Strategies to Improve Chimeric Antigen Receptor Therapies for Neuroblastoma

Chimeric antigen receptors (CARs) are among the curative immunotherapeutic approaches that exploit the antigen specificity and cytotoxicity function of potent immune cells against cancers. Neuroblastomas, the most common extracranial pediatric solid tumors with diverse characteristics, could be a promising candidate for using CAR therapies. Several methods harness CAR-modified cells in neuroblastoma to increase therapeutic efficiency, although the assessment has been less successful. Regarding the improvement of CARs, various trials have been launched to overcome insufficient capacity. However, the reasons behind the inadequate response against neuroblastoma of CAR-modified cells are still not well understood. It is essential to update the present state of comprehension of CARs to improve the efficiency of CAR therapies. This review summarizes the crucial features of CARs and their design for neuroblastoma, discusses challenges that impact the outcomes of the immunotherapeutic competence, and focuses on devising strategies currently being investigated to improve the efficacy of CARs for neuroblastoma immunotherapy

Homodimers of Vanillin and Apocynin Decrease the Metastatic Potential of Human Cancer Cells by Inhibiting the FAK/PI3K/Akt Signaling Pathway

The spread of cancer cells to distant organs, in a process called metastasis, is the main factor that contributes to most death in cancer patients. Vanillin, the vanilla flavoring agent, has been shown to suppress metastasis in a mouse model. Here, we evaluated the antimetastatic potential of the food additive divanillin, the homodimer of vanillin, and their structurally related compounds, apocynin and diapocynin, in hepatocellular carcinoma cells. The Transwell invasion assay showed that the dimeric forms exhibited a potency higher than those of vanillin and apocynin in inhibiting invasion, with IC₅₀ values of 23.3 ± 7.4 to 41.3 ± 4.2 μM for the dimers, which are 26–34-fold lower than IC₅₀ values of vanillin and apocynin (<i>p</i> < 0.05). Both monomeric and dimeric forms target regulation of the invasion process by inhibiting phosphorylation of FAK and Akt. Molecular docking studies suggested that the dimers should bind more tightly than vanillin and apocynin to the Y397 pocket of the FAK FERM domain. Thus, the food additive divanillin has antimetastatic potential greater than that of the flavoring agent vanillin

A novel anti-membrane CD30 single-chain variable fragment discovered from the human phage library: A potential targeted immunotherapy.

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

Immunophenotype of the engineered T cells detected by flow cytometry.

Each group of T cells consisted of almost 100% CD3+ T cells, divided into CD8+ T cells and CD4+ T cells. The proportion of CD8+ T cells was higher than that of CD4+ T cells. Each population was classified into four subpopulations: naïve T cells (CD62L+, CD45RA+), effector T cells (CD62L-, CD45RA+), effector memory T cells (CD62L-, CD45RA-), and central memory T cells (CD62L+, CD45RA-). Effector memory and central memory T cells in CD4+ and CD8+ T cells showed higher levels than naïve and effector T cells. (TIF)</p

Clonal selection of mCD30-bound ScFvs.

(A) The screening of E. coli clones that produced mCD30-bound ScFvs by indirect ELISA used the mCD30 peptide as a peptide-coated plate and the blocking buffer as a non-coated plate (negative control) in the side-by-side wells. Bound ScFvs were selected from the OD405nm signal above the mean + 3 SD of the background binding control (lysate of original E. coli HB2151; HB) (cut off = 0.03875). Lysates of 12 scFv-positive E. coli clones (#A3, A4, A8, A10, A14, A15, A17, A31, A32, A35, A36, and A37) produced soluble ScFvs bound to the mCD30 peptide. (B) The statistical significance between the bound, unbound ScFvs groups and the HB control was analyzed by one-way ANOVA and Tukey’s post hoc test.</p

The screening of membrane CD30 (mCD30)-bound phages.

The screening of E. coli HB2151 clones that were transfected with mCD30-bound phages used direct PCR. The size of PCR amplicons was separated by gel electrophoresis. The sizes of a human single-chain Fv (huscFv), a human single-domain (husdFv) fragment, and an empty vector are about 1,000 bp, 500–700 bp, and 250 bp, respectively. Thirty-three clones, as indicated with red arrows, were carrying huscFv of mCD30-bound phages, which were clone #A2, A3, A4, A6, A8, A9, A10, A11, A12, A14, A15, A16, A17, A19, A21, A22, A23, A24, A26, A31, A32, A33, A35, A36, A37, A41, A43, A46, A49, A50, A56, A58, and A59. (TIF)</p

The detection of CD30 expression.

The CD30 antigen on the cell surface of healthy target cell lines (K562 and SupB15) was stained with PE anti-human CD30 antibody, and the expression was detected by flow cytometry. CD30 expresses at a consistently high level on the cell surface of K562 cells but not of SupB15 cells. The black-shaded histogram represented the unstain, and the blue showed CD30 staining. (TIF)</p

The thermodynamic parameters of the purified protein binding determined by the isothermal titration calorimetry (ITC).

Titrations of membrane CD30 (mCD30) and recombinant CD30 (rCD30) peptides into the ScFv#A4 and the ScFv#AK (positive control) revealed an exothermic association based on favorable enthalpy and unfavorable entropy with a binding affinity (Kd) of 421 nM and 1 pM, respectively. The stoichiometry (N) of the ScFv#A4 and the ScFv#AK showed 1.66 ± 1.93 and 1.10 ± 2.87, respectively. (n = 3). (DOCX)</p

Computerized simulation of the 3D structure of mCD30-ScFv complex and presumptive contact interfaces.

(A) The complexing of the mCD30 peptide (cyan) and the ScFv (grey) of E. coli clone #A4 after simulation from the molecular docking showed the contact interaction at the complementarity-determining regions (CDRs) binding sites of the ScFv. (B) Contact interfaces with hydrogen bonding between mCD30 and CDRs of ScFv#A4 at G102, T104, F105, Y109, D113, N172, L173, T196, and a salt bridge at E100).</p

The confirmation of soluble-bound human single-chain Fv proteins (HuscFvs) expression in <i>E</i>. <i>coli</i> fraction.

The expression of soluble bound HuscFvs in E. coli fraction extracted from bacteria cells was confirmed by SDS-PAGE (upper panels) and western blot (lower panels) using an anti-E tag antibody as the E-tagged-HuscFv tracer. The soluble bound-HuscFvs (approximately 35 kDa) from human single-chain Fv (huscfv)-positive E. coli clone #A3, A4, A8, A10, A14, A15, A17, A31, A32, A35, A36, and A37 were detected. (TIF)</p