Development of CAR‐T therapy led to immediate success in the treatment of B cell leukemia. Manufacturing of therapy‐competent functional CAR‐T cells needs robust protocols for ex vivo/in vitro expansion of modified T‐cells. This step is challenging, especially if non‐viral low‐efficiency delivery protocols are used to generate CAR‐T cells. Modern protocols for CAR‐T cell expansion are imperfect since non‐specific stimulation results in rapid outgrowth of CAR‐negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. To develop a specific and improved protocol for CAR‐T cell expansion, cell‐derived membrane vesicles are taken advantage of, and the simple structural demands of the CAR‐antigen interaction. This novel approach is to make antigenic microcytospheres from common cell lines stably expressing surface‐bound CAR antigens, and then use them for stimulation and expansion of CAR‐T cells. The data presented in this article clearly demonstrate that this protocol produced antigen‐specific vesicles with the capacity to induce stronger stimulation, proliferation, and functional activity of CAR‐T cells than is possible with existing protocols. It is predicted that this new methodology will significantly advance the ability to obtain improved populations of functional CAR‐T cells for therapy.

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.

The leukocyte common antigen CD45 is a receptor tyrosine phosphatase and one of the most prevalent antigens found on the surface of blood cells. CD45 plays a crucial role in the initial stages of signal transmission from receptors of various immune cell types. Immunodeficiency, autoimmune disorders, and oncological diseases are frequently caused by gene expression disorders and imbalances in CD45 isoforms. Despite extensive research into the structure and functions of CD45, the molecular mechanisms behind its role in transmitting signals from T-cell receptors and chimeric antigen receptors remain not fully understood. It is of utmost importance to comprehend the structural features of CD45 and its function in regulating immune system cell activation to study oncological diseases and the impact of CD45 on lymphocytes and T cells modified by chimeric antigen receptors.

Background: Neuroblastoma is a childhood tumor that is usually fatal after relapse in high-risk patients. Most clinical trials of CAR-T therapy for neuroblastoma are based on targeting the disialoganglioside GD2. B7-H3, a protein from the immunoglobulin superfamily, is a specific marker for neuroblastoma and a number of other solid tumors. We conducted a preclinical study of three variants of anti-B7-H3 CAR-T cells in order to justify the selection of the best candidate for subsequent clinical trials. Methods: The expression level of B7-H3 was measured in a number of cell lines and neuroblastoma tissue samples via flow cytometry. The functional activity of CAR-T cells was compared using an NFAT-inducible reporter assay, a cytotoxicity test, cytokine production, and a repeated stimulation assay. Results: The obtained CAR-T cells carrying all resulting CAR variants specifically recognized and killed B7-H3-positive tumor cells in vitro. Nevertheless, TE9-28z and 8H9-28BBz demonstrated superior activation and cytokine production compared to the second-generation 8H9-BBz construct. TE9-28z and 8H9-28BBz exhibited functional differences in expansion, exhaustion markers, and cytokine secretion in co-cultures with target cells in vitro. In particular, TE9-28z induced higher IFNγ production, while 8H9-28BBz showed increased TNFα release. Despite comparable cytotoxicity, TE9-28z and 8H9-28BBz CAR-T cells exhibited varying persistence depending on the tumor type, and showed signs of functional exhaustion upon prolonged exposure to the target antigen. Conclusion: TE9-28z and 8H9-28BBz were selected for further preclinical development as promising candidates for the effective targeting of B7-H3-expressing malignancies.

Adoptive cell therapy (ACT) with T cells modified with a chimeric antigen receptor (CAR T cells) has dramatically improved outcomes in hematologic cancers. However, its efficacy in solid tumors, such as melanoma, is hampered by several factors. These include heterogeneous expression of tumor-associated antigens (TAA) and an immunosuppressive, profibrotic tumor microenvironment (TME), which restricts cytotoxic CAR T cells trafficking into the tumor, as well as their persistence and cytolytic activity. As a result, responses to CAR T cell monotherapy in melanoma and other solid tumors are typically weak, transient or even absent. Emerging evidence suggests that combining traditional chemotherapy with CAR T cell therapy can enhance the antitumor activity of CAR T cells in solid malignancies. Partial tumor cell killing by chemotherapy improves access to TAA and disrupts the TME by affecting the global structure of the tumor tissue. Here, we developed an immunocompetent syngeneic B16 melanoma mouse model to test a combination of classical dacarbazine (DTIC) chemotherapy with ACT with murine CAR T cells. B16-F10 (next as B16) melanoma cells were modified to express a human/murine hybrid epidermal growth factor receptor (EGFR) recognized by a murine CAR bearing a single-chain variable fragment (scFv) derived from cetuximab, an anti-EGFR monoclonal antibody approved for the treatment of colorectal and certain other solid tumors. Prior to CAR T cells administration, cyclophosphamide (CPA) pre-conditioning was used. We demonstrated that DTIC therapy followed by infusion of murine CAR T cells targeting the human/murine hybrid EGFR (EGFR mCAR T cells) provided superior tumor control and prolonged survival compared to monotherapy with either DTIC or EGFR mCAR T cells alone. These findings support the potential feasibility of a combined therapeutic strategy for human melanoma involving DTIC treatment followed by EGFR CAR T cells infusion after CPA pre-conditioning.