Bispecific CAR-T cells targeting FAP and GPC3 have the potential to treat hepatocellular carcinoma.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated robust efficacy against hematological malignancies, but there are still some challenges regarding treating solid tumors, including tumor heterogeneity, antigen escape, and an immunosuppressive microenvironment. Here, we found that SNU398, a hepatocellular carcinoma (HCC) cell line, exhibited high expression levels of fibroblast activation protein (FAP) and Glypican 3 (GPC3), which were negatively correlated with patient prognosis. The HepG2 HCC cell line highly expressed GPC3, while the SNU387 cell line exhibited high expression of FAP. Thus, we developed bispecific CAR-T cells to simultaneously target FAP and GPC3 to address tumor heterogeneity in HCC. The anti-FAP-GPC3 bispecific CAR-T cells could recognize and be activated by FAP or GPC3 expressed by tumor cells. Compared with anti-FAP CAR-T cells or anti-GPC3 CAR-T cells, bispecific CAR-T cells achieved more robust activity against tumor cells expressing FAP and GPC3 in vitro. The anti-FAP-GPC3 bispecific CAR-T cells also exhibited superior antitumor efficacy and significantly prolonged the survival of mice compared with single-target CAR-T cells in vivo. Overall, the use of anti-FAP-GPC3 bispecific CAR-T cells is a promising treatment approach to reduce tumor recurrence caused by tumor antigen heterogeneity.

T lymphocytes expressing the switchable chimeric Fc receptor CD64 exhibit augmented persistence and antitumor activity.

Chimeric antigen receptor (CAR) T cell therapy lacks persistent efficacy with "on-target, off-tumor" toxicities for treating solid tumors. Thus, an antibody-guided switchable CAR vector, the chimeric Fc receptor CD64 (CFR64), composed of a CD64 extracellular domain, is designed. T cells expressing CFR64 exert more robust cytotoxicity against cancer cells than CFR T cells with high-affinity CD16 variant (CD16v) or CD32A as their extracellular domains. CFR64 T cells also exhibit better long-term cytotoxicity and resistance to T cell exhaustion compared with conventional CAR T cells. With trastuzumab, the immunological synapse (IS) established by CFR64 is more stable with lower intensity induction of downstream signaling than anti-HER2 CAR T cells. Moreover, CFR64 T cells exhibit fused mitochondria in response to stimulation, while CARH2 T cells contain predominantly punctate mitochondria. These results show that CFR64 T cells may serve as a controllable engineered T cell therapy with prolonged persistence and long-term antitumor activity.

Nanoparticles Hitchhike on Monocytes for Glioblastoma Treatment after Low-Dose Radiotherapy.

Glioblastomas (GBMs) are aggressive primary brain tumors with fatal outcome. Traditional chemo-radiotherapy has poor therapeutic effect and significant side effects, due to the drug and radiotherapy (RT) resistance, natural blood-brain barrier, and high-dose RT damage. Even more, tumor-associated monocytes (macrophages and microglia, TAMs) constitute up to 30%-50% of the GBM cellular content, and the tumor microenvironment (TME) in GBM is extremely immunosuppressive. Here, we synthesized nanoparticles (D@MLL) that hitchhike on circulating monocytes to target intracranial GBMs with the assistance of low-dose RT. The chemical construction of D@MLL was DOX·HCl loaded MMP-2 peptide-liposome, which could target monocytes by the surface modified lipoteichoic acid. First, low-dose RT at the tumor site increases monocyte chemotaxis and induces M1 type polarization of TAMs. Subsequently, the intravenous injected D@MLL targets circulating monocytes and hitchhikes with them to the central site of the GBM area. DOX·HCl was then released by the MMP-2 response, inducing immunogenic cell death, releasing calreticulin and high-mobility group box 1. This further contributed to TAMs M1-type polarization, dendritic cell maturation, and T cell activation. This study demonstrates the therapeutic advantages of D@MLL delivered by endogenous monocytes to GBM sites after low-dose RT, and it provides a high-precision treatment for GBMs.

Deep-penetration functionalized cuttlefish ink nanoparticles for combating wound infections with synergetic photothermal-immunologic therapy.

The challenge of wound infections post-surgery and open trauma caused by multidrug-resistant bacteria poses a constant threat to clinical treatment. As a promising antimicrobial treatment, photothermal therapy can effectively resolve the problem of drug resistance in conventional antibiotic antimicrobial therapy. Here, we report a deep-penetration functionalized cuttlefish ink nanoparticle (CINP) for photothermal and immunological therapy of wound infections. CINP is decorated with zwitterionic polymer (ZP, namely sulfobetaine methacrylate-methacrylate copolymer) to form CINP@ZP nanoparticles. Natural CINP is found to not only exhibit photothermal destruction of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli), but also trigger macrophages-related innate immunity and enhance their antibacterial functions. The ZP coating on the surface of CINP enables nanoparticles to penetrate into deeply infected wound environment. In addition, CINP@ZP is further integrated into the thermosensitive Pluronic F127 gel (CINP@ZP-F127). After in situ spraying gel, CINP@ZP-F127 is also documented notable antibacterial effects in mice wound models infected with MRSA and E. coli. Collectively, this approach combining of photothermal therapy with immunotherapy can promote delivery efficiency of nanoparticles to the deep foci of infective wounds, and effectively eliminate wound infections.

Inhalable Capsular Polysaccharide-Camouflaged Gallium-Polyphenol Nanoparticles Enhance Lung Cancer Chemotherapy by Depleting Local Lung Microbiota.

Local lung microbiota is closely associated with lung tumorigenesis and therapeutic response. It is found that lung commensal microbes induce chemoresistance in lung cancer by directly inactivating therapeutic drugs via biotransformation. Accordingly, an inhalable microbial capsular polysaccharide (CP)-camouflaged gallium-polyphenol metal-organic network (MON) is designed to eliminate lung microbiota and thereby abrogate microbe-induced chemoresistance. As a substitute for iron uptake, Ga3+ released from MON acts as a "Trojan horse" to disrupt bacterial iron respiration, effectively inactivating multiple microbes. Moreover, CP cloaks endow MON with reduced immune clearance by masquerading as normal host-tissue molecules, significantly increasing residence time in lung tissue for enhanced antimicrobial efficacy. In multiple lung cancer mice models, microbe-induced drug degradation is remarkably inhibited when drugs are delivered by antimicrobial MON. Tumor growth is sufficiently suppressed and mouse survival is prolonged. The work develops a novel microbiota-depleted nanostrategy to overcome chemoresistance in lung cancer by inhibiting local microbial inactivation of therapeutic drugs.

The onco-embryonic antigen ROR1 is a target of chimeric antigen T cells for colorectal cancer.

Colorectal cancer is globally ranked second in both incidence and mortality rate. It usually develops during the middle or late stages of diagnosis, and is characterized by easy metastasis, poor prognosis, and a significant decline in postoperative quality of life. ROR1 is an excellent oncoembryonic antigen in numerous immunotherapy treatments for tumors. Additionally, it is overexpressed in colorectal cancer. To fill the void in CRC treatment with ROR1 as a target of CAR-T immunotherapy, we designed and prepared antiROR1-CART. This third-generation CAR-T cell can effectively inhibit the growth of colorectal cancer in vitro and in vivo.

A Microbial Community Cultured in Gradient Hydrogel for Investigating Gut Microbiome-Drug Interaction and Guiding Therapeutic Decisions.

Despite the recognition that the gut microbiota acts a clinically significant role in cancer chemotherapy, both mechanistic understanding and translational research are still limited. Maximizing drug efficacy requires an in-depth understanding of how the microbiota contributes to therapeutic responses, while microbiota modulation is hindered by the complexity of the human body. To address this issue, a 3D experimental model named engineered microbiota (EM) is reported for bridging microbiota-drug interaction research and therapeutic decision-making. EM can be manipulated in vitro and faithfully recapitulate the human gut microbiota at the genus/species level while allowing co-culture with cells, organoids, and isolated tissues for testing drug responses. Examination of various clinical and experimental drugs by EM reveales that the gut microbiota affects drug efficacy through three pathways: immunological effects, bioaccumulation, and drug metabolism. Guided by discovered mechanisms, custom-tailored strategies are adopted to maximize the therapeutic efficacy of drugs on orthotopic tumor models with patient-derived gut microbiota. These strategies include immune synergy, nanoparticle encapsulation, and host-guest complex formation, respectively. Given the important role of the gut microbiota in influencing drug efficacy, EM will likely become an indispensable tool to guide drug translation and clinical decision-making.

Disruption of CISH promotes the antitumor activity of human T cells and decreases PD-1 expression levels.

Tumor cells and the immunosuppressive tumor microenvironment suppress the antitumor activity of T cells through immune checkpoints, including the PD-L1/PD-1 axis. Cytokine-inducible SH2-containing protein (CISH), a member of the suppressor of cytokine signaling (SOCS) family, inhibits JAK-STAT and T cell receptor (TCR) signaling in T and natural killer (NK) cells. However, its role in the regulation of immune checkpoints in T cells remains unclear. In this study, we ablated CISH in T cells with CRISPR-Cas9 and found that the sensitivity of T cells to TCR and cytokine stimulation was increased. In addition, chimeric antigen receptor T cells with CISH deficiency exhibited longer survival and higher cytokine secretion and antitumor activity. Notably, PD-1 expression was decreased in activated CISH-deficient T cells in vitro and in vivo. The level of FBXO38, a ubiquitination-regulating protein that reduces PD-1 expression, was elevated in activated T cells after CISH ablation. Hence, this study reveals a mechanism by which CISH promotes PD-1 expression by suppressing the expression of FBXO38 and proposes a new strategy for augmenting the therapeutic effect of CAR-T cells by inhibiting CISH.

In Situ Sprayed Biotherapeutic Gel Containing Stable Microbial Communities for Efficient Anti-Infection Treatment.

Systematic administration of antibiotics to treat infections often leads to the rapid evolution and spread of multidrug-resistant bacteria. Here, an in situ-formed biotherapeutic gel that controls multidrug-resistant bacterial infections and accelerates wound healing is reported. This biotherapeutic gel is constructed by incorporating stable microbial communities (kombucha) capable of producing antimicrobial substances and organic acids into thermosensitive Pluronic F127 (polyethylene-polypropylene glycol) solutions. Furthermore, it is found that the stable microbial communities-based biotherapeutic gel possesses a broad antimicrobial spectrum and strong antibacterial effects in diverse pathogenic bacteria-derived xenograft infection models, as well as in patient-derived multidrug-resistant bacterial xenograft infection models. The biotherapeutic gel system considerably outperforms the commercial broad-spectrum antibacterial gel (0.1% polyaminopropyl biguanide) in pathogen removal and infected wound healing. Collectively, this biotherapeutic strategy of exploiting stable symbiotic consortiums to repel pathogens provides a paradigm for developing efficient antibacterial biomaterials and overcomes the failure of antibiotics to treat multidrug-resistant bacterial infections.

CD318 is a target of chimeric antigen receptor T cells for the treatment of colorectal cancer.

Colorectal cancer (CRC) currently has a poor prognosis with a 6.9-year median survival time; to relieve this malignant cancer, we proposed to establish CRC xenografts that can be used to evaluate the cytotoxicity of adoptive chimeric antigen receptor (CAR)-T cells and accelerate the clinical translation of CAR-T cells for use against CRC. We first verified that CD318 had a higher expression level in primary human CRC tissues than in normal tissues based on hundreds of clinical samples. Then, we redirected CAR-T cells containing anti-CD318 single-chain variable fragment (anti-CD318 scFv), CD3ζ, CD28, and Toll-like receptor 2 (TLR2) domains. Next, we evaluated the function of these CAR-T cells in vitro in terms of surface phenotype changes, cytotoxicity and cytokine secretion when they encountered CD318+ CRC cells. Finally, we established two different xenograft mouse models to assess in vivo antitumor activity. The results showed that CAR318 T cells were significantly activated and exhibited strong cytotoxicity and cytokine-secreting abilities against CRC cells in vitro. Furthermore, CAR318 T cells induced CRC regression in different xenograft mouse models and suppressed tumors compared with CAR19 T cells. In summary, our work demonstrates that CAR318 T cells possess strong antitumor capabilities and represent a promising therapeutic approach for CRC.

Targeting to Tumor-Harbored Bacteria for Precision Tumor Therapy.

The differential tumor environment guides various antitumor drug delivery strategies for efficient cancer treatment. Here, based on the special bacteria-enriched tumor environment, we report a different drug delivery strategy by targeting bacteria inhabiting tumor sites. With a tissue microarray analysis, it was found that bacteria amounts displayed significant differences between tumor and normal tissues. Bacteria-targeted mesoporous silica nanoparticles decorated with bacterial lipoteichoic acid (LTA) antibody (LTA-MSNs) could precisely target bacteria in tumors and deliver antitumor drugs. By the intravenous administration of bacteria-targeted nanoparticles, we showed in mice with colon cancer, lung cancer, and breast cancer that LTA-MSNs exhibited a high tumor-targeting ability. As a proof-of-concept study, tumor microbes as some of the characteristics of a tumor environment could be utilized as potential targets for tumor targeting. This bacteria-guided tumor-targeting strategy might have great potential in differential drug delivery and cancer treatment.

Tumor Antigen Loaded Nanovaccine Induced NIR-Activated Inflammation for Enhanced Antigen Presentation During Immunotherapy of Tumors.

Although anticancer vaccines have achieved certain effects in early clinical practice, T cell immunity as the most common responsive pattern of anticancer vaccines is still limited by unsatisfied tumor recognition and inhibition efficiency. As the critical step of T cell immunity, uptake and presentation of specific antigen by antigen-presenting cells (APC) can be activated by inflammation for enhancing the response of T cells to the antigen source. Here, a hybrid nanovaccine named PTh/MnO2 @M activated with a near-infrared ray (NIR) is prepared by coating an autologous tumor cell membrane on the surface of a polythiophene/MnO2 composite core. The photoelectrical material polythiophene can produce local microinflammation under NIR radiation and activate specific T cell antitumor immunity by promoting APC maturation and autologous tumor antigens presentation. Moreover, the synthesized nanovaccine PTh/MnO2 @M is shown to induce a significant antitumor immune response, effectively inhibit the progression of melanoma in mice, and significantly prolong the survival time of mice in vivo. This strategy aims to enhance T-cell immune responses by promoting antigen presentation, leading to effective and specific cancer therapy.

DAP10 integration in CAR-T cells enhances the killing of heterogeneous tumors by harnessing endogenous NKG2D.

Although chimeric antigen receptor T (CAR-T) cells have achieved remarkable successes in hematological malignancies, the efficacies of CAR-T cells against solid tumors remains unsatisfactory. Heterogeneous antigen expression is one of the obstacles on its effective elimination of solid cancer cells. DNAX-activating protein 10 (DAP10) interacts with natural killer group 2D (NKG2D), acting as an adaptor that targets various malignant cells for surveillance. Here, we designed a DAP10 chimeric receptor that utilized native NKG2D on T cells to target NKG2D ligand-expressing cancer cells. We then tandemly incorporated it with anti-glypican 3 (GPC3) single-chain variable fragment (scFv) to construct a dual-antigen-targeting system. T cells expressing DAP10 chimeric receptor (DAP10-T cells) displayed with an enhancement on both cytotoxicity and cytokine secretion against solid cancer cell lines, and its tandem connection with anti-GPC3 scFv (CAR GPC3-DAP10-T cells) exhibited a dual-antigen-targeting capacity on eliminating heterogeneous cancer cells in vitro and suppressing the growth of heterogeneous cancer in vivo. Thus, this novel dual-targeting system enabled a high efficacy on killing cancer cells and extended the recognition profile of CAR-T cells toward tumors, which providing a potential strategy on treatment of solid cancer clinically.

The Chemokine Receptor CCR8 Is a Target of Chimeric Antigen T Cells for Treating T Cell Malignancies.

Chimeric antigen receptor (CAR) T cells have been successfully used in the therapy of B cell leukemia and lymphoma, but still have many challenges in their use for treating T cell malignancies, such as the lack of unique tumor antigens, their limitation of T cell expansion, and the need for third party donors or genome editing. Therefore, we need to find novel targets for CAR T cell therapy to overcome these challenges. Here, we found that both adult T-cell leukemia/lymphoma (ATLL) patients and ATLL cells had increased CCR8 expression but did not express CD7. Moreover, targeting CCR8 in T cells did not impair T cell expansion in vitro. Importantly, anti-CCR8 CAR T cells exhibited antitumor effects on ATLL- and other CCR8-expressing T-ALL cells in vitro and in vivo, and prolonged the survival of ATLL and Jurkat tumor-bearing mouse models. In conclusion, these collective results show that anti-CCR8 CAR T cells possess strong antitumor activity and represent a promising therapeutic approach for ATLL and CCR8+ tumors.

Bioinspired nano-vaccine construction by antigen pre-degradation for boosting cancer personalized immunotherapy.

Cancer vaccines-based cancer immunotherapy has drawn widespread concern. However, insufficient cancer antigens and inefficient antigen presentation lead to low immune response rate, which greatly restrict the practical application of cancer vaccines. Here, inspired by intracellular proteasome-mediated protein degradation pathway, we report an antigen presentation simplification strategy by extracellular degradation of antigen proteins into peptides with proteolytic enzyme for improving the utilization of cancer antigens and arousing restricted cancer immunity. The pre-degraded antigen peptides are first validated to exhibit an increased capacity on antigen-presenting cell (APC) stimulation compared with proteins and still reserve antigen specificity and major histocompatibility complex (MHC) affinity. Furthermore, by coordinating the pre-degraded peptides with calcium phosphate nanoparticles (CaP), a CaP-peptide vaccine (CaP-Pep) is constructed, which is verified to induce an efficient personalized immune response in vivo for multi-model anti-cancer therapy. Notably, this bioinspired strategy based on extracellular enzymatic hydrolysis for vaccine construction is not only applicable for multiple types of cancers, but also shows great potential in expanding immunology fields and translational medicine.

Human induced-T-to-natural killer cells have potent anti-tumour activities.

BACKGROUND: Adoptive cell therapy (ACT) is a particularly promising area of cancer immunotherapy, engineered T and NK cells that express chimeric antigen receptors (CAR) are being explored for treating hematopoietic malignancies but exhibit limited clinical benefits for solid tumour patients, successful cellular immunotherapy of solid tumors demands new strategies. METHODS: Inactivation of BCL11B were performed by CRISPR/Cas9 in human T cells. Immunophenotypic and transcriptional profiles of sgBCL11B T cells were characterized by cytometer and transcriptomics, respectively. sgBCL11B T cells are further engineered with chimeric antigen receptor. Anti-tumor activity of ITNK or CAR-ITNK cells were evaluated in preclinical and clinical studies. RESULTS: We report that inactivation of BCL11B in human CD8+ and CD4+ T cells induced their reprogramming into induced T-to-natural killer cells (ITNKs). ITNKs contained a diverse TCR repertoire; downregulated T cell-associated genes such as TCF7 and LEF1; and expressed high levels of NK cell lineage-associated genes. ITNKs and chimeric antigen receptor (CAR)-transduced ITNKs selectively lysed a variety of cancer cells in culture and suppressed the growth of solid tumors in xenograft models. In a preliminary clinical study, autologous administration of ITNKs in patients with advanced solid tumors was well tolerated, and tumor stabilization was seen in six out nine patients, with one partial remission. CONCLUSIONS: The novel ITNKs thus may be a promising novel cell source for cancer immunotherapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03882840 . Registered 20 March 2019-Retrospectively registered.

Temulence Therapy to Orthotopic Colorectal Tumor via Oral Administration of Fungi-Based Acetaldehyde Generator.

Taking inspiration from percutaneous ethanol injection (PEI) for tumor ablation, an acetaldehyde generator (SC@ZIF@ADH) is constructed for tumor treatment by modifying a metal-organic framework nanocarrier (ZIF), which is loaded with alcohol dehydrogenase (ADH), onto the surface of Saccharomyces cerevisiae (SC). Oral administration of SC@ZIF@ADH can target tumor via mannose-mediated targeting to tumor associated macrophages (TAMs) and generate ethanol at the hypoxic tumor areas. Ethanol is subsequently catalyzed to toxic acetaldehyde by ADH, inducing tumor cells apoptosis and polarizing TAMs toward the anti-tumor phenotype. In vivo animal results show that this acetaldehyde generator can cause a temulence-like reaction in the tumor, significantly inhibiting tumor progression, and might provide an intelligent and nonsurgical substitute for PEI therapy.

Neisseria meningitidis Opca Protein/MnO2 Hybrid Nanoparticles for Overcoming the Blood-Brain Barrier to Treat Glioblastoma.

The major hurdle in glioblastoma therapy is the low efficacy of drugs crossing the blood-brain barrier (BBB). Neisseria meningitidis is known to specifically enrich in the central nervous system through the guidance of an outer membrane invasion protein named Opca. Here, by loading a chemotherapeutic drug methotrexate (MTX) in hollow manganese dioxide (MnO2 ) nanoparticles with surface modification of the Opca protein of Neisseria meningitidis, a bionic nanotherapeutic system (MTX@MnO2 -Opca) is demonstrated to effectively overcome the BBB. The presence of the Opca protein enables the drug to cross the BBB and penetrate into tumor tissues. After accumulating in glioblastoma, the nanotherapeutic system catalyzes the decomposition of excess H2 O2 in the tumor tissue and thereby generates O2 , which alleviates tumor hypoxia and enhances the effect of chemotherapy in the treatment of glioblastoma. This bionic nanotherapeutic system may exhibit great potential in the treatment of glioblastoma.

Biomaterial-mediated modulation of oral microbiota synergizes with PD-1 blockade in mice with oral squamous cell carcinoma.

Because a host's immune system is affected by host-microbiota interactions, means of modulating the microbiota could be leveraged to augment the effectiveness of cancer therapies. Here we report that patients with oral squamous cell carcinoma (OSCC) whose tumours contained higher levels of bacteria of the genus Peptostreptococcus had higher probability of long-term survival. We then show that in mice with murine OSCC tumours injected with oral microbiota from patients with OSCCs, antitumour responses were enhanced by the subcutaneous delivery of an adhesive hydrogel incorporating silver nanoparticles (which inhibited the growth of bacteria competing with Peptostreptococcus) alongside the intratumoural delivery of the bacterium P. anaerobius (which upregulated the levels of Peptostreptococcus). We also show that in mice with subcutaneous or orthotopic murine OSCC tumours, combination therapy with the two components (nanoparticle-incorporating hydrogel and exogenous P. anaerobius) synergized with checkpoint inhibition with programmed death-1. Our findings suggest that biomaterials can be designed to modulate human microbiota to augment antitumour immune responses.

Chimeric CTLA4-CD28-CD3z T Cells Potentiate Antitumor Activity Against CD80/CD86-Positive B Cell Malignancies.

The adoptive transfer of chimeric antigen receptor T (CAR T) cells have been recognized as a promising therapeutic strategy for the treatment of hematological malignancies; however, clinical success using CAR T cells for the treatment of solid tumors are still limited since the T-cell function is inhibited by negative signals in the microenvironment of solid tumors. CTLA4 is a well-known immune checkpoint molecule, thus we developed a novel CAR by converting this negative signal to positive signal. The CAR developed consists of the extracellular and transmembrane domains of CTLA4 and the cytoplasmic domains of CD28 and CD3z (CTLA4-CAR T). CTLA4-CAR T cells exhibited superior cytokine secreting activities and cytotoxic to tumor cells in vitro and in xenograft models. CTLA4-CAR T cells were found to accumulate in tumors and are toxic to myeloid-derived suppressor cells (MDSCs) without signs of severe GVHD and CRS in preclinical models. Thus, this chimeric CTLA4-CAR can enhance the antitumor activity of CAR T cells and shed light on the strategy of using armed CAR T cells to target the immunomodulatory tumor microenvironment.