TREM1/DAP12 based novel multiple chain CAR-T cells targeting DLL3 show robust anti-tumour efficacy for small cell lung cancer.

Small cell lung cancer (SCLC), recognized as the most aggressive subtype of lung cancer, presents an extremely poor prognosis. Currently, patients with small cell lung cancer face a significant dearth of effective alternative treatment options once they experience recurrence and progression after first-line therapy. Despite the promising efficacy of immunotherapy, particularly immune checkpoint inhibitors in non-small cell lung cancer (NSCLC) and various other tumours, its impact on significantly enhancing the prognosis of SCLC patients remains elusive. DLL3 has emerged as a compelling target for targeted therapy in SCLC due to its high expression on the membranes of SCLC and other neuroendocrine carcinoma cells, with minimal to no expression in normal cells. Our previous work led to the development of a novel multiple chain chimeric antigen receptor (CAR) leveraging the TREM1 receptor and DAP12, which efficiently activated T cells and conferred potent cell cytotoxicity. In this study, we have developed a DLL3-TREM1/DAP12 CAR-T (DLL3-DT CAR-T) therapy, demonstrating comparable anti-tumour efficacy against SCLC cells in vitro. In murine xenograft and patient-derived xenograft models, DLL3-DT CAR-T cells exhibited a more robust tumour eradication efficiency than second-generation DLL3-BBZ CAR-T cells. Furthermore, we observed elevated memory phenotypes, induced durable responses, and activation under antigen-presenting cells in DLL3-DT CAR-T cells. Collectively, these findings suggest that DLL3-DT CAR-T cells may offer a novel and potentially effective therapeutic strategy for treating DLL3-expressing SCLC and other solid tumours.

Effects of methionine deficiency on B7H3-DAP12-CAR-T cells in the treatment of lung squamous cell carcinoma.

Lung squamous cell carcinoma (LUSC) is a subtype of lung cancer for which precision therapy is lacking. Chimeric antigen receptor T-cells (CAR-T) have the potential to eliminate cancer cells by targeting specific antigens. However, the tumor microenvironment (TME), characterized by abnormal metabolism could inhibit CAR-T function. Therefore, the aim of this study was to improve CAR-T efficacy in solid TME by investigating the effects of amino acid metabolism. We found that B7H3 was highly expressed in LUSC and developed DAP12-CAR-T targeting B7H3 based on our previous findings. When co-cultured with B7H3-overexpressing LUSC cells, B7H3-DAP12-CAR-T showed significant cell killing effects and released cytokines including IFN-γ and IL-2. However, LUSC cells consumed methionine (Met) in a competitive manner to induce a Met deficiency. CAR-T showed suppressed cell killing capacity, reduced cytokine release and less central memory T phenotype in medium with lower Met, while the exhaustion markers were up-regulated. Furthermore, the gene NKG7, responsible for T cell cytotoxicity, was downregulated in CAR-T cells at low Met concentration due to a decrease in m5C modification. NKG7 overexpression could partially restore the cytotoxicity of CAR-T in low Met. In addition, the anti-tumor efficacy of CAR-T was significantly enhanced when co-cultured with SLC7A5 knockdown LUSC cells at low Met concentration. In conclusion, B7H3 is a prospective target for LUSC, and B7H3-DAP12-CAR-T cells are promising for LUSC treatment. Maintaining Met levels in CAR-T may help overcome TME suppression and improve its clinical application potential.

A novel TREM1/DAP12-based multiple chain CAR-T cell targets PTK7 in ovarian cancer therapy.

While CAR-T cell therapy has shown success against hematological tumors, its effectiveness for solid tumors, including ovarian cancer, remains unsatisfactory. This study aimed to develop and evaluate the efficacy of novel chimeric antigen receptor T (CAR-T) cells targeting PTK7 through TREM1/DAP12 signaling against ovarian cancer. The expression of PTK7 in ovarian cancer tissues and cells was evaluated using immunohistochemical staining and flow cytometric analysis. The anti-tumor effects of PTK7 CAR-T cells were assessed in vitro using real-time cell analysis and enzyme-linked immunosorbent assay, and in vivo using a xenograft tumor model. PTK7 was significantly expressed in ovarian cancer tissues and cells. PTK7-targeting CAR-T cells based on TREM1/DAP12 signaling exhibited potent cytotoxicity against ovarian cancer cells expressing PTK7 in vitro, and effectively eradicated tumors in vivo. Our findings suggest that TREM1/DAP12-based PTK7 CAR-T cells have potential as a treatment strategy for ovarian cancer. Further studies are needed to evaluate the safety and efficacy of this approach in clinical trials.

Junin virus infects mouse cells and induces innate immune responses.

Junín virus is the causative agent for Argentine hemorrhagic fever, and its natural host is the New World rodent Calomys musculinus. The virus is transmitted to humans by aerosolization, and it is believed that many of the clinical symptoms are caused by cytokines produced by sentinel cells of the immune system. Here we used the Junín virus vaccine strain Candid 1 to determine whether mouse cells could be used to study virus entry and antiviral innate immune responses. We show that Candid 1 can infect and propagate in different mouse-derived cell lines through a low-pH-dependent, transferrin receptor 1-independent mechanism, suggesting that there is a second entry receptor. In addition, Candid 1 induced expression of the antiviral cytokines tumor necrosis factor alpha and beta interferon in macrophages, and this induction was independent of viral replication. Using Candid 1, as well as virus-like particles bearing the viral glycoprotein, to infect different primary cells and established macrophage cell lines with deletions in the Toll-like receptor (TLR) pathway, we show that TLR2 is a cellular sensor of both the Parodi and Candid 1 viral glycoproteins. Because Junín virus is highly lethal in humans, the use of an experimentally tractable model system, such as the mouse, could provide a better understanding of the antiviral innate cellular responses to Junín virus and the role of these responses in pathogenesis.

Coexpression of c-Jun in multiple-chain DAP-CAR-engineered T-cells for solid tumor therapy.

Aim: This work was designed to explore whether c-Jun overexpression could improve the persistence and antitumor efficacy of DAP chimeric antigen receptor T-cell (CAR-T) cells. Methods: The in vitro and in vivo antitumor effects of mesothelin (MSLN) targeting DAP-CAR-T cells were verified by ELISA, real-time cell analysis and in a xenograft model. Results: c-Jun overexpression did not affect DAP-CAR-T cell expansion while slightly increasing IL-2 secretion. Moreover, c-Jun did not improve the antitumor efficacy of DAP-CAR-T cells in vitro or in vivo, but reduced LAG3 expression and increased the ratio of Tcm and Tn/Tscm cells in vivo. Conclusion: The findings indicate that coexpression with c-Jun in DAP-CAR-T cells slightly improves T-cell exhaustion and central memory phenotype maintenance, which may be useful for DAP-CAR-T cell therapy in solid tumors.

Chimeric antigen receptor (CAR) T-cell therapy has achieved great success in treating patients with hematological tumors such as b-acute lymphoblastic leukemia and lymphoma. However, a growing number of clinical trials show that most of the second-generation CAR-T cells with different targeting single-chain fragment variables (scFv) did not exhibit comparable therapeutic effects with CD19-targeting CAR-T cells in solid tumors. To overcome this challenge, scientists have developed several methods to optimize the structure of CARs, including coexpression of a transcription factor called c-Jun in CAR-T cells. The authors previously developed a novel multiple-chain DAP-CAR that shows promising solid tumor eradication capacity. In this study, overexpression of c-Jun only slightly improved the antitumor activity of DAP-CAR-T cells, suggesting other optimization methods are needed.

Receptor determinants of zoonotic transmission of New World hemorrhagic fever arenaviruses.

Transferrin receptor 1 (TfR1) is a cellular receptor for the New World hemorrhagic fever arenaviruses Machupo (MACV), Junín (JUNV), and Guanarito (GTOV). Each of these viruses is specifically adapted to a distinct rodent host species, but all cause human disease. Here we compare the ability of these viruses to use various mammalian transferrin receptor 1 (TfR1) orthologs, including those of the South American rodents that serve as reservoirs for MACV, JUNV, and GTOV (Calomys callosus, Calomys musculinus, and Zygodontomys brevicauda, respectively). Retroviruses pseudotyped with MACV and JUNV but not GTOV glycoproteins (GPs) efficiently used C. callosus TfR1, whereas only JUNV GP could use C. musculinus TfR1. All three viruses efficiently used Z. brevicauda TfR1. TfR1 orthologs from related rodents, including house mouse (Mus musculus) and rat (Rattus norvegicus), did not support entry of these viruses. In contrast, these viruses efficiently used human and domestic cat TfR1 orthologs. We further show that a local region of the human TfR1 apical domain, including tyrosine 211, determined the efficiency with which MACV, JUNV, and GTOV used various TfR1 orthologs. Our data show that these New World arenaviruses are specifically adapted to the TfR1 orthologs of their respective rodent hosts and identify key commonalities between these orthologs and human TfR1 necessary for efficient transmission of these viruses to humans.

Pseudoprogression of extramedullary disease in relapsed acute lymphoblastic leukemia after CAR T-cell therapy.

Background: CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has emerged as a powerful immunotherapy in relapsed or refractory B-cell acute lymphoblastic leukemia. The changes in extramedullary (EM) disease in pediatric relapsed or refractory B-cell acute lymphoblastic leukemia after CAR T-cell therapy have rarely been reported. Materials & methods: A child with relapsed B-ALL was treated with CAR T-cell therapy. Bone marrow morphological examination, minimal residual disease, fusion mutation and radiological evaluation of the EM disease were performed before and after CAR T-cell infusion. Results: Radiological assessment revealed a distinct asymptomatic pseudo progression of EM involvements on day 16 after CAR T-cell infusion. Conclusion: Pseudoprogression of EM disease indicates heterogeneous immune-related patterns of response in patients treated with CAR-T therapy. Such patients should be closely monitored and practical immune-related response criteria should be developed for them.

Novel two-chain structure utilizing KIRS2/DAP12 domain improves the safety and efficacy of CAR-T cells in adults with r/r B-ALL.

Engineered T cells that express chimeric antigen receptors (CARs) have been a promising therapy for hematologic malignancies. The optimization of CAR structure using different signaling domains can alter a wide range of CAR-T cell properties, including anti-tumor activity, long-term persistence, and safety. In this study, we developed a novel CAR structure based on KIRS2/Dap12 for B cell acute lymphoblastic leukemia (B-ALL) antigen CD19 and compared the anti-tumor efficacy and safety of this construct in transduced T cells with standard second-generation CAR-T cells targeting CD19 for B-ALL in vitro and in vivo and in adult relapsed/refractory (r/r) B-ALL patients. We discovered that KIRS2/Dap12 receptor infused with 4-1BB co-stimulation domain could enhance anti-tumor efficacy by remarkably increasing the production of pro-inflammatory interleukin-2 (IL-2), especially when co-cultured with antigen-positive tumor cells. In addition, CD19-KIRS2/Dap12-BB CAR-T cells showed the inspiring outcome that complete responses were seen in 4 of 4 (100%) patients without neurotoxicity and a high rate of severe cytokine release syndrome (CRS) after CAR-T infusion in a phase I clinical trial. Given these encouraging findings, CD19-KIRS2/Dap12-BB CAR-T cells are safe and can lead to clinical responses in adult patients with r/r B-ALL, indicating that further assessment of this therapy is warranted.

TREM1/Dap12-based CAR-T cells show potent antitumor activity.

Aim: Chimeric antigen receptor-engineered T (CAR-T) cells have gained huge success in treating hematological malignancies, yet the CD3ζ-based CAR-T therapies have not shown comparable clinical benefits in solid tumors. We designed an alternative chimeric immunoreceptor in which a single-chain variable fragment was fused to the transmembrane-cytoplasmic domains of triggering receptor expressed on myeloid (TREM1), which may show potent antitumor activity. Methods: To generate TREM1/DNAX activation protein of 12 kDa (Dap12)-based CAR-T cells, TREM1 along with DAP12 was transduced into T cells. Results: TREM1/Dap12-based CAR-T cells showed more lysis in vitro and a similar antitumor effect in mouse models compared with CD19BBζ CAR-T cells. Conclusion: In this study, we designed a TREM1/Dap12-based CAR, which was not reported previously and demonstrated that TREM1/Dap12-based CAR-T cells had potent antitumor activity in vitro and in vivo.

Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors.

Chimeric antigen receptors (CAR) bearing an antigen-binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors have provided a potent method for engineering T-cell cytotoxicity toward B-cell leukemia and lymphoma. However, resistance to immunotherapy due to loss of T-cell effector function remains a significant barrier, especially in solid malignancies. We describe an alternative chimeric immunoreceptor design in which we have fused a single-chain variable fragment for antigen recognition to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR). We show that this simple, KIR-based CAR (KIR-CAR) triggers robust antigen-specific proliferation and effector function in vitro when introduced into human T cells with DAP12, an immunotyrosine-based activation motifs-containing adaptor. T cells modified to express a KIR-CAR and DAP12 exhibit superior antitumor activity compared with standard first- and second-generation CD3ζ-based CARs in a xenograft model of mesothelioma highly resistant to immunotherapy. The enhanced antitumor activity is associated with improved retention of chimeric immunoreceptor expression and improved effector function of isolated tumor-infiltrating lymphocytes. These results support the exploration of KIR-CARs for adoptive T-cell immunotherapy, particularly in immunotherapy-resistant solid tumors.

Aurora B dynamics at centromeres create a diffusion-based phosphorylation gradient.

Aurora B kinase is essential for successful cell division and regulates spindle assembly and kinetochore-microtubule interactions. The kinase localizes to the inner centromere until anaphase, but many of its substrates have distinct localizations, for example on chromosome arms and at kinetochores. Furthermore, substrate phosphorylation depends on distance from the kinase. How the kinase reaches substrates at a distance and how spatial phosphorylation patterns are determined are unknown. In this paper, we show that a phosphorylation gradient is produced by Aurora B concentration and activation at centromeres and release and diffusion to reach substrates at a distance. Kinase concentration, either at centromeres or at another chromosomal site, is necessary for activity globally. By experimentally manipulating dynamic exchange at centromeres, we demonstrate that the kinase reaches its substrates by diffusion. We also directly observe, using a fluorescence resonance energy transfer-based biosensor, phosphorylation spreading from centromeres after kinase activation. We propose that Aurora B dynamics and diffusion from the inner centromere create spatial information to regulate cell division.

Molecular control of kinetochore-microtubule dynamics and chromosome oscillations.

Chromosome segregation in metazoans requires the alignment of sister kinetochores on the metaphase plate. During chromosome alignment, bioriented kinetochores move chromosomes by regulating the plus-end dynamics of the attached microtubules. The bundles of kinetochore-bound microtubules alternate between growth and shrinkage, leading to regular oscillations along the spindle axis. However, the molecular mechanisms that coordinate microtubule plus-end dynamics remain unknown. Here we show that centromere protein (CENP)-H, a subunit of the CENP-A nucleosome-associated and CENP-A distal complexes (CENP-A NAC/CAD), is essential for this coordination, because kinetochores lacking CENP-H establish bioriented attachments but fail to generate regular oscillations, as a result of an uncontrolled rate of microtubule plus-end turnover. These alterations lead to rapid erratic movements that disrupt metaphase plate organization. We also show that the abundance of the CENP-A NAC/CAD subunits CENP-H and CENP-I dynamically change on individual sister kinetochores in vivo, because they preferentially bind the sister kinetochore attached to growing microtubules, and that one other subunit, CENP-Q, binds microtubules in vitro. We therefore propose that CENP-A NAC/CAD is a direct regulator of kinetochore-microtubule dynamics, which physically links centromeric DNA to microtubule plus ends.

Enhancement of humoral and cellular immunity in mice against Japanese encephalitis virus using a DNA prime-protein boost vaccine strategy.

A synthetic multi-epitope gene containing critical epitopes of the Japanese encephalitis virus (JEV) envelope gene was cloned into both prokaryotic and eukaryotic expression vectors. The recombinant plasmid and purified recombinant protein (heterologously expressed in Escherichia coli) were used as immunogens in a mouse model. The results indicate that both the recombinant protein and the DNA vaccine induce humoral and cellular immune responses. Neutralising antibody titres in mice in the pcDNA-TEP plus rEP group increased considerably relative to mice immunised using either pcDNA-TEP or rEP alone (P<0.05). Furthermore, the highest levels of interleukin (IL)-2, interferon-gamma and IL-4 were induced following priming with the DNA vaccine and boosting with the recombinant protein. Together these findings demonstrate that a DNA-recombinant protein prime-boost vaccination strategy can produce high levels of antibody and trigger significant T cell responses in mice, highlighting the potential value of such an approach in the prevention of JEV infection.

Mouse mammary tumor virus uses mouse but not human transferrin receptor 1 to reach a low pH compartment and infect cells.

Mouse mammary tumor virus (MMTV) is a pH-dependent virus that uses mouse transferrin receptor 1 (TfR1) for entry into cells. Previous studies demonstrated that MMTV could induce pH 5-dependent fusion-from-with of mouse cells. Here we show that the MMTV envelope-mediated cell-cell fusion requires both the entry receptor and low pH (pH 5). Although expression of the MMTV envelope and TfR1 was sufficient to mediate low pH-dependent syncytia formation, virus infection required trafficking to a low pH compartment; infection was independent of cathepsin-mediated proteolysis. Human TfR1 did not support virus infection, although envelope-mediated syncytia formation occurred with human cells after pH 5 treatment and this fusion depended on TfR1 expression. However, although the MMTV envelope bound human TfR1, virus was only internalized and trafficked to a low pH compartment in cells expressing mouse TfR1. Thus, while human TfR1 supported cell-cell fusion, because it was not internalized when bound to MMTV, it did not function as an entry receptor. Our data suggest that MMTV uses TfR1 for all steps of entry: cell attachment, induction of the conformational changes in Env required for membrane fusion and internalization to an appropriate acidic compartment.

Immune responses of recombinant adenoviruses expressing immunodominant epitopes against Japanese encephalitis virus.

Japanese encephalitis virus (JEV), which belongs to the family Flaviviridae, causes infection of the central nervous system in humans and equines and stillbirths in swine. In the present report, we constructed and characterized the immune responses conferred by recombinant adenoviruses expressing JEV E epitopes (six amino acid residues 60-68, 327-333, 337-345, 373-399, 397-403 and 436-445 in E, designated TEP). Seven groups (n = 10) of female BALB/c mice received intramuscular (IM) or oral immunization with the recombinant adenoviruses twice at 2-week intervals. Intramuscular immunization of mice with rAd-TEP generated greater titers of anti-JEV antibodies and JEV neutralizing activity than in animals with oral injection. It statistically significant differences were found in anti-JEV antibody titers and JEV neutralizing activity induced by IM immunization with rAd-TEP at a dose of 1 x 10(8.0)TCID50 when compared with the doses tested (3 x 10(7.0) and 1 x 10(7.0)TCID50) IM inoculation of rAd-TEP. Splenocytes from mice immunized intramuscularly with rAd-TEP secreted the largest amounts of interferon-gamma and interleukin-2 and moderate amounts of interleukin-4 in the presence of JEV. It demonstrates that IM immunization with rAd-TEP induced the highest level of cell-mediated immune responses and the higher level of JEV-specific humoral immune responses than oral immunization. Then we further evaluated the protective efficacy of the recombinants in swine. All swine were protected from viral challenge with IM rAd-TEP at 1 x 10(10.0)TCID50, even though the neutralizing antibody titers were lower than those in the group inoculated with inactivated vaccine. Our findings indicate that rAd-TEP might be an attractive candidate vaccines for preventing JEV infection.

Identification of the segments of the mouse transferrin receptor 1 required for mouse mammary tumor virus infection.

Most enveloped viruses enter cells through binding of virion surface envelope proteins to receptors found on the plasma membrane of the cell. The beta retrovirus mouse mammary tumor virus (MMTV) uses transferrin receptor 1 (TfR1) to enter cells in a pH-dependent mechanism, probably co-trafficking with TfR1 to an acidic compartment where virus entry occurs. We have shown here that, although mouse and rat TfR1 function as entry receptors, cat, dog, hamster, or human TfR1s do not support MMTV infection. We also demonstrated that MMTV entry is independent of transferrin, iron, and the TfR1 cofactor hereditary hematochromatosis HFE protein. Using chimeric mouse/human hybrid TfR1 constructs, we determined the site of interaction with MMTV and found that it maps to two segments physically disparate from the TfR and HFE binding sites. Thus, MMTV has apparently evolved to enter cells independently of the iron status of the host.

Both heptad repeats of human respiratory syncytial virus fusion protein are potent inhibitors of viral fusion.

Heptad repeat regions (HR1 and HR2) are highly conserved peptides located in F(1) of paramyxovirus envelope proteins. They are important in the process of virus fusion and form six-helix bundle structure (trimer of HR1 and HR2 heterodimer) post-fusion, similar to those found in the fusion proteins of other enveloped viruses, such as retrovirus HIV. Both HR1 and HR2 show potent inhibition for virus fusion in some members of paramyxovirus. However, in other members, only HR2 gives strong inhibition whereas HR1 does not. Human respiratory syncytial virus (hRSV) is a member of paramyxovirus and its crystal structure of HR1 and HR2 six-helix bundle was solved lately. Although hRSV HR2 inhibition was reported, nevertheless the effect of HR1 on virus fusion is not known. In this study, hRSV HR1 and HR2 were expressed as fusion protein separately in Escherichia coli system and their complex assembly and virus fusion inhibition effect have been analysed. It shows that both HR1 and HR2 (in the fusion form with 50-amino-acid fusion partner) of hRSV F protein give strong inhibition on virus fusion (IC(50) values are 1.68 and 2.93 microM, respectively) and they form stable six-helix bundle in vitro with both in the fusion protein form.

Six-helix bundle assembly and characterization of heptad repeat regions from the F protein of Newcastle disease virus.

Paramyxoviruses may adopt a similar fusion mechanism to other enveloped viruses, in which an anti-parallel six-helix bundle structure is formed post-fusion in the heptad repeat (HR) regions of the envelope fusion protein. In order to understand the fusion mechanism and identify fusion inhibitors of Newcastle disease virus (NDV), a member of the Paramyxoviridae family, we have developed an E. coli system that separately expresses the F protein HR1 and HR2 regions as GST fusion proteins. The purified cleaved HR1 and HR2 have subsequently been assembled into a stable six-helix bundle heterotrimer complex. Furthermore, both the GST fusion protein and the cleaved HR2 show virus-cell fusion inhibition activity (IC(50) of 1.07-2.93 microM). The solubility of the GST-HR2 fusion protein is much higher than that of the corresponding peptide. Hence this provides a plausible method for large-scale production of HR peptides as virus fusion inhibitors.