Hodgkin’s lymphoma and anaplastic large cell lymphoma, especially relapsed or refractory diseases, could recently be cured by CD30-targeted immunotherapy. However, the CD30 antigen releases the soluble ectodomain of CD30, which might obscure the targeted therapy. Therefore, the membrane epitope of CD30 (mCD30), left on the cancer cells, might be a prospective target for lymphoma treatment. The discovery of novel mCD30 monoclonal antibodies (mAbs) using phage technology yielded 59 potential human single-chain variable fragments (HuscFvs). Ten candidate HuscFv clones have been selected based on various methods, i.e., direct PCR, ELISA and western blot assays, and nucleotide sequencing techniques. Fortunately, only one potential HuscFv clone, clone #A4, was determined by the prediction of HuscFv-peptide molecular docking and the binding affinity test using isothermal titration calorimetry. Finally, we proved that the HuscFv #A4, which had a binding affinity (Kd) of 421e-9 ± 2.76e-6 M, might be the novel mCD30 mAb. We generated chimeric antigen receptor-modified T lymphocytes using HuscFv #A4 as an antigen detection part (anti-mCD30-H4CART). The cytotoxicity assay of anti-mCD30-H4CART cells showed significant eradication of the CD30-expressing cell line, K562 (p = 0.0378). We found a novel mCD30 HuscFv using human phage technology. We systematically examined and proved that our HuscFv #A4 could specifically eradicate CD30-expressing cancers.
ObjectivesMultiple myeloma (MM) accounts for 10% of hematologic malignancies. However, most of the patients suffered from relapsed/refractory disease. We would like to expand CAR T cell therapy to treat MM using our current platform.MethodsBCMA CAR T lymphocytes were generated for volunteers or MM patients. The transduction efficiency was detected by the ddPCR technique. Immunophenotyping and exhaustion markers were monitored by flow cytometry. The efficacy of BCMA CAR T cells was tested using coculturing with BCMA CAR or mock, and the positive and negative targets, K562/hBCMA‐ECTM and K562, respectively.ResultsBCMA CAR T cells were generated from consented volunteers or MM patients and could be detected CAR BCMA expression at a mean of 4.07 ± 1.95 or 4.65 ± 1.21 copies/cell, respectively. Those modified T cells were primarily effector memory T cells. Our BCMA CAR T cells could explicitly eradicate the K562/hBCMA‐ECTM cell line while the K562 cell line survived. Interestingly, the BCMA CAR, mock T cells, and peripheral blood mononuclear cells from MM patients expressed similar levels of the exhaustion makers, TIM‐3, LAG‐3, and PD1.ConclusionsOur BCMA CAR T cells, mainly effector/effector memory, could eliminate BCMA‐expressing cells in vitro and had similar levels of exhaustion markers among different populations.
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