MULTIPLEX GENE EDITING IN CAR T CELLS Jafarzadeh, L.; Boudreau, G.; Rulleau, C. ...
Cytotherapy (Oxford, England),
June 2024, 2024-06-00, Letnik:
26, Številka:
6
Journal Article
Recenzirano
Chimeric Antigen Receptor (CAR) T-cell therapy for cancer is limited by several mechanisms that impact T-cell function. The pan T-cell receptor CD5 is an inhibitory co-receptor that can be targeted ...through CRISPR-Cas9 gene editing techniques to improve T-cell function. We found that CD5 deletion alone had little impact on T-cell functionality and aimed to assess whether the co-deletion of other immune checkpoints alongside CD5 was feasible in CAR T cells. Focusing on the inhibitory receptor TIM-3, we assessed the impact of multiple gene editing in CAR T cells.
Human CD3+ T cells isolated from healthy peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 and anti-CD28 antibodies. After a three-day activation, T cells were transduced with a lentivirus carrying a second-generation CAR construct. Two days post-transduction, CRISPR-Cas9-mediated electroporation induced Tim3 and/or CD5 knockout in CAR T cells. CD5/Tim3 knockout CAR T cells underwent additional activation, and after one week, their phenotype and functionality were assessed using flow cytometry.
Our study confirms the efficacy of single and double knockout strategies targeting CD5 and TIM-3 in CAR T cells. Initial investigations revealed comparable effectiveness of editing both genes to single deletions in CD3/CD28-stimulated T cells. This pattern extended to anti-CD19 and anti-BCMA CAR T cells, where edited CAR T cells mirrored single deletions' phenotype and functionality. Despite a slight reduction in expansion post-gene knockout, both single and double knockout cells exhibited similar differentiation, primarily favoring effector memory T cells, and consistently expressed PD-1. Functionally, edited CAR T cells displayed high levels of IFN-g and TNF-a expression, with no discernible differences among single or double CD5 and TIM-3 knockout CAR T cells in comparison with unedited CAR T cells. Importantly, all CAR T cells maintained robust cytotoxicity, confirming specific antigen-dependent cytotoxicity through specific target killing. In conclusion, our findings affirm the integrity of our production procedure, demonstrating that single or double deletions do not compromise CAR T cell phenotype or function. Future studies will involve serial stimulation of unedited, single, and double knockout CAR T cells to investigate CAR T-cell exhaustion/dysfunction and the expansion of antigen-specific CAR T cells with single and double editions.
Free-floating car-sharing schemes operate without fixed car-sharing stations, ahead reservations or return-trip requirements. Providing fast and convenient motorization, they attract both public ...transportation users and (former) car-owners. However, given their highly flexible nature and different pricing structures, previous findings on user groups and environmental impact of station-based car-sharing may not be easily transferable. Therefore, this research uses survey data to compare user groups and usage patterns of a free-floating and station-based car-sharing service both operating in the city of Basel, Switzerland. The findings suggest, that the schemes indeed attract different user groups and are also used differently. Moreover, we see, that car-sharing membership is governed by other factors than car-sharing activity.
Chimeric antigen receptor (CAR) T cell expansion and persistence emerged as key efficacy determinants in cancer patients. These features are typical of early-memory T cells, which can be enriched ...with specific manufacturing procedures, providing signal one and signal two in the proper steric conformation and in the presence of homeostatic cytokines. In this project, we exploited our expertise with paramagnetic beads and IL-7/IL-15 to develop an optimized protocol for CAR T cell production based on reagents, including a polymeric nanomatrix, which are compatible with automated manufacturing
via
the CliniMACS Prodigy. We found that both procedures generate similar CAR T cell products, highly enriched of stem cell memory T cells (T
SCM
) and equally effective in counteracting tumor growth in xenograft mouse models. Most importantly, the optimized protocol was able to expand CAR T
SCM
from B-cell acute lymphoblastic leukemia (B-ALL) patients, which in origin were highly enriched of late-memory and exhausted T cells. Notably, CAR T cells derived from B-ALL patients proved to be as efficient as healthy donor-derived CAR T cells in mediating profound and prolonged anti-tumor responses in xenograft mouse models. On the contrary, the protocol failed to expand fully functional CAR T
SCM
from patients with pancreatic ductal adenocarcinoma, suggesting that patient-specific factors may profoundly affect intrinsic T cell quality. Finally, by retrospective analysis of
in vivo
data, we observed that the proportion of T
SCM
in the final CAR T cell product positively correlated with
in vivo
expansion, which in turn proved to be crucial for achieving long-term remissions. Collectively, our data indicate that next-generation manufacturing protocols can overcome initial T cell defects, resulting in T
SCM
-enriched CAR T cell products qualitatively equivalent to the ones generated from healthy donors. However, this positive effect may be decreased in specific conditions, for which the development of further improved protocols and novel strategies might be highly beneficial.
CAR T cells recognizing CD19 effectively treat relapsed and refractory B-ALL and DLBCL. However, CD19 loss is a frequent cause of relapse. Simultaneously targeting a second antigen, CD22, may ...decrease antigen escape, but is challenging: its density is approximately 10-fold less than CD19, and its large structure may hamper immune synapse formation. The characteristics of the optimal CD22 CAR are underexplored. We generated 12 distinct CD22 antibodies and tested CARs derived from them to identify a CAR based on the novel 9A8 antibody, which was sensitive to low CD22 density and lacked tonic signaling. We found no correlation between affinity or membrane proximity of recognition epitope within Ig domains 3–6 of CD22 with CART function. The optimal strategy for CD19/CD22 CART co-targeting is undetermined. Co-administration of CD19 and CD22 CARs is costly; single CARs targeting CD19 and CD22 are challenging to construct. The co-expression of two CARs has previously been achieved using bicistronic vectors. Here, we generated a dual CART product by co-transduction with 9A8-41BBζ and CAT-41BBζ (obe-cel), the previously described CD19 CAR. CAT/9A8 CART eliminated single- and double-positive target cells in vitro and eliminated CD19- tumors in vivo. CAT/9A8 CART is being tested in a phase I clinical study (NCT02443831).
Display omitted
Pule et al. designed a high-sensitivity CD22 CAR after generating 12 novel CD22 antibodies. The candidate, 9A8, was co-transduced with CAT, a clinically relevant CD19 CAR, for a dual CART product. The efficiency of the CAT/9A8 CART product in vitro and in vivo led to an ongoing phase I trial (NCT02443831).
Adoptive cell therapy with chimeric antigen receptor (CAR) immunotherapy has made tremendous progress with five CAR T therapies approved by the US Food and Drug Administration for hematological ...malignancies. However, CAR immunotherapy in solid tumors lags significantly behind. Some of the major hurdles for CAR immunotherapy in solid tumors include CAR T cell manufacturing, lack of tumor-specific antigens, inefficient CAR T cell trafficking and infiltration into tumor sites, immunosuppressive tumor microenvironment (TME), therapy-associated toxicity, and antigen escape. CAR Natural Killer (NK) cells have several advantages over CAR T cells as the NK cells can be manufactured from pre-existing cell lines or allogeneic NK cells with unmatched major histocompatibility complex (MHC); can kill cancer cells through both CAR-dependent and CAR-independent pathways; and have less toxicity, especially cytokine-release syndrome and neurotoxicity. At least one clinical trial showed the efficacy and tolerability of CAR NK cell therapy. Macrophages can efficiently infiltrate into tumors, are major immune regulators and abundantly present in TME. The immunosuppressive M2 macrophages are at least as efficient as the proinflammatory M1 macrophages in phagocytosis of target cells; and M2 macrophages can be induced to differentiate to the M1 phenotype. Consequently, there is significant interest in developing CAR macrophages for cancer immunotherapy to overcome some major hurdles associated with CAR T/NK therapy, especially in solid tumors. Nevertheless, both CAR NK and CAR macrophages have their own limitations. This comprehensive review article will discuss the current status and the major hurdles associated with CAR T and CAR NK therapy, followed by the structure and cutting-edge research of developing CAR macrophages as cancer-specific phagocytes, antigen presenters, immunostimulators, and TME modifiers.
Cancer has recently been identified as the leading cause of mortality worldwide. Several conventional treatments and cytotoxic immunotherapies have been developed and made available to the market. ...Considering the complex behavior of tumors and the involvement of numerous genetic and cellular factors involved in tumorigenesis and metastasis, there is a need to develop a promising immunotherapy that targets tumors at both the cellular and genetic levels. Chimeric antigen receptor (CAR) T cell therapy has emerged as a novel therapeutic T cell engineering practice, in which T cells derived from patient blood are engineered in vitro to express artificial receptors targeted to a specific tumor antigen. These directly identify the tumor antigen without the involvement of the major histocompatibility complex. The use of this therapy in the last few years has been successful, with a reduction in remission rates of up to 80% for hematologic cancer, particularly for acute lymphoblastic leukemia (ALL) and non‑Hodgkin lymphomas, such as large B cell lymphoma. Recently, anti‑CD19 CAR therapy, or UCART19, has been shown to be efficacious in treating relapsed/refractory hematologic cancer. Several other cell surface tumor antigens, such as CD20 and CD22, found in the majority of leukemias and lymphomas are considered potential targets by pharmaceutical companies and research organizations, and trials have been ongoing in this direction. Although this therapeutic regimen is currently confined to treating hematologic cancer, the increasing involvement of several auxiliary techniques, such as bispecific CAR, Tan‑CAR, inhibitory‑CAR, combined antigens, the clustered regularly interspaced short palindromic repeats gene‑editing tool and nanoparticle delivery, may substantially improve its overall anticancer effects. CAR therapy has the potential to offer a rapid and safer treatment regime to treat non‑solid and solid tumors. The present review presents an insight into the advantages and the advances of CAR immunotherapy and presents the emerging discrepancy of CAR therapy over usual forms of therapy, such as chemotherapy and radiotherapy.
Chimeric antigen receptor (CAR) T cell immunotherapy have demonstrated great response rate in patients with B cell malignancies. However, 20-30% of patients relapse after currently available CD19 CAR ...T cells therapy due to treatment-related antigen loss and lack of therapeutic persistence. More autologous CAR T cell products are made available for clinical application in academia nowadays. The research team has developed MC10029 product, a second-generation CAR product derived from a novel human B-cell activating factor receptor (BAFF-R), with the goal of targeting relapse and refractory diseases. This report presents a summary of the three BAFF-R CAR T manufacturing runs performed at Advanced Manufacturing Facility, Center for Regenerative Biotherapeutics Mayo Clinic Jacksonville Florida.
Three leukapheresis products collected from healthy donors were processed according to the standard operating procedures between September 2023-December 2023. In briefly, T cells were selected from peripheral blood mononuclear cells, activated using Dynabeads, lentiviral transduced, followed by expansion until the day of harvest for cryopreservation.
First engineering run used 85.5 × 10^6 T cells for activation, lentiviral transduction, and expansion. On day 15, 563 × 10^6 cells were harvested with 7-fold expansion cell product. In the second run, 195.7 × 10^6 T cells were used and expanded until day 15, when 2,520 × 10^6 cells were harvested prior to cryopreservation (with a 12-fold expansion). Third qualification run produced 2,490 × 10^6 cells on day of cryopreservation (14-fold expansion). All three manufacturing runs final products were tested by Quality Control (QC) team and results showed all release criteria were met.
Advanced Biomanufacturing Facility, Mayo Clinic, Jacksonville Florida is ready for manufacturing MC10029 CAR T cell products for FDA approved Phase 1a/1b trial in subjects with relapsed or refractory BAFF-R-expressing B-cell hematologic malignancies.
Chimeric antigen receptor (CAR)-T cell therapy has led to remarkable clinical outcomes in the treatment of hematological malignancies. However, challenges remain, such as limited infiltration into ...solid tumors, inadequate persistence, systemic toxicities, and manufacturing insufficiencies. The use of alternative cell sources for CAR-based therapies, such as natural killer cells (NK), macrophages (MΦ), invariant Natural Killer T (iNKT) cells, γδT cells, neutrophils, and induced pluripotent stem cells (iPSC), has emerged as a promising avenue. By harnessing these cells’ inherent cytotoxic mechanisms and incorporating CAR technology, common CAR-T cell-related limitations can be effectively mitigated. We herein present an overview of the tumoricidal mechanisms, CAR designs, and manufacturing processes of CAR-NK cells, CAR-MΦ, CAR-iNKT cells, CAR-γδT cells, CAR-neutrophils, and iPSC-derived CAR-cells, outlining the advantages, limitations, and potential solutions of these therapeutic strategies.
CAR-T: What Is Next? Chen, Yi-Ju; Abila, Bams; Mostafa Kamel, Yasser
Cancers,
01/2023, Letnik:
15, Številka:
3
Journal Article
Recenzirano
Odprti dostop
The year 2017 was marked by the Food and Drug Administration (FDA) approval of the first two chimeric antigen receptor-T (CAR-T) therapies. The approved indications were for the treatment of relapsed ...or refractory diffuse large B-cell lymphoma (DLBCL) and for the treatment of patients up to 25 years of age with acute lymphoblastic leukemia (ALL) that is refractory or in a second or later relapse. Since then, extensive research activities have been ongoing globally on different hematologic and solid tumors to assess the safety and efficacy of CAR-T therapy for these diseases. Limitations to CAR-T therapy became apparent from, e.g., the relapse in up to 60% of patients and certain side effects such as cytokine release syndrome (CRS). This led to extensive clinical activities aimed at overcoming these obstacles, so that the use of CAR-T therapy can be expanded. Attempts to improve on efficacy and safety include changing the CAR-T administration schedule, combining it with chemotherapy, and the development of next-generation CAR-T therapies, e.g., through the use of CAR-natural killer (CAR-NK) and CAR macrophages (CAR-Ms). This review will focus on new CAR-T treatment strategies in hematologic malignancies, clinical trials aimed at improving efficacy and addressing side effects, the challenges that CAR-T therapy faces in solid tumors, and the ongoing research aimed at overcoming these challenges.
Chimeric antigen receptor (CAR)-T cell therapy has been shown to be an effective treatment for hematological tumors, but the treatment of solid tumors still lacks effectiveness. In the tumor ...microenvironment, macrophages are the innate immune cells with the highest infiltration rate. Tumor-associated macrophages (TAMs) stimulate angiogenesis, increase tumor invasion, and mediate immunosuppression. Because macrophages can infiltrate solid tumor tissue and interact with almost all cellular components in the tumor microenvironment (including tumor cells, immune cells such as T-cells, NK cells, DCs, and other resident non-immune cells), researchers are trying to use macrophages modified with CAR (CAR-M) against solid tumors. This review describes recent reports of CAR-M-based tumor treatments and summarizes their shortcomings and future applications.
Display omitted
PDF
