A Novel Siglec-4 Derived Spacer Improves the Functionality of CAR T Cells Against Membrane-Proximal Epitopes

A domain that is often neglected in the assessment of chimeric antigen receptor (CAR) functionality is the extracellular spacer module. However, several studies have elucidated that membrane proximal epitopes are best targeted through CARs comprising long spacers, while short spacer CARs exhibit highest activity on distal epitopes. This finding can be explained by the requirement to have an optimal distance between the effector T cell and target cell. Commonly used long spacer domains are the CH2-CH3 domains of IgG molecules. However, CARs containing these spacers generally show inferior in vivo efficacy in mouse models compared to their observed in vitro activity, which is linked to unspecific Fcγ-Receptor binding and can be abolished by mutating the respective regions. Here, we first assessed a CAR therapy targeting membrane proximal CD20 using such a modified long IgG1 spacer. However, despite these mutations, this construct failed to unfold its observed in vitro cytotoxic potential in an in vivo model, while a shorter but less structured CD8α spacer CAR showed complete tumor clearance. Given the shortage of well-described long spacer domains with a favorable functionality profile, we designed a novel class of CAR spacers with similar attributes to IgG spacers but without unspecific off-target binding, derived from the Sialic acid-binding immunoglobulin-type lectins (Siglecs). Of five constructs tested, a Siglec-4 derived spacer showed highest cytotoxic potential and similar performance to a CD8α spacer in a CD20 specific CAR setting. In a pancreatic ductal adenocarcinoma model, a Siglec-4 spacer CAR targeting a membrane proximal (TSPAN8) epitope was efficiently engaged in vitro, while a membrane distal (CD66c) epitope did not activate the T cell. Transfer of the TSPAN8 specific Siglec-4 spacer CAR to an in vivo setting maintained the excellent tumor killing characteristics being indistinguishable from a TSPAN8 CD8α spacer CAR while outperforming an IgG4 long spacer CAR and, at the same time, showing an advantageous central memory CAR T cell phenotype with lower release of inflammatory cytokines. In summary, we developed a novel spacer that combines cytotoxic potential with an advantageous T cell and cytokine release phenotype, which make this an interesting candidate for future clinical applications

Immunotherapy of solid tumors: multimodal imaging strategies for chimeric antigen receptor T cell tracking in the tumor microenvironment

Immunotherapy is an emerging building block of modern oncology, after chimeric antigen receptor (CAR) T cells demonstrated groundbreaking survival rates in hematological malignancies. However, therapy success in more common solid tumors has not been achieved yet, due to a variety of obstacles, such as a limited availability of suitable targets and decreased CAR T cells trafficking to the tumor. One of these barriers is the tumor microenvironment (TME), which is most pronounced in pancreatic ductal adenocarcinoma (PDAC). Combinatorial 2D and 3D preclinical multimodal imaging and cell tracking strategies can help to understand the mechanism that play a role in the solid tumor-specific barriers for CAR T cell migration. To this end, three non-solid CARs were characterized in vitro for killing and cytokine expression as well as for in vivo efficacy and tumor control. While in vitro results showed a potent killing and cytokine profile for all three CARs, in vivo analysis revealed a diminished killing potential of one CAR carrying an unspecifically bound IgG1-based spacer. This demonstrates the importance of imaging techniques to identify the most promising CARs for clinical transfer, to depict problematic CAR components and to unravel the underlying mechanisms. Furthermore, in vivo imaging identified the relevance of the CAR spacer domain, which is normally neglected. Favorable targeting of membrane-proximal epitopes with spacer, structural comparable to IgG1, encouraged the development of a novel spacer class, derived from sialic acid-binding immunoglobulin-type lectin (Siglec). Next, CAR T cells, incorporating the new spacers, were evaluated in vitro and ex vivo in solid and hematological malignancies. The functionality of the novel Siglec-4 derived spacer was superior to the established IgG4 and CD8α spacers in terms of the cytotoxic potential and a more potent anti-tumor marker and cytokine expression profile in comparison to IgG1-based spacers, supportive for future clinical trials. These results displayed the general functionality of CAR T cells against PDAC under the optimal conditions, in terms of target specificity, CAR composition and cell number and emphasize the implication of advanced imaging strategies for preclinical CAR T cell research. Thus, a rational multimodal imaging workflow was established and evaluated in a xenograft PDAC mouse model. First, optical 3D in vivo tracking of modified luciferase-expressing CAR T cells was applied. 3D Bioluminescence tomography (BLT) enabled the analysis of whole-body CAR T cells biodistribution and detection of pronounced CAR T cell accumulation in tumor and spleen in PDAC bearing mice. Subsequent combination with ex vivo light-sheet fluorescence microscopy (LSFM) of xenografts facilitated the generation of data visualizing whole-body and intratumoral T cell distribution of two different CARs. The addition of cyclic immunofluorescence staining (IF) provided an in-depth characterization of tumor-infiltrating CAR T cells and surrounding tumor cells, revealing strong activation and proliferation of target-specific CAR T cells. The multi-modal imaging strategy enabled the evaluation of locally applied interleukin-2 (IL-2) as a support for CAR T cells in the immunosuppressive TME of PDAC. IL-2, repeatedly injected at the tumor site was shown to negatively impact intratumoral T cell distribution and phenotype. IL-2 co-treated CAR T cells infiltrated the tumor tissue less deep and showed a more overstimulated phenotype. These cells were no longer able to perform sufficient tumor eradication and local IL-2 did not translate into an enhanced anti-tumor efficacy. Taken together, this project established optical 3D CAR T cell tracking as part of a combined in vivo and ex vivo workflow for solid tumor cell therapy, TME-redirected treatment protocols and safety-orientated research. This preclinical imaging strategy enables the in-depth characterization of combinatorial CAR T cell approaches against solid tumors and TME in a mouse model of PDAC.2022-07-1

Arthroscopic Debridement of Triangular Fibrocartilage Complex Tears

Arthroscopic Debridement of Triangular Fibrocartilage Complex Tear

Enhancing the Efficacy of CAR T Cells in the Tumor Microenvironment of Pancreatic Cancer

Pancreatic cancer has the worst prognosis and lowest survival rate among all types of cancers and thus, there exists a strong need for novel therapeutic strategies. Chimeric antigen receptor (CAR)-modified T cells present a new potential option after successful FDA-approval in hematologic malignancies, however, current CAR T cell clinical trials in pancreatic cancer failed to improve survival and were unable to demonstrate any significant response. The physical and environmental barriers created by the distinct tumor microenvironment (TME) as a result of the desmoplastic reaction in pancreatic cancer present major hurdles for CAR T cells as a viable therapeutic option in this tumor entity. Cancer cells and cancer-associated fibroblasts express extracellular matrix molecules, enzymes, and growth factors, which can attenuate CAR T cell infiltration and efficacy. Recent efforts demonstrate a niche shift where targeting the TME along CAR T cell therapy is believed or hoped to provide a substantial clinical added value to improve overall survival. This review summarizes therapeutic approaches targeting the TME and their effect on CAR T cells as well as their outcome in preclinical and clinical trials in pancreatic cancer

A multimodal imaging workflow for monitoring CAR T cell therapy against solid tumor from whole-body to single-cell level

CAR T cell research in solid tumors often lacks spatiotemporal information and therefore, there is a need for a molecular tomography to facilitate high-throughput preclinical monitoring of CAR T cells. Furthermore, a gap exists between macro-and microlevel imaging data to better assess intratumor infiltration of therapeutic cells. We addressed this challenge by combining 3D mu Computer tomography bioluminescence tomography (mu CT/BLT), light-sheet fluorescence microscopy (LSFM) and cyclic immunofluorescence (IF) staining. Methods: NSG mice with subcutaneous AsPC1 xenograft tumors were treated with EGFR CAR T cell (+/- IL-2) or control BDCA-2 CAR T cell (+/- IL-2) (n = 7 each). Therapeutic T cells were genetically modified to co-express the CAR of interest and the luciferase CBR2opt. IL-2 was administered s.c. under the xenograft tumor on days 1, 3, 5 and 7 post-therapy-initiation at a dose of 25,000 Ill/mouse. CAR T cell distribution was measured in 2D BLI and 3D mu CT/BLT every 3-4 days. On day 6, 4 tumors were excised for cyclic IF where tumor sections were stained with a panel of 25 antibodies. On day 6 and 13, 8 tumors were excised from rhodamine lectin-preinjected mice, permeabilized, stained for CD3 and imaged by LSFM. Results: 3D mu CT/BLT revealed that CAR T cells pharmacokinetics is affected by antigen recognition, where CAR T cell tumor accumulation based on target-dependent infiltration was significantly increased in comparison to target-independent infiltration, and spleen accumulation was delayed. LSFM supported these findings and revealed higher T cell accumulation in target-positive groups at day 6, which also infiltrated the tumor deeper. Interestingly, LSFM showed that most CAR T cells accumulate at the tumor periphery and around vessels. Surprisingly, LSFM and cyclic IF revealed that local IL-2 application resulted in early-phase increased proliferation, but long-term overstimulation of CAR T cells, which halted the early added therapeutic effect. Conclusion: Overall, we demonstrated that 3D mu CT/BLT is a valuable non-isotope-based technology for whole-body cell therapy monitoring and investigating CAR T cell pharmacokinetics. We also presented combining LSFM and MICS for ex vivo 3D-and 2D-microscopy tissue analysis to assess intratumoral therapeutic cell distribution and status

Clinical Predictors of Neurogenic Lower Urinary Tract Dysfunction in Persons with Multiple Sclerosis

Background: Multiple sclerosis patients often develop neurogenic lower urinary tract dysfunction with a potential risk of upper urinary tract damage. Diagnostic tools are urodynamics, bladder diary, uroflowmetry, and post-void residual, but recommendations for their use are controversial. Objective: We aimed to identify clinical parameters indicative of neurogenic lower urinary tract dysfunction in multiple sclerosis patients. Methods: 207 patients were prospectively assessed independent of the presence of lower urinary tract symptoms. We analyzed Expanded Disability Status Scale scores, uroflowmetry, post-void residual, rate of urinary tract infections, standardized voiding frequency, and voided volume in correlation with urodynamic findings. Results: We found a significant correlation between post-void residual (odds ratio (OR) 4.17, confidence interval (CI) 1.20–22.46), urinary tract infection rate (OR 3.91, CI 1.13–21.0), voided volume (OR 4.53, CI 1.85–11.99), increased standardized voiding frequency (OR 7.40, CI 2.15–39.66), and urodynamic findings indicative of neurogenic lower urinary tract dysfunction. Expanded Disability Status Scale shows no correlation. Those parameters (except post-void residual) are also associated with reduced bladder compliance, as potential risk for kidney damage. Conclusion: Therefore, bladder diary and urinary tract infection rate should be routinely assessed to identify patients who require urodynamics