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

Schäfer, Daniel; Henze, Janina; Pfeifer, Rita; Schleicher, Anna; Brauner, Janina; Mockel-Tenbrinck, Nadine; Barth, Carola; Gudert, Daniela; Rawashdeh, Wa’el Al; Johnston, Ian; Hardt, Olaf

doi:10.3389/fimmu.2020.01704

Cited by 26 publications

(18 citation statements)

References 88 publications

(150 reference statements)

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“…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), was used to build a CAR directed against a membrane-proximal (TSPAN8) epitope of a pancreatic ductal adenocarcinoma model. In vivo settings using these CAR-T cells led to the generation of advantageous T CM cells that released low levels of inflammatory cytokines and retained excellent tumor-killing functions ( 111 ).…”

Section: Strategies To Improve the Persistence Of Car-t Cellsmentioning

confidence: 99%

CAR-T Cell Performance: How to Improve Their Persistence?

López-Cantillo¹,

Urueña

Camacho³

et al. 2022

Front. Immunol.

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Adoptive cell therapy with T cells reprogrammed to express chimeric antigen receptors (CAR-T cells) has been highly successful in patients with hematological neoplasms. However, its therapeutic benefits have been limited in solid tumor cases. Even those patients who respond to this immunotherapy remain at risk of relapse due to the short-term persistence or non-expansion of CAR-T cells; moreover, the hostile tumor microenvironment (TME) leads to the dysfunction of these cells after reinfusion. Some research has shown that, in adoptive T-cell therapies, the presence of less differentiated T-cell subsets within the infusion product is associated with better clinical outcomes. Naive and memory T cells persist longer and exhibit greater antitumor activity than effector T cells. Therefore, new methods are being studied to overcome the limitations of this therapy to generate CAR-T cells with these ideal phenotypes. In this paper, we review the characteristics of T-cell subsets and their implications in the clinical outcomes of adoptive therapy with CAR-T cells. In addition, we describe some strategies developed to overcome the reduced persistence of CAR T-cells and alternatives to improve this therapy by increasing the expansion ability and longevity of modified T cells. These methods include cell culture optimization, incorporating homeostatic cytokines during the expansion phase of manufacturing, modulation of CAR-T cell metabolism, manipulating signaling pathways involved in T-cell differentiation, and strategies related to CAR construct designs.

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Section: Strategies To Improve the Persistence Of Car-t Cellsmentioning

confidence: 99%

CAR-T Cell Performance: How to Improve Their Persistence?

López-Cantillo¹,

Urueña

Camacho³

et al. 2022

Front. Immunol.

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“…Lentiviral vector production was performed as previously described 51 . In brief, HEK293T cells at a confluency of 70-90% were transfected with DNA:MACSfectin™ (Miltenyi Biotec) complexes using a total amount of 50µ DNA per T175 flask.…”

Section: Methodsmentioning

confidence: 99%

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

Pfeifer¹,

Henze²,

Wittich³

et al. 2022

Theranostics

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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 µComputer tomography bioluminescence tomography (µCT/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 IU/mouse. CAR T cell distribution was measured in 2D BLI and 3D µCT/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 µCT/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 µCT/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.

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“…More interestingly, besides the brain, the interaction between MAG and cancer-associated MUC-1 is found to be sialic-acid dependent in pancreatic cancer perineural invasion [63]. Additionally, in CAR functionality, a Siglec-4-derived long spacer has similar antitumor efficacy as a CD8α spacer for CAR-T therapy but is proposed to better target membrane-proximal epitopes with minimized CNS toxicities [64].…”

Section: Siglec-4 (Myelin Associated Glycoprotein Mag)mentioning

confidence: 99%

Siglecs as Therapeutic Targets in Cancer

Lim

Sari-Ak

Bagga

2021

Biology

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Hypersialylation is a common post-translational modification of protein and lipids found on cancer cell surfaces, which participate in cell-cell interactions and in the regulation of immune responses. Sialic acids are a family of nine-carbon α-keto acids found at the outermost ends of glycans attached to cell surfaces. Given their locations on cell surfaces, tumor cells aberrantly overexpress sialic acids, which are recognized by Siglec receptors found on immune cells to mediate broad immunomodulatory signaling. Enhanced sialylation exposed on cancer cell surfaces is exemplified as “self-associated molecular pattern” (SAMP), which tricks Siglec receptors found on leukocytes to greatly down-regulate immune responsiveness, leading to tumor growth. In this review, we focused on all 15 human Siglecs (including Siglec XII), many of which still remain understudied. We also highlighted strategies that disrupt the course of Siglec-sialic acid interactions, such as antibody-based therapies and sialic acid mimetics leading to tumor cell depletion. Herein, we introduced the central roles of Siglecs in mediating pro-tumor immunity and discussed strategies that target these receptors, which could benefit improved cancer immunotherapy.

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

Cited by 26 publications

References 88 publications

CAR-T Cell Performance: How to Improve Their Persistence?

CAR-T Cell Performance: How to Improve Their Persistence?

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

Siglecs as Therapeutic Targets in Cancer

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