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B Background: b The redundancy of CD28 and CD3 signaling in a CAR design incorporating all 3 immunoreceptor tyrosine-based activation motifs (ITAMs) might negatively affect T cell differentiation and promote exhaustion. In mouse models, 19-28z1XX CAR successfully induced tumor eradication at low CAR T cell doses with improved survival compared to conventional 19-28z CAR and with enhanced persistence of functional CAR T cells. A Phase I Study of CD19-targeted 19(T2)28z1xx CAR T Cells in Adult Patients with Relapsed or Refractory Diffuse Large B-cell Lymphoma. [Extracted from the article]

The lack of persistence of infused T cells is a principal limitation of adoptive immunotherapy in man. Interleukin (IL)-15 can sustain memory T cell expansion when presented in complex with IL-15Rα (15Rα/15). We developed a novel in-vitro system for generation of stable 15Rα/15 complexes. Immunologically quantifiable amounts of IL-15 were obtained when both IL-15Rα and IL-15 genes were co-transduced in NIH 3T3 fibroblast-based artificial antigen-presenting cells expressing human leucocyte antigen (HLA) A:0201, β2 microglobulin, CD80, CD58 and CD54 [A2-artificial antigen presenting cell (AAPC)] and a murine pro-B cell line (Baf-3) (A2-AAPC15Rα/15and Baf-315Rα/15). Transduction of cells with IL-15 alone resulted in only transient expression of IL-15, with minimal amounts of immunologically detectable IL-15. In comparison, cells transduced with IL-15Rα alone (A2-AAPCRα) demonstrated stable expression of IL-15Rα; however, when loaded with soluble IL-15 (sIL-15), these cells sequestered 15Rα/15 intracellularly and also demonstrated minimal amounts of IL-15. Human T cells stimulated in vitro against a viral antigen (CMVpp65) in the presence of 15Rα/15 generated superior yields of high-avidity CMVpp65 epitope-specific T cells [cytomegalovirus-cytotoxic T lymphocytes (CMV-CTLs)] responding to ≤ 10− 13 M peptide concentrations, and lysing targets cells at lower effector : target ratios (1 : 10 and 1 : 100), where sIL-15, sIL-2 or sIL-7 CMV-CTLs demonstrated minimal or no activity. Both soluble and surface presented 15Rα/15, but not sIL-15, sustained in-vitro expansion of CD62L+ and CCR7+ central memory phenotype CMV-CTLs (TCM). 15Rα/15 complexes represent a potent adjuvant for augmenting the efficacy of adoptive immunotherapy. Such cell-bound or soluble 15Rα/15 complexes could be developed for use in combination immunotherapy approaches. [ABSTRACT FROM AUTHOR]

The article presents the collaborative Italy-U.S.. project study for gene therapy of hematopoietic stem cells (HSCs). The study aims to produce globin lentiviral (LV) vectors with improved titer gene transfer and expression to improve efficiency and safety of the TNS9 lentiviral vector for gene therapy of beta-thalassemia major. It also demonstrates the critical aspect of gene therapy which is the efficiency of the systems of gene transduction.

Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumor rejection. CD19 targeted CAR T cells have showed remarkable effacy for chemorefractory/relapsed B cell malignancies. Recombinant retroviral vectors mediated- CAR gene transfer is currently the standard method to generate cGMP grade CAR T cells. However, CAR gene expression is highly variable, owing to position effects and vector incorporation variations. CAR T cells engineered in this manner are capable of tumor eradication, but are prone to tonic signaling and accelerated exhaustion. We have developed a novel genetic engineering strategy to insert CAR genes into a precise genomic location in human peripheral blood T cells. T cells are electroporated with Cas9 mRNA or Cas9 protein and a guide RNA, followed by transduction with a recombinant Adeno-Associated Virus encoding the CAR sequence to facilitate the insertion of the CAR gene upstream of the constant region of TCR alpha chain. This results in the endogenous TCR promoter (TRAC) controlled CAR expression and abrogation of TCR surface expression. This strategy not only allows uniform CAR expression, but also delays T-cell differentiation and exhaustion, leading to enhanced T cell function and anti-tumor efficacy. The edited cells vastly outperformes retrovirally modified CAR T cells in a pre-B ALL NALM6 mouse model. The targeting of CARs to the TCR locus also provides a safer therapeutic T cell by minimizing the risks of insertional oncogenesis, and TCR-induced autoimmunity and alloreactivity (thus spanning both autologous and allogeneic T cell applications). In addition, we have incorporated in the CAR a mutant CD3z chain encoding a single immunoreceptor tyrosine-based activation motif that improves the balance between effector and memory T cells composition. We are in the process of translating this novel approach into the clinical setting by establishing cGMP conditions and protocols for the manufacturing of TRAC-CAR T cells. Using the 4D-LV electroporator, we have demonstrated that we can knockout the TCR at large scale (100 E06 CD3+ T cells) with high efficiency (70-80%) as that obtained at small scale. We have evaluated AAV6 to deliver the CAR transgene in the TCR locus. Data will be presented on optimizing the manufacturing of the TRAC-CAR T cells and on evaluating their anti-tumor efficacy in vivo. [ABSTRACT FROM AUTHOR]

A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (microPET) designed specifically for studies of small animals. We review “marker/reporter gene” imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (D2R) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications. Neoplasia (2000) 2, 118–138. [ABSTRACT FROM AUTHOR]

In order to conduct a clinical trial of adoptive therapy with genetically modified T cells in patients with CLL, we are investigating the feasibility of ex vivo T cell transduction and expansion using Xcyte™ Dynabeads® to generate sufficient numbers of biologically active tumor-reactive T cells. T cells are genetically modified to express 19-28z chimeric antigen receptor (CAR). The 19-28z CAR is derived from a single chain fragment length murine antibody specific to the human CD19 antigen expressed on CLL tumor cells, fused to the transmembrane and cytoplasmic domains of the human CD28 co-stimulatory receptor and the cytoplasmic domain of the CD3ζ chain of the human T cell receptor.In four experiments, we find that transduction efficiencies of healthy donor-derived T cells initially activated with Xcyte™ Dynabeads® range from 50% to 88% 19-28z+ T cells as measured by flow cytometry analysis. The degree of 19-28z+ T cell expansion ranges from 2 to 3 log over 14 days of culture. Significantly, we find that T cells activated with Xcyte™ Dynabeads® efficiently lyse CD19+ tumor cells in vitro (70 to 90% killing at 25:1 E:T ratio) similarly to T cells activated with PHA and subsequently restimulated on artificial antigen presenting cells (PHA/AAPCs) as previously demonstrated (Brentjens et al. Nature Medicine 2002). To determine whether 19-28z+ T cells activated with Xcyte™ Dynabeads® have cytotoxic activity in vivo, SCID-Beige mice bearing disseminated Raji cell lymphoma were treated with a single intravenous injection of either 2 × 107 19-28z transduced T cells (n=22) or 2 × 107 control Pz1 transduced T cells (n=15) or 2 × 107 1928z T cells activated on PHA/AAPC (n=8). The latter group was used as a positive control as we have previously shown that PHA/AAPC-stimulated T cells could eradicate established CD19+ tumor cells in vivo (Brentjens et al. Nature Medicine 2002). After treatment, mice were routinely checked for tumor progression as determined by hind-limb paralysis. Mice with hind-limb paralysis were sacrificed. As expected, all control mice treated with Pz1 transduced T cells all developed hind-limb paralysis 4-5 weeks after tumor cell injection. In contrast, mice treated with 19-28z+ T cells activated with Xcyte™ Dynabeads® had either delayed progression of disease (n=2) or remain disease free (n=20) at >180 days. Mice treated with 19-28z activated with PHA/AAPC showed either delayed progression of disease (n=1) or remain disease free (n=7) at >180 days. These results demonstrate that 19-28z+ T cells activated with Xcyte™ Dynabeads® are as potent as 19-28z+ T cells activated with PHA/AAPCs. Subsequently, we have demonstrated that T cells derived from patients with CLL can also be transduced and expanded with Xcyte™ Dynabeads® and that mice (n=14) treated with these cells have delayed progression of disease (n=7) or remain disease free for > 120 days. These results suggest that a Xcyte™ Dynabeads® -based approach should be suitable to transduce and expand T cells for our planned clinical trial.Molecular Therapy (2006) 13, S102–S102; doi: 10.1016/j.ymthe.2006.08.322 [ABSTRACT FROM AUTHOR]

In order to optimize the transduction of CD34+ cells, CD34+ derived Langerhans cells (LCs) and T lymphocytes for upcoming clinical trials, we determined which medium is best suited for each cell type. Our aim is to identify the formulation providing the best cell proliferation, survival and the highest transduction efficiency (TE) while maintaining the biological activity of the cells. To this end, we harvested and screened PG13-derived retroviral vector stocks (VS) collected in various media formulations. High-titer VS were consistently obtained by controlling PG13 cell-plating densities, time of cell growth and vector harvests. We also periodically determined the titer of the frozen VS to determine how long these VS can be stored for clinical applications.CD34+ cells and CD34+-derived LCs. We investigated the vector titers and ex vivo transduction efficiencies obtained with VS collected in six serum-free media formulations (IMDM,QBSF 60, XVivo 10, XVivo 15, DMEM low and high glucose) and their serum containing counterparts. In all experiments, CD34+ cells were pre-stimulated with rhMDGF, rhSCF and rhFLT-3. PG13-derived VS collected in either DMEM or XVivo 10 gave the best results and the transgene expression averaged 45.6±6.03% and 55.3±6.6%, respectively in the total cell population. However, the percentage of cells co- expressing both CD34 and eGFP were significantly lower when the transductions were performed in DMEM (14.3±4.6%) as compared to XVivo 10 (21.8±4%). This data led us to select XVivo10 for pre-stimulating CD34+ cells and manufacturing VS for the transduction of CD34+ cells and CD34+-derived LCs. We have shown that upon freezing these VS are stable for at least 12 months.T lymphocytes. We investigated the vector titers and TE obtained with three media formulations (XVivo10, RPMI and DMEM) in presence or absence of 10% serum. PBMCs were activated with PHA and 20U IL-2 in RPMI+FBS. PG13-derived VS collected in either DMEM+FBS, RPMI+FBS gave better TE (55-56% range) than VS collected in either DMEM, RPMI or XVivo10+FBS (44-49% range) or than VS collected in X-Vivo 10 alone (26% range). Subsequently, we found that at least 5% of serum (FBS or human AB) was required for optimal proliferation and transduction of T lymphocytes and that activation of the PBMCs in XVivo15+5% AB serum gave consistently higher TE (45% ± 8% NIT+) than RPMI (37% ± 4% NIT+) or Yssel’s(33% ± 3% NIT+) using VS collected in DMEM+10% FBS. Finally, we compared the TE in PBMCs using frozen VS collected in either DMEM alone or supplemented with 5%HSA or DMEM+FBS. We observe that some of the VS generated in DMEM+FBS can be stored for up to 41 months with no apparent decrease in TE on T lymphocytes while other VS stored for 24 months show a 3 fold decrease of TE on T lymphocytes. These results will be discussed in the context of various VS coming from PG13 packaging cell clones carrying different transgenes and of various collection media.In conclusion, the collection of VS can be optimized with various media but the optimum medium varies for each cell type.Molecular Therapy (2006) 13, S119–S120; doi: 10.1016/j.ymthe.2006.08.369 [ABSTRACT FROM AUTHOR]

Allogeneic cellular immunotherapies hold promise for broad clinical implementation but face limitations due to potential rejection of donor cells by the host immune system. Silencing of beta-2 microglobulin (B2M) expression is commonly employed to evade T cell-mediated rejection by the host, although the absence of B2M is expected to trigger missing-self responses by host natural killer (NK) cells. Here, we demonstrate that genetic deletion of the adhesion ligands CD54 and CD58 in B2M-deficient chimeric antigen receptor (CAR) T cells and multi-edited induced pluripotent stem cell (iPSC)-derived CAR NK cells reduces their susceptibility to rejection by host NK cells in vitro and in vivo. The absence of adhesion ligands limits rejection in a unidirectional manner in B2M-deficient and B2M-sufficient settings without affecting the antitumor functionality of the engineered donor cells. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection by host immune cells, facilitating the implementation of universal immunotherapy., Competing Interests: Declaration of interests K.-J.M. is a consultant and has research support from Fate Therapeutics. K.-J.M. has research support from Oncopeptides. K.-J.M. and Q.H. are consultants at Vycellix. All relationships have been approved by Oslo University Hospital, the University of Oslo, and the Karolinska Institute. R.M.M., E.V., Y.P., M.J., B.G., R.A., T.L., A.W., J.P.G., and B.V. are employees at Fate Therapeutics. Memorial Sloan Kettering has licensed IP (unrelated to this study) to Fate Therapeutics, and M.S. receives research support (for unrelated studies) from Fate Therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Allogeneic cellular immunotherapies hold a great promise for cancer treatment owing to their potential cost-effectiveness, scalability and on-demand availability. However, immune rejection of adoptively transferred allogeneic T and natural killer (NK) cells is a substantial obstacle to achieving clinical responses that are comparable to responses obtained with current autologous chimeric antigen receptor T cell therapies. In this Perspective, we discuss strategies to confer cell-intrinsic, immune-evasive properties to allogeneic T cells and NK cells in order to prevent or delay their immune rejection, thereby widening the therapeutic window. We discuss how common viral and cancer immune escape mechanisms can serve as a blueprint for improving the persistence of off-the-shelf allogeneic cell therapies. The prospects of harnessing genome editing and synthetic biology to design cell-based precision immunotherapies extend beyond programming target specificities and require careful consideration of innate and adaptive responses in the recipient that may curtail the biodistribution, in vivo expansion and persistence of cellular therapeutics., (© 2024. Springer Nature Limited.)

The tumor microenvironment presents many obstacles to effective chimeric antigen receptor (CAR) T cell therapy, including glucose competition from tumor and myeloid cells. Using mouse models of acute lymphoblastic leukemia (ALL), renal cell carcinoma (RCC), and glioblastoma (GBM), we show that enforced expression of the glucose transporter GLUT1 enhances anti-tumor efficacy and promotes favorable CAR-T cell phenotypes for two clinically relevant CAR designs, 19-28z and IL13Rα2-BBz. In the NALM6 ALL model, 19-28z-GLUT1 promotes T stem cell-like memory formation and prolongs survival. RNA sequencing of these CAR-T cells reveals that the overexpression of GLUT1, but not GLUT3, enriches for genes involved in glycolysis, mitochondrial respiration, and memory precursor phenotypes. Extending these data, 19-28z-GLUT1 CAR-T cells improve tumor control and response to rechallenge in an RCC patient-derived xenograft model. Furthermore, IL13Rα2-BBz CAR-T cells overexpressing GLUT1 prolong the survival of mice bearing orthotopic GBMs and exhibit decreased exhaustion markers. This novel engineering approach can offer a competitive advantage to CAR-T cells in harsh tumor environments where glucose is limiting., Competing Interests: Declaration of interests The authors declare no financial competing interests. M.S. reports research funding from Takeda Pharmaceuticals, Atara Biotherapeutics, Fate Therapeutics, and Mnemo Therapeutics, all of which are unrelated to the present research. M.S. is a scientific cofounder of Mnemo Therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

We designed a CD19-targeted CAR comprising a calibrated signaling module, termed 1XX, that differs from that of conventional CD28/CD3z and 4-1BB/CD3z CARs. Here we report the first-in-human, phase 1 clinical trial of 19(T2)28z-1XX CAR T cells in relapsed/refractory large B-cell lymphoma. We hypothesized that 1XX CAR T cells may be effective at low doses and investigated 4 doubling dose levels starting from 25×10 6 CAR T cells. The overall response rate (ORR) was 82% and complete response (CR) rate 71% in the entire cohort (n=28) and 88% ORR and 75% CR in 16 patients treated at 25×10 6 . With the median follow-up of 24 months, the 1-year EFS was 61% (95% CI: 45-82%). Overall, grade ≥3 CRS and ICANS rates were low at 4% and 7%. The calibrated potency of the 1XX CAR affords excellent efficacy at low cell doses and may benefit the treatment of other hematological malignancies, solid tumors and autoimmunity., Competing Interests: Competing Interests J.H.P. received consulting fees from Affyimmune Therapeutics, Amgen, Autolus, Be Biopharma, Beigene, Bright Pharmaceutical Services, Inc., Caribou Biosciences, Curocell, Galapagos, In8Bio, Kite, Medpace, Minerva Biotechnologies, Pfizer, Servier, Sobi, and Takeda; received honoraria from OncLive, Physician Education Resource, and MJH Life Sciences; serves on scientific advisory board of Allogene Therapeutics, Artiva Biotherapeutics and Green Cross Biopharma; and received institutional research funding from Autolus, Genentech, Fate Therapeutics, InCyte, Servier, and Takeda. M.L.P. received consulting fees from BMS, Cellectis, Kite, Mustang Bio and Synthekine. G.Sh. received research funding to the institution from Amgen, BMS, Beyond Spring, Janssen, and GPCR, and serves on DSMB for Arcellx. P.D. received consulting fees from Kite. M.Sc. served as a paid consultant for McKinsey & Company, Angiocrine Bioscience, Inc., Kite, and Omeros Corporation; received research funding from Angiocrine Bioscience, Inc., Omeros Corporation, and Amgen, Inc.; served on ad hoc advisory boards for Kite-A Gilead company; and received honoraria from i3Health, Medscape and CancerNetwork for CME-related activity. G.Sa. has received financial compensations for participating in advisory boards or consulting for Abbvie, Atbtherapeutics, Beigene, BMS, Debiopharm Genentech/Roche, Genmab, Incyte, Ipsen, Janssen, Kite/Gilead, Loxo/Lilly, Merck, Molecular Partners, Nordic Nanovector, Novartis, Nurix and Orna. He is also a shareholder of: Owkin; he received Research Support managed by his institution from Genentech, Janssen, Ipsen, Nurix. Y.K.V. received one time consulting fee from EastRx. J.M.S. is an inventor on 1XX related IP. I.R. reports grants from Takeda Pharmaceuticals and Atara, personal fees from Mnemo Therapeutics, Akron, the Centre for Commercialization of Cancer, and Oribiotech, and other support from Bristol Myers Squibb outside the submitted work. M. S. reports grants from Atara Biotherapeutics outside the submitted work, as well as patent 8389282 issued and licensed to Juno Therapeutics, patent 11242375 issued and licensed to Atara Biotherapeutics, patent 10370452 issued, licensed, and with royalties paid from Fate Therapeutics, patent 11377637 issued and licensed to Takeda Pharmaceuticals, patent 11377637 issued and licensed to Mnemo Therapeutics, and patent 11377637 issued and licensed to Minerva Biotechnologies. MSK has licensed 1XX technology to Atara Biotherapy, Fate Therapeutics, Minerva Therapeutics, and Takeda Pharmaceuticals.

Chimeric antigen receptor T cells have dramatically improved the treatment of hematologic malignancies. T cell antigen receptor (TCR)-based cell therapies are yet to achieve comparable outcomes. Importantly, chimeric antigen receptors not only target selected antigens but also reprogram T cell functions through the co-stimulatory pathways that they engage upon antigen recognition. We show here that a fusion receptor comprising the CD80 ectodomain and the 4-1BB cytoplasmic domain, termed 80BB, acts as both a ligand and a receptor to engage the CD28 and 4-1BB pathways, thereby increasing the antitumor potency of human leukocyte antigen-independent TCR (HIT) receptor- or TCR-engineered T cells and tumor-infiltrating lymphocytes. Furthermore, 80BB serves as a switch receptor that provides agonistic 4-1BB co-stimulation upon its ligation by the inhibitory CTLA4 molecule. By combining multiple co-stimulatory features in a single antigen-agnostic synthetic receptor, 80BB is a promising tool to sustain CD3-dependent T cell responses in a wide range of targeted immunotherapies., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)