Universal Chimeric Antigen Receptor Research Articles

Evaluation of Anti-CAR Linker mAbs for CAR T Monitoring after BiTEs/bsAbs and CAR T-Cell Pretreatment

For the monitoring of chimeric antigen receptor (CAR) T-cell therapies, antigen-based CAR detection methods are usually applied. However, for each target-antigen, a separate detection system is required. Furthermore, when monitored CAR T-cells in the blood of patients treated with bispecific antibodies or T-cell engagers (bsAbs/BiTEs) recognize the same antigen, these methods produce false-positive results in clinical diagnostics. Anti-CAR-linker monoclonal antibodies (mAbs) targeting the linker sequence between the variable domains of the antigen binding CAR fragment promise a universal and unbiased CAR detection. To test this, we analyzed clinical specimens of all BCMA- and CD19-targeting CAR T-cell products currently approved for clinical use. We found a highly specific and sensitive CAR detection using anti-CAR-linker mAb in blood cells from patients treated with Ide-cel, Tisa-cel, Axi-cel, Brexu-cel, and Liso-cel. For Ide-cel and Tisa-cel, the sensitivity was significantly lower compared to that for antigen-based CAR detection assays. Strikingly, the specificity of anti-CAR linker mAb was not affected by the simultaneous presence of bispecific blinatumomab or teclistamab for Axi-cel, Brexu-cel, Liso-cel, or Ide-cel, respectively. Cilta-cel (containing a monomeric G4S-CAR linker) could not be detected by anti-CAR linker mAb. In conclusion, anti-CAR-linker mAbs are highly specific and useful for CAR T-cell monitoring but are not universally applicable.

Universal CAR 2.0 to overcome current limitations in CAR therapy.

Chimeric antigen receptor (CAR) T cell therapy has effectively complemented the treatment of advanced relapsed and refractory hematological cancers. The remarkable achievements of CD19- and BCMA-CAR T therapies have raised high expectations within the fields of hematology and oncology. These groundbreaking successes are propelling a collective aspiration to extend the reach of CAR therapies beyond B-lineage malignancies. Advanced CAR technologies have created a momentum to surmount the limitations of conventional CAR concepts. Most importantly, innovations that enable combinatorial targeting to address target antigen heterogeneity, using versatile adapter CAR concepts in conjunction with recent transformative next-generation CAR design, offer the promise to overcome both the bottleneck associated with CAR manufacturing and patient-individualized treatment regimens. In this comprehensive review, we delineate the fundamental prerequisites, navigate through pivotal challenges, and elucidate strategic approaches, all aimed at paving the way for the future establishment of multitargeted immunotherapies using universal CAR technologies.

Acidity-targeting transition-aided universal chimeric antigen receptor T-cell (ATT-CAR-T) therapy for the treatment of solid tumors

The use of CAR-T cells in treating solid tumors frequently faces significant challenges, mainly due to the heterogeneity of tumor antigens. This study assessed the efficacy of an acidity-targeting transition-aided universal chimeric antigen receptor T (ATT-CAR-T) cell strategy, which is facilitated by an acidity-targeted transition. Specifically, the EGFRvIII peptide was attached to the N-terminus of a pH-low insertion peptide. Triggered by the acidic conditions of the tumor microenvironment, this peptide alters its structure and selectively integrates into the membrane of solid tumor cells. The acidity-targeted transition component effectively relocated the EGFRvIII peptide across various tumor cell membranes; thus, allowing the direct destruction of these cells by EGFRvIII-specific CAR-T cells. This method was efficient even when endogenous antigens were absent. In vivo tests showed marked antigen modification within the acidic tumor microenvironment using this component. Integrating this component with CAR-T cell therapy showed high effectiveness in combating solid tumors. These results highlight the capability of ATT-CAR-T cell therapy to address the challenges presented by tumor heterogeneity and expand the utility of CAR-T cell therapy in the treatment of solid tumors.

The construction of modular universal chimeric antigen receptor T (MU-CAR-T) cells by covalent linkage of allogeneic T cells and various antibody fragments

BackgroundChimeric antigen receptor-T (CAR-T) cells therapy is one of the novel immunotherapeutic approaches with significant clinical success. However, their applications are limited because of long preparation time, high cost, and interpersonal variations. Although the manufacture of universal CAR-T (U-CAR-T) cells have significantly improved, they are still not a stable and unified cell bank.MethodsHere, we tried to further improve the convenience and flexibility of U-CAR-T cells by constructing novel modular universal CAR-T (MU-CAR-T) cells. For this purpose, we initially screened healthy donors and cultured their T cells to obtain a higher proportion of stem cell-like memory T (TSCM) cells, which exhibit robust self-renewal capacity, sustainability and cytotoxicity. To reduce the alloreactivity, the T cells were further edited by double knockout of the T cell receptor (TCR) and class I human leukocyte antigen (HLA-I) genes utilizing the CRISPR/Cas9 system. The well-growing and genetically stable universal cells carrying the CAR-moiety were then stored as a stable and unified cell bank. Subsequently, the SDcatcher/GVoptiTag system, which generate an isopeptide bond, was used to covalently connect the purified scFvs of antibody targeting different antigens to the recovered CAR-T cells.ResultsThe resulting CAR-T cells can perform different functions by specifically targeting various cells, such as the eradication of human immunodeficiency virus type 1 (HIV-1)-latenly-infected cells or elimination of T lymphoma cells, with similar efficiency as the traditional CAR-T cells did.ConclusionTaken together, our strategy allows the production of CAR-T cells more modularization, and makes the quality control and pharmaceutic manufacture of CAR-T cells more feasible.

Protocol for Efficient Generation of Chimeric Antigen Receptor T Cells with Multiplexed Gene Silencing by Epigenome Editing.

Multiplex gene regulation enables the controlled and simultaneous alteration of the expression levels of multiple genes and is generally pursued to precisely alter complex cellular pathways with a single intervention. Thus far, this has been typically exploited in combination with genome editing tools (i.e., base-/prime-editing, designer nucleases) to enable simultaneous genetic alterations and modulate complex physiologic cellular pathways. In the field of cancer immunotherapy, multiplex genome editing has been used to simultaneously inactivate three genes (i.e., TRAC, B2M, and PDCD1) and generate universal chimeric antigen receptor (CAR) T cells resistant to the inhibitory activity of the PD-1 ligand. However, the intrinsic risk of genomic aberrations driven by such tools poses concerns because of the generation of multiple single-strand or double-strand DNA breaks followed by DNA repair. Modulating gene expression without DNA damage using epigenome editing promises a safer and efficient approach to alter gene expression. This method enables for simultaneous activation and/or repression of target genes, offering superior fine-tuning capabilities with reduced off-targeting effects and potential reversibility as compared to genome editing. Here we describe a detailed protocol for achieving multiplexed and sustainable gene silencing in CAR T cells. In an exemplary approach, we use designer epigenome modifiers (DEMs) for the simultaneous inactivation of two T cell inhibitory genes, PDCD1 and LAG3 to generate CAR T cells with increased resistance to tumor-induced exhaustion.

Functional Characterization and Optimization of Switchable Allogeneic Chimeric Antigen Receptor T Cells for Targeting CD19 and CD20 in B Cell Malignancies

Chimeric antigen receptor (CAR) T cell therapy has demonstrated significant efficacy in B cell malignancies. However, despite clinically manageable on-target/off-tumor toxicity when targeting antigens like CD19 and BCMA, severe cytokine release and neurotoxicity remain a clinical challenge. Additionally, patients may require immunoglobulin replacement therapy, and antigen-loss is recognized as a cause of relapse (PMID: 30275569, 31798590, 36263838, 37051246 and 37122700). To overcome these limitations, we developed a novel switchable CAR-T cell therapy that simultaneously targets both CD19 and CD20 in B cell malignancies. This innovative therapy is based on our reverse universal chimeric antigen receptor platform (RevCAR), a 2-component CAR-T technology (Figure 1; PMID: 34638268 and 32923149). The first component is a universal CAR-T cell, incorporating a CAR based on a short, non-immunogenic peptide motif derived from the human nuclear La/SSB autoantigen. The CAR itself does not recognize any human cell surface antigen. However, it is specifically targeted by an antibody-based binder in the second component, a soluble adaptor called targeting module (TM). This TM confers specificity against the desired cancer antigens of choice. Consequently, the availability of the TM effectively controls the activity of RevCAR-T cells. We have recently reported preclinical development of an allogeneic RevCAR-T cell therapy for CD123-positive hematologic malignancies (AVC-201; Ehninger et al. 2023, AACR Abstract 4095; NCT05949125). To target CD123, which is also expressed on normal hematopoietic progenitor cells, a TM with a short half-life was selected (R-TM123), enabling a rapid switch-off of the RevCAR system by TM withdrawal to avoid acute toxicity and continued aplasia. As CD19 and CD20 have a substantially more favorable safety profile and autologous conventional CAR-T cells and bispecific T cell engagers with respective target-specificities are approved in certain B cell malignancies, a half-life extended TM was developed to allow for dosing every 1-2 weeks. After an extensive screen for an optimized TM targeting CD19 and CD20, we screened multiple RevCAR variants differing in hinge, transmembrane and costimulatory domains for optimal anti-tumor potency using both preclinical in vitro and in vivo models. Profound efficacy and complete tumor control could be observed for optimized RevCAR constructs (Figure 2). While short-term in vitro cytotoxicity assays already gave a small hint towards in vivo efficacy, surprisingly, long-term in vitro rechallenge assay results did not correlate with in vivo efficacy. A summary of functional characterization data will be presented at the meeting with a focus on the lack of predictability of certain in vitro models and underlying hypotheses. Insights from these studies are now flowing into preclinical development of a novel switchable allogeneic CAR-T cell product candidate targeting CD19 and CD20 in B cell malignancies engineered to fully overcome graft-versus-host disease as well as graft rejection by host T and NK cells.

Nursing care of patients with relapsed and refractory multiple myeloma treated with B-cell mature antigen-targeted universal chimeric antigen receptor T cells.

To investigate the efficacy of a nursing approach using B-cell maturation antigen (BCMA)-targeted universal chimeric antigen receptor T-cell (BCMA-UCART) immunotherapy in the treatment of 8 patients with relapsed refractory multiple myeloma (MM). In this study, 16 patients with relapsed and refractory MM who were treated with BCMA-targeted UCART in our department from May 2020 to November 2022 were selected, and were divided into a control group and an experimental group of 8 cases each according to the difference in the nursing methods, and the control group adopted the conventional universal nursing program. The experimental group used the nursing protocol that cooperated with the immunotherapy of this study, and the main points of nursing care included timely assessment of organ functional status, safe and accurate infusion of BCMA-UCART, identification and management of hyperthermia, hypotension, arrhythmia and central nervous system adverse reactions caused by cytokine release after BCMA-UCART infusion, as well as management of fluid imbalance, maintenance of stable blood pressure, and cooperation with physicians to effectively control of inflammatory factors. In addition, patients were provided with psychological and dietary support. The duration of hospitalization was compared between the two groups after the intervention. The discharge time of the experimental group was significantly shorter than that of the control group (P he.05), and the experimental group effectively controlled cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome and acute graft-versus-host disease. The nursing program with BCMA-UCART immunotherapy is effective in intervening MM patients and promotes their early recovery and discharge from the hospital.

Unlocking the potential of Tregs: innovations in CAR technology.

Regulatory T cells (Tregs) adoptive immunotherapy is emerging as a viable treatment option for both autoimmune and alloimmune diseases. However, numerous challenges remain, including limitations related to cell number, availability of target-specific cells, stability, purity, homing ability, and safety concerns. To address these challenges, cell engineering strategies have emerged as promising solutions. Indeed, it has become feasible to increase Treg numbers or enhance their stability through Foxp3 overexpression, post-translational modifications, or demethylation of the Treg-specific demethylated region (TSDR). Specificity can be engineered by the addition of chimeric antigen receptors (CARs), with new techniques designed to fine-tune specificity (tandem chimeric antigen receptors, universal chimeric antigen receptors, synNotch chimeric antigen receptors). The introduction of B-cell targeting antibody receptor (BAR) Tregs has paved the way for effective regulation of B cells and plasma cells. In addition, other constructs have emerged to enhance Tregs activation and function, such as optimized chimeric antigen receptors constructs and the use of armour proteins. Chimeric antigen receptor expression can also be better regulated to limit tonic signaling. Furthermore, various opportunities exist for enhancing the homing capabilities of CAR-Tregs to improve therapy outcomes. Many of these genetic modifications have already been explored for conventional CAR-T therapy but need to be further considered for CAR-Tregs therapies. This review highlights innovative CAR-engineering strategies that have the potential to precisely and efficiently manage immune responses in autoimmune diseases and improve transplant outcomes. As these strategies are further explored and optimized, CAR-Treg therapies may emerge as powerful tools for immune intervention.

Conditional Control of Universal CAR T Cells by Cleavable OFF-Switch Adaptors.

As living drugs, engineered T cell therapies are revolutionizing disease treatment with their unique functional capabilities. However, they suffer from limitations of potentially unpredictable behavior, toxicities, and nontraditional pharmacokinetics. Engineering conditional control mechanisms responsive to tractable stimuli such as small molecules or light is thus highly desirable. We and others previously developed "universal" chimeric antigen receptors (CARs) that interact with coadministered antibody adaptors to direct target cell killing and T cell activation. Universal CARs are of high therapeutic interest due to their ability to simultaneously target multiple antigens on the same disease or different diseases by combining with adaptors to different antigens. Here, we further enhance the programmability and potential safety of universal CAR T cells by engineering OFF-switch adaptors that can conditionally control CAR activity, including T cell activation, target cell lysis, and transgene expression, in response to a small molecule or light stimulus. Moreover, in adaptor combination assays, OFF-switch adaptors were capable of orthogonal conditional targeting of multiple antigens simultaneously, following Boolean logic. OFF-switch adaptors represent a robust new approach for the precision targeting of universal CAR T cells with potential for enhanced safety.

Epitope base editing CD45 in hematopoietic cells enables universal blood cancer immune therapy.

In the absence of cell surface cancer-specific antigens, immunotherapies such as chimeric antigen receptor (CAR) T cells, monoclonal antibodies, or bispecific T cell engagers typically target lineage antigens. Currently, such immunotherapies are individually designed and tested for each disease. This approach is inefficient and limited to a few lineage antigens for which the on-target/off-tumor toxicities are clinically tolerated. Here, we sought to develop a universal CAR T cell therapy for blood cancers directed against the pan-leukocyte marker CD45. To protect healthy hematopoietic cells, including CAR T cells, from CD45-directed on-target/off-tumor toxicity while preserving the essential functions of CD45, we mapped the epitope on CD45 that is targeted by the CAR and used CRISPR adenine base editing to install a function-preserving mutation sufficient to evade CAR T cell recognition. Epitope-edited CD45 CAR T cells were fratricide resistant and effective against patient-derived acute myeloid leukemia, B cell lymphoma, and acute T cell leukemia. Epitope-edited hematopoietic stem cells (HSCs) were protected from CAR T cells and, unlike CD45 knockout cells, could engraft, persist, and differentiate in vivo. Ex vivo epitope editing in HSCs and T cells enables the safe and effective use of CD45-directed CAR T cells and bispecific T cell engagers for the universal treatment of hematologic malignancies and might be exploited for other diseases requiring intensive hematopoietic ablation.

Chimeric antigen receptor T cell therapy for the treatment of systemic rheumatic diseases: a comprehensive review of recent literature.

Systemic rheumatoid diseases (SRDs) are autoimmune and inflammatory disorders that affect multiple organ systems, impacting patients' quality of life, and survival rates. Standard treatment requires continuous drug therapy and immunosuppression. Chimeric antigen receptor (CAR) T cell therapy has the potential to target and eliminate pathologically activated immune cells and re-establish tolerance in organs affected by dysregulated immunity, making them a promising treatment option for autoimmune diseases. In autoimmune diseases, CAR T cells have the advantage of being able to kill B cells effectively without the need for an accessory cell type. Additionally, CAR T cells targeting CD19 have shown promise in comprehensive B cell aplasia, preserving pre-existing humoral immunity, and specifically eliminating pathogenic B cells. CAR T cell therapy's limited use in SRDs is due to its inability to effectively target the various autoreactive lymphocytes present. Researchers are developing a universal CAR T cell therapy that detects and targets autoreactive lymphocytes using major epitope peptides, though further studies are required. Moreover, adoptive transfer of CAR-Tregs has shown promise for effectively reducing inflammation and treating autoimmunity. Through this exploration, the authors hope to provide a comprehensive understanding of the current state of research on this topic, identify areas for further study, and promote the advancement of CAR T cell therapy as a treatment option for SRDs.

Facing challenges with hope: universal immune cells for hematologic malignancies.

Many patients have achieved a favorable overall survival rate since allogenic hematopoietic stem cell transplantation (allo-HSCT) has been widely implemented to treat hematologic malignancies. However, graft-versus-host disease (GVHD) and complications of immunosuppressive drugs after allo-HSCT are the main causes of non-relapse mortality and a poor quality of life. In addition, GVHD and infusion-induced toxicity still occur with donor lymphocyte infusions (DLIs) and chimeric antigen receptor (CAR) T-cell therapy. Because of the special immune tolerance characteristics and anti-tumor ability of universal immune cells, universal immune cell therapy may strongly reduce GVHD, while simultaneously reducing tumor burden. Nevertheless, widespread application of universal immune cell therapy is mainly restricted by poor expansion and persistence efficacy. Many strategies have been applied to improve universal immune cell proliferation and persistence efficacy, including the use of universal cell lines, signaling regulation and CAR technology. In this review we have summarized current advances in universal immune cell therapy for hematologic malignancies with a discussion of future perspectives.

Hypoimmune anti-CD19 chimeric antigen receptor T cells provide lasting tumor control in fully immunocompetent allogeneic humanized mice

Manufacturing autologous chimeric antigen receptor (CAR) T cell therapeutics is complex, and many patients experience treatment delays or cannot be treated at all. Although current allogeneic CAR products have the potential to overcome manufacturing bottlenecks, they are subject to immune rejection and failure to persist in the host, and thus do not provide the same level of efficacy as their autologous counterparts. Here, we aimed to develop universal allogeneic CAR T cells that evade the immune system and produce a durable response. We generated human hypoimmune (HIP) T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-CD19 CAR were expressed using lentiviral transduction. These allogeneic HIP CD19 CAR T cells were compared to allogeneic CD19 CAR T cells that only expressed the anti-CD19 CAR (allo CAR T). In vitro assays for cancer killing and exhaustion revealed no differences between allo CAR T and HIP CAR T cells, confirming that the HIP edits did not negatively affect T cell performance. Clearance of CD19+ tumors by HIP CAR T cells in immunodeficient NSG mice was comparable to that of allo CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, showed improved persistence and expansion, and provided lasting cancer clearance. Furthermore, CD47-targeting safety strategies reliably and specifically eliminated HIP CAR T cells. These findings suggest that universal allogeneic HIP CAR T cell-based therapeutics might overcome the limitations associated with poor persistence of allogeneic CAR T cells and exert durable anti-tumor responses.

Abstract 1787: Design of bispecific adapters for universal CAR T cell therapies of solid tumors

Abstract Recent success in the use of chimeric antigen receptor (CAR) T cell therapies to treat hematologic cancers has encouraged the exploration of CAR T cell technologies to treat solid tumors. For establishment of a productive immunologic synapse between the CAR T cell and cancer cell, multiple CARs on the T cell surface must engage multiple tumor antigens on the cancer cell surface. Whereas these binding interactions are mediated in classical CAR T cells by direct recognition of the tumor antigen by the scFv on the CAR, in the case of universal CAR T cells this recognition must be created by a bispecific adapter that can bridge between the tumor antigen and the CAR. While a diversity of bispecific adapters have been explored in the literature, the bispecific adapter used here is comprised of a low molecular weight ligand that binds avidly to the desired tumor antigen, the yellow dye fluorescein (FL) that binds tightly to an anti-fluorescein scFv on the CAR, and a polyethylene glycol (PEG) spacer that spans between the two. The length of this PEG spacer appears to be critical to ensure that 1) the bispecific adapter is long enough to mediate engagement of the universal CAR T cell with the cancer cell, and 2) the resulting intercellular distance is optimal for activating the necessary co-receptor interactions.In our study, we have utilized protein structure data and molecular modeling to estimate the optimal adapter spacer lengths for bridging between our CAR T cell and two important target cells, namely PSMA positive prostate cancer cells and fibroblast activation protein (FAP) expressing cancer associated fibroblasts. We then synthesized the desired bispecific adapters with a variety lengths of PEG spacer and evaluated their effects in mediating the cytotoxicity of our universal anti-FL CAR T cells both in vitro and in vivo. Our results reveal that the optimal spacer lengths in the bispecific adapters for targeting PSMA (DUPA-FL) and FAP (FAPL-FL) are different and depend on the locations of the adapter binding pockets on their respective protein targets, as predicted by molecular modeling. Specifically, a shorter PEG spacer is required for maximal cytoxicity when employing the PSMA-targeted adapter, whereas a much longer PEG spacer is needed to achieve maximal cell killing with the FAP-targeted adapter. In addition, spacer length can also be adjusted to avoid overactivation-induced T cell exhaustion. Taken together, our studies demonstrate that instead of optimizing the hinge length of a CAR for each antigen epitope, our bispecific adapter/universal CAR platform provides a highly flexible strategy for optimizing the intermembrane distance between the universal CAR T cell and cancer cell to achieve maximal target cell killing with minimal CAR T cell exhaustion. Citation Format: Suilan Zheng, Bo Huang, Yashapal Singh, Haiyan Chu, Suresh K. Bowroju, Md Sazzadul Bari, Madduri Srinivasarao, Sudarsan R. Kasireddy, Ramesh Mukkamala, Laurie Beitz, Byoung Ryu, Andrew M. Scharenberg, Michael Jensen, Philip S. Low. Design of bispecific adapters for universal CAR T cell therapies of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1787.

Abstract 4095: Preclinical characterization of switchable allogeneic chimeric antigen receptor T cells to support first in human clinical study in CD123-positive hematologic and lymphatic malignancies

Abstract Approximately 80% of AML patients express CD123 on their leukemic blasts and CD123 is frequently expressed on other hematologic and lymphatic malignancies. Chimeric antigen receptor (CAR) T-cell therapy has demonstrated significant efficacy in B cell malignancies. Breakthrough of conventional CAR-T technology in AML has been hampered by expression of suitable target antigens on normal hematopoietic progenitor cells posing a risk for continued aplasia. Thus, innovative approaches putting the power of CAR-T technology under the control of reliable and fast-acting on/off switches to avoid and/or abrogate acute and long-term side effects are required. We have recently reported clinical proof-of-concept for autologous switchable CAR-T cell therapy in AML (Wermke et al. Blood 2021). However, manufacturing of autologous products is very costly and rrAML patients are often in urgent need for a product and multiple prior lines of treatment may be detrimental for product quality. While current clinical allogeneic approaches successfully prevent graft-versus-host disease, observed persistence has been limited due to rejection of the grafted T cells by host immune cells. Here, we report preclinical development of a donor-derived allogeneic switchable CAR-T therapy for CD123-positive hematologic and lymphatic malignancies (AVC-201). The reverse universal chimeric antigen receptor platform (RevCAR) is a 2-component CAR-T platform. The first component is a universal CAR-T cell. Its binding domain is a short, non-immunogenic peptide that by itself does not recognize any human cell surface antigen but is specifically bound by an scFv included in the second component, a soluble adaptor called targeting module (TM). To target CD123, a TM with a short half-life was selected (R-TM123), enabling a rapid switch-off of the RevCAR system by TM withdrawal to avoid acute and long-term toxicity usually associated with continuous activation of CAR-T cells. The allogeneic cells are generated by three edits with CRISPR-Cas9, which is meant to fully overcome graft-versus-host disease as well as graft rejection by host T and NK cells. Therefore, we expect a persistent T cell product that can be re-expanded with additional TM cycles. A summary of preclinical characterization of AVC-201 will be presented at the meeting including in vitro and in vivo pharmacology and toxicology experiments. Activation of Allo-RevCAR-T cells is strictly dependent on the presence of CD123-positive target cells and R-TM123. Allo-RevCAR-T cells redirected by R-TM123 efficiently lyse CD123-positive AML cells in vitro and in vivo, and in vitro EC50 values are in the low picomolar range of R-TM123 concentrations. In conclusion, preclinical data support the clinical exploration of AVC-201 in a first in human study. Citation Format: Armin Ehninger, Johannes Spehr, Simon Loff, Anika Langer, Julia Riewaldt, Julia Reinhardt, Jan-Erik Meyer, Josephine Dietrich, Gabriel Jurado, Reynald Lescarbeau, Marc Cartellieri. Preclinical characterization of switchable allogeneic chimeric antigen receptor T cells to support first in human clinical study in CD123-positive hematologic and lymphatic malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4095.

Palbociclib impairs the proliferative capacity of activated T cells while retaining their cytotoxic efficacy.

The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor palbociclib is an emerging cancer therapeutic that just recently gained Food and Drug Administration approval for treatment of estrogen receptor (ER)-positive, human epidermal growth factor receptor (Her)2-negative breast cancer in combination with the ER degrader fulvestrant. However, CDK4/6 inhibitors are not cancer-specific and may affect also other proliferating cells. Given the importance of T cells in antitumor defense, we studied the influence of palbociclib/fulvestrant on human CD3+ T cells and novel emerging T cell-based cancer immunotherapies. Palbociclib considerably inhibited the proliferation of activated T cells by mediating G0/G1 cell cycle arrest. However, after stopping the drug supply this suppression was fully reversible. In light of combination approaches, we further investigated the effect of palbociclib/fulvestrant on T cell-based immunotherapies by using a CD3-PSCA bispecific antibody or universal chimeric antigen receptor (UniCAR) T cells. Thereby, we observed that palbociclib clearly impaired T cell expansion. This effect resulted in a lower total concentration of interferon-γ and tumor necrosis factor, while palbociclib did not inhibit the average cytokine release per cell. In addition, the cytotoxic potential of the redirected T cells was unaffected by palbociclib and fulvestrant. Overall, these novel findings may have implications for the design of treatment modalities combining CDK4/6 inhibition and T cell-based cancer immunotherapeutic strategies.

Induced pluripotent stem cell-derived hematopoietic stem and progenitor cells: potential, challenges, and future perspectives

Hematopoietic stem and progenitor cells (HSPCs) are responsible for the lifetime dynamics of hematopoiesis, as they are well known for their self-renewing ability and multipotency to differentiate into all types of blood cells, including both myeloid and lymphoid lineages. However, due to their limited amount and accessibility, there is a strong need to search out alternative methods to produce HSPCs. In this review, we suggest induced pluripotent stem cells (iPSCs) as a new viable source for HSPC production because these cells have the potential to self-renew while being relatively easy to modify. Recent studies have revealed that the recapitulation of definitive hematopoiesis is the key to the successful in vitro production of HSPCs with multilineage potential. Therefore, we summarized recent progress in establishing the generation of definitive HSPCs with high maturity and functionality in vitro. Definitive HSPCs can be used in disease modeling and gene therapy for genetic blood disorders via gene modification in iPSCs, applied in cellular immunotherapy in the form of a universal chimeric antigen receptor system, and may recapitulate the intricate immune system within the iPSC-derived organoids that closely mimic the in vivo pathophysiological environment. In summary, this review provides an overview of the generation of HSPCs from iPSCs, in terms of the developmental process of hematopoiesis, in vitro attempts to produce iPSC-derived definitive HSPCs, and the following applications of these cells in numerous areas. This review sheds light on the concept of iPSC-derived definitive HSPCs, setting a milestone for artificial blood production in the near future.

Multivalent adaptor proteins specifically target NK cells carrying a universal chimeric antigen receptor to ErbB2 (HER2)-expressing cancers.

Chimeric antigen receptor (CAR)-engineered immune effector cells constitute a promising approach for adoptive cancer immunotherapy. Nevertheless, on-target/off-tumor toxicity and immune escape due to antigen loss represent considerable challenges. These may be overcome by adaptor CARs that are selectively triggered by bispecific molecules that crosslink the CAR with a tumor-associated surface antigen. Here, we generated NK cells carrying a first- or second-generation universal CAR (UniCAR) and redirected them to tumor cells with so-called target modules (TMs) which harbor an ErbB2 (HER2)-specific antibody domain for target cell binding and the E5B9 peptide recognized by the UniCAR. To investigate differential effects of the protein design on activity, we developed homodimeric TMs with one, two or three E5B9 peptides per monomer, and binding domains either directly linked or separated by an IgG4 Fc domain. The adaptor molecules were expressed as secreted proteins in Expi293F cells, purified from culture supernatants and their bispecific binding to UniCAR and ErbB2 was confirmed by flow cytometry. In cell killing experiments, all tested TMs redirected NK cell cytotoxicity selectively to ErbB2-positive tumor cells. Nevertheless, we found considerable differences in the extent of specific cell killing depending on TM design and CAR composition, with adaptor proteins carrying two or three E5B9 epitopes being more effective when combined with NK cells expressing the first-generation UniCAR, while the second-generation UniCAR was more active in the presence of TMs with one E5B9 sequence. These results may have important implications for the further development of optimized UniCAR and target module combinations for cancer immunotherapy.

Generating universal chimeric antigen receptor expressing cell products from induced pluripotent stem cells: beyond the autologous CAR-T cells.

Adoptive therapeutic immune cells, such as chimeric antigen receptor (CAR)-T cells and natural killer cells, have established a new generation of precision medicine based on which dramatic breakthroughs have been achieved in intractable lymphoma treatments. Currently, well-explored approaches focus on autologous cells due to their low immunogenicity, but they are highly restricted by the high costs, time consumption of processing, and the insufficiency of primary cells in some patients. Induced pluripotent stem cells (iPSCs) are cell sources that can theoretically produce indefinite well-differentiated immune cells. Based on the above facts, it may be reasonable to combine the iPSC technology and the CAR design to produce a series of highly controllable and economical "live" drugs. Manufacturing hypoimmunogenic iPSCs by inactivation or over-expression at the genetic level and then arming the derived cells with CAR have emerged as a form of "off-the-shelf" strategy to eliminate tumor cells efficiently and safely in a broader range of patients. This review describes the reasonability, feasibility, superiority, and drawbacks of such approaches, summarizes the current practices and relevant research progress, and provides insights into the possible new paths for personalized cell-based therapies.

Simultaneous editing of TCR, HLA-I/II and HLA-E resulted in enhanced universal CAR-T resistance to allo-rejection.

The major challenge for universal chimeric antigen receptor T cell (UCAR-T) therapy is the inability to persist for a long time in patients leading to inferior efficacy clinically. The objective of this study was to design a novel UCAR-T cell that could avoid the occurrence of allo-rejection and provide effective resistance to allogeneic Natural Killer (NK) cell rejection, together with the validation of its safety and efficacy ex vivo and in vivo. We prepared T-cell receptor (TCR), Human leukocyte antigen (HLA)-I/II triple-edited (TUCAR-T) cells and evaluated the anti-tumor efficacy ex vivo and in vivo. We measured the resistance of exogenous HLA-E expressing TUCAR-T (ETUCAR-T) to NK rejection by using an enhanced NK. Furthermore, we established the safety and efficacy of this regimen by treating Nalm6 tumor-bearing mice with a repeated high-dose infusion of ETUCAR-T. Moreover, we analyzed the effects of individual gene deficiency CAR-T on treated mice and the changes in the transcriptional profiles of different gene-edited T cells via RNA-Seq. Data showed that HLA-II editing didn't impair the anti-tumor efficacy of TUCAR-T ex vivo and in vivo and we found for the first time that HLA-II deficiency could facilitate the persistence of CAR-T. Contrastively, as the most commonly eliminated target in UCAR-T, TCR deficiency was found to be a key disadvantageous factor for the shorter-term anti-tumor efficacy in vivo. Our study demonstrated ETUCAR-T could effectively resist allogeneic NK rejection ex vivo and in vivo. Our research provided a potential and effective strategy for promoting the persistence of UCAR-T cells in clinical application. And it reveals the potential key factors of the poor persistence of UCAR-T along with new insights for future development.