Genetic Engineering Publications - GEG Tech top picks

CAR T cells have become an established treatment option for children with a rare form of relapsed or incurable leukemia. One of the key factors determining whether treatment will lead to lasting remission of leukemia is how long the CAR T cells live in the body. One team was able to study the cells of 10 children enrolled in a pioneering clinical trial (CARPALL) for up to five years after their initial CAR T cell treatment. This has enabled them to better understand why some of these CAR T cells remain in a patient's bloodstream, and why others disappear early, potentially allowing the cancer to recur. Using techniques that analyze individual cells at the genetic level to understand what they do, the scientists were able to identify a unique "signature" in long-lived CAR T cells. The signature suggested that long-lived CAR T cells in the blood transformed into a different state that allowed them to continue monitoring the patient's body for cancer cells. As part of the study, the researchers identified key genes in CAR T cells that appeared to enable them to persist in the body for a long time. These genes will provide a starting point for future studies to identify markers of persistence in CAR T-cell products as they are manufactured, and ultimately to improve their efficacy.

A new study shows that some effective cancer treatments, such as CAR-T cells, significantly improve quality of life six months after receiving therapy. To conduct the study, researchers recruited 103 patients aged 23 to 90 years with a diagnosis of blood cancer from April 2019 to November 2021. The researchers administered self-reported questionnaires measuring quality of life variables at time intervals including before CAR-T cell infusion and one week, one month, three months, and six months after CAR-T cell infusion. Quality of life was measured using a 27-item questionnaire known as the General Cancer Therapy Functional Assessment, which is composed of four different subscales (physical, functional, emotional, and social). Psychological distress was measured using the Hospital Anxiety and Depression Scale. Finally, major depressive symptoms were measured using the PHQ-9, and symptoms of posttraumatic stress disorder were measured using the Posttraumatic Stress Checklist. While most study participants eventually experienced an improvement in quality of life, approximately 20% of patients experienced persistent physical and psychological symptoms, which at times interfered with their quality of life.

Many people are excluded from CAR T cell-based treatments because of its cost. One reason for the high cost is that the manufacturing process is complex, time-consuming and must be individually tailored to each cancer patient. So to address this challenge, the researchers created a biotechnology called Multifunctional Alginate Scaffolds for T cell Engineering and Release (MASTER) that is a biocompatible sponge-like material. To begin treatment, the researchers isolate the patient's T cells and mix these naive T cells with the modified virus. The researchers pour this mixture onto MASTER, which absorbs it. MASTER is decorated with the antibodies that activate the T cells, so the cell activation process begins almost immediately. Meanwhile, MASTER is surgically implanted into the patient. After implantation, as the T cells become activated, they begin to respond to the modified viruses, which reprogram them into CAR-T cells. MASTER is also imbued with interleukin factors that promote cell proliferation. After implantation, these interleukins begin to leach out, promoting rapid proliferation of CAR-T cells. In a proof-of-concept study involving lymphoma in mice, researchers found that this treatment was faster and more effective than conventional CAR-T cell cancer treatment. 

Researchers at Seidman Cancer Center and Case Western Reserve University Hospitals have developed a new approach to CAR T cell therapy for B-cell cancers that triples targeted antigens on cancer cells. This approach promises to significantly reduce the potential for antigen escape currently found in CAR T therapies that target only CD19. The novel B-cell activating factor (BAFF) CAR T product developed here specifically binds to each of three receptors instead of one - BAFF-R, BCMA and TACI, providing more therapeutic options. At least two of these three receptors are found in almost all B-cell cancers, with some cancers expressing all three. Experimental results reported in Nature Communications show that BAFF CAR T is effective in killing several B-cell cancers. In addition, studies show robust in vitro and in vivo cytotoxicity exerted by CAR T BAFFs against mantle cell lymphoma, multiple myeloma, and mouse xenograft models of acute lymphoblastic leukemia. An Investigational New Drug application with the U.S. Food and Drug Administration will be filed in the coming weeks with Luminary Therapeutics and the team plans to initiate a clinical trial of BAFF CAR T therapy in patients with non-Hodgkin's lymphoma within the next few months. 

According to Jing Pan's article in the Journal of Clinical Oncology titled: Donor-derived CD7 chimeric antigen receptor T cells for T-cell acute lymphoblastic leukemia: first-in-human phase I trial, CAR T cells are reported to be remarkably effective in patients with B-cell acute lymphoblastic leukemia (ALL) but have not been successful to date in patients with T-cell ALL (T-ALL). Now, data from Pan and colleagues demonstrate the safety and impressive short-term efficacy of allogeneic donor-derived anti-CD7 CAR T cells in an early phase clinical trial involving patients with relapsed and/or refractory T-ALL.

T cells that fight cancer for too long stop fighting. Researchers have named this phenomenon: T cell exhaustion. This exhaustion disrupts even the most modern cancer immunotherapies such as CAR T cells. A new study addresses this problem by giving T cells the ability to fight exhaustion itself. Researchers found that BATF, a transcription factor, cooperates with another transcription factor called IRF4 to counteract the T cell depletion program. In mouse models of melanoma and colorectal carcinoma, modifying CAR T cells to overexpress BATF by about 20-fold compared to normal cells eliminated the tumor without causing T-cell exhaustion. CAR T therapy worked against solid tumors.

Further testing showed that while IRF4 is important, it should not be overexpressed to the same degree as BATF. In addition, modified T cells also remained in place and became memory T cells. This is important because T cell depletion often prevents them from mounting a strong memory response against recurrent cancers.

The "T-Cell Immunotherapy Market, 2018-2030 (3rd edition)" report features an extensive study of the current market landscape and the future potential of T-cell immunotherapies (focusing particularly on CAR-T therapies, TCR therapies and TIL therapies). One of the key objectives of the study was to review and quantify the future opportunities associated with the ongoing development programs of both small and big pharmaceutical firms.

The immunosuppressive nature of tumor microenvironment is considered one of the key factors limiting CAR-T efficacy. One negative regulator of Tcell activity is lymphocyte activation gene-3 (LAG-3). In this study, scientists successfully generated LAG-3 knockout Tand CAR-T cells with high efficiency using CRISPR-Cas9 mediated gene editing and found that the viability and immune phenotype were not dramatically changed during in vitro culture. LAG-3 knockout CAR-T cells displayed robust antigen-specific antitumor activity in cell culture and in murine xenograft model, which is comparable to standard CAR-T cells. This study demonstrates an efficient approach to silence immune checkpoint in CAR-T cells via gene editing.

Here scientists show that directing a CD19-specific CAR to the T-cell receptor α constant (TRAC) locus not only results in uniform CAR expression in human peripheral blood T cells, but also enhances T-cell potency, with edited cells vastly outperforming conventionally generated CAR T cells in a mouse model of acute lymphoblastic leukaemia.

Researchers have developed a universal receptor system that allows T cells to recognize any cell surface target, enabling highly customizable CAR T cell and other immunotherapies for treating cancer and other diseases. The new approach involves engineering T cells with receptors bearing a universal "SNAPtag" that fuses with antibodies targeting different proteins. By tweaking the type or dose of these antibodies, treatments could be tailored for optimal immune responses. The researchers showed that their SNAP approach works in two important receptors: CAR receptors, a synthetic T cell receptor that coordinates a suite of immune responses, and SynNotch, a synthetic receptor that can be programmed to activate just about any gene. In a mouse model of cancer, treatment with SNAP-CAR T cells shrunk tumors and greatly prolonged survival, an important proof-of-concept that sets the stage to test this approach in clinical trials in partnership with Coeptis Therapeutics, which has licensed the SNAP-CAR technology from Pitt. The discovery could extend into solid tumors and give more patients access to the game-changing results CAR T cell therapy has produced in certain blood cancers. With the addition of SNAP, the possibilities for customized therapies become almost endless.

T-cell transfer therapies have not yet been successfully applied to solid tumors because T cells do not readily penetrate and persist in solid tumor masses for long periods of time, and because their activity is attenuated by an immunosuppressive tumor microenvironment. One way to overcome these limitations could be to couple T cell transfer therapies with cytokine therapy. However, a serious drawback of this approach is the significant side effects resulting from cytokines circulating freely in the body, leading to toxicity and potentially fatal inflammatory syndromes. Now, researchers have developed a nanotechnology-based solution to these problems. The method uses an unnatural sugar that is absorbed and embedded in the outer coating of T cells, which can then be used to anchor cytokines. The concentrated cytokines improve T-cell function locally without producing unwanted systemic side effects. In mice with melanoma, the approach also stimulated the host immune system against tumor cells, which inhibited tumor growth. As an adjunct to CAR-T cell therapy, it resulted in complete regression of lymphoma tumors at otherwise non-curative cell doses.

CRISPR genome editing has served as a powerful tool to delete or modify DNA sequences and study the resulting effect. Now, researchers at the Gladstone Institutes and UC San Francisco (UCSF) have co-opted the CRISPR-Cas9 system to forcibly turn on genes rather than edit them in human immune cells. The method, known as CRISPRa, allowed them to discover genes that play a role in immune cell biology more thoroughly and quickly than before. The study, published in the journal Science, is the first to successfully use CRISPRa on a large scale in primary human cells, which are cells isolated directly from a person. In the new work, Marson, Steinhart and co-first author Ralf Schmidt, MD, worked with their colleagues to adapt CRISPRa and CRISPRi to work at high efficiency in primary T cells, something never done before. Improving the efficiency of delivery of the CRISPRa or CRISPRi machinery into cells was essential to enable genome-wide experiments and accelerate discovery. Marson's lab is currently studying some of the individual genes identified in their screen, and working to further leverage CRISPRa and CRISPRi to discover genes that control other critical traits in human immune cells.  

The race to the clinic is reviving for a ready-made alternative to autologous CAR-T cell therapy, even as concerns about chromosomal abnormalities persist. The Advanced Regenerative Medicine Therapy designation, which makes the therapy eligible for accelerated approval, will also help remove a veil that has hung over standard CAR-T cell therapies since October, when the FDA put all trials of competitor Allogene Therapeutics on hold following the detection of a chromosomal abnormality in a patient who received ALLO-501A in a Phase 2 trial. The FDA's green light for CRISPR Therapeutics dispels broader concerns that the agency views this type of genotoxic safety event as an intractable problem for the entire class of allogeneic CAR-T therapies. Today, many companies are eliminating loci associated with the MHC-I to avoid host T cell recognition of transplanted CAR-T cells. Companies also equip their T cells with a variety of safety switches and performance enhancers.

However, as the complexity of the assembly increases, the risk of off-target effects also increases. This may be important from a safety perspective, given that most cancers lack unique antigens. Achieving rapid remission and re-dosing if necessary, can minimize the toxic effects that CAR-T cells can have on healthy tissues expressing the targeted antigen.

For 17 years, Thomas Blankenstein has been working on his Berlin-based start-up T-knife and is finally going to be able to test his T-cell receptor (TCR) cancer immunotherapy. To make this immunotherapy, they replaced the TCR genes of a mouse with those of a human and inserted genes to encompass both the human TCR repertoire and human major histocompatibility complex (MHC) molecules. They eliminated genes from related mice, generating mouse lines carrying human genes, and then subjected them to a succession of crosses to bring all the genes together in a single mouse, the HuTCR mouse. Designing transgenic mice with such efficient humanized TCRs has been very difficult and laborious. However, TCR-T cells are capable of more extensive signaling and killing than CAR-T cells. The engineered TCRs integrate seamlessly into the signal transduction pathways of T cells: there are ten subunits in a TCR versus one subunit in a CAR, they have ten immunoreceptor tyrosine activation motifs versus three in a CAR, and they are associated with more co-stimulatory receptors: CD3, CD4, CD2. In addition, unlike CARs that only bind to antigens on the cell surface, TCRs can target intracellular as well as extracellular tumor antigens.

Scientists in Japan have used CRISPR-Cas9 technology to stop human immunodeficiency virus type 1 (HIV-1) replication in latently infected T cells that can’t be controlled using existing drug treatments. The gene-editing approach effectively disrupts two regulatory HIV-1 genes, tat and rev, which are essential for viral replication. Describing their in vitro studies in Scientific Reports, the researchers at Kobe University Graduate School of Medicine and Kobe University Graduate School of Health Sciences say initial results indicate that using CRISPR-Cas9 to target HIV-1 regulatory genes may offer a new approach to achieving “functional cures.”

In this work, the authors  assembled 12 transcription activator-like effector nucleases (TALENs) and five guide RNAs (gRNAs) for CRISPR system to knock out endogenous TCR expression. Using nuclease-expressing plasmid DNA, they achieved up to 19.9% and 12.2% knockout of TCR expression in primary T cells with CRISPR/Cas9 and TALENs, respectively. In contrast, delivery of TALEN mRNA by electroporation resulted in high viability and TCR knockout efficiencies of up to 78.8% for the TCR α chain and 81.2% for the β chain on day 6 after electroporation. 

In this study, the scientists evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. They found that SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients. These results support further clinical development of this nonviral gene therapy approach.

In the report Kochenderfer et al discuss the efficacy of autologous T cells expressing a CD19-specific chimeric antigen receptor (CAR) in patients with relapsed diffuse large B-cell lymphoma.