Switchable targeting of solid tumors by BsCAR T cells Stepanov, Alexey V; Kalinin, Roman S; Shipunova, Victoria O ...
Proceedings of the National Academy of Sciences - PNAS,
11/2022, Letnik:
119, Številka:
46
Journal Article
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The development of chimeric antigen receptor (CAR) T cell therapy has become a critical milestone in modern oncotherapy. Despite the remarkable in vitro effectiveness, the problem of safety and ...efficacy of CAR T cell therapy against solid tumors is challenged by the lack of tumor-specific antigens required to avoid on-target off-tumor effects. Spatially separating the cytotoxic function of CAR T cells from tumor antigen recognition provided by protein mediators allows for the precise control of CAR T cell cytotoxicity. Here, the high affinity and capability of the bacterial toxin-antitoxin barnase-barstar system were adopted to guide CAR T cells to solid tumors. The complementary modules based on (1) ankyrin repeat (DARPin)-barnase proteins and (2) barstar-based CAR (BsCAR) were designed to provide switchable targeting to tumor cells. The alteration of the DARPin-barnase switches enabled the targeting of different tumor antigens with a single BsCAR. A gradual increase in cytokine release and tunable BsCAR T cell cytotoxicity was achieved by varying DARPin-barnase loads. Switchable BsCAR T cell therapy was able to eradicate the HER2
ductal carcinoma in vivo. Guiding BsCAR T cells by DARPin-barnase switches provides a universal approach for a controlled multitargeted adoptive immunotherapy.
The human ErbB3 receptor confers resistance to the pharmacological inhibition of EGFR and HER2 receptor tyrosine kinases in cancer, which makes it an important therapeutic target. Several anti-ErbB3 ...monoclonal antibodies that are currently being developed are all classical immunoglobulins. We took a different approach and discovered a group of novel heavy-chain antibodies targeting the extracellular domain of ErbB3 via a phage display of an antibody library from immunized llamas. We first produced three selected single-domain antibodies, named BCD090-P1, BCD090-M2, and BCD090-M456, in E. coli, as SUMO fusions that yielded up to 180 mg of recombinant protein per liter of culture. Then, we studied folding, aggregation, and disulfide bond formation, and showed their ultimate stability with half-denaturation of the strongest candidate, BCD090-P1, occurring in 8 M of urea. In surface plasmon resonance experiments, two most potent antibodies, BCD090-P1 and BCD090-M2, bound the extracellular domain of ErbB3 with 1.6 nM and 15 nM affinities for the monovalent interaction, respectively. The receptor binding was demonstrated by immunofluorescent confocal microscopy on four different ErbB3+ cancer cell lines. We observed that BCD090-P1 and BCD090-M2 bind noncompetitively to two distinct epitopes on the receptor. Both antibodies inhibited the ErbB3-driven proliferation of MCF-7 breast adenocarcinoma cells and HER2-overexpressing SK-BR-3 cells, with the EC50 in the range of 0.1–25 μg/mL. BCD090-M2 directly blocks ligand binding, whereas BCD090-P1 does not compete with the ligand and presumably acts through a distinct allosteric mechanism. We anticipate that these llama antibodies can be used to engineer new biparatopic anti-ErbB3 or bispecific anti-ErbB2/3 antibodies.
Single-wavelength anomalous diffraction (SAD) is the most common method for
de novo elucidation of macromolecular structures by X-ray crystallography. It requires an anomalous scatterer in a crystal ...to calculate phases. A recent study by Panneerselvam
et al. emphasized the utility of cadmium ions for SAD phasing at the standard synchrotron wavelength of 1 Å. Here we show that cadmium is also useful for phasing of crystals collected in-house with CuKα radiation. Using a crystal of single-domain antibody as an experimental model, we demonstrate how cadmium SAD can be conveniently employed to solve a CuKα dataset. We then discuss the factors which make this method generally applicable.
During the ongoing coronavirus disease COVID-19 pandemic, many individuals were infected with and have cleared the virus, developing virus-specific antibodies and effector/memory T cells. An ...important unanswered question is what levels of T cell and antibody responses are sufficient to protect from the infection.
In 5340 Moscow residents, we evaluated anti-SARS-CoV-2 IgM/IgG titers and frequencies of the T cells specific to the membrane, nucleocapsid, and spike proteins of SARS-CoV-2, using IFNγ ELISpot assay. Additionally, we evaluated the fractions of virus-specific CD4+ and CD8+ T cells using intracellular staining of IFNγ and IL2 followed by flow cytometry. We analyzed the COVID-19 rates as a function of the assessed antibody and T cell responses, using the Kaplan-Meyer estimator method, for up to 300 days post-inclusion.
We showed that T cell and antibody responses are closely interconnected and are commonly induced concurrently. Magnitudes of both responses inversely correlated with infection probability. Individuals positive for both responses demonstrated the highest levels of protectivity against the SARS-CoV-2 infection. A comparable level of protection was found in individuals with antibody response only, while the T cell response by itself granted only intermediate protection.
We found that the contribution of the virus-specific antibodies to protection against the SARS-CoV-2 infection is more pronounced than that of the T cells. The data on the virus-specific IgG titers may be instructive for making decisions in personalized health care and public anti-COVID-19 policies.
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