Toroidal-Spiral Particles as a CAR-T Cell Delivery Device for Solid Tumor Immunotherapy

Abstract

Chimeric antigen receptor (CAR) T cell therapy has resulted in positive effects on patients with hematologic malignancy but shows limited efficacy in solid tumor treatments due to insufficient trafficking and tumor infiltration, intensive CAR-T-related toxicities, and antigen escape. In this work, we developed and investigated a biodegradable and biocompatible polymeric toroidal-spiral particles (TSPs) as a in vivo cell incubator and delivery device that can be implanted near tumor through a minimally invasive procedure or injected near or into solid tumors by using a biopsy needle. The main matrix structure of the millimeter-sized TSP is made from crosslinking of gelatin methacrylamine (GelMA) and poly (ethylene glycol) diacrylate (PEGDA) with a tunable degradation rate from a few days to months, providing appropriate mechanical properties and sustained release of co-encapsulated drugs and/or stimulation compounds. The toroid-spiral layer of the particles, present an internal void volume for high-capacity cell loading and flexibility of co-encapsulating small and large molecular compounds with individually manipulated release schedules, is filled with collagen and suspended T cells. The TSPs promote cell proliferation, activation, and migration in the tumor micro-environment in a prolonged and sustained manner. In this study, the efficacy of mesothelin (MSLN) CAR-T cells released from the TSPs was tested in preclinical mouse tumor models. Compared to systemic and intratumoral injection, peritumoral delivery of MSLN CAR-T cells using the TSPs resulted in a superior antitumor effect. The TSPs made of FDA approved materials as an in vivo reactor may provide an option for efficiently local delivery of CAR-T cells to solid tumors for higher efficacy and lower toxicity, with a minimally invasive administration procedure.

INTRODUCTION

Adoptive cellular therapy (ACT), using ex vivo expanded patient tumor antigen-specific T cells or genetically modified patient T cells, with a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that can recognize a tumor-associated antigen on the surface of tumor cells, holds promise for the treatment of many cancers. Since CAR-T cell therapy was approved by the FDA in 2017, it has led to considerable clinical results in treating hematologic malignancies.[1-4] However, the success of using CAR-T cells for treatment of solid tumors remains unsatisfactory because of the cells’ inefficient expansion in the immunosuppressive tumor microenvironment. To improve the tumor targeting capabilities of CAR-T cell therapy, it is essential to find a unique antigen which is overexpressed in the targeted tumor and preferably shows low expression in normal tissues.[5] ACT often involves the co-administration of agents (e.g. immunosuppressive chemotherapy, cytokines such as Interleukin-2 (IL-2), and/or vaccines) to address these problems. However, its effectiveness is limited by the toxicity associated with the systemic delivery of these co-administered agents.[6, 7]

An efficient cell delivery system that improves the persistence and functionality of transferred T cells and immune suppression/exhaustion in the tumor microenvironment is necessary. Stephan et al. reported significantly improved anti-tumor potency of the transplanted lymphocytes by harboring them in centimeter-size polymeric disks and placing them in tumor resection sites.[8, 9] Recently, Grosskopf el at. reported an injectable hydrogel for the controlled co-delivery of CAR-T cells and stimulatory cytokines that acted as an inflammatory niche and improved the treatment of the medulloblastoma solid tumor.[10] The study shows great promises for loco-regional delivery of the CAR-T therapy and in vivo expansion of more tumor-reactive T cells to crucially reduce the number of the required cells. Here, we introduce a millimeter-size cell delivery device, toroidal spiral particles (TSPs), composed of FDA-approved biodegradable and biocompatible polymers for CAR-T cell in vivo proliferation, activation, and release. The main polymer matrix of the particles, made of gelatin methacrylamine (GelMA) and poly (ethylene glycol) diacrylate (PEGDA), is generated through a self-assembly process of polymer drop sedimentation and crosslinking.[11, 12] The degradation rate and the mechanical properties of the TSPs are tunable by varying the ratio of GelMA and PEGDA. TSPs featuring inner channels in a toroidal-spiral shape can provide a large void volume cell encapsulation.[13, 14] Co-encapsulation of the cytokine near the surface of the inner toroidal-spiral channel results in extremely slow release of the cytokine to the surrounding environment but effective stimulation of the T cell proliferation and activation.

The cell-delivery platform should be universal for different types of lymphocytes. In this study, we used the mouse models of breast tumor or colorectal carcinoma to evaluate the effectiveness of delivering anti-mesothelin (MSLN) CAR-T cells compared with systemic and intratumoral injection. MSLN expression is significantly upregulated in various cancers, including breast, lung, ovarian, and pancreatic cancers.[15, 16] Although the function of mesothelin in normal cells is not fully understood, it has been shown that MSLN might promote tumor progression through stimulating cancer cell proliferation.[17-20] Several vaccines and antibody-based therapies have been developed to target MSLN as one of the most attractive tumor-associated antigens.[21] However, utilizing anti-MSLN CAR-T cells for solid tumor treatment requires improvements in cell trafficking, reducing off-targeting and antigen escape.[21] Therefore, an investigation is warranted to find a sustained and long-term CAR-T cell release system that could provide high therapeutic efficiency, evade multiple administrations, and reduce toxicities and side effects.

MATERIALS AND METHODS

Materials

Poly (ethylene glycol) diacrylate (PEGDA) M_w 700, glycerol, gelatin (Type B from bovine skin), ethanol, phosphate buffer saline 1X (PBS 1X), methacrylic anhydride (MA), and fetal bovine serum were purchased from Sigma Aldrich (St. Louis, MO). Irgacure 2959 (2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone) (I-2959) was kindly provided by BASF (Florham Park, NJ). Collagenase Type II was purchased from Worthington Biochemicals (Lakewood, NJ). Rat tail collagen (4 mg/mL) was purchased from Advanced BioMatrix (Carlsbad, CA). Molecular grade water was purchased from Corning (Corning, NY). Spectra/Pro dialysis membrane MWCO 10 kDa was purchased from LubioScience. Water used in all experiments was deionized to 18.2 Ωcm (Nanopure II, Barnstead). Human IL-2 ELISA kit was purchased from Abcam (Waltham, MA).

Cell lines

A549, CanPan2, HCC1806, HCT116, OVCAR3, PanC1, and SKOV3 cells were purchased from ATCC and cultured in media as indicated in the manufacturer's protocols. For all experiments, cells were recovered from frozen aliquots and cultured for 1 to 2 weeks prior to the inoculation of mice or in vitro treatment.

CAR-T cell culture

MSLN (Mesothelin) CAR-T cells (PM-CAR1014-1M) and control Mock CAR-T cells (PM-CAR1000-1M) were purchased from PROMAB and cultured in ImmunoCult^™-XF T Cell Expansion Medium (STEM CELL, 10981) containing 10 ng/mL human IL-2 (BIOLEGEND, 570804) with initial activation by ImmunoCult^™ Human CD3/CD28 T Cell Activator (STEM CELL, 10971) for 3 days. CAR-T cells were then washed and expanded in T cell expansion media for 7 to 10 days prior to the in vitro and in vivo experiments. Cells were counted and cultured in fresh media every two days at an appropriate concentration as indicated in the manufacturer's protocol.

Mice

C57BL6/ mice and NSG mice at 8- to 12-weeks-old were purchased from the Jackson Lab. Mice were maintained by the animal facility of the Northwestern University Cancer Center, with a 12 light/12 dark cycle, at 18–23 °C with 40–60% humidity and maintained in pathogen-free barrier facilities. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at the Northwestern University.

GelMA Synthesis

GelMA was synthesized based on the protocol previously reported.[22, 23] Briefly, gelatin was first dissolved in phosphate buffer (pH 7.4) at 50°C and stirred until it was fully dissolved. Methacrylic anhydride was then added to the gelatin solution in a dropwise manner while stirring. After one hour of reaction at 50°C, the solution was diluted 3 folds with phosphate buffer and dialyzed against deionized water through a dialysis tubing (10 kDa MWCO) at 40°C for 4 days to remove the unreacted gelatin, methacrylic anhydride, and methacrylic acid byproducts. Finally, the dialyzed solution was lyophilized and stored under refrigeration until use.

The degree of substitution of GelMA, which determines the physicochemical and mechanical properties of the hydrogel, is defined as the percentage of the amino groups on the gelatin replaced by the methacrylate groups. The degree of substitution was analyzed using an H nuclear magnetic resonance spectrometer (H NMR).[24] Briefly, gelatin and GelMA were dissolved in deuterium oxide (D2O, 50 mg/mL). The degree of substitution of GelMA can be calculated by measuring the peak area of modified amino groups (C═C bond) and primary amino groups (−CH─NH bond) in the H NMR spectra of GelMA and gelatin respectively as,

$$\text{degree of substitution}(\%)=\frac{\text{peak area of modified amino groups}}{\text{peak area of primary amino groups}}\times 100.$$

A comparison between gelatin and GelMA H NMR spectra shows a decrease in lysine signal at around 3 ppm in GelMA, the appearance of two peaks at around 5.5 ppm associated with methacrylamide groups, and another peak at around 1.8 ppm related to methyl protons in GelMA H NMR spectra (Supplementary Fig. S1). H NMR analysis on several batches showed a consistent methacrylation, which is essential for obtaining consistent mechanical properties and degradation rates after crosslinking into a GelMA hydrogel.

TSP formation

TSPs were prepared by a self-assembly process of droplet sedimentation and solidification (Fig. 1A). GelMA was dissolved at 37°C in DI water containing PEGDA M_w 700, glycerol and photoinitiator Irgacure 2959. Droplets of the mixed solution were generated by the pulsating motion of the syringe driven by a syringe pump. Specifically, 20 μL of the polymer solution was injected into a bulk solution of glycerol and ethanol. The bulk solution was kept in a heated water bath to avoid gelation at room temperature due to the presence of GelMA. When a droplet sediments in a miscible solution with minimal surface tension, the viscous force from the surrounding fluid shapes the droplet into a toroidal-spiral structure (Fig. 1B). Solidification was initiated by the exposure of high intensity UV light (~10 W/cm2) (Bluewave 75, Dymax, Torrington, CT), at a desired stage monitored from a high-speed camera (Prosilica GX 1050, Allied Vission Technology, Germany) with a magnification lens (MLH-10X, computer, 269 Commack, NY). The TSPs were collected, washed using a mixture of DI water and ethanol to remove unreacted polymers and free radicals (Fig. 1C). TSPs were dehydrated for storage until being used for cell encapsulation.

Fig. 1. Peritumor implantation of the TSPs as a reactor for in vivo T-cell expansion, activation, and local release.

(A) Schematic of the setup to generate TSPs by droplet sedimentation in a miscible bulk solution followed by UV light crosslinking. A polymer drop is injected into a bulk solution placed on a plate heater. The droplets are solidified by using a UV light at a certain time point for an ideal particle morphology. (B) High-speed camera images of the droplet shape evolution. The scale bars represent 1 mm. (C) Microscopic image of the solidified TSP (top) and stereoscope image of the TSPs in a petri dish (bottom). The scale bar in the top image represents 500 μm. (D) Images of sequentially loading the cytokine and then the cells in the TSP. (E) Schematic illustrations of the cell-encapsulated TSP and in vivo and in vitro characterization and evaluation of the particles.

TSP Mechanical property characterization

To measure the mechanical properties of TSP, a compression test was done with a Shimadzu EZ-Test Compact Bench Testing Machine (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan). After the particles were rehydrated in DI water for 24 hours, they were compressed till failure at a rate of 1 mm/min with a load cell. Due to the heterogeneous shape of the TSP, the tests were repeated with mechanical pressure applied to various sides of the particles.

TSP in vitro degradation measurements

After the initial weights of the dehydrated particles were measured, they were placed in a solution of 2 U/mL collagenase type II and kept at 37°C. The collagenase solution was collected and replaced with a fresh solution every 2 days. At desired time points, the particles were collected, rinsed with DI water, and lyophilized. The weight of the lyophilized particles was then measured, and the weight loss of the particles was calculated. The degradation rate of the particles with the presence of collagenase is defined as the percentage weight loss of the particles.

IL-2 loading into TSP

Prior loading IL-2, the tail of the TSP was cut off. The loading of the IL-2 to the dehydrated TSPs is by adding 2 μl of IL-2 solution at the top opening of the channel using a micropipette (Fig. 1D). The TSPs are placed on a custom-made plate for precise operation. IL-2 solution quickly goes into the void TS channel by capillary force and then water was absorbed to the dehydrated polymer matrix. After the IL-2 solution was completely absorbed, the TSP was dehydrated again and prepared for cell loading.

IL-2 in vitro release measurement

IL-2 release from the TSPs was measured in a 96-well plate. Three TSPs were transferred into a well containing 200 μl of RPMI 1640 medium. The plate was then maintained at 37°C. All the measurements were tripled. At the specific time points, the medium was collected and replaced with a fresh medium. A commercial ELISA kit was used to quantify the amount of IL-2 in the collected medium.

IL-2 & CAR-T in vivo release measurement

Blood was collected from HCT116 tumor-bearing mice that had received CAR-T cells and IL-2 therapy via TSP delivery or intravenous injection at different time points. To obtain serum, the collected blood was centrifuged, and the supernatant was collected. The human IL-2 levels in the serum were quantified using a human IL-2 ELISA kit (Biolegend, 431804) as per the manufacturer’s protocol. The cell count of human CD3⁺ CAR-T cells was calculated using flow cytometry and was normalized to 100 μL of peripheral blood. Peripheral blood samples from three mice were pooled together to obtain one sample.

Loading of T cells into the TSPs and detection of the in vitro T cell release

Mouse splenic T cells were activated by anti-CD3 (0.5 μg/mL), anti-CD28 (0.5 μg/mL) antibodies and mIL-2 (10 ng/mL). The cells were incubated in a humidified CO₂ incubator at 37°C for 24 hours and then collected and washed by using pH 7.4 phosphate buffer saline (PBS). Human CAR-T cells were activated and expanded as shown above as well. Activated mouse T cells or CAR-T cells were resuspended in RPMI 1640 medium at a concentration of 10⁸ cells per mL prior to the loading. The cell suspension was then mixed with rat tail collagen (4 mg/mL) at a 1:1 ratio and then drop wisely loaded into a dehydrated TSP pre-loaded with IL-2. A total volume of 8-10 μL of cell and collagen suspension was added into the particles to reach 0.5 million cells loaded in each particle. Collagen was chosen based on our previous study of fast cell active migration through collagen matrix.[14] Three particles were transferred to a well of a 96-well plate containing 200 μl of RPMI 1640 medium and then incubated in a humidified CO₂ incubator at 37 °C. Every 2 days, the medium was collected for cell counting and was replaced with fresh medium.

T cell Proliferation assay

Mouse splenocytes were isolated from C57BL/6 mice and activated with mouse anti-CD3 and anti-CD28 antibodies in cell culture plates overnight. Cells were then stained with proliferation dye eFluor^™ 450 according to the manufacturer's protocol. 5×10⁵ cells were loaded to each TSP with 200 ng or 400 ng mouse IL-2. The released cells were collected every two days. The TSP particles were broken on day 10, and the unreleased cells were collected. For the re-stimulation assay, 5×10⁵ pre-activated cells were loaded to each TSP with 200 ng mouse IL-2. The released cells were collected on day 3, the TSP particles were then broken to collect the unreleased cells. 1×10⁶/mL cells cultured in medium with 10ng/mL mouse IL-2 was used as control. The cells were then stained with proliferation dye eFluor^™ 450 and restimulated with anti-CD3 and anti-CD28 antibodies for 3 days. The proliferation (eFluor^™ 450 dilution) of T cells was determined by flow cytometry as described previously.[25]

Flow cytometry analysis

Single-cell suspensions were prepared at appropriate concentrations, then stained with zombie-yellow or zombie-red for 30 minutes at room temperature. Cells were then washed with PBS and stained with antibodies according to specific detecting panels for 30 minutes at 4°C. For apoptosis analysis, cells were then washed with Annexin V binding solution and stained with Annexin V and 7-AAD for an extra 30 minutes. The phenotype of the cells was carried out on the LSRII (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc.).

In vivo degradation and biocompatibility of the TSP

To evaluate in vivo degradation, 3 TSPs were peritumorally implanted in C57BL/6 Mice. After 2 weeks and 4 weeks, mice were sacrificed and TSPs along with surrounding tissues were harvested. Samples were then fixed with 10% formaldehyde for 24 hours and stored in 70% alcohol. The fixed samples were submitted to the Research Resources Center at the University of Illinois Chicago (UIC) to be embedded, sectioned into 5 μm thick slides, and hematoxylin and eosin (H&E) stained following standard protocol for histological analysis.

Tumor challenge and treatment

Tumor challenge was done by subcutaneous injection of 1x10⁶ HCC1806 and HCT116 cells in 100 μl of PBS into NSG female and male mice separately. 2 to 3 weeks after injection, mice with a tumor sized around 100mm³ were injected intravenously or intratumorally with PBS or MSLN CAR-T cells w/wo 800 ng human IL-2. For the TSP group, 4 particles each loaded with 5 x 10⁵ MSLN CAR-T cells and 200 ng human IL-2 were peritumorally engrafted. The tumor volume was determined by calipers at 2-day intervals. Tumor volumes were calculated as 1/2 x L x W². The end point for the in-vivo studies was determined as when the tumor size reached 1500 mm³.

Cell cytotoxicity assay

Target cells (tumor cells) and effector cells (CAR-T cells) were cultured one to two weeks prior to the co-culture cytotoxicity assay. 1x10⁵ target cells were gently mixed with 1x10⁵, 3x10⁵ or 9x10⁵ CAR-T cells at final E:T ratios of 1:1, 3:1, or 9:1 and cultured at 37°C in an incubator for 4 to 6 hours. Cell apoptosis was indicated by Annexin V, 7-AAD staining, and flow cytometry analysis.

Statistical Analysis

The data was analyzed by GraphPad Prism Software (Version 9.5.1). For datasets involving two groups, Student’s t-test was performed. The statistical differences in tumor growth between groups of mice were determined by ANOVA analysis. The statistical differences between the survival of groups of mice were calculated according to the log-rank test. Probability values > 0.05 were considered non-significant.

RESULTS AND DISCUSSIONS

TSP design, formation, and characterization

CAR-T cell delivery to solid tumors requires unique design of the polymeric reactor for a minimum invasive procedure and in vivo cell proliferation, expansion, and release. For this purpose, the main polymer matrix of the TSPs is fabricated from a mixture of GelMA (a synthetic biodegradable polymer) and poly (ethylene glycol) diacrylate (PEGDA) to manipulate particle degradation rates and mechanical properties for intratumoral or peritumoral implantation. The heterogeneous structure and component have the flexibility for co-encapsulation and delivery of multiple immunostimulants and checkpoint inhibitors with individually manipulated kinetics. The toroidal-spiral void space near the surface of the particles enables a large volume of cell loading.

The formation of the TSP is through a self-assembly process of droplet sedimentation in a miscible solution and solidification (Fig. 1A and 1B). More details of the process can be found from the previous section of “TSP formation”. The process is scalable and highly reproducible, which is essential for eventual clinical translation and commercialization. The particles can be massively generated, sterilized, and ready to be use (Fig. 1C). A cytokine, IL-2, is loaded to the topical layer of the internal channel to promote T cell activation, proliferation, and long-term survival (Fig. 1D). The CAR-T cells are suspended in collagen and loaded into the internal toroidal-spiral channel (Fig. 1D). After peritumoral injection of the TSPs near the solid tumor, the CAR-T cells are expanded and activated in the device and actively climb out of the collagen matrix toward the tumor cells (Fig. 1E).

Degradation of TSP plays an important role in deciding cell release kinetics while also eliminating the need for a surgery to retrieve the particles after implantation. The cell release kinetics is a result of the cells climbing out from the collagen matrix and the degradation of the main particle structure. Fluid in living organisms contains many types of proteinase enzymes, such as collagenase and gelatinolytic proteinase. The identity and function of many of these metalloproteinases are still unknown, but studies show that the matrix metalloproteinase (MMPs) family is responsible for degrading extracellular matrix compounds like collagen and gelatin. Collagenase type II, which consists of many MMPs such as MMP-2 (also known as gelatinase A) and MMP-9 (also known as gelatinase B) to break the peptide bond in gelatin, is used to study the TSP in vitro degradation. To evaluate the degradation rate of hydrogels composed of PEGDA and GelMA, disks composed of different PEGDA and GelMA contents were prepared, and after washing with DI water they were incubated in 2 U/mL of collagenase type II at 37°C .[26] Pristine GelMA hydrogels containing 5-20 wt% of GelMA degraded within hours in the presence of collagenase, but adding PEGDA resulted in a slower degradation rate (Fig. 2A). It is desirable to have the particles degraded in a few weeks for the delivery of CAR-T cells to the solid tumor. Based on the degradation kinetics of the hydrogel and the TSP formation process (matching the density and viscosity of the polymer droplets with the bulk solution), the composition of the TSPs was chosen to be 8.2 wt% PEGDA and 8.9 wt% GelMA. The in vitro degradation of the TSPs was measured by recording the weight loss of the particles over time under the similar condition by incubating the particles in the 2 U/mL collagenase solution, resulting in a comparable degradation rate to the disk of similar polymer composition (Fig. 2B). The photos of the TSPs after 13 and 26 days of being incubated in collagenase solution (2 U/mL) are shown in Fig. 2B. After 13 days, particle structure started to collapse, and after 26 days they turned into small pieces due to degradation.

Fig. 2. Degradation of the main polymer matrix of the TSPs.

(A) Weight loss curves of diskshaped hydrogels with different compositions of PEGDA and GelMA due to degradation in the presence of 2 U/mL collagenase solution in vitro. For the legend, P is for PEGDA, G is for GelMA, and the numbers represent weight percentages. (B) The weight loss of the TSPs with 8.2 wt% PEGDA and 8.9 wt% GelMA due to TSP degradation in vitro over 60 days (n=3). The red arrows point to the corresponding microscopic images of the TSPs after 13 days and 26 days of in vitro degradation. After 26 days, the particle broke into small pieces. The scale bars represent 500 μm. (C & D) H&E staining of the TSP and surrounding tissue retrieved after 2-week (C) and 4-week (D) peritumoral implantation of the particle. The scale bars on the top images represent 500 μm and the scale bars on the bottom images represent 100 μm. The arrows in (D) point to the remaining undegraded polymer.

In vivo degradation of TSPs encapsulating CAR T cells and IL-2 was followed for four weeks after peritumoral implantation of the particles. No sign of weight loss or scratching was observed in mice implanted with the TSPs. The retrieved particles were histologically analyzed following hematoxylin and eosin (H&E) stain. The H & E images shows that after two weeks the particles kept relatively integral (Fig. 2C) and after four weeks they were further degradated, which appears faster than the in vitro degradation as expected (Fig. 2D). Both two-week and four-week H&E images show formation of a thin fibrous capsule layer (about 30 μm thickness) produced by fibroblasts present at the implantation site.

The millimeter-size TSPs may be implanted or injected into solid tumors or tumor surrounding tissues by using a large biopsy needle or catheter. The response of the particles to deformation under compression is critical for particle injection and long-term biomedical functionality.[27] Young’s modulus measures the material’s stiffness; the higher stiffness of the material, the higher its ability to maintain its shape after impact of the applied forces. The main polymer matrix of the TSPs, made from the mixture of PEGDA and GelMA, provides mechanical strength. In general, hydrogels with higher polymer concentration and lower molecular weight are less elastic.[13] The mechanical properties of the TSPs can be manipulated by varying the composition and molecular weight of PEGDA (Mw 700) and GelMA. In addition, because of the heterogeneous structure of the TSPs, the stress-strain mechanical tests need to be performed by applying the compression force from two different directions to the particles. The stress-strain response of the TSPs with 8.2 wt% PEGDA and 8.9 wt% GelMA is presented in Supplementary Fig. S2. The values of Young’s modulus are determined by the slopes of the elastic region (the initial linear portion) of the stress-strain curves. The Young’s modulus of the TSP with the compression added from the side, which was 0.02 MPa, is smaller compared to the case with the compression added from the top, which was 0.044 MPa, because the toroidal-spiral layer is filled with collagen and the particles are less stiff from the side. When applying the compression from the top, the TSP failed at 8.4 MPa stress and 44 % strain, while when applying the compression from the side, the TSPs failed at a lower stress but a higher strain.

IL-2 and Cell in vitro and in vivo release from TSPs

T cell-based cancer treatment requires activation processes normally evoked by signals from antigen-presenting cells, including membrane-bound costimulatory ligands and secreted factors. IL-2 was loaded to the TSP in the main polymer matrix, close to the internal surface of the TS layer to support the proliferation of the T cells (Fig. 3A). Other cytokines (such as IL-15/IL-15Rα) and antibodies can be co-encapsulated to optimize cell proliferation and activation. Although the co-encapsulation of IL-2 augments cell proliferation and growth and studies show that co-delivery of IL-2 enhances T cell efficacy, an extra amount of IL-2 can cause serious side effects, such as reparatory distress and arrhythmias.[28, 29] Therefore, the release of IL-2 needs to be carefully quantified. In this case, the pore size of the main polymer matrix of the TSP (containing low molecular weight PEGDA) is too small for IL-2 (Mw 15.4 kDa) to diffuse through, so the release of IL-2 is mainly by enhanced diffusion through the revolving channel, which is expected to be very slow. In this process, the IL-2 molecule disengages from the thin layer of the inner toroidal-spiral channel, then passively diffused out the particle through the long revolving channel filled with collagen. Therefore, IL-2 molecules behave more like a surface catalyst to activate the T-cells (Fig. 3A). The in vitro release of IL-2 from the TSP is quantified and reported in Fig. 3B.

Fig. 3. In vitro and in vivo release of the cells and IL-2.

(A) A schematic of cells expanded and activated in the TSP and trafficking toward the tumor cells by migrating through the collagen matrix. The mIL-2 molecules slowly released from the inner surface of the TS channel by passive diffusion and polymer degradation. (B) The amount of mIL-2 released from each TSP initially loaded with 50, 200, and 400 ng mIL-2 over time (n=3). (C) In vitro release of mouse splenic T cells from the TSPs preloaded with 0, 200, and 400 ng mIL-2 in each particle (n=3). (D) The amount of IL-2 in blood over time, for the comparison of i.v. injection and TSP peritumoral implantation. (E) The amount of CAR T cells in blood over time, for the comparison of i.v. injection and TSP peritumoral implantation. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired Student’s two-tailed t test was used (D&E). Data (mean ± SD) are representative of at least 2 independent experiments with 3–5 independently analyzed mice/group.

The cells are expanded and activated in the TSP and roll out of the polymer matrix toward the tumor cells. The in vitro release rate of mouse splenocyte T cells with various amounts of IL-2 (0, 200, and 400 ng per particle) from the TSPs was measured. About 0.5 million cells suspended in rat tail collagen were loaded into each TSP. Three TSPs were used for each study with total ~ 1.5 million initial cell loading. Without IL-2 preloaded in the TSPs, about 1 million cells released from the particles within the first seven days followed by a nearly flat curve. This may indicate that the cells have limited growth and one third of the loaded cells could not be released or died during the release measurement. TSPs loaded with IL-2 prior to cell loading showed a significant increase in number of cells released from the TSPs compared to the ones without IL-2 (Fig. 3C). IL-2 promotes cell activation, and growth, while the cells continuously migrate out of the collagen matrix. Doubling the amount of IL-2 from 200 ng to 400 ng had minimal effect on cell release rate, indicating enough IL-2 molecules in the collagen matrix for their functions. There was a burst release for the first 7 days with about 2 million cells released and total about 2.5 million cells in 4 weeks, which was beyond the number of cells initially loaded into the TSPs as the cells continued to expand in the TSP. The in vitro cell release measurement indicates that microenvironment of the TSP promotes cell proliferation followed by migration out from the particles actively. The continuous expansion of the cells in the particles may potentially enable a reduced dose of the cells for effective treatment, therefore reducing the cost and time to generate sufficient number of cells. The study of the in vitro release of IL-2 and mouse T cells are consistent with our previous study using human IL-2 and T cells.[14] Based on the in vitro cell release study, TSPs containing 200 ng IL-2 were used for all the in vivo studies.

The in vivo pharmacokinetics of IL-2 and CAR-T cells were measured after peritumor implantation of the particles and compared to the intravenous (i.v.) injection of T-cells and IL-2 800 ng to NSG mice. Despite the concerns of the immune-mediated side effects of IL-2, no conspicuous weight loss was observed in both groups of the mice with peritumor implantation of the particles and the one with a single i.v. dose of 800 ng IL-2 (Fig. S3). Blood was withdrawn from the animals and the amount of IL-2 and CAR-T were quantified. Compared to i.v. injection, both IL-2 and CAR-T cells show prolonged release from the TSPs (Fig. 3D and 3E). A small amount (a few hundred pg/mL) of IL-2 were constantly detected in the mice which received the TSP peritumor implantation. For the group of mice with i.v. injection, a relatively large amount of IL-2 was detected in a transient time for the first day and then the amount of IL-2 quickly dropped a low level. Similar pharmacokinetics is displayed for cell count in blood.

Activated T cells released from TSP show greater proliferative ability, survival, and memory phenotype

Activated T cells were loaded into TSP with mIL-2 at appropriate doses and cultured in the medium in vitro. After 10 days, the released and unreleased cells were harvested, and cellular proliferation and apoptosis were measured using flow cytometry. As shown in Fig. 4A, T cell proliferation was significantly increased when loaded with 200 ng or 400 ng mIL-2 in both CD4 and CD8 subsets compared with the control cells without mIL-2 treatment. To assess the impact of TSP on cell proliferation, we collected both released and unreleased cells on day 3 and restimulated them with anti-CD3/CD28 for additional 3 days in the medium. The unreleased cells exhibited a significantly lower proliferative capacity compared to the released cells and unloaded cells, while the released cells expanded faster than the unloaded cells (Fig. 4B). These findings suggest that more proliferative cells are likely to be released easily from TSP, and TSP may restrain transiently the proliferation capacity of certain subpopulations of loaded cells, allowing slow release of the proliferative cells. On the other hand, activated T cells directly cultured in complete RPMI-1640 medium containing 10 ng/mL mIL-2 showed greater apoptosis, compared to the cells released from TSP (Fig. 4C). As expected, in both released and unreleased populations, the counts of total CD3 T cells when the TSPs were loaded with 200 ng or 400 ng mIL-2 were dramatically higher than the counts of cells directly cultured in medium without TSP (Fig. 4D).

Fig. 4. Flow analysis of proliferation, apoptosis and activation of T cells transported by TSP.

(A) Mouse splenocytes were activated with anti- CD3 overnight and activated T cells were then loaded to TSP with indicated amounts of mIL-2 for 10 days. The proliferation (eFluor^™ 450 dilution) of released T cells and unreleased T cells was detected by flow cytometry (mean±SD). (B) Mouse splenocytes were activated with anti-CD3 overnight and activated T cells were then loaded to TSP with indicated amounts of mIL-2 for 3 days. Released cells and unreleased cells were collected and re-stimulated with anti-CD3 and anti-CD28 for 3 days. Proliferation was detected via flow cytometry (mean±SD). (C) Apoptosis of T cells released from TSP on day 10 was determined by flow cytometry. The activated T cells cultured directly in RPMI1640 medium with 10ng/mL mIL-2 were used as a control medium group. (D) The cell count of CD3+ T cells on day 10 was summarized. (E&F) Different subsets (Tn, naïve T cells; Tscm, stem cell-like memory T cells; Tcm, central memory T cells; Tem/eff, effector memory and effector T cells) among CD4 T cells (E) and CD8 T cells (F) were analyzed by flow cytometry. (G-I) MSLN CAR-T cells were activated and expanded for 7 to 10 days. 5x10⁵ cells and 200ng human IL-2 were loaded to each particle, and 3 particles were cultured in each well of a 96-well plate in T cell expansion medium. 2x10⁵ cells with 10 ng/mL human IL-2 cultured in T cell expansion medium were used as the control medium group. The percentage of CD4 and CD8 T cell subsets (G) and apoptosis (H) of CD3⁺ T cells released from TSP on day 10 were detected by flow cytometry, and total CD3⁺ T cell counts were summarized (I). *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired Student’s two-tailed t test was used. Data (mean ± SD) are representative of at least 2 independent experiments with 3–5 independently analyzed mice/group.

Given the importance of memory T cells for adoptive cell therapy [30, 31], we examined the percentage of central memory T cells (Tcm), effector memory T cells (Tem), effector T cells (Teff), naïve T cells (Tn), and stem cell-like memory T cells (Tscm) of CD4 and CD8 T cells subsets in the TSP-released cells, comparing with activated T cells directly cultured in medium (Supplementary Fig. S4A). The percentages of both Tcm and Tem/eff were significantly increased in cells released from the TSP, while the Tn population was decreased in both CD4 and CD8 subsets (Fig. 4E and 4F). The percentage of CD8 Tscm was also decreased (Fig. 4F).

Next, MSLN CAR-T cells were loaded to TSP with 200 ng recombination human IL-2, and cell proliferation and apoptosis were compared with cells cultured in complete RPMI-1640 medium containing 10 ng/mL hIL-2. Similar to the activated mouse T cells above, CAR-T cells released from TSP exhibited increased percentages of both CD4 and CD8 subsets (Fig. 4G and Supplementary Fig. S4B) and decreased apoptosis (Fig. 4H). The number of total CD3 T cells released from the TSP group was significantly higher when compared with the medium control (Fig. 4I).

Characterization of MSLN CAR-T cells

We used the 2^nd generation of CAR-T cells, which target the MSLN antigens and contain a Flag tag for detection (Fig. 5A). After the recognition of MSLN-expressing tumor cells by the ScFv domain, CAR-T cells will be activated through the primary and co-stimulatory signals (Fig. 5A).

Fig. 5. Characterization of MSLN CAR-T cells.

(A) MSLN-targeted CAR constructs with CD3ζ endodomain, the co-stimulatory domain 4-1BB, and Flag as a tag transduced into human T cells recognizing MSLN-expressing tumor cells. The Mock scFv Control CAR-T cells were included in the experiments as a negative control. (B) Detection of MSLN CAR constructs by flow cytometry stained with FLAG-PE antibody. Percentage of the FLAG expressing cells was compared between non-stained, isotype, and flag-stained samples in both CD4 and CD8 T cell subsets (mean±SD). (C) Effector cells (MSLN CAR-T cells) and Target cells (HCC1806) were co-cultured at different ratios as indicated. Annexin V and 7-AAD were stained, and apoptosis was measured by flow cytometry (mean±SD). (D) Quantification of early, late, and total apoptotic tumor cells. Effector cells (MSLN CAR-T cells) and Target cells (HCC1806) were co-cultured at different ratios as indicated. Mock CAR-T cells were used as the control for MSLN CAR-T cells and co-cultured with Target cells at 3:1 ratio. (E) Effector cells (MSLN CAR-T cells) and Target cells (HCT116) were co-cultured at different ratios as indicated. The percentage of early apoptosis, late apoptosis, and total apoptosis were summarized. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired Student’s two-tailed t test was used. Data (mean ± SD) are representative of at least 2 independent experiments with 3–5 independently analyzed mice/group.

The purity of CAR constructs was detected via flow cytometry. Almost all the cells (>98% in CD4 subsets and >99% in CD8 subsets) were Flag-PE positive cells when compared to non-stained and isotype control (Fig. 5B), indicating the extremely high purity of mesothelin CARs.

To measure the anti-tumor effect of MSLN CAR-T cells, we performed cytotoxicity assays by co-culture of MSLN CAR-T cells with multiple mesothelin expressing tumor cell lines, including HCC1806, HCT116, A549, CanPan2, OVCAR3, PanC1, and SKOV3 (Supplementary Fig. S5A). As shown in Fig. 5C and 5D, CAR-T induced the apoptosis of HCC1806 in a dose-dependent manner. By contrast, as a control, the mock CAR-T cells showed no significant toxicity against HCC1806 cells. Furthermore, CAR-T-induced cytotoxicity was also found in HCT116 (Fig. 5E) and other mesothelin expressing tumor cell lines (Supplementary Fig. S5B). Accordingly, these results confirmed the cytotoxicity of MSLN CAR-T cells on MSLN-expressing tumor cells.

Local delivery of CAR-T cells by TSP shows superior anti-tumor effect in vivo

The anti-tumor activity of MSLN CAR-T cells via TSP-mediated peritumoral delivery was compared to the intratumoral injection of MSLN CAR-T cells and the conventional i.v. injection of MSLN CAR-T cells (Supplementary Fig. S6A) in the established HCT116 tumor model. As expected, compared to the PBS treatment, a greater antitumor effect and increased survival was observed in mice that received MSLN CAR-T cells, whichever routes they were administrated (Fig. 6A and Supplementary Fig. S6B). Notably, TSP-mediated peritumoral delivery was more effective in inhibiting tumor growth and prolonging mice survival as compared to either intratumoral injection or conventional i.v. injection of MSLN CAR-T cells (Fig. 6A and Supplementary Fig. S6B). Similar anti-tumor effects were observed in an additional tumor model with HCC1806 cells (Fig. 6B and Supplementary Fig. S6C).

Fig. 6. TSP enhanced the anti-tumor capacity of MSLN CAR-T cells.

(A) Tumor growth (left) and mice survival (right) of HCT116-bearing mice treated with TSP-mediated peritumoral delivery, intratumoral (I.T.) injection, intravenous (I.V.) injection of MSLN CAR-T, or PBS. (B) Tumor growth in HCC1806-bearing mice treated with TSP-mediated peritumoral delivery of MSLN CAR-T, I.T., I.V. injection of MSLN CAR-T, or PBS. (C) Tumor weight in HCT116-bearing mice on day 14 (top) and day 21 (bottom) after delivery of CAR-T+IL-2 by TSP versus I.V. injection. (D) Flow cytometry analysis of the accumulation of peripheral CD3⁺ CAR-T cells from HCT116 tumor-bearing mice 7 days after treatment. (E) Normalized cell counts of CAR-T cells in tumor from HCT116 tumor-bearing mice 14 days (left) and 21 days (right) after treatment. Flow cytometry analysis of PD-1⁺ cells (mean±SD) (F) and expression levels of PD-1 (mean fluorescence intensity, MFI) (G) in transferred CAR-T cells from HCT116 tumor-bearing mice 7 days after treatment. Flow cytometry analysis of CD39⁺, LAG3⁺, and TIM3⁺ cells (mean±SD) (H) as well as their expression levels (MFI) (I) in transferred CAR-T cells from HCT116 tumor-bearing mice 14 days after treatment. *p < 0.05, **p < 0.01, ***p < 0.001. ANOVA analysis, log-rank test and unpaired Student’s two-tailed t test were used. Data (mean ± SD) are representative of at least 2 independent experiments with 3–5 independently analyzed mice/group.

The immune checkpoint molecules PD-1 [32] on T cells impair antitumor T cell immunity. We detected the expression levels PD-1 in the CAR-T cells isolated from tumor-bearing mice. In both tumor models, more peripheral CD3⁺ MSLN CAR-T cells were detected in mice through the TSP-mediated peritumoral delivery compared to other routes (Fig. 6D and Supplementary Fig. S6D). Consistently, increased tumor infiltration of CAR-T cells was detected through the TSP-mediated peritumoral delivery compared to the intravenous (IV) delivery on day 14 and day 21 after tumor implantation (Fig. 6E). Likewise, transferred CD3⁺ MSLN CAR-T cells through the TSP-mediated peritumoral delivery expressed lower levels of PD-1 than those through other routes (Fig. 6F-G and Supplementary Fig. S6E). Furthermore, reduced expression levels of additional exhaustion markers including CD39 [33], LAG3, and TIM3 [34] were observed in CAR-T cells delivered via TSP-mediated peritumoral delivery compared to those delivered via the IV route (Fig. 6H and 6I).

Collectively, these results suggest that compared to conventional administration, TSP-mediated peritumoral delivery elicits a more effective antitumor effect in association with increased persistence of transferred MSLN CAR-T cells with a less-exhausted phenotype.

CONCLUSIONS

Biodegradable and biocompatible heterogenous TSPs can seed cells for in vivo proliferation, activation, and active release. Besides delivering T lymphocytes, the platform can easily be adapted for the co-encapsulation and delivery of a wide range of macromolecules and small molecular drugs. We have demonstrated the formation of a biodegradable and biocompatible TSP as well as the utilization of TSPs for co-delivery of CAR-T cells and IL-2, a growth factor for cell expansion and proliferation for solid tumor treatment. Biodegradable TSPs are generated using GelMA, a synthesized biodegradable material that in mixture with PEGDA, could control degradation and physical properties of the particles and provide suitable biocompatibility and mechanical properties. The large inner void volume of the particles could enhance cell encapsulation capacity and the polymer matrix can act as a platform for prolonged cytokine release. Co-encapsulation of IL-2 and mouse T cells showed a sustained release of the cytokines and cells in vitro while maintaining high viability of the cells. Persistent release of IL-2 could support the adequate proliferation of both CD4 and CD8 T cells, but was more favorable towards CD8 T cells proliferation. In addition, TSPs loaded with IL-2 and MSLN-CAR-T cells could more effectively decelerate several solid tumor outgrowths in vivo compared to intratumoral and intravenous injection of CAR-T cells. Our study suggests that using biodegradable TSP as a localized immunotherapy method can potentially improve solid tumor treatment.

In both mouse tumor models of HCT116 and HCC1806, although tumor growth was significantly slower for the groups of mice that received TSP implantation, the tumor could not be completely eradicated. More investigation on the dosage is necessary in future. Additionally, using TSP as a bioreactor for CAR-T in vivo expansion and release may have advantages for preventing relapses of incompletely resected tumors, which needs further evaluation.