A recent advancement in the delivery of CAR-T: Use lyophilized lymph nodes

Abstract

A new study by Shi et al. in Nature Materials utilized lyophilized lymph nodes (L-LNs) as carriers for the delivery of chimeric antigen receptor (CAR) T cells targeting mesothelin (MSLN) to effectively suppress local recurrence following the resection of solid tumors.¹ They demonstrated significant antitumor efficacy in preclinical models. The work proposed a novel delivery strategy for CAR-T cells and highlighted the pivotal role of tumor-draining lymph nodes (tdLNs) in immunotherapy for solid tumors.

CAR-T cell therapy has demonstrated remarkable efficacy in the treatment of B cell malignancies and multiple myeloma in recent years. Due to the poor infiltration of CAR-T cells within the tumor, its effectiveness in solid tumor remains limited. However, Shi's work offered a novel CAR-T cells' delivery approach which exhibits a distinct clinical application scenario and holds significant potential for clinical implementation (Figure 1).

Shi et al. washed LNs in ice-cold phosphate-buffered saline and lyophilized the frozen LNs quickly for 4 h or overnight. Then they infused CAR-T cells into L-LNs to construct CAR-T@L-LNs, and characterized the CAR-T@L-LNs using scanning electron microscope (SEM) and immunofluorescence staining, demonstrating its successful construction with a CAR-T cells loading efficiency of up to 93%. They subsequently confirmed in vitro that L-LNs could preserve CAR-T cells activity and sustain their proliferation, as well as maintain a continuous release profile of CAR-T cells. Finally, partial resection models were used to validate the therapeutic efficacy of CAR-T@L-LNs in suppressing postoperative recurrence of solid tumors.

In terms of the material preparation, L-LNs exhibit the following characteristics: (1) minimal presence of viable cells, (2) preservation of suitable pores (~3–10 μm in size) for CAR-T cells loading, and (3) maintenance of a cytokine environment akin to that found in fresh lymph nodes. These attributes elucidate the outcomes observed in subsequent analyses investigating the biological functionalities of L-LNs. The elimination of living cells through lyophilizing prevents tumor cells' infiltration and mitigates any potential impact from immune cells on CAR-T cells' function within the lymph nodes. The retained structures postlyophilizing ensures a high CAR-T cells loading rate. Moreover, the presence of a cytokine milieu resembling that found in fresh lymph nodes ensures the robust proliferation of CAR-T cells.

In terms of antitumor functions, Shi's treatment modality involving loading CAR-T cells onto L-LNs has demonstrated significant inhibition of residual tumor growth, exhibiting superior therapeutic effects compared to both direct intravenous injection and encapsulating CAR-T cells in hydrogel with cytokines. Interestingly, mere placement of L-LNs on the surgical site also exhibited favorable therapeutic effects, which can be attributed to the direct action of cytokines on the tumor site as validated by cytokine elimination. Notably, the CAR-T@L-LNs implantation on surgical site significantly influences therapeutic efficacy.

In summary, this study has successfully developed a novel delivery system for CAR-T cells, utilizing L-LNs as effective carriers. The maintenance of CAR-T cell function was demonstrated through in vitro, while the antitumor efficacy was validated via in vivo. Importantly, this work led us to profound reflection.

Several clinical studies have explored intratumoral administration of CAR-T cell therapy. Unfortunately, these studies did not achieve improved therapeutic efficacy. Additionally, the local administration method of CAR-T cells presents difficulties in delivering them to different organs based on tumor type.² Furthermore, after local injection, CAR-T cells struggle to persistently infiltrate and remain within the tumor locality, resulting in a desired effect. To overcome these limitations, research have been conducted on delivery methods utilizing porous microneedles,³ toroidal-spiral particles,⁴ and hydrogel-containing cytokines.⁵ Although they have demonstrated antitumor advantages over direct local injection in preclinical models, compared to these studies, some concerns still exist.

Firstly, the cytokine milieu of L-LNs exhibits a higher degree of similarity to that in normal lymph nodes compared to other materials. Combined with the pores that can “firmly hold” CAR-T cells, CAR-T@L-LNs create a pattern that T cells can stably proliferate and gradually release. This pattern has two advantages. Compared to porous microneedles and toroidal-spiral particles, it provides an environment for the proliferation of CAR-T cells in vivo. More CAR-T cells can infiltrate the tumor microenvironment. Compared to hydrogel, the sustained release pattern of CAR-T cells prevents their simultaneous release into the residual tumor. T cells are continuously produced, and they are continuously released and kill tumor cells.

Secondly, due to the excellent biocompatibility of L-LNs, it is better equipped to preserve the cytokine environment within lymph nodes, thereby preventing local tissue and organ damage caused by cytokine storms. This aspect seems to have minimal impact on the efficacy of tumor treatment in preclinical models, and can even be considered as an enhancement in antitumor treatment efficacy. However, when considering CAR-T cells postoperative local delivery for preventing recurrence in numerous solid tumors, avoiding local cytokine storms becomes crucial. The repair process of tissues at the surgical site after tumor resection plays a vital role since inflammation-induced tissue damage can lead to severe complications such as postoperative bleeding and infections. Consequently, this issue poses a challenge that hydrogel and other CAR-T cells' local delivery methods must confront during actual clinical applications.

Notably, L-LNs are easily extractable and preparable because of lymph node dissection which is essential for curative resection of malignant tumors in actual clinical scenarios. Additionally, as postoperative drainage tubes are commonly used after surgery, ultrasound-guided placement of CAR-T@L-LNs through the drainage tube port can be performed to minimize patient trauma and optimize wound healing.

Furthermore, this study provides additional theoretical support for the crucial role of tdLNs in solid tumor immunotherapy. Initially, surgeons were influenced by the theory of surgery-first and thus pursued wider resection range and more comprehensive lymph node dissection. However, subsequent clinical studies involving extensive resection techniques such as pancreatic cancer D4 dissection have demonstrated that patients could not derive any additional benefits from more comprehensive lymph node dissection. In recent years, some studies have demonstrated that tdLNs of a tumor serve not only as an indicator for tumor node metastasis we staging and prognosis assessment but also play a crucial role in facilitating the efficacy of tumor immunotherapy. For example, immune checkpoint inhibitors (ICIs) therapy are ineffective without tdLNs. This is a finding consistent with the significant improvement observed when CAR-T cells loaded in L-LNs in this study. Unfortunately, this study lacks in vivo analysis of L-LNs' immune cells. They missed an opportunity to gain a more comprehensive understanding of the diverse physiological functions of L-LNs in vivo.

Moreover, the proposed treatment model in this study exhibits promising clinical application prospects. As stated by the authors, it can be effectively delivered to the operative area through ultrasound-guided positioning of the postoperative drainage tube. We eagerly anticipate the implementation of this treatment model in clinical practice and are highly intrigued by its actual therapeutic effects. Furthermore, this study also offers valuable insights for administering CAR-T therapy via the lymphatic system in subsequent treatments. Can CAR-T cells be directly injected into tdLNs through laparoscopic surgery to downstage locally advanced tumors? For patients with resectable solid tumors, can we inject CAR-T cells into nonmetastatic lymph nodes during surgery to prevent postoperative local recurrence and metastasis? The validation of these hypotheses necessitates further experiments to verify.

Manuscript writing: Xinze Du, Keman Cheng, and Xiao Zhao. Supervision: Xinze Du, Keman Cheng, and Xiao Zhao. All authors have read and approved the final manuscript.

The authors declare no conflict of interest.

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