
==== Front
J Nanobiotechnology
J Nanobiotechnology
Journal of Nanobiotechnology
1477-3155
BioMed Central London

39272205
2781
10.1186/s12951-024-02781-1
Research
A nanobody-guided multifunctional T cell engager promotes strong anti-tumor responses via synergistic immuno-photothermal effects
Xie Shenxia 12
Shi Wei 1
Duan Siliang 15
Huang Xianing 1
Liu Aiqun 1
Hou Xiaoqiong 13
Lin Xuandong 1
Zhong Dani 1
Sun Shuyang 1
Ding Ziqiang 1
Yang Xiaomei yangxiaomei6009@sina.com

13
Chen Xiaoyuan chen.shawn@nus.edu.sg

4
Lu Xiaoling luxiaoling@gxmu.edu.cn

1
1 https://ror.org/03dveyr97 grid.256607.0 0000 0004 1798 2653 Guangxi Key Laboratory of Nanobody Research, Guangxi Nanobody Engineering Research Center, College of Stomatology, Hospital of Stomatology Guangxi Medical University, Nanning, Guangxi 530021 P. R. China
2 https://ror.org/03dveyr97 grid.256607.0 0000 0004 1798 2653 Pharmaceutical College, Guangxi Medical University, Nanning, Guangxi 530021 P. R. China
3 https://ror.org/03dveyr97 grid.256607.0 0000 0004 1798 2653 School of Basic Medical Science, Guangxi Medical University, Nanning, Guangxi 530021 P. R. China
4 https://ror.org/01tgyzw49 grid.4280.e 0000 0001 2180 6431 Nanomedicine Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117597 Singapore
5 https://ror.org/02fj6b627 grid.440719.f 0000 0004 1800 187X Department of Medicine, Guangxi University of Science and Technology, Liuzhou, Guangxi 545005 P. R. China
14 9 2024
14 9 2024
2024
22 56130 4 2024
14 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

T cell-based immunotherapies are facing great challenges in the recruitment and activation of tumor-specific T cells against solid tumors. Among which, utilizing nanobody (Nb) or nanobodies (Nbs) to construct T cell engager has emerged as a more practical potential for enhancing the anti-tumor effectiveness of T cells. Here, we designed a new Nb-guided multifunctional T cell engager (Nb-MuTE) that not only recruited effector T cells into the tumor tissues, but also efficiently activated T cells anti-tumor immunity when synergies with photothermal effect.

Results

The Nb-MuTE, which was constructed based on an indocyanine green (ICG)-containing liposome with surface conjugation of CD105 and CD3 Nbs, and showed excellent targetability to both tumor and T cells, following enhancement of activation, proliferation and cytokine secretion of tumor-specific T cells. Notably, the immunological anti-tumor functions of Nb-MuTE-mediated T cells were further enhanced by the ICG-induced photothermal effect in vitro and in vivo.

Conclusions

Such a new platform Nb-MuTE provides a practical and “all-in-one” strategy to potentiate T cell responses for the treatment of solid tumor in clinic.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-024-02781-1.

Keywords

Nanobody
T cell
T cell engager
Solid tumor immunotherapy
Photothermal therapy
Guangxi Natural Science Foundation Youth Fund Project of ChinaNo.2023GXNSFBA026178 NUS School of Medicine Nanomedicine Translational Research ProgrammeNUHSRO/2021/034/TRP/09/Nanomedicine the International (Regional) Cooperation and Exchange Program of National Natural Science Foundation of ChinaNo. 82220108003 issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcBackground

T cell-based immunotherapy has revolutionized cancer treatment by effectively mobilizing T-cells to eradicate malignant cells, and one promising therapeutic approach is the bispecific T-cell engager (BiTE) [1]. Currently, an increasing number of BiTEs are undergoing clinical or preclinical studies and have shown substantial activity in several hematological malignancies, but progress with solid tumors has been slower, partly due to their heterogeneity and complexity [2, 3]. In addition, the ordinary formation of T-cell engagers is based on single-chain antibody fragments (scFvs) that may not fold efficiently and are prone to aggregation, which can interfere with T-cell engager function [4]. Furthermore, the production of these traditional T-cell engagers can be challenging due to the difficulty in assembling the correct heavy-light chain fragments [5].

Nanobodies (Nbs or VHHs) are the smallest naturally occurring antigen-binding functional fragments that have emerged as therapeutic agents in antibody engineering. Unlike monoclonal antibodies, scFvs, or other antibody fragments, the minimal molecular weight (∼ 15 kDa) of Nbs makes them rapidly distributed and able to diffuse deeply into solid tissues [6, 7]. Nbs are also less affected by the shifting of the heavy and light chains and have better functionality, solubility, and thermostability compared to scFvs, making them much easier to fuse into bispecific or trivalent constructs with stable effects [8, 9]. Our group had previously proposed a Nb-based BiTE (Nb-BiTE) platform targeting tumor antigens including CD105 (also known as endoglin) and fibroblast activation protein (FAP) [10, 11]. This platform has proven to be highly effective in mediating specific tumor lysis in vitro and in mouse xenograft models. However, there is still room for improvement of Nb-BiTE-primed T cell therapy in the treatment of solid tumors. These Nb-based fusion molecules have brought activated T cells into the tumor tissue, but solid tumors also have a complex and immunosuppressive microenvironment that blocks T cells from being active inside [12–14]. Based on the above research, the key issue to be addressed is how to further promote the activated T cells to infiltrate the tumor tissue faster, more accurately, and more adequately is still the key issue to be overcome. To achieve this goal, a new strategy that combines BiTEs for synergistic enhancement may need to be considered.

Photothermal therapy (PTT) is a potent local therapeutic technique that uses photothermal agents (PTAs) to convert light energy into heat under near-infrared light (NIR) light irradiation, which can disrupt and destroy the tumor microenvironment through the photothermal effect [15]. The resulting local heating at the tumor site not only degrades the extracellular matrix of the tumor but also dilates local vessels and accelerates blood flow, which in turn leads to more rapid recruitment of immune cells into the tumor tissues [16]. These would first remodel the tumor environment from a “cold” state to a “hot” state with higher susceptibility to immune attack [17]. As a consequence of the tumor cell death, PTT then triggers the release of tumor-associated antigen, which can cause a secondary but more robust T cell response with higher tumor-specificity and activity [18]. The antitumor immune response induced by the PTT can also be triggered by activating immune cells (NK cells, T cells and DCs), promoting the release of exosomes from tumor cells, and upregulating the expression of inflammatory cytokines [19]. Therefore, it would be a smart and effective way to integrate PTT with Nb-based T cell engager-mediated immunotherapy to achieve a synergistic treatment of solid tumors.

Conjugation of nanobodies in drug-loaded nanoparticles has exhibited an efficient potential in new drug delivery systems in cancer therapy [20, 21]. Therefore, we used liposome nanoparticle materials as a carrier to integrate the Nb-based T cell engagers with PTT into a bundle system. Liposomes (Lipo) have been widely used as drug nanocarriers or molecular imaging agents in the field of biomedicine during the last decades, which is attributed to their easier preparation, low immunogenicity, favorable biocompatibility, extended circulation half-life and endoplasmic reticulum (ER) effect-mediated passive targeting [22, 23]. Recently, PEGylated Lipo with antibodies or other antibody-like peptides conjugated to their surfaces are rapidly developed which can effectively improve drug targetability, accessibility as well as drug sustained release [24, 25].

Herein, we propose to construct a novel multifunctional T cell engager (MuTE) by utilizing liposomal nanoparticles to load Nb and photosensitizers to form an “all-in-one” system. MuTE differs from the traditional fusion protein BiTE or Nb-BiTE structures, we assembled anti-CD3 Nb and anti-CD105 Nb on the surface of Lipo to specifically attract T cells to CD105-expressing tumors to activate T cell killing, similar to the function of CD105-CD3/ Nb-BiTE reported previously. Meanwhile, for the photothermal reaction, we encapsulated indocyanine green (ICG), an FDA-approved fluorescent dye/photosensitizer into our Lipo, which has been increasingly applied to near-infrared fluorescence imaging (NFI)-based surgery and PTT in the clinic because of its high photothermal conversion efficiency, optical stability and biocompatibility [26, 27]. Since the photothermal effect exerted by ICG requires NIR excitation, the entire course of the immunotherapy can be well controlled by the location and timing of laser irradiation according to the design of the treatment plan, to achieve “all-in-control”. Another predictive benefit of using ICG as the core of the MuTE complex here was non-invasively observing the targeted transport of drugs in real-time in vivo, and indirectly judging the status of tumor growth through its specific binding to tumors. As shown in Fig. 1, this so-called CD105/CD3 Nb-LipoICG as an example of MuTE platform, would engage T cells and tumor cells via the dual Nbs’ specificity and stability, whereas provides both photothermic effect and dynamic visibility via ICG, therefore enhances the tumor-specific infiltration and antitumor activity of T cells in dense solid tumor tissue based on the functional synergy of Nb-BiTE-primed T cell immunotherapy and PTT. This work has built up an “all-in-one” and “all-in-control” platform that integrates PTT with T cell immunotherapy for better therapeutic outcomes and in situ visual monitoring of tumor progression to attain the most desirable effect, thus providing a new potential option for the T cell immunotherapy for clinical solid tumor therapy.

Fig. 1 Schema for the synthesis and design of Nb-MuTE (CD105/CD3 Nb-LipoICG) and the antitumor effects in vivo

Methods

Animals and cells

Non-obese diabetes/SCID (NOD/SCID) mice and BALB/c mice (female, 4–6 weeks old, 16–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. and raised under specific pathogen-free (SPF) conditions. Animal experimental protocols were reviewed and approved by the Ethical Committee of the Guangxi Medical University. The present study is compliant with all relevant ethical regulations regarding animal research.

Peripheral blood mononuclear cells (PBMCs) were obtained from healthy human volunteers in accordance with the Declaration of Helsinki. All volunteers signed informed consents approved by the Human Research Ethics Committee of the Guangxi Medical University. CD3+ T cells were further sorted from PBMCs using a BDAriaIII cell-sorting system (BD Biosciences, San Jose, CA). Human hepatocellular carcinoma lines (Bel7407 and Huh7), melanoma line (C8161), and breast cancer line (MCF-7) were purchased from American Type Culture Collection Inc. (ATCC, USA). Human umbilical vein endothelial cells (HUVECs) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All the cell lines used in this study were stored in liquid nitrogen in our laboratory.

To mimic the natural growth condition of tumor endothelial cells, Bel7404 and HUVECs were co-cultured in a MilliCell® 6-well cell culture device (Millipore, USA) to induce the transformation of HUVECs into tumor-derived endothelial cells (TDVECs) [28, 29]. The upregulated expression of CD105 in TDVEC was detected by flow cytometry. All cells mentioned above were cultured in RPMI-1640 complete medium (CM) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), 100 U/mL penicillin, and 100 µg/mL streptomycin (GIBCO) at 37 oC and 5% CO2.

Nanobody expression and purification

Standard procedures of recombinant protein expression were performed to obtain CD105 and CD3 Nbs. Briefly, the cryopreserved E.coli bacteria containing the target plasmid were resuscitated in an LB medium, induced by 1 mM of isopropyl-β-d-thiogalactoside (IPTG) in LB medium containing 1% ampicillin for 14 h at 25 °C. After centrifugation, the bacterial cell pellet was collected, resuspended in non-denatured lysis buffer (2 mL of non-denatured lysate with 2 mg lysozyme per gram wet weight of pellet), incubated on ice for at least 30 min, and then broken by ultrasonication utilizing a sonicator (Sonicator 3000, Misonix, USA) at the constant power of 4 W/cm2. The clear supernatant was collected and purified in a chromatographic column filled with BeyoGold™ His-tag purification resin (Beyotime, Beijing, China) through the built-in His6-tag of Nb, according to the manufacturer’s instruction. The purified fractions were separated by SDS-PAGE on a 12% gel, followed by western blot (WB) assay.

Preparation and purification of CD105/CD3 Nb-LipoICG

Liposome (Lipo) was prepared by the thin film hydration with slight modification. In brief, distearyl phosphatidyl choline (DSPC; Aladdin, China), cholesterol succinate monoester (Chol; Aladdin, China), and distearyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG-2000; Aladdin, China) were dissolved at a molar ratio of 3:2:0.015 in a mixed solution of chloroform and methanol in 4:1 (v/v) in a round-bottom flask. The mixture solvent was completely evaporated at 50 oC by a rotary evaporator. The formed lipid film was rehydrated with 1×PBS buffer to a final concentration of 2 mg/mL of total lipids, and then filtered with a 0.22 μm needle filter. The liposome size was uniformed by multiple extrusion steps (at least 10 times) through polycarbonate membranes (Nuclepore, Pleasanton, CA, USA) with a pore size of 100 nm.

ICG-encapsulating nanoliposome (LipoICG) was prepared by adding ICG-methanol solution (5 mg/mL) into the lipid mixture in a mass gradient ratio, followed by the same procedure. Sephadex G-50 glucan gel column was applied to remove the free ICG that was not encapsulated. To modify the LipoICG with functional Nbs (CD105/CD3 Nb-LipoICG), CD105 Nb and CD3 Nb were incubated with DSPE-PEG2000-NHS, the NHS group underwent nucleophilic substitution reaction with the amino group on CD105 Nb and CD3 Nb to form stable amide bonds at 4 oC, DSPE-PEG-Nb micelles were formed after continuous stirring for 24 h.

The formed DSPE-PEG-Nb micelles were added to LipoICG and reacted for 10 min under the protection of nitrogen, and incubated in a microplate thermostatic oscillator (Eppendorf, Germany) with a slow shaking speed at 4 oC for 12 h. Free unbound Nbs were removed by the Sephadex G-50 column.

Identification and characterization of CD105/CD3 Nb-LipoICG

The successful encapsulation of ICG in the prepared CD105/CD3 Nb-LipoICG was verified by adding 20 µM of Dil® (DOJINDO, Japan) to stain the phospholipid bilayer. Unbounded free Dil was removed by passing through the Sephadex G-50 column to obtain purified Dil-labeled CD105/CD3 Nb-Lipo (Dil)ICG, and the fluorescences were detected by laser scanning confocal microscope (LSCM; Olympus, USA). The coupling of Nb(s) into the Liposomes was determined by adding fluorescein isothiocyanate (FITC; BD, USA) and observed under the fluorescent microscope based on the chemical reaction between N = C = S group of FITC and the NH2 group of Nb. The absorbance of ICG in liposomes was measured in a UV-Vis spectrophotometer (PerkinElmer, USA) at 400–900 nm after ultrasonic breaking of the liposomes in methanol to estimate the encapsulation efficiency of the Nb-modified liposome complexes. The encapsulation amount was indirectly calculated according to the mass ratio of ICG concentration before and after ultrasonication.

The photothermal property of CD105/CD3 Nb-LipoICG was assessed based on the fluorescence intensity. Each liposomal sample in various PBS dilutions was irradiated with an 808 nm laser at a certain power and the solution temperature was monitored by a thermocouple thermometer (FLIR, USA). The fluorescence intensity was measured in the Odyssey® CLx imaging system (Li-COR, USA). PBS and unmodified Lipo samples served as background and negative control, respectively. The particle size, zeta potential and basic surface characteristics of CD105/CD3 Nb-LipoICG were analyzed with the transmission electron microscope (TEM; Hitachi, Japan), Zetasizer Nano ZS (Nano ZS, Britain), and Laser scanning confocal microscope.

To evaluate the thermostability of Nbs, purified CD105 Nb or CD3 Nb was diluted in PBS (100 µg/mL) and incubated at different temperatures for 5 min, while an irrelevant (irr) Nb and two monoclonal antibodies (mAbs), i.e. CD3 mAb (CD3; ThermoFisher, USA) and CD105 mAb (CD105; ThermoFisher, USA), served as isotypic and positive controls. The protein concentration in the Nb and mAb dilution was measured using an anti-His-tag ELISA kit (R&D, USA), and an anti-IgG ELISA kit (Elabscienc, China), respectively, according to the manufacturers’ instructions.

The stability and binding property of CD105/CD3 Nb-LipoICG were evaluated based on the specific affinity to different cell types. CD105/CD3 Nb-LipoICG resuspended in PBS was left untreated or pretreated with CD3 blocking or NIR exposure (808 nm laser for 5 min), and stained with PE-conjugated anti-His tag antibody (ThermoFisher, USA). All Lipo samples were incubated with T cells, TDVECs or Bel7404/Huh7 tumor cells (50 nM per 106 cells) on ice for 30 min. The fluorescent signals of PE on these cells were detected by flow cytometry (BDAriaIII, USA), and the data were analyzed using BD FACS Diva software. The colloidal stability of CD105/CD3 Nb-LipoICG at different media (PBS/CM), temperatures (4 oC / 25 oC / 37 oC), and storage durations was also determined by measuring the alterations of basic characteristics including the particle size, zeta potential and the polydispersity using a Zetasizer Nano ZS. In addition, the ICG leakage was analyzed using a UV-vis spectrophotometer.

T cell activity and target cell killing assays

Sorted T cells were pre-labeled with 5 mmol/L Carboxyfluorescein succinimidyl amino ester (CFSE; Sigma Aldrich, USA). Bel7404 tumor cells were pre-incubated in a medium containing 25 µg/mL of mitomycin C (Gibco, USA) to limit proliferation. The CFSE-pre-labeled T cells and tumor cells were mixed at an effector target ratio (E: T) of 1:1, then co-cultured at 37 oC and 5% CO2 for 3 days in media containing CD105/CD3 Nb-LipoICG (the concentration of ICG was 20 µg/mL). An 808 nm laser irradiation (2 W/cm2, 5 min) was applied to investigate the synergy of photothermal effect upon liposomes. T cells were incubated with PE-conjugated anti-CD25 and anti-CD69 mAbs (BD, USA). The CFSE-fluorescence intensity and the expression of CD25 and CD69 markers were then analyzed by flow cytometry to determine the level of T cell proliferation and activation, respectively. The co-culture supernatant was collected for the measurement of cytokines including IFN-γ, TNF-α and IL-2 using commercial ELISA kits (LiankeBio, China), according to the manufacturer’s instruction.

To assess the direct killing effect against target cells, the target cells (Bel7404, TDVECs, or Huh7) were pre-incubated with CD105/CD3 Nb-LipoICG (ICG, 20 µg/mL) for 10 min, and then co-cultured with human T cells for 6 h at the E: T ratios of 1:1, 5:1 and 10:1. The target cells were irradiated by NIR (2 W/cm2) for 5 min before co-culture with T cells to assess the synergic effect of NIR upon CD105/CD3 Nb-LipoICG in vitro. The target cell killing efficacy was determined by measurement of apoptosis/necrosis and mitochondrial membrane potential change after treatment, using the Annexin V-APC/7-AAD Apoptosis Detection Kit (Elabscience, China) and the Mitochondrial Membrane Potential (MMP) Assay Kit with JC-1 (Solarbio, China), followed by flow cytometry analysis.

Cellular migration and tubule formation assay

The effect of CD105/CD3 Nb-LipoICG on TDVEC migration was determined by a transwell migration assay. Bel7404 tumor cells were resuspended in CM containing 10% FBS, and seeded in a 24-well plate for 6 h before the assay. An equal number of TDVECs was pre-incubated with CD105/CD3 Nb-LipoICG (ICG, 20 µg/mL) in CM containing 1% FBS. After 2 h incubation, TDVECs were mixed with T cells at the equivalent number ratio and transferred into a Transwell® filter insert (8 μm pores; Millipore, USA), and co-cultured with TDVECs at 37 oC and 5% CO2 for 12 h with NIR irradiation (2 W/cm2, 5 min). The filter insert was removed from the transwell, and the bottom side of the insert membrane was washed and stained with 0.1% crystal violet for 20 min. Cell migration was observed and determined in a microscope by counting the number of violet-staining cell spots that migrated across the transwell membrane to the bottom.

The in vitro effect of CD105/CD3 Nb-LipoICG on angiogenesis was assessed by tubule formation assay. TDVECs were incubated in a medium containing 20 µg/mL of CD105/CD3 Nb-LipoICG with NIR irradiation at different powers for 5 min. The treated TDVECs cells were then transferred into the individual well of an angiogenic slide pre-coated with Matrigel® matrix (Corning, USA) at a density of 104 cells per well and then cultured at 37 oC and 5% CO2 for another 6 h. After washing, 5 µM Calcein-AM fluorescent dye (20 µL per well) was added to stain the cells for 30 min. Tubule formation was observed and photographed in a fluorescence microscope. The length and area of the TDVEC tubular structures were quantified, and calculated using the ImagePro Plus 6.0 software and statistically analyzed Table 1.

Xenograft tumor animal modeling and in vivo imaging

Female NOD/SCID mice were subcutaneously injected with Bel7404, MCF-7, or C8161 tumor cells (2 × 106 cells per mouse) on the right flank. The survival and body weight of mice were monitored every 2 days. The tumor volume was calculated using the following formula: tumor volume = length×width2/2. When the subcutaneous tumor volume reached about 80–100 mm3, the tumor-bearing mice were used for in vivo imaging or therapy. Nine tumor-bearing mice were randomized into three groups (n = 3) and injected with the equivalent concentration (ICG, 5 mg/kg) of LipoICG, CD105/CD3 Nb-LipoICG, or CD105/CD3 Nb-LipoICG with CD105 blockade, through the caudal vein. Mice were anesthetized and photographed in a small animal optical imaging system (Princeton, PIXIS 1024, USA) at 0–72 h to monitor the in vivo distribution of LipoICG or CD105/CD3 Nb-LipoICG. For further understanding of the biodistribution within specific organs, another set of tumor-bearing mice (n = 3) received the same treatments and were euthanized at 12 h. The subcutaneous tumor, heart, liver, spleen, lung and kidney were dissected and photographed ex vivo.

In vivo photothermal effect of CD105/CD3 Nb-LipoICG was assessed by thermal imaging. Mice (n = 3) received similar treatments as above and were anesthetized at 12 h after injection. The tumor site was irradiated with 808 nm NIR at different powers for 5 min. The tumor regional maximum temperature and infrared thermograph were obtained by the infrared thermal imaging camera (FLIR, China).

In vivo treatment and tumor analysis

In vivo therapeutic efficacy of CD105/CD3 Nb-LipoICG was evaluated for various types of xenograft tumors in mice, which were successfully modeled. These tumor-bearing mice received treatment of T cells and/or CD105/CD3 Nb-LipoICG via the caudal vein and NIR irradiation. Five mice from each group were euthanized at 28 d post-treatment for tumor analysis, while the other mice remained to monitor their survival. The whole blood, subcutaneous tumors, and main organs (including heart, liver, spleen, lung and kidney) were freshly sampled from the executed animals for further analyses of serum biochemistry/cytokine profile, histology and immunohistochemistry, respectively.

A fresh tumor sample was homogenized, pieced, and digested with collagenase II (2 mg/mL in 3 times the volume of PBS). The digestion was stopped by adding an equal volume of FBS, and the precipitate and the supernatant of the homogenates were separated. To analyze the intratumoral blood vessel and T cell infiltration, the digestive juice was passed through a 200-µm metal mesh to obtain the single-cell suspension of tumors. Fixable Viability Dye (ThermoFisher, USA) and PE-conjugated anti-mouse (m) CD31 mAb (ThermoFisher, USA) were used for tumor blood vessel staining, and FITC-conjugated anti-human (h) CD3, APC-conjugated anti-hCD4 and PerCP-Cy5.5-conjugated anti-hCD8 mAb for T cell subgroup staining. The percentage of living mCD31+ cells, hCD4+ T cells and hCD8+ T cells was determined by flow cytometry FACS data to evaluate the intratumoral mouse-original blood vessel density and CD4+/CD8+ T cells of the transferred human T cells. The supernatant of the tumor homogenate was used to measure the intratumoral cytokine profile. The levels of mIL-6, hIL-2 and hIFN-γ and hTNF-α were detected by commercial ELISA kits (LiankeBio, China), according to the manufacturer’s instruction.

Tumor tissues fixed with 4% paraformaldehyde (PFA; Solarbio, Beijing, China) were then paraffin-embedded for histology and immunohistochemistry staining. The ultra-thin sections were stained with eosin-hematoxylin dye to observe histological changes and cellular morphology in the tumor tissues. The number of necrotic or apoptotic cells in each treatment group was counted, using the normal cell number of the untreated control as a reference. The tumor inhibition rate was calculated as the percentage of necrotic or apoptotic cells in total cells per field. For apoptosis assay and proliferation assay, immunohistology staining was conducted using a TUNEL assay kit (Roche, Switzerland) and an anti-Ki67 antibody (Maixin Biotech, China), respectively.

Toxicity tests

Cytotoxicity of CD105/CD3 Nb-LipoICG was evaluated in HUVEC cells. CD105/CD3 Nb-LipoICG was diluted in RPMI 1640 complete medium at 6 gradient dilutions (calculated based on the ICG concentration from 2.5, 5, 10, 20, 40, to 80 µg/mL), and incubated with HUVECs at 37 oC and 5% CO2 for 24–48 h, respectively. The cell viability was determined using a CCK-8 assay kit (DOJINDO, Japan).

In vivo toxicity was assessed based on serum biochemistry and histology. BALB/c mice (complete immunity) were injected with CD105/CD3 Nb-LipoICG (ICG, 5 mg/kg) in PBS or an equal volume of PBS vehicle (n = 5) via the caudal vein, once every 7 days for 4 times. After 28 d post-injection, these mice were euthanized. Serum was separated from the coagulated blood of mice for the measurement of major biochemical indices, including aspartate aminotransferase (AST), globulin (GLB), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and albumin (ALB), using a biochemical analyzer (HITACHI, Japan). For histology, the heart, liver, spleen, lung and kidney of each mouse were sampled, PFA-fixed, paraffin-embedded, and ultra-thin-sectioned, followed by hematoxylin and eosin (H&E) staining for the histology observation.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Prism Software Inc., USA). Data in each figure represent the mean ± standard deviation (S.D.) collected in at least three independent experiments. Differences between the two groups were compared using the two-tailed Student’s t post-hoc test and One-way analysis of variance (ANOVA). For the survival analysis, the survival rate of each group was calculated by the Kaplan-Meier method, and the differences between the groups were calculated using a weighted log-rank test. p < 0.05 was considered statistically significant in the difference.

Results

Characteristics of CD105/CD3 Nb-LipoICG

SDS-PAGE and western blot assay validated the purity and molecular weight (∼ 17 kDa) of CD3 and CD105 Nbs (Fig. S1). CD105/CD3 Nb-LipoICG was constructed by post-insertion of CD3 and CD105 Nbs on LipoICG, and the size and morphology of these liposomes were visualized in TEM images (Fig. 2A). CD105/CD3 Nb-LipoICG had a round-shape morphology and appeared more stereoscopic and hierarchical than Lipo and LipoICG. CD105/CD3 Nb-LipoICG possessed a uniform particle size distribution with an average size of ∼ 116 nm, polydispersity index (PDI) of ∼ 0.204, and zeta potential of ∼-36 mV, suggesting good dispersion (Fig. 2B). The successful encapsulation of ICG and the conjugation between Nbs and LipoICG was confirmed based on the fluorescence co-localization of the FITC-labeled Nbs or encapsulated ICG with the Dil-stained phospholipid membrane of Lipo (Fig. 2C and D).

The encapsulation rate of ICG in CD105/CD3 Nb-LipoICG was calculated to be 95% (Fig. S2A and S2B) when the mass ratio of ICG: Lipo (MICG: MLipo) was 1:60. The binding efficiency between CD105/CD3 Nb-LipoICG and the target cell is dependent on the Nb density on the liposome surface, and the optimal cell binding capability is achieved when one 100 nm-liposome has about 60 Nb molecules on the surface, as previously reported [30, 31]. Based on comprehensive analyses of the DSPC concentration (Stewart method), the Nb concentration (BCA method), and the particle size (Zetasizer Nano ZS), we estimated that the coupling efficiency between Nbs and Lipo was both ∼ 65% in CD105/CD3 Nb-LipoICG. When the Nb: DSPC mass ratio was 1:40, each CD105/CD3 Nb-LipoICG particle had ∼ 68 Nb molecules on the surface, very close to the optimal density (Supplementary Table S1). The particle size, potential, PDI value, and the ICG leakage rate of CD105/CD3 Nb-LipoICG in PBS solution at 4 oC (within 30 d) (Fig. 2E-H) and serum-containing medium solution at 37 oC (up to 72 h) (Fig. S3A-D) had no significant alteration, suggesting good colloidal and structural stability during storage and applications in vitro and in vivo.

CD105/CD3 Nb-LipoICG had also good photothermal and fluorescent properties. As shown in Fig. 2I and J, the temperature of CD105/CD3 Nb-LipoICG (ICG, 20 µg/mL) was 40 ∼ 45 oC upon NIR irradiation at 2 W/cm2 for 100 ∼ 300 s, which is optimal for PTT according to the literature [32]. Fluorescence detection by the Odyssey® DLx imaging system revealed that 20–50 µg/mL of ICG in CD105/CD3 Nb-LipoICG achieved a higher intensity of ICG-excited fluorescence (Fig. 2K).

Fig. 2 Characteristics of CD105/CD3 Nb-LipoICG. A The TEM images of nanoliposomes. Scale bars are shown. B The size distribution, zeta potential and PDI of nanoliposomes dissolved in PBS. C and D The laser confocal microscopy images of CD105/CD3 Nb-LipoICG. (ICG: excited at 808 nm, Dil-labeled lipid: excited at 633 nm, FITC-labeled Nb, excited at 488 nm). Scale bars are shown. E-H The stability of CD105/CD3 Nb-LipoICG in different storage conditions (including 4 oC and 25 oC in PBS) were detected by Zetasizer Nano ZS. Variation of particle size (E), zeta potential (F), PDI value (G) and leakage rate (H) were detected and analyzed. I The corresponding temperature variation curve of CD105/CD3 Nb-LipoICG at the laser power density of 2 W/cm2. J Corresponding temperature variation curve of CD105/CD3 Nb-LipoICG at the ICG concentration of 20 µg/mL. K The fluorescent property of nanoliposomes was recorded by Odyssey CLx Imager Dual-color Infrared Imaging at gradient dilutions in PBS solution (ranging from 0.75 µg/mL to 96 µg/mL)

CD105/CD3 Nb-LipoICG simultaneously binds T cell and CD105-expressing cell with high specificity

The highly specific binding of CD3 and CD105 Nbs to recombinant human CD3 and CD105 proteins is similar to that of their counterparts CD3 and CD105 mAbs, as confirmed by ELISA data (Fig. 3A and B). When the pretreatment temperature of Nbs increased to 50 ℃ for 5 min, the specific binding rate of CD3 and CD105 Nbs was only slightly reduced (Fig. 3C and D). In contrast to CD3 and CD105 Nbs, the binding ability of CD3 and CD105 mAbs to CD3 and CD105 proteins significantly decreased at the same heating condition. The high-temperature tolerance of Nbs was demonstrated to surpass that of mAb.

The binding specificity of CD105/CD3 Nb-LipoICG to different cell types was examined by flow cytometry. The high purity and viability of CD3-expressing T cells were sorted from human PBMCs (Fig. S4) and the binding rate of CD105/CD3 Nb-LipoICG to the sorted T cells was 60-70%. The addition of recombinant CD3 protein resulted in a significant reduction in the binding rate, thereby demonstrating the specific targeting of CD105/CD3 Nb-LipoICG towards CD3 (Fig. 3E). CD105/CD3 Nb-LipoICG was also found to bind specifically to the cells with high expression of CD105 antigen, such as Bel7404 cells (Fig. 3F) and TDVECs (Fig. 3G), reaching over 80% and 90% positive population, respectively. Similarly, adding recombinant CD105 protein also led to a significant reduction of the binding rate of CD105/CD3 Nb-LipoICG to either Bel7404 cells or TDVECs. In contrast, this Lipo is hardly bound to Huh7 cells (Fig. 3H), which express a much lower level of CD105 (Fig. S5). Similarly, adding recombinant CD105 protein also led to a significant reduction of the binding rate of CD105/CD3 Nb-LipoICG to either Bel7404 cells or TDVECs. Intriguingly, the binding rate of CD105/CD3 Nb-LipoICG to the tested cells remained unaffected after 5 min of NIR irradiation, indicating excellent thermostability of this novel liposome complex.

Fig. 3 The binding specificity and thermostability of CD105/CD3 Nb-LipoICG. A-B The binding specificities of CD3 Nb and CD105 Nb at variable concentrations were measured by ELISA. C-D The thermostability of Nbs was analyzed by ELISA after treatment with variable temperature. E-H The binding property of CD105/CD3 Nb-LipoICG to T cell (E), Bel7404 (F), TDVECs (G) or Huh7(H) was analyzed by flow cytometry. Representative results of data from three independent experiments are shown. Data are presented as mean ± SD of three replicates of corresponding independent samples. Ns, not significant, * P < 0.05, *** P < 0.001, **** P < 0.0001

CD105/CD3 Nb-LipoICG promotes proliferation, activation and cytokines secretion of T cells

To evaluate the impact of CD105/CD3 Nb-LipoICG on T cell proliferation, CFSE-labeled T cells were co-cultured with Bel7404 cells in a medium supplemented with CD105/CD3 Nb-LipoICG. Flow cytometry data showed that the proliferation rate of T cells upon Bel7404 co-incubation with CD105/CD3 Nb-LipoICG was about 65%, which is significantly higher than other controls. The proliferation rate was further increased to about 84% after NIR exposure at 2 W/cm2 for 5 min (Fig. 4A). Upon co-stimulation of Bel7404 and CD105/CD3 Nb-LipoICG, the expression of lymphocytic activation markers CD25 and CD69 on the T cell surface were significantly elevated compared to the control groups. The application of NIR further promoted the CD25+ and CD69+ levels to ∼ 45% and ∼ 39% on T cells treated with CD105/CD3 Nb-LipoICG (Fig. 4B). Furthermore, the concentrations of hIFN-γ, hTNF-α and hIL-2 in the culture supernatant of co-incubated Bel7407 and T cells in the presence of CD105/CD3 Nb-LipoICG exhibited significantly elevated levels compared to the control groups. Moreover, upon the NIR irradiation, there was a further augmentation in the levels of all these cytokines (Fig. 4C). These findings suggest that CD105/CD3 Nb-LipoICG facilitates the proliferation and activation of T cells co-culture with CD105+ tumor cells, exhibiting further enhancement upon NIR laser irradiation.

Fig. 4 CD105/CD3 Nb-LipoICG promotes antitumor activities of T cells in vitro. A Proliferation of T cells was evaluated by measuring the fluorescent signal of CFSE using flow cytometry. B Activation of T cells was analyzed by determination of the expression of CD25 and CD69 markers using flow cytometry. One representative experiment out of three independent experiments is shown. C The levels of IFN-γ, TNF-α and IL-2 in the co-culture supernatant of the T cells and Bel7404 cells in the presence of CD105/CD3 Nb-LipoICG with or without NIR laser-induced PTT. Data are presented as mean ± SD of three replicates of corresponding independent samples. * P < 0.05, *** P < 0.001, **** P < 0.0001

CD105/CD3 Nb-LipoICG enhances the cytotoxicity of T cell against CD105+ tumor cells

To assess the efficacy of CD105/CD3 Nb-LipoICG in enhancing T cell cytotoxicity against CD105+ tumor cells, two types of tumor cells (Bel7404 and Huh7) were investigated at various effector-to-target (E: T) ratios with NIR irradiation. Notably, the cytotoxicity of CD105/CD3 Nb-LipoICG mediated T cells against Bel7404 cells was significantly enhanced at the E: T ratio of 10:1 (Fig. 5A). Additionally, CD105/CD3 Nb-LipoICG marginally augmented killing efficiency of Huh7 cells at the same E: T ratio (Fig. 5B). The efficiency, however, is significantly lower against Huh7 cells compared to Bel7404 cells due to the limited surface expression of CD105 on Huh7 cells. (Fig. 5S), indicating a positive correlation between the CD105 expression level of the tumor cell and the cytotoxicity of T cells mediated by CD105/CD3 Nb-LipoICG. Both tumor cell lines showed clear increasing tendencies when the E: T ratio increased from 1:1 to 10:1. Moreover, NIR laser irradiation significantly increased the killing efficiency of T cells (Fig. 5A and B).

The MMP variation further provided evidence for the cytotoxicity of T cells against tumor cells, which is mediated by CD105/CD3 Nb-LipoICG. CD105/CD3 Nb-LipoICG plus NIR laser exposure significantly declined MMP (JC-1 aggregate) of CD105+ target cells (Bel7404) in the presence of T cells. The MMP changes observed in the Huh7 cells exhibited an almost identical pattern, albeit with overall lower values compared those of Bel7404 (Fig. 5C). These data again support our hypothesis that the killing efficacy of T cells mediated by CD105/CD3 Nb-LipoICG is proportionally dependent on the CD105 level on the tumor cell surface and can be further enhanced by the NIR application.

Fig. 5 CD105/CD3 Nb-LipoICG significantly enhances the CD105+ cell lysis mediated by T cells in vitro. A-B Flow cytometric analysis for the killing effect of T cells against Bel7404 (A) or Huh7 (B) mediated by CD105/CD3 Nb-LipoICG or the control materials at the E: T ratio of 10:1, with or without NIR (2 W/cm2, 5 min). C Flow cytometric analysis for mitochondrial membrane potential (MMP) declines of Bel7404 and Huh7 target cells. Data are presented as mean ± SD of three replicates of corresponding independent samples. ** P < 0.01, *** P < 0.001, **** P < 0.0001

CD105/CD3 Nb-LipoICG enhances the functional inhibition and apoptosis of TDVECs

Similarly, the co-administration of CD105/CD3 Nb-LipoICG significantly augmented T cell-mediated cytotoxicity against TDVECs, and the additional NIR irradiation further potentiated the killing efficacy (Fig. 6A). Such a combination treatment induced a much higher MMP decline in TDVECs in the presence of T cells (Fig. 6B). These data also indicated that the CD105/CD3 Nb-LipoICG together with NIR laser irradiation increased the toxicity of T cells against TDVECs.

We further elucidated the T cell-mediated suppression of TDVEC tubulogenesis and migration, which would provide evidence for the inhibition of tumor angiogenesis in vivo. The data of tubule formation (Fig. 6C) revealed that CD105/CD3 Nb-LipoICG significantly reduced formation of the tubule structure of TDVECs. The relative vessel area and length both significantly decreased along with the increase of laser irradiation power, suggesting a positive correlation between the NIR intensity and the damage of vascularization. The transwell migration assay demonstrated that the inhibitory effect mediated by CD105/CD3 Nb-LipoICG was improved and further enhanced by NIR laser irradiation. The addition of CD105 protein as a blocker of CD105/CD3 Nb-LipoICG partly affected the migration of TDVECs, indicating CD105/CD3 Nb-LipoICG targeted both T cells and vascular endothelial cells (Fig. 6D). Collectively, CD105/CD3 Nb-LipoICG increased the suppressive capacity of T cells towards CD105-associated tumor vascularization, which was enhanced by NIR laser irradiation.

Fig. 6 CD105/CD3 Nb-LipoICG significantly induces cytotoxicity of T cells on TDVECs and inhibits the tubulogenesis and migration of TDVECs in vitro. A Flow cytometric analysis for the killing effect of T cells against TDVECs mediated by CD105/CD3 Nb-LipoICG or the control materials at the E: T ratio of 10:1, with or without NIR (2 W/cm2, 5 min). B Flow cytometric analysis for MMP decline of TDVECs. C Representative microscopic photograph of the inhibitory tubule formation induced of TDVECs by T cells and CD105/CD3 Nb-LipoICG or the indicated controls in a combination of NIR with different laser power density for 5 min. Statistical results of relative tube area and relative tube length in each group. D Representative microscopic photograph of the inhibition of TDVECs migration after the co-culture of T cells and CD105/CD3 Nb-LipoICG or the indicated controls in transwell devices, with or without NIR irradiation at the same condition (2 W/cm2, 5 min). Statistical results of the migration TDVECs number in each group. Data are presented as mean ± SD of three replicates of corresponding independent samples. ** P < 0.01, *** P < 0.001, **** P < 0.0001

CD105/CD3 Nb-LipoICG has strong targetability and photothermal effect in vivo

In vivo tumor targetability of CD105/CD3 Nb-LipoICG was evaluated by dynamic comparison of ICG fluorescence intensity in Bel7404 xenograft tumor tissues, which were inoculated in NOD/SCID mice, using a small animal in vivo imager at various time points after injection. Figure 7A shows the time-dependent accumulation of CD105/CD3 Nb-LipoICG and the controls in the tumor tissue. In specific, the CD105/CD3 Nb-LipoICG fluorescence intensity reached the highest at 12 h and was still strong even at 72 h (the test endpoint). In contrast, the fluorescence intensity of ICG or LipoICG and CD105-blocking group in the tumor tissue decreased rapidly after 12 h, especially for ICG group.

In another set of experiments, were similarly with CD105/CD3 Nb-LipoICG was injected into Bel7404 tumor-bearing mice, followed by euthanization at 12 h to collect major organs for assessing in vivo distribution and tissue retention in vitro. As shown in Fig. 7B and C, the fluorescence intensity of CD105/CD3 Nb-LipoICG treated group was the highest in tumor tissues among all treatments, while the fluorescence intensity of ICG, LipoICG or CD105-blocking CD105/CD3 Nb-LipoICG treated groups was relatively low in tumor tissues but higher in liver, lung and kidney. Compared to free ICG, the accumulation of LipoICG was much higher in tumor tissue but lower in the liver, further evidencing the tumor targetability of CD105/CD3 Nb-LipoICG together with the enhanced permeation and retention (EPR) effect.

The optimal NIR power was investigated by employing an NFI camera for continuous in vivo monitoring of the temperature surrounding tumor sites during laser irradiation. After 12 h post intravenous injection, the temperature in the tumor region was exhibited a gradual increase with prolonged irradiation time, reaching a plateau after approximately 120 s (Fig. 7D and E). The tumor surface temperature in CD105/CD3 Nb-LipoICG-treated mice reached 43 oC ∼ 47 oC upon the laser irradiation at an intensity of 1.0 W/cm2 for 2–5 min. In contrast, the tumor temperature of mice injected with PBS remained unchanged, further demonstrating the excellent tumor site-specific photothermal effect of CD105/CD3 Nb-LipoICG.

Fig. 7 CD105/CD3 Nb-LipoICG exhibits robust retention, targetability and photothermal effect in NOD/SCID mice bearing Bel7404 tumors. A Tumoral fluorescence intensity measured at 0, 6, 12, 24, 48, and 72 h post-injection. Data are shown as the mean ± SD (n = 3). B The subcutaneous tumor and major organs were dissected and imaged 12 h post-injection of the indicated materials. C Statistical results of the fluorescence intensity of subcutaneous tumor and major organs in each group. D Temperature variation curves of the tumor sites in Bel7404-bearing mice after caudal veins injection with PBS or CD105/CD3 Nb-LipoICG, followed by NIR laser irradiation. E Data are presented as mean ± SD of three replicates of corresponding independent samples. *** P < 0.001, **** P < 0.0001

CD105/CD3 Nb-LipoICG enhances the antitumor effect of T cells in xenograft tumor-bearing mice

The in vivo antitumor effect of CD105/CD3 Nb-LipoICG was then evaluated in three well-established xenograft tumor models using NOD/SCID mice, which were derived by Bel7404, MCF-7 and C8161 tumor cell lines. CD105/CD3 Nb-LipoICG was given together with human T cells to treat the tumor, with direct NIR laser irradiation over the tumor nodules (therapeutic scheme shown in Fig. 8A). In the Bel7404 tumor model, the treatment of CD105/CD3 Nb-LipoICG with either T cells or NIR significantly inhibited tumor growth and extended the survival in comparison with PBS control (Fig. 8B, C and D), indicating a high antitumor effect of CD105/CD3 Nb-LipoICG in the Bel7404 tumor-bearing mice in combination with either T cells or PTT. Three-modular combination, i.e. CD105/CD3 Nb-LipoICG, T cells and NIR laser irradiation, almost completely inhibited tumor growth (Fig. 8B and C) and significantly prolonged the survival of mice (Fig. 8D), suggesting a clear synergy among NIR, T cells and CD105/CD3 Nb-LipoICG. Similar antitumor effects were also observed in both MCF-7 and C8161-derived tumor models (Fig. S6). These data demonstrate that CD105/CD3 Nb-LipoICG exhibits targeted PTT efficacy and enhances antitumor activity of T cells against multiple solid tumor types.

It’s noteworthy that the fluorescence characteristic of ICG in CD105/CD3 Nb-LipoICG enables its application in vivo for fluorescence imaging, facilitating the monitoring of tusor growth kinetics during treatment. As demonstrated in Fig. 8E, during the experiment in mice that treated exclusively with CD105/CD3 Nb-LipoICG, the fluorescence intensity of tumor, which indicates the tumor size, exhibited a consistent increase. In contrast, the mice treated with CD105/CD3 Nb-LipoICG with T cells or with NIR treatment, either alone or in combination, demonstrated a significant reduction in tumor fluorescence intensity, which corresponded to a consistent tumor volume during the entire test time. This implied that a potent synergistic photothermal effect and anti-tumor immune response were generated over the tumor growth by the co-treatment of NIR radiation and T cells. Such real-time in vivo fluorescence imaging provides an option for the integrating tumor diagnosis and therapy.

Fig. 8 CD105/CD3 Nb-LipoICG enhanced the inhibition of xenograft tumor in mice. A Schematic diagram of the design for the tumor modeling and therapeutic trial in NOD/SCID mice. Mice were randomized into four groups (5 mice per group) and gave 100 µL injection per mouse of the following treatments: PBS (untreated control), CD105/CD3 Nb-LipoICG (ICG, 5 mg/kg) + T cells (1 × 107), CD105/CD3 Nb-LipoICG (ICG, 5 mg/kg ) + NIR (irradiation with NIR laser at 1.0 W/cm2 for 5 min after 12 h post-injection), and CD105/CD3 Nb-LipoICG (ICG, 5 mg/kg) + T cells (1 × 107) + NIR (irradiation with NIR laser at 1.0 W/cm2 for 5 min after 12 h post-injection). B Photographs show the individual tumors separated from the mice at 28 d after different treatments. C The volumes of Bel7404 xenograft tumors in NON/SCID mice were periodically measured, and the growth curves of the tumors were drawn. D The survival of Bel7404 tumor-bearing mice was analyzed using the Kaplan-Meier method. Data are expressed as the mean percentage of each group of mice that survived throughout the period. Data are presented as mean ± SD. E Tumoral fluorescence intensity of mice after injection of CD105/CD3 Nb-LipoICG was dynamically monitored by a small animal optical imaging system. Data are presented as mean ± SD of five replicates of corresponding independent samples. *** P < 0.001, **** P < 0.0001

CD105/CD3 Nb-LipoICG promotes tumor cell apoptosis, suppresses angiogenesis, and increases infiltration and cytokines secretion of T cells in the tumor tissues

Immunochemistry data also showed that CD105/CD3 Nb-LipoICG caused extensive tumor tissue necrosis and apoptosis-like lesions around the necrotic area, including varying degrees of fibrous hyperplasia and nuclear pyknosis upon NIR laser irradiation (Fig. 9A). In the CD105/CD3 Nb-LipoICG + NIR + T cell group, the number of apoptotic cells was significantly higher than that in the other control groups (Fig. 9B), while the number of proliferating cells was significantly lower (Fig. 9C). Therefore, CD105/CD3 Nb-LipoICG and targeted PTT effect together mediated T cells to promote cellular apoptosis/necrosis and inhibited cell proliferation in the tumor, thus preventing tumor growth. Furthermore, the proportion of mCD31+ cells in the tumor tissue was significantly fewer in CD105/CD3 Nb-LipoICG + PTT + T cell treatment group than any other controls (Fig. 9D). These data suggest that the reduction of tumor neovascularization is caused by an inhibitory mechanism on the growth of tumor vascular endothelial cells through a synergy of immunotherapeutic T cells and photothermal effect of CD105/CD3 Nb-LipoICG.

As shown in Fig. 9E, CD105/CD3 Nb-LipoICG treatment brought about a higher density of T cells (CD3+) in tumor tissues, and the proportion of CD8+ T cells as a major cytotoxic T cell subset was also significantly greater, suggesting that CD105/CD3 Nb-LipoICG enhanced the recruitment of tumor-reactive T cells in vivo. As expected, NIR laser irradiation and CD105/CD3 Nb-LipoICG further increased the infiltration of T cells and the proportion of CD8+ T cells intratumorally. By measuring the cytokines in the tumor homogenates of tumor-bearing mice, the administration of CD105/CD3 Nb-LipoICG and human T cells with NIR irradiation significantly increased the intratumoral levels of hIFN-γ, hTNF-α, hIL-2 and mIL6 (Fig. 9F-I). Seemingly, the cytokine secretion is consistent with that of target cell apoptosis, so these two events could be directly related to each other. Taken together, CD105/CD3 Nb-LipoICG recruited more T cells into the tumor tissue and enhanced the antitumoral cytokine production, while suppressing tumor angiogenesis to inhibit tumor growth.

Fig. 9 CD105/CD3 Nb-LipoICG enhances the in vivo antitumor effect by promoting the apoptosis of tumor cells and the intratumoral T cell infiltration and activity but suppressing tumor angiogenesis. A-C The intratumoral cell apoptosis and proliferation were assessed by histology and immunohistochemistry (IHC). HE-staining (A) and TUNEL-staining (B) sections showed the apoptotic/necrotic profile, while an anti-Ki67 antibody (C) was used to stain proliferate cells intratumorally. Representative tissue histology and IHC are shown. The ratio of apoptotic/necrotic cells versus alive cells (inhibition rate), TUNEL-positive cells, and Ki67-positive cells in each field were respectively determined by computerized image processing. The numbers of apoptotic cells, proliferate cells, and alive cells from five separate microscopic fields were recorded, respectively. Statistics are presented as mean ± SD. Scale bars are shown. D-G The fresh tumor tissue was sampled and collagenase-digested into single cells. The cell pellets were stained with anti-mCD31 mAb, anti-hCD3 mAb, anti-hCD4 mAbs, and anti-hCD8 mAbs for flow cytometry, to analyze the intratumoral vessel density and T cell infiltration, respectively. Representative flow cytometry analysis is shown. F-I The supernatant of tumor homogenate was used to detect the intratumoral mIL-6 (F), hIL-2 (G), hIFN-γ (H), and hTNF-α (I) by ELISA. Data are presented as mean ± SD of five replicates of corresponding independent samples. The quantitative analysis was used by one-way ANOVA with multiple comparisons test. Ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

CD105/CD3 Nb-LipoICG has no significant cytotoxicity and histotoxicity

To ensure the safety of CD105/CD3 Nb-LipoICG as a therapeutic agent for tumor treatment, toxicity to normal cells and mouse organs was assessed. The CCK-8 assay demonstrated that the proportion of living HUVECs was close to 100% after incubation with CD105/CD3 Nb-LipoICG in a series of dilutions ranging from 5 to 80 µg/mL for 24 and 48 h, suggesting that this new agent was not toxic to normal human cells (Fig. S7A). Histology of the organ tissues (heart, liver, spleen, lung and kidney) showed that CD105/CD3 Nb-LipoICG barely caused visible inflammatory cell infiltration or pathological feature in the treated mice, compared with that treated with PBS vehicle (Fig. S7B). A series of serum biochemical indices that reflect liver and kidney biofunctions were also detected. The data showed no significant difference in sera between treatments of CD105/CD3 Nb-LipoICG and PBS control from either tumor-free BALB/c mice (Fig. S7C). In combination with the above data, we demonstrate that CD105/CD3 Nb-LipoICG has no cytotoxicity and histoxicity, thereby establishing its potential as a safe therapeutic agent for antitumor treatment.

Discussion

Concerning the failure of effector T cells when engaging the suppressive TME of solid tumors, PTT-mediated immunotherapies offer the benefits, which not only ablating the tumor to allow T cells to penetrate the dense tumor tissue but also remodeling the tumor milieu to activate local immune responses for more effective antitumor effect [33]. Taking advantage of the peculiarities of Nb, we propose a new Nb-MuTE platform, which more precisely targets the TME and engages the effectors (T cells), and further enhances antitumor effects through the NIR-induced photothermal effect. By targeting rich tumoral antigen CD105, this new platform can inhibit the growth of solid tumors by mediating cytotoxic T cells to infiltrate into TME and suppressing tumor neoangiogenesis [34, 35]. The present study developed and demonstrated CD105/CD3 Nb-LipoICG as a tumoral CD105-targeting Nb-MuTE, which bundles PTT with live imaging and immunotherapeutic effect of T cells and shows excellent tumor growth inhibition.

The outstanding characteristics of this Nb-MuTE new platform in recruiting tumor-reactive T cells and developing a photothermal effect are attributed to the modification of Nbs on LipoICG. Nbs as a new type of miniaturizing antibody overcomes many drawbacks of traditional antibody types, and provides greater advantages in targeting tumor antigens [7, 21]. To the best of our knowledge, most investigations on Nbs-based PTT focus on the targeted delivery of photosensitizer to the disease loci, while no nanoplatforms integrate immunoreactive Nbs and photothermal agents. Here, for the first time, we have developed an “all-in-one” and controllable strategy by integrating the target binding capabilities of specific Nbs and the NIR laser-induced PTT in a novel Lipo platform, as exemplified as CD105/CD3 Nb-LipoICG. Based on our data, the anti-tumor effect of CD105/CD3 Nb-LipoICG-mediated T cells was further improved, through synergy between the bispecific Nb-based T cell enhancement and ICG-mediated photothermal effect. Upon interaction with tumor cells, CD105/CD3 Nb-LipoICG generates a photothermal effect under laser irradiation, leading to tumor lysis and apoptosis. Consequently, this process triggers the release of tumor-associated antigens and enhances the immune-mediated anti-tumor responses [16]. In the present study, we also observed a significant induction of activation, proliferation, and secretion of inflammatory cytokines in T cells following photothermal treatment. The infiltration of T cells into the tumor tissue was significantly enhanced in tumor-bearing mice. This phenomenon can be attributed to the fact that targeted local heating not only induces degradation of the extracellular matrix within the tumor but also promotes vasodilation and enhances local blood flow, thereby facilitating expedited recruitment of T cells into the tumor microenvironment. Moreover, due to the remarkable thermostability of Nbs on Nb-MuTE, the immunocompetence of Nbs can be maintained even after NIR laser irradiation.

The CD105/CD3 Nb-LipoICG developed in this study had a uniform size, low leakage rate of ICG and excellent stability. The PEGylated liposome prolongs the tumoral retention of Nbs and ICG in Nb-MuTE. Owing to the extremely good biocompatibility and low immunogenicity, CD105/CD3 Nb-LipoICG caused almost no systemic toxicity in mice. To sum up, CD105/CD3 Nb-LipoICG would no doubt benefit the reduction of the therapeutic dosage and the incidence of adverse side effects. Taken together, our innovative concept centered on such a novel Nb-MuTE platform may break through the technical bottleneck of current tumor immunotherapies in the aspects of both efficacy and safety.

Notably, it would be an ideal strategy to realize the real-time monitoring of the development and prognosis of the disease in the clinical therapeutic process. The encapsulated ICG allows CD105/CD3 Nb-LipoICG to display fluorescence upon NIR laser excitation, therefore makes it not only a therapeutic agent but also a NIF-guided molecular imaging media during treatments, which makes the tumor growth can be simultaneously monitored without excessive and excessive invasive procedures, realizing a simpler and more convenient “all-in-one” operation [36, 37]. As we observed in this study, CD105/CD3 Nb-LipoICG was prone to specifically accumulate and retain at the tumor site, achieving both effective tumor treatment and real-time monitoring of tumor volume change. Both the therapeutic effect and fluorescence imaging potency of such Nb-MuTE allow the clinicians to keep abreast of the changes of treated tumors and to accordingly adjust, optimize or decide the drug dosage, duration or regimen in the next stage, so that the entire process could be better controlled by the doctors. This “all-in-control” procedure may be expected to improve the therapeutic outcome of tumor patients, especially for personalized treatment.

Besides, we also need to highlight that the Nb-MuTE platform was designed to be expandable and flexible. As an example of Nb-MuTE, the CD105/CD3 Nb-LipoICG was specifically designed to guide T cells toward the TME in the present study. Even so, we can still easily replace the CD105 Nb with any other Nb specific to some alternative target of the TME in else case. Similarly, CD3 Nb can also be changed to any other Nb against the maker of other immune cells, such as CD56 Nb to recruit NK and NKT cells [38]. The basic skeleton and admissibility of various nanomaterials (not limited to Lipo complexes) offer convenience to insert more than two Nbs to expand the targetability of this Nb-MuTE platform but without a significant increase in operation difficulty and cost. Using Nb-MuTE as a targeted carrier for delivering other radiotherapeutic and chemotherapeutic drugs may also help superimpose their advantages onto immunotherapy. Increasing knowledge and technologies over tumor neoantigens, new photothermal materials and new molecular imaging systems will certainly expand the applicability of this Nb-MuTE platform for monitorable targeted immunotherapies.

Conclusions

This is the first report of a new “all-in-one” and “all-in-control” theranostic system based on our newly designed Nb-MuTE platform, which successfully integrated Nbs and photothermal agent to play the dual role of immune and photothermal therapeutic effects for both targeted therapy and diagnosis of tumors. Gathering the advantages of Nbs and liposomal nanocarrier (as described above), this multifunctional theranostic approach represents an improvement over many current similar approaches in terms of solid tumor imaging and treatment. Our study lays a reliable foundation for developing Nbs-based multifunctional immunophotothermal therapy mode and has a particular value for inspiring similar research and applications in the future, showing potential translation of this kind of T cell immunotherapy for solid tumor treatment.

Electronic supplementary material

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Supplementary Material 1

Acknowledgements

Not applicable.

Author contributions

All authors contributed to the study’s conception and design. SX and XL conceived the study, designed, and supervised the experiments. SX, WS and SD together designed, prepared, and performed the major experiments, and data analysis. The first draft of the manuscript was written by SX, WS and SD. XH, AL and XH reviewed and made significant revisions to the manuscript. DZ, SS, and ZD revised the manuscript. XL, XC and XY provided direction and guidance throughout the preparation of this manuscript. All authors have read and discussed the manuscript and approved the submission for publication.

Funding

The work reported in this article was partly funded by the International (Regional) Cooperation and Exchange Program of National Natural Science Foundation of China (No. 82220108003), Guangxi Natural Science Foundation Youth Fund Project of China (No.2023GXNSFBA026178), Project of National Natural Science Foundation of China ( No. 82260614), NUS School of Medicine Nanomedicine Translational Research Programme (NUHSRO/2021/034/TRP/09/Nanomedicine), the Youth Science Foundation of Guangxi Medical University (No.GXMUYSF202325).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The study was approved by the Animal Ethics Committee of Guangxi Medical University (approval number 0014). All animal experiments were performed by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the guidelines of Animal Care and Use of Guangxi Medical University. Whole blood from the healthy registered donors were collected from the First Affiliated Hospital of Guangxi Medical University (Guangxi, China), and informed consent from participants.

Consent for publication

All authors agree to be published.

Competing interests

The authors declare no competing interests.

Abbreviations

Nb Nanobody

Nb-MuTE Nanobody-based Multifunctional T cell Engager

TME Tumor microenvironment

FBS Fetal Bovine Serum

mAb Monclone Antibody

PTT Photothermal Therapy

NIR Near-infrared

ICG Indocyanine Geen

IPTG Isopropyl-beta-D-Thiogalactopyranoside

CFSE Carboxyfluorescein Succinimidyl Amino Ester

VHH Variable Domain of Heavy Chain of Heavy-Chain Antibody

PDI Polymer Dispersity Index

PEG Polyethylene Glycol

IFN Interferon

TNF Tumor Necrosis Factor

ELISA Enzyme-linked Immunosorbent Assay

MMP Mitochondrial Membrane Potential

Publisher’s note

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Shenxia Xie, Wei Shi and Siliang Duan contributed equally to this work.
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