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Chimeric antigen receptor (CAR) T cell therapeutic responses are hampered by limited T cell trafficking, persistence, and durable anti-tumor activity in solid tumors. However, these challenges can be largely overcome by relatively unconstrained synthetic engineering strategies. Here, we describe CAR T cells targeting tumor-associated glycoprotein-72 (TAG72), utilizing the CD28 transmembrane domain upstream of the 4-1BB co-stimulatory domain as a driver of potent anti-tumor activity and IFNγ secretion. CAR T cell-mediated IFNγ production facilitated by IL-12 signaling is required for tumor cell killing, which is recapitulated by engineering an optimized membrane-bound IL-12 (mbIL12) molecule in CAR T cells. These T cells show improved antigen-dependent T cell proliferation and recursive tumor cell killing in vitro, with robust in vivo efficacy in human ovarian cancer xenograft models. Locoregional administration of mbIL12-engineered CAR T cells promotes durable anti-tumor responses against both regional and systemic disease in mice. Safety and efficacy of mbIL12-engineered CAR T cells is demonstrated using an immunocompetent mouse model, with beneficial effects on the immunosuppressive tumor microenvironment. Collectively, our study features a clinically-applicable strategy to improve the efficacy of locoregionally-delivered CAR T cells engineered with antigen-dependent immune-modulating cytokines in targeting regional and systemic disease., (© 2023. Springer Nature Limited.)

Short hairpin RNAs (shRNAs) have emerged as a powerful tool for gene knockdown in various cellular systems, including chimeric antigen receptor (CAR) T cells. However, the elements of shRNAs that are crucial for their efficacy in developing shRNA-containing CAR T cells remain unclear. In this study, we evaluated the impact of different shRNA elements, including promoter strength, orientation, multiple shRNAs, self-targeting, and sense and antisense sequence composition on the knockdown efficiency of the target gene in CAR T cells. Our findings highlight the importance of considering multiple shRNAs and their orientation to achieve effective knockdown. Moreover, we demonstrate that using a strong promoter and avoiding self-targeting can enhance CAR T cell functionality. These results provide a framework for the rational design of CAR T cells with shRNA-mediated knockdown capabilities, which could improve the therapeutic efficacy of CAR T cell-based immunotherapy.

Background: Adoptive transfer of CD19-specific chimeric antigen receptor (CD19CAR) T cells can induce dramatic disease regression in patients with B cell malignancies. CD19CAR T cell therapy may be limited by insufficient engraftment and persistence, resulting in tumor relapse. We previously demonstrated a proof of principle that cytomegalovirus (CMV)-specific T cells can be isolated and enriched prior to CD19CAR transduction to produce CMV-CD19CAR T cells, and that these CMV-CD19CAR T cells can be expanded in vivo through CMV vaccination, resulting in better tumor control in a murine model. Here we developed a clinical platform for generating CMV-CD19CAR T cells., Methods: Peripheral blood mononuclear cells (PBMCs) collected from CMV-seropositive healthy donors were stimulated with a good manufacturing practices-grade PepTivator overlapping CMVpp65 peptide pool and enriched for CMV-responsive interferon γ (IFNγ)+T cells using IFNγ Catchmatrix, within the CliniMACS Prodigy Cytokine Capture System (Miltenyi Biotec). Resulting CMV-specific T cells were transduced with a lentiviral vector encoding a second generation CD19R:CD28:ζ/EGFRt CAR and expanded with interleukin 2 (IL-2) and IL-15 for 15 days before characterization., Results: CMV-specific T cells were enriched from 0.8%±0.5 of input PBMC to 76.3%±11.6 in nine full-scale qualification runs (absolute yield of 4.2±3.3×10 6 IFNγ+T cells from an input of 1×10 9 PBMCs). Average CD19CAR transduction efficiency of CMV-specific T cells was 27.0%±14.2 in the final products, which underwent rapid expansion, resulting in a total cell dose of 6.2±0.9 × 10 6 CD19CAR-tranduced T cells with CMV specificity (ie, functionally bispecific). CMV-CD19CAR T cells were polyclonal, expressed memory markers but had low expression of exhaustion markers, responded to both CD19 and CMVpp65 stimulation with rapid proliferation and exhibited antigen-specific effector functions against both CD19-expressing tumors and CMVpp65 antigen. The final products passed release criteria for clinical use., Conclusions: We demonstrated the feasibility of our large-scale platform for generating CMV-CD19CAR T cells for clinical application. We plan to initiate a clinical trial at City of Hope using CMV-CD19CAR T cells for patients with intermediate/high-grade B cell non-Hodgkin's lymphoma immediately after autologous hematopoietic cell transplantation followed by vaccination with a novel CMV vaccine based on Modified Vaccinia Ankara (Triplex) 28 days and 56 days post-T cell infusion., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Forelimb-hindlimb coordination in adult rats moving freely along 2m long runway was investigated using the method of footprint recording. Rats were divided into 3 groups with different extent of spinal lesions (T(9)). Before surgery rats moved with a mean locomotor speed of 73±20 to 96±18cms(-1), stride lengths of 17.5±2.0 to 21.2±2.0cm, and trot like coordination. Early after surgery the locomotor speed and the stride lengths were decreased. The forelimb steps were shorter than hindlimb steps, which led to the occurrence of unpaired forelimb steps. Unpaired steps occurred when the hind paw print lay more than half the hindlimb stride length in front of the ipsilateral paw. The number of unpaired steps was negatively correlated with the difference between the fore- and hindlimb step lengths. The recovery of locomotor speed, stride length, and step sequence patterns took up to 3.5 months depending on the extent of lesion. In the last testings the coordination was characterized by increased distances between ipsilateral footprints leading to a change from an almost synchronized trot to a lesion-dependent walk. This change was accompanied by a switch from the use of both patterns A and C to the most frequent use of the Aa pattern that is better adapted to maintain the body balance. All locomotor changes depended on the extent of the injury of lateral and ventral funiculi. These results demonstrate that footprint analysis can be used for the evaluation of forelimb-hindlimb coordination after spinal lesion in rats., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In six rats with incomplete low thoracic spinal cord lesions of different extent, basic gait indices such as locomotor speed, step cycle duration, soleus (Sol) muscle activity duration, the interval between the onsets of Sol and tibialis anterior (TA) muscle activities and interlimb coordination were investigated by EMG analysis of the Sol and TA muscles recorded using chronic electrodes. The operated animals were divided into two subgroups: (1) those with a smaller lesion involving the dorsal quadrants of the spinal cord and, to a variable extent, the ventrolateral funiculi, and (2) those with an extensive lesion sparing only parts of the ventral funiculi. The locomotion of all rats was tested once a week for the first 5 weeks postsurgery and then once or twice a month, up to 3.5 months. The surgical lesions affected all analyzed gait indices: the locomotor speed decreased, while all other indices increased compared to recordings made preoperatively. In both subgroups the major improvement in locomotion occurred within the first 5 weeks following surgery and the rats reached a plateau in their recovery at around 2 months postoperatively. The late effects of injury depended on the severity of the spinal lesion: in the subgroup of rats with a smaller lesion, the postoperative changes in the different indices amounted to approximately 20%, while in the subgroup with extensive lesions this was increased by 20-50%, with changes in various indices being strongly correlated with the extent of the injury in individual animals. These postoperative changes were partly due to alterations in the relationships between the analyzed variables.

In rats with incomplete low thoracic spinal cord lesions of different extents, the basic indices of gait such as locomotor velocity, step and stance phase duration and the duty factor (i.e., the relative duration of the stance phase) during overground runway locomotion were analyzed using contact electrodes on each paw for data recording. In animals with lesions confined to the dorsal columns (DC), tested 3 weeks postsurgery, these gait indices were essentially unchanged compared to the preoperative period. After the same recovery period, rats with larger lesions, comprising the dorsal columns plus a major part of the dorsolateral funiculi (DL), showed a transient increase in the hindlimb stance phase duration and the duty factor. More extensive injuries, with additional damage to parts of the ventrolateral and ventral funiculi (VL), produced increments in the stance phase duration and duty factor much above that which would be expected from changes in step cycle duration due to slowing down of locomotion. These changes, which lasted for at least 3 months, were more conspicuous in animals with extensive spinal cord injuries and were due to an altered relationship between the stance phase and step cycle duration. It is suggested that the excessive increment in the hindlimb stance phase and the duty factor constitute a reliable indicator of impairment in locomotor movements, which is correlated with the extent of spinal cord injury.

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