Fcγ Receptor-Dependent Internalization and Off-Target Cytotoxicity of Antibody-Drug Conjugate Aggregates

Purpose Antibody-drug conjugates (ADCs), which are monoclonal antibodies (mAbs) conjugated with highly toxic payloads, achieve high tumor killing efficacy due to the specific delivery of payloads in accordance with mAbs’ function. On the other hand, the conjugation of payloads often increases the hydrophobicity of mAbs, resulting in reduced stability and increased aggregation. It is considered that mAb aggregates have potential risk for activating Fcγ receptors (FcγRs) on immune cells, and are internalized into cells via FcγRs. Based on the mechanism of action of ADCs, the internalization of ADCs into target-negative cells may cause the off-target toxicity. However, the impacts of aggregation on the safety of ADCs including off-target cytotoxicity have been unclear. In this study, we investigated the cytotoxicity of ADC aggregates in target-negative cells. Methods The ADC aggregates were generated by stirring stress or thermal stress. The off-target cytotoxicity of ADC aggregates was evaluated in several target-negative cell lines, and FcγR-activation properties of ADC aggregates were characterized using a reporter cell assay. Results Aggregation of ADCs enhanced the off-target cytotoxicity in several target-negative cell lines compared with non-stressed ADCs. Notably, ADC aggregates with FcγR-activation properties showed dramatically enhanced cytotoxicity in FcγR-expressing cells. The FcγR-mediated off-target cytotoxicity of ADC aggregates was reduced by using a FcγR-blocking antibody or Fc-engineering for silencing Fc-mediated effector functions. Conclusions These results indicated that FcγRs play an important role for internalization of ADC aggregates into non-target cells, and the aggregation of ADCs increases the potential risk for off-target toxicity.

Glutamine Availability Controls BCR/Abl Protein Expression and Functional Phenotype of Chronic Myeloid Leukemia Cells Endowed with Stem/Progenitor Cell Potential

This study was directed to characterize the role of glutamine in the modulation of the response of chronic myeloid leukemia (CML) cells to low oxygen, a main condition of hematopoietic stem cell niches of bone marrow. Cells were incubated in atmosphere at 0.2% oxygen in the absence or the presence of glutamine. The absence of glutamine markedly delayed glucose consumption, which had previously been shown to drive the suppression of BCR/Abl oncoprotein (but not of the fusion oncogene BCR/abl) in low oxygen. Glutamine availability thus emerged as a key regulator of the balance between the pools of BCR/Abl protein-expressing and -negative CML cells endowed with stem/progenitor cell potential and capable to stand extremely low oxygen. These findings were confirmed by the effects of the inhibitors of glucose or glutamine metabolism. The BCR/Abl-negative cell phenotype is the best candidate to sustain the treatment-resistant minimal residual disease (MRD) of CML because these cells are devoid of the molecular target of the BCR/Abl-active tyrosine kinase inhibitors (TKi) used for CML therapy. Therefore, the treatments capable of interfering with glutamine action may result in the reduction in the BCR/Abl-negative cell subset sustaining MRD and in the concomitant rescue of the TKi sensitivity of CML stem cell potential. The data obtained with glutaminase inhibitors seem to confirm this perspective.

Detection and infectivity of SARS-CoV-2 in exhumated corpses

Postmortem detection of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) after the exhumation of a corpse can become important, e.g. in the case of subsequent medical malpractice allegations. To date, data on possible detection periods [e.g. by reverse transcription polymerase chain reaction (RT-PCR)] or on the potential infectivity of the virus after an exhumation are rare. In the present study, these parameters were examined in two cases with a time span of approximately 4 months between day of death and exhumation. Using SARS-CoV-2 RT-PCR on swabs of both lungs and the oropharynx detection was possible with cycle threshold (Ct) values of about 30 despite signs of beginning decay. RT-PCR testing of perioral and perinasal swabs and swabs collected from the inside of the body bag, taken to estimate the risk of infection of those involved in the exhumation, was negative. Cell culture-based infectivity testing was negative for both, lung and oropharyngeal swabs. In one case, RT-PCR testing at the day of death of an oropharyngeal swab showed almost identical Ct values as postmortem testing of an oropharyngeal swab, impressively demonstrating the stability of viral RNA in the intact corpse. However, favorable climatic conditions in the grave have to be taken into account, as it was wintertime with constant low temperatures. Nevertheless, it was possible to demonstrate successful postmortem detection of SARS-CoV-2 infection following exhumation even after months in an earth grave.

Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development

Background Human oligodendrocyte precursor cells (hOPCs) are an important source of myelinating cells for cell transplantation to treat demyelinating diseases. Myelin oligodendrocytes develop from migratory and proliferative hOPCs. It is well known that NG2 and A2B5 are important biological markers of hOPCs. However, the functional differences between the cell populations represented by these two biomarkers have not been well studied in depth. Objective To study the difference between NG2 and A2B5 cells in the development of human oligodendrocyte progenitor cells. Methods Using cell sorting technology, we obtained NG2+/−, A2B5+/− cells. Further research was then conducted via in vitro cell proliferation and migration assays, single-cell sequencing, mRNA sequencing, and cell transplantation into shiverer mice. Results The proportion of PDGFR-α + cells in the negative cell population was higher than that in the positive cell population. The migration ability of the NG2+/−, A2B5+/− cells was inversely proportional to their myelination ability. The migration, proliferation, and myelination capacities of the negative cell population were stronger than those of the positive cell population. The ability of cell migration and proliferation of the four groups of cells from high to low was: A2B5− > NG2− > NG2+ > A2B5+. The content of PDGFR-α+ cells and the ability of cell differentiation from high to low was: NG2− > A2B5− > A2B5+ > NG2+. Conclusion In summary, NG2+ and A2B5+ cells have poor myelination ability due to low levels of PDGFR-α+ cells. Therefore, hOPCs with a higher content of PDGFR-α+ cells may have a better effect in the cell transplantation treatment of demyelinating diseases.

Pseudogene-mediated gene conversion after CRISPR-Cas9 editing demonstrated by partial CD33 conversion with SIGLEC22P

Although gene editing workflows typically consider the possibility of off-target editing, pseudogene-directed homology repair has not, to our knowledge, been reported previously. Here, we employed a CRISPR-Cas9 strategy for targeted excision of exon 2 in CD33 in U937 human monocytes. Candidate clonal cell lines were screened by using a clinically relevant antibody known to label the IgV domain encoded by exon 2 (P67.6, gemtuzumab). In addition to the anticipated deletion of exon 2, we also found unexpected P67.6-negative cell lines which had apparently retained CD33 exon 2. Sequencing revealed that these lines underwent gene conversion from the nearby SIGLEC22P pseudogene during homology repair that resulted in three missense mutations relative to CD33. Ectopic expression studies confirmed that the P67.6 epitope is dependent upon these amino acids. In summation, we report that pseudogene-directed homology repair can lead to aberrant CRISPR gene editing.

Mutational Landscape of Virus- and UV-Associated Merkel Cell Carcinoma Cell Lines Is Comparable to Tumor Tissue

Merkel cell carcinoma (MCC) is a rare, highly aggressive cutaneous malignancy that is either associated with the integration of the Merkel cell polyomavirus or chronic UV exposure. These two types of carcinogenesis are reflected in characteristic mutational features present in MCC tumor lesions. However, the genomic characteristics of MCC cell lines used as preclinical models are not well established. Thus, we analyzed the exomes of three virus-negative and six virus-positive MCC cell lines, all showing a classical neuroendocrine growth pattern. Virus-negative cell lines are characterized by a high tumor mutational burden (TMB), UV-light-induced DNA damage, functionally relevant coding mutations, e.g., in RB1 and TP53, and large amounts of copy number variations (CNVs). In contrast, virus-positive cell lines have a low TMB with few coding mutations and lack prominent mutational signatures, but harbor characteristic CNVs. One of the virus-negative cell lines has a local MYC amplification associated with high MYC mRNA expression. In conclusion, virus-positive and -negative MCC cell lines with a neuroendocrine growth pattern resemble mutational features observed in MCC tissue samples, which strengthens their utility for functional studies.

[18F]PARPi Imaging Is Not Affected by HPV Status In Vitro

Background. Human papillomavirus- (HPV-) associated oropharyngeal squamous cell carcinomas (OPSCCs) are clinically and pathologically distinct from HPV-negative tumors. Here, we explore whether HPV affects functional biomarkers, including γH2AX, RAD51, and PARP1. Moreover, the role of [18F]PARPi as a broadly applicable imaging tool for head and neck carcinomas is investigated. Methods. HPV-positive and HPV-negative cell lines were used to evaluate the γH2AX, RAD51, and PARP1 expression with immunoblotting and immunofluorescence. Effects of external beam ionizing radiation were investigated in vitro, and survival was investigated via colony-formation assay. [18F]PARPi uptake experiments were performed on HPV-negative and HPV-positive cell lines to quantify PARP1 expression. PARP1 IHC and γH2AX foci were quantified using patient-derived oropharyngeal tumor specimens. Results. Differences in DNA repair were detected, showing higher RAD51 and γH2AX expression in HPV-positive cell lines. Clonogenic assays confirm HPV-positive cell lines to be significantly more radiosensitive. PARP1 expression levels were similar, irrespective of HPV status. Consequently, [18F]PARPi uptake assays demonstrated that this tracer is internalized in cell lines independently from their HPV status. Conclusion. The HPV status, often used clinically to stratify patients, did not affect PARP1 levels, suggesting that PARP imaging can be performed in both HPV-positive and HPV-negative patients. This study confirms that the PET imaging agent [18F]PARPi could serve as a general clinical tool for oropharyngeal cancer patients.