Engineering of SauriCas9 with enhanced specificity.

Several allogeneic chimeric antigen receptor (CAR) T-cell therapies in clinical trials rely on CRISPR-Cas genome editing, but the enzyme’s random repair mechanism increases the risk of undesired off-target effects, challenging safe CAR T-cell generation. To address this, we developed a novel CRISPR RNA (crRNA) targeting the T-cell receptor beta constant (TRBC) gene. Combined with AsCas12a Ultra, this crRNA edits primary human T-cells via a predictable microhomology-mediated end joining (MMEJ) DNA repair pathway, significantly lowering off-target risks. During evaluation, we sequestered a unique T-cell subset with disrupted T-cell receptor (TCR), retained CD3 expression, and no in vivo alloreactivity. Termed CD3-retained, allogeneically functioning T-cells (CRAFT-cells), these cells exhibited growth kinetics comparable to unedited T-cells. When engineered with CD19- or BAFF-R-targeted CARs, CRAFT CAR T-cells showed strong antigen-specific cytotoxicity and significant ex vivo expansion compared to conventional CD3-disrupted CAR T-cells. Moreover, CRAFT CAR T-cells effectively served as effector cells for bispecific T-cell engagers (BiTEs), enabling CD3-dependent tumor cell killing. Our CRAFT crRNA platform offers a novel strategy to generate safer allogeneic CAR T-cells. The distinct properties of CRAFT CAR T-cells, combined with BiTE therapy, represent a promising and potentially more durable approach for next-generation allogeneic CAR T-cell therapies in clinical applications.

HLA-E single-chain trimer shields gene-edited allogeneic CAR T cells from NK cell attack

Introduction Allogeneic chimeric antigen receptor (CAR) T-cell therapy is a promising yet underexplored treatment for clear cell renal cell carcinoma (ccRCC), potentially more effective than existing treatment options. This review compares several ongoing preclinical and clinical trials using CAR T-cell therapy. Areas covered This review discusses the development of CAR T-cell therapy in ccRCC, covering four significant themes: (1) optimizing therapeutic efficacy through combination strategies, (2) the translation pathway from preclinical development to clinical application, (3) safety and toxicity management, and (4) immune response modulation in the tumor microenvironment. Finally, this review highlights opportunities to overcome current limitations and guide future therapeutic approaches. We conducted a structured review of existing research using the PubMed and Google Scholar databases from the past 5 years, compiled relevant studies on CAR T-cell therapies for ccRCC, and categorized them into four key themes, which were then cross-analyzed to identify trends, challenges, and emerging limitations. Expert opinion Development of universal CAR T-cells may be more affordable, more accessible, and easier to administer in less time with fewer mechanical failures than autologous CAR T-cell therapy. Some challenges persist, including patient toxicities, depletion of CAR T-cells in vivo, and an immunosuppressive tumor microenvironment.

Long-Term Outcomes and Adverse Events of CAR T-19 Cell Therapy in Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia - a Systematic Review and Meta-Analysis

<div>Abstract<p>Therapeutic approaches for clear cell renal cell carcinoma (ccRCC) remain limited; however, chimeric antigen receptor (CAR) T-cell therapies may offer novel treatment options. CTX130, an allogeneic CD70-targeting CAR T-cell product, was developed for the treatment of advanced or refractory ccRCC. We report that CTX130 showed favorable preclinical proliferation and cytotoxicity profiles and completely regressed RCC xenograft tumors. We also report results from 16 patients with relapsed/refractory ccRCC who received CTX130 in a phase I, multicenter, first-in-human clinical trial. No patients encountered dose-limiting toxicity, and disease control was achieved in 81.3% of patients. One patient remains in a durable complete response at 3 years. Finally, we report on a next-generation CAR T construct, CTX131, in which synergistic potency edits to CTX130 confer improved expansion and efficacy in preclinical studies. These data represent a proof of concept for the treatment of ccRCC and other CD70<sup>+</sup> malignancies with CD70<sup>−</sup> targeted allogeneic CAR T cells.</p><p><b>Significance:</b> Although the role of CAR T cells is well established in hematologic malignancies, the clinical experience in solid tumors has been disappointing. This clinical trial demonstrates the first complete response in a patient with RCC, reinforcing the potential benefit of CAR T cells in the treatment of solid tumors.</p><p>See corresponding author Sumanta K. Pal discuss this research article, published simultaneously at the AACR Annual Meeting 2024: <a href="https://vimeo.com/932606570/887520f9cb" target="_blank">https://vimeo.com/932606570/887520f9cb</a></p></div>

<div>Abstract<p>Therapeutic approaches for clear cell renal cell carcinoma (ccRCC) remain limited; however, chimeric antigen receptor (CAR) T-cell therapies may offer novel treatment options. CTX130, an allogeneic CD70-targeting CAR T-cell product, was developed for the treatment of advanced or refractory ccRCC. We report that CTX130 showed favorable preclinical proliferation and cytotoxicity profiles and completely regressed RCC xenograft tumors. We also report results from 16 patients with relapsed/refractory ccRCC who received CTX130 in a phase I, multicenter, first-in-human clinical trial. No patients encountered dose-limiting toxicity, and disease control was achieved in 81.3% of patients. One patient remains in a durable complete response at 3 years. Finally, we report on a next-generation CAR T construct, CTX131, in which synergistic potency edits to CTX130 confer improved expansion and efficacy in preclinical studies. These data represent a proof of concept for the treatment of ccRCC and other CD70<sup>+</sup> malignancies with CD70<sup>−</sup> targeted allogeneic CAR T cells.</p><p><b>Significance:</b> Although the role of CAR T cells is well established in hematologic malignancies, the clinical experience in solid tumors has been disappointing. This clinical trial demonstrates the first complete response in a patient with RCC, reinforcing the potential benefit of CAR T cells in the treatment of solid tumors.</p></div>

Chimeric antigen receptor (CAR) T-cell therapy has ushered in substantial advancements in the management of various B-cell malignancies. However, its integration into chronic lymphocytic leukemia (CLL) treatment has been challenging, attributed largely to the development of very effective chemo-free alternatives. Additionally, CAR T-cell responses in CLL have not been as high as in other B-cell lymphomas or leukemias. However, a critical void exists in therapeutic options for patients with high-risk diseases who are resistant to the current CLL therapies, underscoring the urgency for adoptive immunotherapies in these patients. The diminished CAR T-cell efficacy within CLL can be traced to factors such as compromised T-cell fitness due to persistent antigenic stimulation inherent to CLL. Resistance mechanisms encompass tumor-related factors like antigen escape, CAR T-cell-intrinsic factors like T-cell exhaustion, and a suppressive tumor microenvironment (TME). New strategies to combat CAR T-cell resistance include the concurrent administration of therapies that augment CAR T-cell endurance and function, as well as the engineering of novel CAR T-cells targeting different antigens. Moreover, the concept of "armored" CAR T-cells, armed with transgenic modulators to modify both CAR T-cell function and the tumor milieu, is gaining traction. Beyond this, the development of readily available, allogeneic CAR T-cells and natural killer (NK) cells presents a promising countermeasure to innate T-cell defects in CLL patients. In this review, we explore the role of CAR T-cell therapy in CLL, the intricate tapestry of resistance mechanisms, and the pioneering methods studied to overcome resistance.

Therapeutic approaches for clear cell renal cell carcinoma (ccRCC) remain limited; however, chimeric antigen receptor (CAR) T-cell therapies may offer novel treatment options. CTX130, an allogeneic CD70-targeting CAR T-cell product, was developed for the treatment of advanced or refractory ccRCC. We report that CTX130 showed favorable preclinical proliferation and cytotoxicity profiles and completely regressed RCC xenograft tumors. We also report results from 16 patients with relapsed/refractory ccRCC who received CTX130 in a phase I, multicenter, first-in-human clinical trial. No patients encountered dose-limiting toxicity, and disease control was achieved in 81.3% of patients. One patient remains in a durable complete response at 3 years. Finally, we report on a next-generation CAR T construct, CTX131, in which synergistic potency edits to CTX130 confer improved expansion and efficacy in preclinical studies. These data represent a proof of concept for the treatment of ccRCC and other CD70+ malignancies with CD70- targeted allogeneic CAR T cells. Significance: Although the role of CAR T cells is well established in hematologic malignancies, the clinical experience in solid tumors has been disappointing. This clinical trial demonstrates the first complete response in a patient with RCC, reinforcing the potential benefit of CAR T cells in the treatment of solid tumors.

Introduction: Despite therapeutic advances, multiple myeloma remains incurable. BCMA targeting immunotherapies, such as bispecific T-cell engagers (TCE) and autologous CAR-T provide high response rates, but relapses are common. Autologous CAR-T are logistically challenging due to the need for apheresis, prolonged manufacturing and occasional manufacturing failures. Importantly, pts who have progressed after a prior BCMA targeting immunotherapy are an emerging area of high unmet need. Emerging data indicate that autologous CAR-T have lower clinical activity in pts ho have progressed on TCE. Methods: P-BCMA-ALLO1 is an allogeneic CAR-T therapy manufactured from healthy donor T-cells available “off-the-shelf” and being evaluated in a phase 1 clinical trial (P-BCMA-ALLO1-001) in RRMM pts. This primary objective is to determine the maximum tolerated dose of P-BCMA-ALLO1, and the key secondary objective is to investigate the anti-myeloma activity. The pts must have progressed on a prior proteasome inhibitor, immunomodulatory drug and anti-CD38 monoclonal antibody. The study allows enrollment of pts who have received prior BCMA targeting therapy. The study is exploring escalating P-BCMA-ALLO1 doses and several different lymphodepletion chemotherapy (LD) regimens. Here we report the safety and early efficacy results for the 5 pts who were treated with P-BCMA-ALLO1 after having progressed on BCMA targeting CAR-T, TCE or both. These pts were treated in arms P1 (LD: cyclophosphamide (cy) 500 mg/m2 + fludarabine (flu) 30 mg/m2 X 3 days) or arm P2 (LD: cy 1000 mg/m2 + flu 30 mg/m2 X 3 days) at a P-BCMA-ALLO1 dose of > 2 X 106 to <6 X 106 cells/kg. Results: The median pt age was 62 years and median prior lines of therapy was 10. Three pts were treated in arm P2 and 2 in arm P1. Two pts had received prior teclistamab, 2 had received prior CAR-T and 1 had received prior teclistamab and CAR-T. P-BCMA-ALLO1 was well tolerated with no dose limiting toxicities or graft vs. host disease. Three of the five pts developed cytokine release syndrome (all grade (G) 2) and one developed G2 immune effector cell neurotoxicity syndrome. Three of the five pts (60%) achieved clinical responses with all three achieving the best response of very good partial response. The two non-responders had previously received and failed to achieve clinical response with teclistamab. One pt who had previously received both teclistamab and CAR-T achieved VGPR. Conclusion: In conclusion, P-BCMA-ALLO1 is an allogeneic CAR-T that is available “on-demand” with activity in RRMM pts who have progressed following prior BCMA targeted CAR-T and TCE. We believe this is the first such report of an allogeneic CAR-T showing clinical activity in such a pt population with high unmet need. Enrollment is continuing and updated data will be presented at the meeting. Citation Format: Bhagirathbhai Dholaria, Leyla Shune, Andrew Kin, Katherine McArthur, Jeff D. Eskew, Christopher E. Martin, Sabrina Haag, Joanne McCaigue, Hamid Namini, Sam DePrimo, Stacey Cranert, Julia Coronella, Devon Shedlock, Rajesh Belani. Clinical activity of P-BCMA-ALLO1, a B-cell maturation antigen (BCMA) targeted allogeneic chimeric antigen receptor T-cell (CAR-T) therapy, in relapsed refractory multiple myeloma (RRMM) patients (pts) following progression on prior BCMA targeting therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT071.

Pediatric cancer poses a major health challenge globally, especially in low-middle-income countries like Indonesia. The survival rate of pediatric cancer in many high-income countries (HICs) reaches 90%, while it only ranges from 5 to 60% in LMICs. Over 80% of children with cancer live in low-middle-income countries, indicating the urgency to improve the survival rate of pediatric cancer in LMICs [1]. In Indonesia, the prevalence of pediatric cancer was 43.5% in 2020, making it the highest among Southeast Asian countries [2]. According to Dharmais Cancer Hospital (2024), the national cancer referral center for all of Indonesia, the 5-year survival rate of high-risk pediatric acute lymphoblastic leukemia is only 48.8% (unpublished data).One key factor contributing to the low survival rate of pediatric cancer in Indonesia is the lack of effective therapy options, especially for high-risk and relapsed or refractory patients. Several therapeutic approaches, such as immunotherapy, have been widely used in HICs but are still not very popular in Indonesia. CAR (Chimeric Antigen Receptor) T-cell therapy is one of the most promising immunotherapeutic approaches to treat pediatric cancer. Implementing CAR T Cell therapy in Indonesia offers promising prospects for improving the survival rates of pediatric cancer patients.CAR T cell therapy utilizes the body's immune system to specifically target and eliminate cancer cells. This innovative therapy entails extracting a patient's T cells, genetically modifying them to express chimeric antigen receptors specific to tumor-associated antigens, and then reinfusing them into the patient. Once infused, these engineered T cells recognize and eliminate cancer cells bearing the targeted antigen, thereby offering a highly targeted and potentially curative treatment option [3]. This innovative therapy has demonstrated remarkable success in treating certain hematologic malignancies, including pediatric leukemia. The most extensively studied case in childhood patients involves CAR T cells that target CD19, a B cell surface receptor [4].CAR T cell therapy holds great promise for improving survival rates among pediatric cancer patients in Indonesia. Children with refractory or relapsed leukemia, such as B-cell acute lymphoblastic leukemia (B-ALL), who have exhausted standard treatment options, can benefit from CAR T cell therapy. Most relapsed or refractory pediatric cancer patients in Indonesia do not have effective therapy options to treat the disease. CAR T cell therapy emerges as a novel therapy that can significantly improve the survival of this subset of patients. Numerous studies have documented high remission rates (ranging from 70% to 90%) in adults and children diagnosed with refractory B-ALL [4]. A study by Maude et al. [5] reported high remission rates and durable responses in young adults and children with refractory or relapsed B-ALL treated with CAR T cells. Similarly, Park et al. [6] demonstrated long-term remissions and improved survival in pediatric leukemia patients receiving CAR T cell therapy. Several groups also have observed the persistence of CAR T cells and sustained remission lasting over six months in the majority of patients examined [4]. Efforts have been made to implement CAR T cells in Indonesia. Dharmais Cancer Hospital, as a National Cancer Center in Indonesia, has initiated this effort by collaborating with iCarTAB Biomed Inc., a China-based CAR T cell manufacturer with one of its manufacturing sites located in Malaysia. However, this approach involves sending patients' blood samples that have been processed through leukapheresis to Malaysia for CAR T cell manufacturing, followed by the shipment of the manufactured cells back to Indonesia for administration to patients. This process is impractical and incurs intangible costs such as transportation and cryopreservation, ultimately making it more expensive for patients. Regulatory issues related to the shipment of cells across borders in the region and early preparation of patients for CAR T cell therapy soon after relapse before they succumb to treatment-related mortality or relapse-related complications are also challenges that need to be addressed [7]. Reflecting on the abovementioned issue, CAR T cell therapy adoption in Indonesia faces significant challenges. Limited healthcare infrastructure, including specialized facilities for cell therapy manufacturing and administration, poses logistical hurdles. Moreover, cost remains a major barrier, as CAR T cell therapy is often expensive and inaccessible to many patients in Indonesia. Furthermore, the lack of local expertise in cellular immunotherapy may impede the successful implementation of CAR T cell therapy programs.Efforts to address these challenges and maximize the potential of CAR T cell therapy in Indonesia are essential. This requires a multi-faceted approach involving investment in healthcare infrastructure, including establishing specialized centers equipped for CAR T cell therapy manufacturing and administration. Two alternative models have been proposed for manufacturing CAR-T cell therapy: centralized and de-centralized models [8]. In the centralized manufacturing model, point of manufacturing and point of care are located in different geographical areas, while decentralized manufacturing focuses on establishing point of care and manufacturing in close proximity. A decentralized manufacturing model might be the best approach to be implemented in LMICs like Indonesia. Building hospital-based cellular therapy manufacturing reduces the need for transportation and cryopreservation. The decentralized system's geographic proximity improves communication between manufacturing and treatment teams, facilitating the creation of customized products based on a patient's phenotype. This setup also reduces administration time and the risk of delays and mix-ups compared to centralized manufacturing, making hospital-based cellular therapy manufacturing a potentially more cost-effective option [8].In addition, initiatives to reduce the cost of therapy through partnerships with pharmaceutical companies, government subsidies, or philanthropic endeavors can improve affordability and access. Furthermore, capacity-building initiatives aimed at training local healthcare professionals in cellular immunotherapy techniques are essential for ensuring the successful implementation and sustainability of CAR T cell therapy programs in Indonesia. Collaboration between local institutions, international organizations, and industry stakeholders can facilitate knowledge transfer and technology transfer, fostering indigenous expertise in this cutting-edge treatment modality.CAR T cell therapy represents a transformative approach to improving survival rates among pediatric cancer patients in Indonesia. By harnessing the power of immunotherapy, specifically tailored to target cancer cells, CAR T cell therapy offers hope for children with refractory or relapsed leukemia who have limited treatment options. Through continued research, collaboration, and investment in healthcare infrastructure, CAR T cell therapy potentially could greatly improve the prognosis and quality of life for pediatric cancer patients in Indonesia.

Donor-derived and off-the-shelf allogeneic anti-CD19 CAR T-cell therapy for R/R ALL and NHL: A systematic review and meta-analysis

With the success of chimeric antigen receptor (CAR) T-cell therapy in B-cell malignancies, efforts are being made to extend this therapy to other malignancies and broader patient populations. However, limitations associated with the time-consuming and highly personalized manufacturing of autologous CAR T-cells remain. Allogeneic CAR T-cell approaches may overcome these challenges but require further engineering to reduce their alloreactivity. As a means to prevent graft-versus-host disease (GvHD) of allogeneic CAR T-cells, we have selected a micro RNA (miRNA)-based short hairpin RNA (shRNA) targeting CD3ζ which efficiently downregulates the expression of the T-cell receptor (TCR) below detection level. We generated allogeneic anti-B-cell maturation antigen CAR T-cells (CYAD-211) that co-express an anti-CD3ζ miRNA-based shRNA within the CAR construct which efficiently inhibited TCR-mediated signaling in vitro and GvHD in vivo. CYAD-211 was subsequently evaluated in a Phase-I clinical trial (NCT04613557), in patients with relapsed or refractory multiple myeloma. No signs of GvHD were observed despite evidence of engraftment, demonstrating efficient downregulation of the TCR. Our data provide proof of concept that a non-gene-edited technology can generate fully functional allogeneic CAR T-cells, without any signs of GvHD. However, further engineering of the CAR T-cells is needed to improve their persistence and long-term activity.

Acute Kidney Injury After the CAR-T Therapy Tisagenlecleucel

Cardiac involvement in hematological malignancies is uncommon, with only a few cases reported to date, and it often leads to a poor prognosis. Here, we report a case of a 42-year-old woman with a history of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and anti-CD19 chimeric antigen receptor (CAR) T-cell therapy for B-cell lymphoblastic lymphoma/acute lymphoblastic leukemia in whom cardiac mass and myocardial infiltration were detected. Prior to this presentation, massive pericardial effusion had occurred 6 months after CAR T-cell therapy, which was improved via ultrasound-guided pericardiocentesis. We observed elevated cytokine levels and increased copy number of CAR DNA in both pericardial effusion and serum. Upon detecting cardiac mass and myocardial infiltration, the patient was administered tocilizumab (a humanized monoclonal antibody against IL-6 receptor), which controlled the serum cytokine levels, and reduced intensity chemotherapy, including vindesine, cyclophosphamide, and prednisolone. However, the patient finally died of multiple organ failure. To the best of our knowledge, this is the first report on the development of a cardiac mass and occurrence of myocardial infiltration after allo-HSCT and CAR T-cell therapy. This report may provide supporting data for the early diagnosis and immediate treatment of patients with cardiac involvement.

Lymphodepletion (LD) or conditioning is an essential step in the application of currently used autologous and allogeneic chimeric antigen receptor T-cell (CAR-T) therapies as it maximizes engraftment, efficacy and long-term survival of CAR-T. Its main modes of action are the depletion and modulation of endogenous lymphocytes, conditioning of the microenvironment for improved CAR-T expansion and persistence, and reduction of tumor load. However, most LD regimens provide a broad and fairly unspecific suppression of T-cells as well as other hematopoietic cells, which can also lead to severe side effects, particularly infections. We reviewed 1271 published studies (2011-2023) with regard to current LD strategies for approved anti-CD19 CAR-T products for large B cell lymphoma (LBCL). Fludarabine (Flu) and cyclophosphamide (Cy) (alone or in combination) were the most commonly used agents. A large number of different schemes and combinations have been reported. In the respective schemes, doses of Flu and Cy (range 75-120mg/m2 and 750-1.500mg/m2) and wash out times (range 2-5 days) differed substantially. Furthermore, combinations with other agents such as bendamustine (benda), busulfan or alemtuzumab (for allogeneic CAR-T) were described. This diversity creates a challenge but also an opportunity to investigate the impact of LD on cellular kinetics and clinical outcomes of CAR-T. Only 21 studies explicitly investigated in more detail the influence of LD on safety and efficacy. As Flu and Cy can potentially impact both the in vivo activity and toxicity of CAR-T, a more detailed analysis of LD outcomes will be needed before we are able to fully assess its impact on different T-cell subsets within the CAR-T product. The T2EVOLVE consortium propagates a strategic investigation of LD protocols for the development of optimized conditioning regimens.