Dromedary camel nanobodies broadly neutralize SARS-CoV-2 variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is a trimer of S1/S2 heterodimers with three receptor-binding domains (RBDs) at the S1 subunit for human angiotensin-converting enzyme 2 (hACE2). Due to their small size, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. To isolate high-affinity nanobodies, large libraries with great diversity are highly desirable. Dromedary camels (Camelus dromedarius) are natural reservoirs of coronaviruses like Middle East respiratory syndrome CoV (MERS-CoV) that are transmitted to humans. Here, we built large dromedary camel VHH phage libraries to isolate nanobodies that broadly neutralize SARS-CoV-2 variants. We isolated two VHH nanobodies, NCI-CoV-7A3 (7A3) and NCI-CoV-8A2 (8A2), which have a high affinity for the RBD via targeting nonoverlapping epitopes and show broad neutralization activity against SARS-CoV-2 and its emerging variants of concern. Cryoelectron microscopy (cryo-EM) complex structures revealed that 8A2 binds the RBD in its up mode with a long CDR3 loop directly involved in the ACE2 binding residues and that 7A3 targets a deeply buried region that uniquely extends from the S1 subunit to the apex of the S2 subunit regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, 7A3 efficiently protected transgenic mice expressing hACE2 from the lethal challenge of variants B.1.351 or B.1.617.2, suggesting its therapeutic use against COVID-19 variants. The dromedary camel VHH phage libraries could be helpful as a unique platform ready for quickly isolating potent nanobodies against future emerging viruses.

rhIL-7-hyFc, a long-acting interleukin-7, improves efficacy of CAR-T cell therapy in solid tumors.

BACKGROUND: Chimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable remission in patients with B-cell malignancies. However, its efficacy in treating solid tumors remains limited. Here, we investigated a combination therapy approach using an engineered long-acting interleukin (IL)-7 (rhIL-7-hyFc or NT-I7) and CAR-T cells targeting three antigens, glypican-2 (GPC2), glypican-3 (GPC3), and mesothelin (MSLN), against multiple solid tumor types including liver cancer, neuroblastoma, ovarian cancer, and pancreatic cancer in mice. METHODS: CAR-T cells targeting GPC2, GPC3, and MSLN were used in combination with NT-I7 to assess the anticancer activity. Xenograft tumor models, including the liver cancer orthotopic model, were established using NOD scid gamma mice engrafted with cell lines derived from hepatocellular carcinoma, neuroblastoma, ovarian cancer, and pancreatic cancer. The mice were monitored by bioluminescence in vivo tumor imaging and tumor volume measurement using a caliper. Immunophenotyping of CAR-T cells on NT-I7 stimulation was evaluated for memory markers, exhaust markers, and T-cell signaling molecules by flow cytometry and western blotting. RESULTS: Compared with the IL-2 combination, preclinical evaluation of NT-I7 exhibited regression of solid tumors via enhanced occupancy of CD4+ CAR-T, improved T-cell expansion, reduced exhaustion markers (programmed cell death protein 1 or PD-1 and lymphocyte-activation gene 3 or LAG-3) expression, and increased generation of stem cell-like memory CAR-T cells. The STAT5 pathway was demonstrated to be downstream of NT-I7 signaling, mediated by increased expression of the IL-7 receptor expression in CAR-T cells. Furthermore, CAR-T cells improved efficacy against tumors with low antigen density when combined with NT-I7 in mice, presenting an avenue for patients with heterogeneous antigenic profiles. CONCLUSION: This study provides a rationale for NT-I7 plus CAR-T cell combination therapy for solid tumors in humans.

Camel nanobody-based B7-H3 CAR-T cells show high efficacy against large solid tumours.

Rational design of chimeric antigen receptor T (CAR-T) cells based on the recognition of antigenic epitopes capable of evoking the most potent CAR activation is an important objective in optimizing immune therapy. In solid tumors, the B7-H3 transmembrane protein is an emerging target that harbours two distinct epitope motifs, IgC and IgV, in its ectodomain. Here, we generate dromedary camel nanobodies targeting B7-H3 and demonstrate that CAR-T cells, based on the nanobodies recognizing the IgC but not IgV domain, had potent antitumour activity against large tumors in female mice. These CAR-T cells are characterized by highly activated T cell signaling and significant tumor infiltration. Single-cell transcriptome RNA sequencing coupled with functional T-cell proteomics analysis uncovers the top-upregulated genes that might be critical for the persistence of polyfunctional CAR-T cells in mice. Our results highlight the importance of the specific target antigen epitope in governing optimal CAR-T activity and provide a nanobody-based B7-H3 CAR-T product for use in solid tumor therapy.

A novel PD-L1-targeted shark VNAR single-domain-based CAR-T cell strategy for treating breast cancer and liver cancer.

Chimeric antigen receptor (CAR)-T cell therapy shows excellent potency against hematological malignancies, but it remains challenging to treat solid tumors, mainly because of a lack of appropriate antigenic targets and an immunosuppressive tumor microenvironment (TME). The checkpoint molecule programmed death-ligand 1 (PD-L1) is widely overexpressed in multiple tumor types, and the programmed death-ligand 1 (PD-1)/PD-L1 interaction is a crucial mediator of immunosuppression in the TME. Here we constructed a semi-synthetic shark VNAR phage library and isolated anti-PD-L1 single-domain antibodies. Among these VNARs, B2 showed cross-reactivity to human, mouse, and canine PD-L1, and it partially blocked the interaction of human PD-1 with PD-L1. CAR (B2) T cells specifically lysed human breast cancer and liver cancer cells by targeting constitutive and inducible expression of PD-L1 and hindered tumor metastasis. Combination of PD-L1 CAR (B2) T cells with CAR T cells targeted by GPC3 (a liver cancer-specific antigen) regresses liver tumors in mice. We concluded that PD-L1-targeted shark VNAR single-domain-based CAR-T therapy is a novel strategy to treat breast and liver cancer. This study provides a rationale for potential use of PD-L1 CAR-T cells as a monotherapy or in combination with a tumor-specific therapy in clinical studies.

Camel nanobodies broadly neutralize SARS-CoV-2 variants.

With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two V H H nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of ≥5 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants. ONE-SENTENCE SUMMARY: Dromedary camel ( Camelus dromedarius ) V H H phage libraries were built for isolation of the nanobodies that broadly neutralize SARS-CoV-2 variants.