Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology.

BACKGROUND: Chimeric antigen receptor T (CAR-T) cell therapy, as an emerging anti-tumor treatment, has garnered extensive attention in the study of targeted therapy of multiple tumor-associated antigens in hepatocellular carcinoma (HCC). However, the suppressive microenvironment and individual heterogeneity results in downregulation of these antigens in certain patients' cancer cells. Therefore, optimizing CAR-T cell therapy for HCC is imperative. METHODS: In this study, we administered FGFR4-ferritin (FGFR4-HPF) nanoparticles to the alpaca and constructed a phage library of nanobodies (Nbs) derived from alpaca, following which we screened for Nbs targeting FGFR4. Then, we conducted the functional validation of Nbs. Furthermore, we developed Nb-derived CAR-T cells and evaluated their anti-tumor ability against HCC through in vitro and in vivo validation. RESULTS: Our findings demonstrated that we successfully obtained high specificity and high affinity Nbs targeting FGFR4 after screening. And the specificity of Nbs targeting FGFR4 was markedly superior to their binding to other members of the FGFR family proteins. Furthermore, the Nb-derived CAR-T cells, targeting FGFR4, exhibited significantly enhanced anti-tumor efficacy in both experiments when in vitro and in vivo. CONCLUSIONS: In summary, the results of this study suggest that the CAR-T cells derived from high specificity and high affinity Nbs, targeting FGFR4, exhibited significantly enhanced anti-tumor efficacy in vitro and in vivo. This is an exploration of FGFR4 in the field of Nb-derived CAR-T cell therapy for HCC, holding promise for enhancing safety and effectiveness in the clinical treatment of HCC in the future.

The construction of modular universal chimeric antigen receptor T (MU-CAR-T) cells by covalent linkage of allogeneic T cells and various antibody fragments.

BACKGROUND: Chimeric antigen receptor-T (CAR-T) cells therapy is one of the novel immunotherapeutic approaches with significant clinical success. However, their applications are limited because of long preparation time, high cost, and interpersonal variations. Although the manufacture of universal CAR-T (U-CAR-T) cells have significantly improved, they are still not a stable and unified cell bank. METHODS: Here, we tried to further improve the convenience and flexibility of U-CAR-T cells by constructing novel modular universal CAR-T (MU-CAR-T) cells. For this purpose, we initially screened healthy donors and cultured their T cells to obtain a higher proportion of stem cell-like memory T (TSCM) cells, which exhibit robust self-renewal capacity, sustainability and cytotoxicity. To reduce the alloreactivity, the T cells were further edited by double knockout of the T cell receptor (TCR) and class I human leukocyte antigen (HLA-I) genes utilizing the CRISPR/Cas9 system. The well-growing and genetically stable universal cells carrying the CAR-moiety were then stored as a stable and unified cell bank. Subsequently, the SDcatcher/GVoptiTag system, which generate an isopeptide bond, was used to covalently connect the purified scFvs of antibody targeting different antigens to the recovered CAR-T cells. RESULTS: The resulting CAR-T cells can perform different functions by specifically targeting various cells, such as the eradication of human immunodeficiency virus type 1 (HIV-1)-latenly-infected cells or elimination of T lymphoma cells, with similar efficiency as the traditional CAR-T cells did. CONCLUSION: Taken together, our strategy allows the production of CAR-T cells more modularization, and makes the quality control and pharmaceutic manufacture of CAR-T cells more feasible.

Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment.

T cell lymphoma (TCL) is a highly heterogeneous group of diseases with a poor prognosis and low 5-year overall survival rate. The current therapeutic regimens have relatively low efficacy rates. Clinical studies of single-target chimeric antigen receptor T cell (CAR-T cell) therapy in T lymphocytes require large and multiple infusions, increasing the risks and cost of treatment; therefore, optimizing targeted therapy is a way to improve overall prognosis. Despite significant advances in bispecific CAR-T cell therapy to avoid antigen escape in treatment of B cell lymphoma, applying this strategy to TCL requires further investigation. Here, we constructed an alpaca nanobody (Nb) phage library and generated high-affinity and -specificity Nbs targeting CD30 and CD5, respectively. Based on multiple rounds of screening, bispecific NbCD30-CD5-CAR T cells were constructed, and their superior anti-tumor effect against TCL was validated in vitro and in vivo. Our findings demonstrated that Nb-derived bispecific CAR-T cells significantly improved anti-tumor efficacy in TCL treatment compared with single-target CAR-T cells and bispecific single chain variable fragment (scFv)-derived CAR-T cells. Because Nbs are smaller and less immunogenic, the synergistic effect of Nb-based bispecific CAR-T cells may improve their safety and efficacy in future clinical applications.

Enhancement of CAR-T cell activity against cholangiocarcinoma by simultaneous knockdown of six inhibitory membrane proteins.

BACKGROUND: Existing treatments for cholangiocarcinoma have poor efficacy. However, chimeric antigen receptor-T (CAR-T) cells are emerging as a potential therapeutic strategy. Solid tumors possess multiple adverse factors in an immunosuppressive microenvironment that impair CAR-T cell infiltration and function. This study aimed to improve the function of CAR-T cells through knock down immune checkpoints and immunosuppressive molecular receptors. METHODS: We evaluated the expression of epidermal growth factor receptor (EGFR) and B7 homolog 3 protein (B7H3) antigens in cholangiocarcinoma tissues using immunohistochemistry and screened specific immune checkpoints in the cholangiocarcinoma microenvironment via flow cytometry. Subsequently, we engineered CAR-T cells targeting EGFR and B7H3 antigens. We simultaneously knocked down immune checkpoints and immunosuppressive molecular receptors in CAR-T cells by constructing two clusters of small hairpin RNAs and evaluated the engineered CAR-T cells for antitumor activity both in vitro, using tumor cell lines and cholangiocarcinoma organoid models, and in vivo, using humanized mouse models. RESULTS: We observed high expression of EGFR and B7H3 antigens in cholangiocarcinoma tissues. EGFR-CAR-T and B7H3-CAR-T cells demonstrated specific anti-tumor activity. We found an abundance of programmed cell death protein 1 (PD-1), T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), and T cell immunoglobulin and ITIM domain (Tigit) on infiltrated CD8+ T cells in the cholangiocarcinoma microenvironment. We then decreased the expression of these 3 proteins on the surface of CAR-T cells, named PTG-scFV-CAR-T cells. Furthermore, we knocked-down the expression of transforming growth factor beta receptor (TGFßR), interleukin-10 receptor (IL-10R), and interleukin-6 receptor (IL-6R) of PTG-scFV-CAR-T cells. Those cells, named PTG-T16R-scFV-CAR-T cells, potently killed tumor cells in vitro and promoted apoptosis of tumor cells in a cholangiocarcinoma organoid model. Finally, the PTG-T16R-scFv-CAR-T cells showed greater inhibitory effect on tumor growth in vivo, and were superior in prolonging the survival of mice. CONCLUSIONS: Our results revealed that PTG-T16R-scFV-CAR-T cells with knockdown of sextuplet inhibitory molecules exhibited strong immunity against cholangiocarcinoma and long-term efficacy both in vitro and in vivo. This strategy provides an effective and personalized immune cell therapy against cholangiocarcinoma.

Identification of CD98 as a Novel Biomarker for HIV-1 Permissiveness and Latent Infection.

Human immunodeficiency virus type 1 (HIV-1) can integrate viral DNA into host cell chromosomes to establish a long-term stable latent reservoir, which is a major obstacle to cure HIV-1 infection. The characteristics of the HIV-1 latent reservoir have not been fully understood. Here, we identified 126 upregulated plasma membrane proteins in HIV-1 latently infected cells by a label-free liquid chromatography-tandem mass spectrometry analysis. The higher levels of CD98 expression in multiple HIV-1 latently infected cell lines and primary CD4+ T cells compared to uninfected cells were further confirmed by quantitative reverse transcription PCR (RT-qPCR) and flow cytometry analyses. In addition, CD98high CD4+ T cells displayed hyper-permissiveness to HIV-1 infection and possessed distinct immune phenotypic profiles associated with Th17 and peripheral follicular T helper (pTFH) characteristics. Notably, the CD98high resting memory CD4+ T cells harbored significantly higher cell-associated viral RNA and intact provirus than CD98low counterparts in HIV-1-infected individuals receiving combined antiretroviral therapy. Furthermore, CD98high CD4+ T cells exhibited a robust proliferative capacity and significantly contributed to the clonal expansion of the HIV-1 latent reservoir. Our study demonstrates that CD98 can be used as a novel biomarker of HIV-1 latently infected cells to indicate the effect of various strategies to reduce the viral reservoir. IMPORTANCE Identification of cellular biomarkers is the crucial challenge to eradicate the HIV-1 latent reservoir. In our study, we identified CD98 as a novel plasma membrane biomarker for HIV-1 permissiveness and latent infection. Importantly, CD98high CD4+ T cells exhibited a hyper-permissiveness to HIV-1 infection and significantly contributed to the clonal expansion of the HIV-1 latent reservoir. CD98 could be targeted to develop therapeutic strategies to reduce the HIV-1 latent reservoir in further research.

Broadly neutralizing antibody-derived CAR T cells reduce viral reservoir in individuals infected with HIV-1.

BACKGROUNDChimeric antigen receptor (CAR) T cells have emerged as an approach to treat malignant tumors. This strategy has also been proposed for the treatment of HIV-1 infection. We have developed a broadly neutralizing antibody-derived (bNAb-derived) CAR T cell therapy that can exert specific cytotoxic activity against HIV-1-infected cells.METHODSWe conducted an open-label trial of the safety, side-effect profile, pharmacokinetic properties, and antiviral activity of bNAb-derived CAR T cell therapy in individuals infected with HIV-1 who were undergoing analytical interruption of antiretroviral therapy (ART).RESULTSA total of 14 participants completed only a single administration of bNAb-derived CAR T cells. CAR T cell therapy administration was safe and well tolerated. Six participants discontinued ART, and viremia rebound occurred in all of them, with a 5.3-week median time. Notably, the cell-associated viral RNA and intact proviruses decreased significantly after CAR T cell treatment. Analyses of HIV-1 variants before or after CAR T cell administration suggested that CAR T cells exerted pressure on rebound viruses, resulting in a selection of viruses with less diversity and mutations against CAR T cell-mediated cytotoxicity.CONCLUSIONNo safety concerns were identified with adoptive transfer of bNAb-derived CAR T cells. They reduced viral reservoir. All the rebounds were due to preexisting or emergence of viral escape mutations.TRIAL REGISTRATIONClinicalTrials.gov (NCT03240328).FUNDINGMinistry of Science and Technology of China, National Natural Science Foundation of China, and Department of Science and Technology of Guangdong Province.

The interferon-stimulated exosomal hACE2 potently inhibits SARS-CoV-2 replication through competitively blocking the virus entry.

Since the outbreak of coronavirus disease 2019 (COVID-19), it has become a global pandemic. The spike (S) protein of etiologic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specifically recognizes human angiotensin-converting enzyme 2 (hACE2) as its receptor, which is recently identified as an interferon (IFN)-stimulated gene. Here, we find that hACE2 exists on the surface of exosomes released by different cell types, and the expression of exosomal hACE2 is increased by IFNα/ß treatment. In particular, exosomal hACE2 can specifically block the cell entry of SARS-CoV-2, subsequently inhibit the replication of SARS-CoV-2 in vitro and ex vivo. Our findings have indicated that IFN is able to upregulate a viral receptor on the exosomes which competitively block the virus entry, exhibiting a potential antiviral strategy.

The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has claimed over 2 million lives worldwide. Although the genetic sequences of SARS-CoV and SARS-CoV-2 have high homology, the clinical and pathological characteristics of COVID-19 differ significantly from those of SARS. How and whether SARS-CoV-2 evades (cellular) immune surveillance requires further elucidation. In this study, we show that SARS-CoV-2 infection leads to major histocompability complex class Ι (MHC-Ι) down-regulation both in vitro and in vivo. The viral protein encoded by open reading frame 8 (ORF8) of SARS-CoV-2, which shares the least homology with SARS-CoV among all viral proteins, directly interacts with MHC-Ι molecules and mediates their down-regulation. In ORF8-expressing cells, MHC-Ι molecules are selectively targeted for lysosomal degradation via autophagy. Thus, SARS-CoV-2-infected cells are much less sensitive to lysis by cytotoxic T lymphocytes. Because ORF8 protein impairs the antigen presentation system, inhibition of ORF8 could be a strategy to improve immune surveillance.

Engineering a Reliable and Convenient SARS-CoV-2 Replicon System for Analysis of Viral RNA Synthesis and Screening of Antiviral Inhibitors.

The etiologic agent of COVID-19 is highly contagious and has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. In this study, we reported a replicon system which consists of four plasmids expressing the required segments of SARS-CoV-2. Our study revealed that the features for viral RNA synthesis and responses to antivirus drugs of the replicon are similar to those of wild-type viruses. Further analysis indicated that ORF6 provided potent in trans stimulation of the viral replication. Some viral variations, such as 5'UTR-C241T and ORF8-(T28144C) L84S mutation, also exhibit their different impact upon viral replication. Besides, the screening of clinically used drugs identified that several tyrosine kinase inhibitors and DNA-Top II inhibitors potently inhibit the replicon, as well as authentic SARS-CoV-2 viruses. Collectively, this replicon system provides a biosafety-worry-free platform for studying SARS-CoV-2 virology, monitoring the functional impact of viral mutations, and developing viral inhibitors.IMPORTANCE COVID-19 has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. We reported a replicon system which consists of four ordinary plasmids expressing the required segments of SARS-CoV-2. Using the replicon system, we developed three application scenarios: (i) to identify the effects of viral proteins on virus replication, (ii) to identify the effects of mutations on viral replication during viral epidemics, and (iii) to perform high-throughput screening of antiviral drugs. Collectively, this replicon system would be useful for virologists to study SARS-CoV-2 virology, for epidemiologists to monitor virus mutations, and for industry to develop antiviral drugs.

Lovastatin Inhibits HIV-1-Induced MHC-I Downregulation by Targeting Nef-AP-1 Complex Formation: A New Strategy to Boost Immune Eradication of HIV-1 Infected Cells.

Current combined antiretroviral therapy (cART) mainly targets 3 of the 15 HIV proteins leaving many potential viral vulnerabilities unexploited. To purge the HIV-1 latent reservoir, various strategies including "shock and kill" have been developed. A key question is how to restore impaired immune surveillance. HIV-1 protein Nef has long been known to mediate the downregulation of cell-surface MHC-I and assist HIV-1 to evade the immune system. Through high throughput screening of Food and Drug Administration (FDA) approved drugs, we identified lovastatin, a statin drug, to significantly antagonize Nef to downregulate MHC-I, CD4, and SERINC5, and inhibit the intrinsic infectivity of virions. In addition, lovastatin boosted autologous CTLs to eradicate the infected cells and effectively inhibit the subsequent viral rebound in CD4+ T-lymphocytes isolated from HIV-1-infected individuals receiving suppressive cART. Furthermore, we found that lovastatin inhibits Nef-induced MHC-I downregulation by directly binding with Nef and disrupting the Nef-AP-1 complex. These results demonstrate that lovastatin is a promising agent for counteracting Nef-mediated downregulation of MHC-I, CD4, and SERINC5. Lovastatin could potentially be used in the clinic to enhance anti-HIV-1 immune surveillance.

Engineered triple inhibitory receptor resistance improves anti-tumor CAR-T cell performance via CD56.

The inhibitory receptors PD-1, Tim-3, and Lag-3 are highly expressed on tumor-infiltrating lymphocytes and compromise their antitumor activity. For efficient cancer immunotherapy, it is important to prevent chimeric antigen receptor T (CAR-T)-cell exhaustion. Here we downregulate these three checkpoint receptors simultaneously on CAR-T cells and that show the resulting PTL-CAR-T cells undergo epigenetic modifications and better control tumor growth. Furthermore, we unexpectedly find increased tumor infiltration by PTL-CAR-T cells and their clustering between the living and necrotic tumor tissue. Mechanistically, PTL-CAR-T cells upregulate CD56 (NCAM), which is essential for their effector function. The homophilic interaction between intercellular CD56 molecules correlates with enhanced infiltration of CAR-T cells, increased secretion of interferon-γ, and the prolonged survival of CAR-T cells. Ectopically expressed CD56 promotes CAR-T cell survival and antitumor response. Our findings demonstrate that genetic blockade of three checkpoint inhibitory receptors and the resulting high expression of CD56 on CAR-T cells enhances the inhibition of tumor growth.