of resistance variants to see if the gene drive could function robustly enough to counter all the genetic ways that resistance could occur in the cancer cell populations.

And it worked: Just a handful of engineered cells can take over the cancer cell population and eradicate high levels of genetic heterogeneity. Pritchard said it’s one of the biggest strengths of the paper, conceptually and experimentally.

“The beauty is that we’re able to target the cancer cells without knowing what they are, without waiting for them to grow out or resistance to develop because at that point it’s too late,” Leighow said.

The researchers are currently working on how to translate this genetic circuit so that it can be delivered safely and selectively into growing tumors and eventually metastatic disease.

The post Cancer Treatment Approach Targets Resistance, Gene Drive Style appeared first on GEN - Genetic Engineering and Biotechnology News.

The number of genetic mutations in a cancer cell was previously thought to be purely down to chance. But a new study “Homopolymer switches mediate adaptive mutability in mismatch repair-deficient colorectal cancer,” published in Nature Genetics, has provided insights into how cancers navigate an evolutionary balancing act.

The scientists found that mutations in DNA repair genes can be repeatedly created and repaired, acting as genetic switches that take the brakes off a tumor’s growth or put the brakes back on, depending on what would be most beneficial for the cancer to develop.

The team believes the findings could potentially be used in personalized cancer medicine to gauge how aggressive an individual’s cancer is so that they can be given the most effective treatment.

DNA repair mechanisms

Disruption of DNA repair mechanisms is a major cause of increased cancer risk. About 20% of bowel cancers, known as mismatch repair deficient (MMRd) cancers, are caused by mutations in DNA repair genes. But disrupting these repair mechanisms is not entirely beneficial to tumors. Though they do allow tumors to develop, each mutation increases the risk that the body’s immune system will be triggered to attack the tumor.

“Cancer cells need to acquire certain mutations to circumvent mechanisms that preserve our genetic code,” says Marnix Jansen, senior author of the study. “But if a cancer cell acquires too many mutations, it is more likely to attract the attention of the immune system, because it’s so different from a normal cell.

“We predicted that understanding how tumors exploit faulty DNA repair to drive tumor growth, while simultaneously avoiding immune detection, might help explain why the immune system sometimes fails to control cancer development.”

In this study, researchers from UCL analyzed whole genome sequences from 217 MMRd bowel cancer samples in the 100,000 Genomes Project database. They looked for links between the total number of mutations and genetic changes in key DNA repair genes.

The team identified a strong correlation between DNA repair mutations in the MSH3 and MSH6 genes, and an overall high volume of mutations.

The theory that these “flip-flop” mutations in DNA repair genes might control cancer mutation rates was then validated in complex cell models, called organoids, grown in the lab from patient tumor samples.

“Our study reveals that DNA repair mutations in the MSH3 and MSH6 genes act as a genetic switch that cancers exploit to navigate an evolutionary balancing act,” notes Suzanne van der Horst from University Medical Center Utrecht.” On one hand, these tumors roll the dice by turning off DNA repair to escape the body’s defense mechanisms. While this unrestrained mutation rate kills many cancer cells, it also produces a few ‘winners’ that fuel tumor development.

“The really interesting finding from our research is what happens afterwards. It seems the cancer turns the DNA repair switch back on to protect the parts of the genome that they too need to survive and to avoid attracting the attention of the immune system. This is the first time that we’ve seen a mutation that can be created and repaired over and over again, adding it or deleting it from the cancer’s genetic code as required.”

The researchers say that this knowledge could potentially be used to gauge the characteristics of a patient’s tumor, which may require more intense treatment if DNA repair has been switched off and there is potential for the tumor to adapt more quickly to evade treatment, particularly to immunotherapies, which are designed to target heavily mutated tumors.

A follow-up study is already underway to find out what happens to these DNA repair switches in patients who receive cancer treatment.

The post Bowel Cancer Cells Can Regulate Their Growth to Avoid Immune System Detection appeared first on GEN - Genetic Engineering and Biotechnology News.

“We hope that this proof-of-concept study is the first of many applications of engineered plasma B cells, and eventually will lead to a single-shot therapeutic,” said Richard James, PhD, at the Seattle Children’s Research Institute. “Because engineered plasma B cells can live for a very long time, greater than 10 years, they could be used as a long-term source of many biologic drugs.” James is senior author of the team’s published paper in Molecular Therapy, titled “Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing,” in which they stated, “These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.”

Immunotherapies such as bispecific antibodies that recruit cytotoxic T cells to kill cancer cells have contributed to improved survival rates for patients with B cell acute lymphoblastic leukemia (B-ALL), the authors wrote. Blinatumomab is a bispecific antibody that has been approved for about a decade, for treating patients with relapsed/refractory B-ALL. The authors explained, “Blinatumomab is an anti-CD19 x anti-CD3 non-immunoglobulin G-like bispecific antibody (non-IgG-like bispecific; also called a bispecific T cell engager) that received approval from the U.S. Food and Drug Administration in 2014 for the treatment of patients with relapsed/refractory B-ALL.” However, a limitation of blinatumomab therapy is that it requires continuous high-dose intravenous infusions to maintain activity. “Bispecific non-immunoglobulin therapies pose stability challenges in patients, necessitating three courses of 20-day steady-state infusion,” James said. This intensive regimen poses challenges for patients because frequent bag changes prove inconvenient, and the use of ports increases the risk of infection. The authors further poited out, “Enhanced drug delivery methods for bispecific antibodies like blinatumomab could improve patient adherence and bolster treatment efficacy.”

Work by James and others has explored using engineered plasma cells (ePCs) for long-term protein drug delivery, the team continued. Engineered B cells have been investigated in proof-of-concept studies to deliver biologic drugs to treat protein deficiency diseases, viral infections and cancer. Such cells are “uniquely suited to deliver biologics over long period” due to their long lifespan and high secretory capacity,” the team continued. “Because a subset of PCs and ePC preferentially localize to bone marrow and other tissue microenvironments where progenitor B-ALL cells reside, we predicted that ePCs could harmonize with local bispecific delivery to induce potent anti-leukemia activity.”

James added, “We think that the first application of engineered plasma B cells will be to produce drugs that are difficult for patients to use. In this study, we wanted to demonstrate proof of concept and efficacy for engineered B cell therapies.”

For their reported study the investigators developed a gene-editing strategy for generating ePCs that produce large quantities of bispecifics to target B-ALL or acute myeloid leukemia. “… we describe a homology-directed repair (HDR)-based gene editing strategy for the generation of ePC that produce large quantities of anti-CD19 x anti-CD3 or anti-CD33 x anti-CD3 non-IgG-like bispecifics to target B-ALL or acute myeloid leukemia (AML), respectively,” they wrote. The combined findings of their experiments demonstrated that ePCs secreting bispecifics can promote T cell-driven killing of primary human cells and human leukemic cell lines.

“One challenge we encountered was that the bispecific antibody used for killing tumor cells can also bind the engineered plasma B cells because they express the same target protein,” James stated. “To overcome this challenge, we deleted the protein target of the antibody, CD19, when we were making the engineered cells. We were surprised that deletion of CD19 did not hinder manufacturing of engineered plasma B cells.”

In addition, the researchers discovered that plasma cells secreting anti-CD19 bispecific antibodies elicited antitumor activity, as demonstrated with acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells.

Notably, the steady-state concentration of anti-CD19 bispecifics in serum one month after cell delivery and tumor eradication was comparable to that observed in patients treated with continuous infusion of blinatumomab. “… we obtained in vivo serum concentrations of the bispecific surpassing that of the steady-state plasma concentration (CPss) seen in patients undergoing continuous infusion of blinatumomab,” the investigators noted.

Based on their results the researchers propose that ePC strategies could increase the functional half-life of bispecifics in patients with acute leukemias and other diseases where treatment half-life is limiting or where plasma cell local delivery could enhance therapeutic efficacy. The results also suggest that prolonged clinically relevant levels of bispecific and perhaps other biologics can be achieved via a single administration of ePCs. “The robust levels of bispecific achieved by ePCs compare favorably with those observed by other bispecific-secreting cell products, including macrophages and T cells, which did not produce detectable levels in serum after in vivo transfer,” the authors stated.

They suggest that their findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias and potentially other cancers. “We created engineered plasma B cells capable of continuously producing bispecific antibodies throughout the treatment period after only one injection,” James noted. “These cells effectively eliminated tumors to a comparable extent as the clinical drug. The key takeaway is that engineered plasma B cells can provide long-lasting drug production in vivo.”

The team acknowledged that ePC bispecifics should be carefully evaluated for several possible toxicities if used clinically. Persistent on-target, off-tumor toxicity to normal bystander B cells is common in patients receiving B cell-targeted therapeutics. “In addition, for treatment of a B cell malignancy, it may be difficult to engineer a patient’s own B cells to be used as the therapy because there is a risk that some of the B cells will be cancerous,” James commented. “We did not test whether we can use a different person’s B cells to produce the bispecific antibody. Studies using such allogeneic products will likely need to be done before this specific therapy can be used to treat B cell cancers.”

As noted by the authors, further studies in humanized mice and in non-human primates are warranted to fully understand the activity, longevity, and tissue localization of ePCs. In the short term they plan to test whether engineered plasma B cells that produce bispecific antibodies are effective in other B cell-mediated diseases, including autoimmunity. These tests will initially be conducted in animal models. Additionally, the researchers are developing engineered plasma B cells to produce other therapeutic drugs, such as those needed in protein deficiency diseases such as hemophilia. They are exploring other potential applications of engineered B cells, including modifying other immune cells to either enhance or suppress the immune system.

In their paper the authors concluded, “Our findings suggest that ePCs may provide benefits for delivery of protein therapeutics beyond delivery of bispecifics as studied here … The potential for ePCs to persist long term and produce robust levels of exogenous protein could be a key to unlocking the therapeutic potential of biologics or therapeutic peptides limited by poor pharmacokinetics.”

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Previously, the Karolinska team developed nanostructures capable of housing six peptides assembled in a hexagonal pattern. Under the right conditions, “this hexagonal nanopattern of peptides becomes a lethal weapon,” said Björn Högberg, PhD, a professor in the department of medical biochemistry and biophysics at Karolinska and senior author on the study. That’s because these peptides can “autonomously and selectively turn on the display of cytotoxic ligand patterns in tumor microenvironments” to trigger the apoptosis machinery in cells.

The challenge, however, is that the peptides can also interact with healthy cells so “if you were to administer it as a drug, it would indiscriminately start killing cells in the body,” Högberg explained. “To get around this problem, we have hidden the weapon inside a nanostructure built from DNA.” 

As described in the paper, the origami that the team designed is “an asymmetric double cylinder with a 24-nm-tall hollow head and a 15-nm-tall solid stem. The head has a 14-nm-deep cavity, where specific oligonucleotides … are located to act as bridges between the typical origami staples and a binding site for ligand-decorated oligos.” These oligonucleotide or mini-scaffolds are used to “hybridize a peptide ligand-functionalized oligo, bearing one sequence that forms the double helix (the hybridization region) with the mini-scaffold as well as bearing an additional sequence of triplex-forming oligo (TFO),” the researchers wrote. 

The key to activating the kill switch is the low pH typically found in the acidic microenvironment surrounding cancer cells. Specifically, “when the pH drops, the TFO of the peptide–DNA conjugate forms a tsDNA that forces the six peptides that were originally hidden in the cavity to get displayed as a hexagonal pattern on the top surface of the origami.”

Using cells in test tubes, the researchers demonstrated that the peptide switch remains hidden inside the nanostructure at normal pH. It is activated when the pH drops to 6.5. In fact, “after 24 and 48 h, less than 20% and 10% of cells survived, respectively,” the researchers wrote. “We have managed to hide the weapon in such a way that it can only be exposed in the environment found in and around a solid tumor,” Högberg said. “This means that we have created a type of nanorobot that can specifically target and kill cancer cells.”

Next, the researchers tested the treatment in human breast cancer mouse models. They reported that the treatment resulted in a 70% reduction in tumor growth compared to control mice that received an inactive version of the nanorobot. 

For their next steps, the scientists plan to investigate whether the treatment works in more advanced cancer models that resemble human disease as well as any potential side effects from administering the treatment in humans. They’ll also look into the feasibility of making the nanorobots more precise by attaching proteins or peptides to the structure’s surface that specifically bind to certain cancer types.

The post DNA Origami Selectively Triggers Cytotoxicity to Make Cancer Fold appeared first on GEN - Genetic Engineering and Biotechnology News.

A new spatial biology is in sight—literally. It is a more vivid, more detailed, and ultimately more informative spatial biology. It can distinguish between cell types that were once indistinguishable, and it can do so while capturing their spatial context. It can even pinpoint the subcellular locations of individual molecules. And it can accomplish these tasks with unprecedented precision because technology is now available that opens a new dimension beyond the usual three spatial dimensions. This new dimension may be called the “plex” dimension.

Plex refers to the number of fluorescence markers that are used with microscopy and other cell analysis platforms. Conventional platforms may accommodate just a handful of markers, constraining investigations of complex biological phenomena. But such investigations may require many markers.

Unfortunately, using more and more markers—and thereby shifting from low-plex to high-plex spatial biology—has been too difficult for most laboratories. They’ve balked at the need for special expertise, complicated workflows, and instrument upgrades. Fortunately, these difficulties can be overcome with new multiplex imaging technologies. For example, there are antibody panels that are compatible with streamlined workflows and automated imaging systems.

To learn more about these technologies, consult the articles in this eBook—especially the article describing organ mapping antibody panels. Also, be sure to read the articles that describe the kinds of spatial biology applications that are bound to become more common as high-plex technology becomes more accessible. Indeed, this technology is democratizing spatial biology.

 Sponsored by:

The post Spatial Biology Colors Outside the Lines appeared first on GEN - Genetic Engineering and Biotechnology News.

“Investment in advanced therapies is dropping, so drug developers are restricting their pipeline development to save costs,” noted Qian Liu, PhD, head of plasmid engineering and lentiviral vectors, WuXi Advanced Therapies. “This means many therapies are slower to reach regulatory approval and commercialization and are usually expensively priced, which restricts market and patient access.”

“With cell therapy, many of the cost and time issues are related to manufacturing complexity,” added Victor Vinci, PhD, global vice president, product development, Catalent Biologics. “There is variability in the initial quality of the patient’s T cells, as well as the reagents, growth media, and range of equipment and automation available for the different production stages, which means there is currently no one-size-fits-all solution for CAR T-cell manufacturing.”

Optimizing the process

Enhancing manufacturing efficiency is crucial for scaling up production, reducing costs, and ultimately making CAR/TCR T-cell therapies more accessible to patients. However, the manufacturing process for CAR/TCR T-cell therapies is complex, involving multiple steps, including steps for apheresis, T-cell selection, genetic modification, transduction, expansion, purification, and fill/finish.

Ali Mohamed, PhD, senior vice president, CMC, Immatics, discussed how evaluating different steps in the process had enhanced manufacturing of the company’s ACTengine IMA203 and IMA203CD8 TCR-engineered T cells (TCR-T cells) for targeting PRAME (PReferentially expressed Antigen in MElanoma). “ACTengine is our personalized cell therapy approach for patients with advanced solid tumors,” he said.

According to Mohamed, Immatics’ scientists have made several alterations to the standard method of producing TCR-T cells to enhance the manufacturing process. For example, they have moved to a serum-free transduction stage, where serum is not added during transduction. This, Mohamed says, has “significantly increased the numbers of T cells transfected without affecting cell viability, cell expansion, or the cell’s phenotype.”

Another process change that Immatics has made is to remove monocytes and adherent cells by resting T cells in plasticware, such as a CellSTACK, for a few hours. “Monocytes can make up as much as 50% of the T cells we collect during apheresis,” Mohamed explained. “They sometimes recognize viral vectors as foreign and destroy them, which can result in their rapid clearance and lower T-cell transduction rates. By removing them, we have seen our transduction rates increase significantly.”

The current manufacturing process implements enrichment of CD4 and CD8 T cells using specific antibodies, thereby replacing the adherent cells that have been depleted. “By selecting CD8 and CD4 cells, we can use a defined T-cell population at the start of the manufacturing process,” Mohamed explained. “This can increase the chances of manufacturing TCR-T cells in sufficient numbers to reach the required cell dose.

“In using these three process optimization steps, we can produce TCR-T cells at the recommended Phase II dose (RP2D, 1–10 × 10⁹ total TCR-T cells) in just 14 days with a 7-day manufacturing process plus 7-day quality control release testing. Using our optimized process, we have increased our seeding density and use fewer vessels. All these features help us reduce costs, shorten the turnaround time, and provide the cell products to patients faster while maintaining a manufacturing success rate of over 95%.”

Catalent’s Vinci also emphasized that process optimization is key for de-risking and streamlining a manufacturing pathway. He added, “We have used a quality-by-design approach for process optimization and have developed our UpTempo CAR T-cell therapy platform for manufacturing autologous cell therapy.”

According to Vinci, the Catalent platform provides a modular, flexible CAR T-cell cGMP workflow that utilizes aseptically connected, closed systems—including the G-Rex, Xuri, and CliniMACS Prodigy—to automate, evaluate, and optimize the manufacturing process. “We produce T-cell therapies that typically have around 90% cell viability at harvest,” Vinci noted. “This ensures our manufacturing is efficient, which reduces costs.”

Improving viral gene delivery

To reduce some of the costs involved with manufacturing CAR T-cell therapies, WuXi Advanced Therapies is developing technologies such as the XOFLX packaging and producer cell lines. These cell lines are designed to reduce the cost of producing lentiviral vectors (LVVs), which are commonly used for delivering therapeutic genes in cell therapy because they can efficiently modify T cells in a permanent manner and have a reliable safety profile for this application.

“The industry standard for LVV manufacture is to use four plasmids—a transfer vector containing the gene of interest, two packaging plasmids, and an envelope plasmid,” Liu pointed out. “What we have done with our XOFLX system is to first develop a Packaging Cell Line, which has all the LVV packaging elements stably integrated into the cells’ genome and requires transfection of only one transfer plasmid for LVV production. Additionally, we developed XOFLX Producer Cell Lines, which have also integrated the LVV genomes containing the therapeutic genes and allow scalable transfection-free LVV production.”

Liu presented data to show that at 10 L scale the XOFLX Packaging Cell Line produced comparable LVV titers when compared to WuXi Advanced Therapies’ conventional LVV production system. A research cell bank and a master cell bank have been created for the Packaging Cell Line. She also showed 1 L LVV production data from XOFLX Producer Cell Lines encoding enhanced green fluorescent protein or a therapeutic transgene. The data suggested that production could be easily scaled up from shake flasks due to the simplified, transfection-free process.

Liu concluded, “As our XOFLX system only uses one transfer plasmid or no plasmid at all for LVV production, this reduces the costs of plasmid use and the complexity of LVV manufacturing, which provides cost and quality benefits for drug developers and ultimately for patients.”

Taking the road less travelled—nonviral delivery

According to Ting-Wan Lin, PhD, director, business development, GenomeFrontier Therapeutics, the firm is focusing on making advanced, affordable cell therapies but is choosing the less well-trodden path of using nonviral cell engineering. “Despite advances in viral vector design, there are some challenges and/or disadvantages associated with virus-based vectors for gene therapy, such as their intrinsic safety concerns, costly vector manufacturing, and limited payload capacity,” she noted. “A nonviral approach for cell engineering can overcome these drawbacks.”

Lin added, however, that the nonviral approach poses other challenges. These include poor gene delivery rate, ineffective gene integration, and low cell expansion capacity caused by electroporation-based gene delivery.

To overcome the challenges currently encountered using either viral or nonviral cell engineering technologies, GenomeFrontier Therapeutics has developed Quantum Engine, a technology for facilitating development and manufacturing of high-quality, clinical-scale, and virus-free cell and gene therapy products. This system integrates four platforms: G-Tailor, Quantum pBac, Quantum Nufect, and iCellar, for candidate gene design, therapeutic gene integration, gene delivery, and cell expansion, respectively.

“Quantum pBac, the key platform of Quantum Engine, is our proprietary piggyBac-based transposon, which is potentially safer and much more effective for integrating larger sized gene compared to hyperactive piggyBac, the commercially available piggyBac vector,” Lin stated. “By finely tuning Quantum pBac along with the other three platforms, we have recently developed a robust Quantum engine, named Quantum CART (qCART), for development and manufacturing of multiplex CAR T cells.”

Lin presented data demonstrating that qCART produced CAR T cells that yielded higher percentages of CAR⁺ stem cell–like memory T cells with both CD4 and CD8 plus low expression of senescence/exhaustion markers and good expansion capacity. Furthermore, these CAR T cells also demonstrated robust antitumor efficacy in both lymphoma and gastric solid tumor mice models.

“Our qCART system not only enables us to produce high-quality and clinical scale CAR T cells with great product consistency in a time- and cost-effective manner, but also is capable of rejuvenating aged and exhausted T-cells in refractory patients,” Lin noted. “Quantum Engine is a powerful technology, enabling us to build cell and gene therapy pipelines by using piggyback and thus riding on the shoulders of giants. Our lead candidate, GF-CART01, a CD20/CD19-targeting CAR T-cell therapy to treat B-cell malignancies, has shown promising results in preclinical studies of mice, and we are looking for partners to work with.”

Enhancing manufacturing efficiency is critical for scaling up production, driving down costs, and increasing the accessibility of CAR/TCR T-cell therapies. Speakers at the CAR-TCR Summit Europe agreed that by embracing closed automated systems and adopting standardization and optimization strategies, manufacturers could overcome existing challenges and realize the transformative potential of CAR T-cell therapies.

The post Streamlining CAR/TCR T-Cell Therapy Manufacturing appeared first on GEN - Genetic Engineering and Biotechnology News.

Chimeric antigen receptor (CAR) T-cell therapy has inspired newfound hope for patients with unresponsive cancer and created widespread scientific enthusiasm spanning oncology to autoimmune disease. At the time of this writing, there are only six FDA-approved CAR T-cell therapies, primarily for oncology indications. Even though the FDA granted orphan drug designation for a CAR T-cell therapeutic for use in lupus earlier this year, there is still much work to be done to bring more drugs to market. The path to furthering these treatments requires mouse models, which can be used for proof-of-concept studies, understanding mechanisms of action, optimizing protein designs, predicting clinical outcomes, and even investigating toxicity and side effects.

Focus on potential

Like most new oncology therapies, CAR T-cell therapy still requires additional scientific investigation and refinement in the preclinical stages. Allogeneic CAR T-cell therapies—using human donor cells—could accelerate timelines and broaden distribution, but they face unique challenges, including graft-versus-host disease and cytokine release syndrome. Modeling these responses and treating any negative side effects in murine models will aid in developing more translatable treatments.

The most translatable platform: mice

Before using CAR T-cell therapies in patients, it’s essential to determine efficacy in animal models that capture disease complexities. Cell-based ex vivo assays alone are unable to recapitulate the intricacies of animal immune systems. FDA guidance issued in January 2024 emphasized the use of animal models in CAR T-cell therapy, noting, “Animal models can be useful in demonstrating proof-of-concept data for CAR T-cell functionality.” The guidance also noted, “If a relevant surrogate product is available, syngeneic tumor animal models can provide information regarding the interaction of the surrogate CAR T-cells with an intact host immune system and potential on-target/off-tumor toxicities.” As the immuno-oncology industry evolves, so must the preclinical animal models that fuel these early stages of drug development. Taconic Biosciences recognizes the importance of breaking down licensing barriers and making available the most translationally relevant preclinical models for CAR T-cell therapy development. The IL-2 NOG mouse, a super-immunodeficient mouse that expresses the human IL-2 cytokine, is one example of a complex animal model that has been successfully employed to model CAR T-cell immunotherapy in HER2+ breast cancer and is available off the shelf.¹ In one recent study using hIL-2 NOG mice, researchers were able to demonstrate significant CAR T-mediated antitumor efficacy in mice transplanted with PDX melanoma cell lines. Additionally, Taconic’s immunodeficient NOG-EXL mice, which express human GM-CSF and IL-3 cytokines, were used to assess CAR T-cell therapy in non-small cell lung cancer.² In addition to the hIL-2 NOG and NOG-EXL models, which are maintained in live colonies for easy access, Taconic has the most experienced model generation team in the industry that partners with researchers to create custom models tailored to their specific CAR T-cell therapy investigations.

Creating advanced CAR T-cell therapies requires extensive preclinical studies. However, by using animal models that approximate human disease, scientists are able to reduce the number of animals needed to elucidate how therapeutic candidates act upon disease in preclinical stages. Taconic’s commitment to the 3R’s principle—Replace, Reduce, and Refine—promotes the ethical use of animals in scientific research and exceeds U.S. FDA and international animal use guidelines. Streamlining the preclinical-to-clinical oncology pipeline necessitates effective tools capable of capturing the intricacies of disease response to treatment in humans. Super immunodeficient and genetically engineered models are uniquely valuable tools for oncology and autoimmune researchers seeking to develop novel therapeutics.

References

1. Cao B, Liu M, Wang L, et al. Remodelling of tumour microenvironment by microwave ablation potentiates immunotherapy of AXL-specific CAR T-cells against non-small cell lung cancer. Nat Commun. 2022;13(1):6203. Published 2022 Oct 19. doi:10.1038/s41467-022-33968-5
2. Forsberg EM, Lindberg MF, Jespersen H, Alsén S, Olofsson Bagge R, Donia M, Svane IM, Nilsson O, Ny L, Nilsson LM, Nilsson JA. HER2 CAR-T cells eradicate uveal melanoma and T cell therapy-resistant human melanoma in interleukin-2 (IL-2) transgenic NOD/SCID IL-2 receptor knockout mice. Cancer Res. 2019 Jan 8. pii: canres.3158.2018

Explore the hIL-2 NOG, NOG-EXL, and other super immunodeficient models for CAR T-cell therapy work, visit: taconic.com/gen.

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Imagine being able to map every cell within a tumor, understanding not just which ones are present, but also their precise locations and how they communicate. This is the promise of spatial biology—a cutting-edge approach to cancer research and precision medicine. By examining cells in their natural context, spatial biology helps researchers gain biological insights into tumor structure, cellular composition, proximity, and morphology, which could lead to faster tumor detection, more accurate diagnoses, and personalized treatment strategies.

A study by Arutha Kulasinghe, PhD, Scientific Director of the Queensland Spatial Biology Center at the University of Queensland, showcased the power of this technology by demonstrating how spatial biology enables cellular phenotyping and functional annotation of every cell in the tumor microenvironment (Figure 1).

Insights revealed at the bench are now being translated into the delivery of precision oncology at an entirely new level in clinical settings. This progress provides deeper insights into the current landscape and shapes the next generation of cancer research.

• “We know where the cells are and what they are doing. Using spatial biology, we can identify subsets of cells and their nuances. This will tell us which are key players in driving resistance or sensitivity to therapy.” —Arutha Kulasinghe, PhD, University of Queensland

A catalyst for spatial biology

An enormous amount of spatial data is being generated and combined with other “omics” data, bringing new clarity to the complexity of cancer. However, deriving actionable insights from these large datasets remains a challenge due to a lack of accessible computational tools. Expanding the use of AI in spatial biology can help pathologists and oncologists identify patterns within the tumor microenvironment, leading to more precise diagnostics, personalized treatment strategies, and a better understanding of how patients respond to treatments.

Spatial biology 2.0 elevates research

Despite its potential, spatial biology still faces hurdles. Current spatial biology multiplex imaging platforms are limited by slow processing speeds, complex workflows that limit throughput, and large data storage requirements. While AI can help overcome some of these challenges, limitations remain.

To address these challenges, Akoya Biosciences has developed Spatial Biology 2.0—end-to-end solutions including PhenoCycler®-Fusion 2.0 and PhenoImager® HT 2.0, designed to generate more data, faster, at any scale. From unlocking breakthroughs from individual samples and generating spatial atlases on a human scale to understanding tumor heterogeneity, and identifying cellular neighborhoods, these solutions are poised to transform our understanding of human biology and disease.

Spatial Biology 2.0 introduces key innovations including whole-slide, highspeed imaging, scalable multiplexing, and simplified workflows (Figure 2).

• “AI can deal better and more reproducibly with large amounts of data from multiplex and hyperplex studies. AI can help analyze data and identify
  patterns we may not be able to discern.” —Suzanne Coberly, MD, Bristol Myers Squibb
• “The intricacies of oncology as revealed by spatial biology, along with its ever-evolving landscape of treatments and methodologies, stand to benefit immensely from AI.” —Doug Flora, MD, St. Elizabeth Healthcare, and a pioneer in utilizing AI in precision oncology

Conclusion

The convergence of spatial biology and AI marks a transformative era in precision oncology. By harnessing the detailed cellular insights provided by spatial biology and the pattern recognition of AI, this approach offers insights into cancer complexity that translate directly to the clinic. The result: more precise diagnoses, personalized treatment strategies, and a deeper understanding of cancer and other diseases.

As we continue to push the boundaries of possibility, the future of cancer treatment has never looked brighter.

To gain exclusive insights from leading experts in spatial biology:

Watch on-demand webinar
www.akoyabio.com/webinar-gen-aacr2024

Download 100-plex application note
www.akoyabio.com/100-plex

Talk to us
www.akoyabio.com/contact

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