The Landscape of Neutralizing Monoclonal Antibodies (nAbs) for Treatment and Prevention of COVID-19.

Purpose: After nearly 3 years of the COVID-19 pandemic, even though a vast body of knowledge and products (including vaccines and treatments) have been developed and disseminated, the virus is still evolving and new variants arising. Consequently, thousands of lives continue to be lost. Neutralizing monoclonal antibodies (nAbs) are promising drugs that emerged to treat SARS-CoV-2. In the uncertainty of the current situation, there is the question of whether organizations should continue to invest in this technology. To help decision-making in scientifical and pharmaceutical organizations, it is of major importance to monitor the development of products and technologies. Therefore, the aim of this study is analyze the landscape of nAbs for COVID-19. Methods: The scenario of 473 biotherapeutics focusing on nAbs was evaluated using foresight techniques and a review of literature. Data were obtained from structured and semi-structured databases and processed for treatment, cleaning, consistency, validation, and enrichment. Results: We identified 227 nAbs and performed an extensive literature review of 16 nAbs in late clinical development, including development technologies, responses to variants of concern (VOCs), manufacturing, and clinical aspects. Conclusions: Even though the emergence of new VOCs is a threat to the effectiveness of this treatment, demanding constant genomic surveillance, the use of nAbs to treat and prevent COVID-19 will probably continue to be relevant due to excellent safety profiles and the possibility of immediate immunity transfer, especially in patients showing inadequate immunological response to vaccination. Therefore, we suggest that organizations should keep investing in improvements in this technology.

Initial development of biosimilar immune checkpoint blockers using HEK293 cells.

Antibodies that block interaction of immune checkpoint receptors with its ligands have revolutionized the treatment of several cancers. Despite the success of this approach, the high cost has been restricted the use of this class of drugs. In this context, the development of biosimilar can be an important strategy for reducing prices and expanding access after patent has been dropped. Here, we evaluated the use of HEK293 cells for transient expression of an immune checkpoint-blocking antibody as a first step for biosimilar development. Antibody light and heavy chain genes were cloned into pCI-neo vector and transiently expressed in HEK293 cells. The culture supernatant was then subjected to protein A affinity chromatography, which allowed to obtain the antibody with high homogeneity. For physicochemical comparability, biosimilar antibody and reference drug were analyzed by SDS-PAGE, isoelectric focusing, circular dichroism and fluorescence spectroscopy. The results indicated that the both antibodies have a high degree of structural similarity. Lastly, the biosimilar antibody binding capacity to target receptor was shown to be similar to reference product in ELISA and flow cytometry assays. These data demonstrate that the HEK293 system can be used as an important tool for candidate selection and early development of biosimilar antibodies.

Immune status of Fanconi anemia patients: decrease in T CD8 and CD56dim CD16+ NK lymphocytes.

Fanconi anemia (FA), a rare genetic disease in which patients' life is compromised mainly by hematological abnormalities and cancer prone, seems to be affected by subtle immune cell irregularities. Knowing that FA presents developmental abnormalities and, based on recent reports, suggesting that natural killer (NK) CD56(dim) and NK CD56(bright) correspond to sequential differentiation pathways, we investigated if there were changes on the total number of NK cells and subsets as well as on T CD4 and T CD8 lymphocytes and their ratio. A large sample of FA patients (n = 42) was used in this work, and the results were correlated to clinical hematological status of these patients. Among FA patients, a decreased proportion of T CD8(+) and NK CD56(dim)CD16(+) cells were observed when compared to healthy controls as well as an imbalance of the subsets NK lymphocytes. Data suggest that FA patients might have a defective cytotoxic response due to the lower number of cytotoxic cells as well as impairment in the differentiation process of the NK cells subsets which may be directly related to impairment of the immune surveillance observed in these patients.

Age-related changes in natural killer cell receptors from childhood through old age.

Most studies on natural killer (NK) cells and aging have focused on overall cell numbers and global cytotoxic activity. NK cell functions are controlled by surface receptors belonging to three major families: killer cell immunoglobulin-like receptors (KIRs), natural cytotoxicity receptors (NCRs), and C-type lectins. The expression of these receptors was investigated from childhood through old age in T, NKT- and NK cells and also in the CD56(dim) (cytotoxic) and CD56(bright) (responsible for cytokine production) NK cell subsets. A decrease in the expression of activating receptors (NKp30 and NKp46) was observed in NK cells in elderly individuals. KIR expression was increased only in the CD56(bright) subset. Children presented similar results regarding expression of NKp30 and KIR, but not NKp46. NKG2D expression was decreased in T cells of elderly subjects. Analysis of KIR genotype revealed that KIR2DL5 and KIR2DS3 were significantly associated with old age. Cytotoxic activity was preserved from childhood through old age, suggesting that the increase of the absolute number of CD56(dim), observed in elderly, may represent a compensatory mechanism for the receptor expression alterations. This initial study provides the framework for more focused studies of this subject, which are necessary to determine whether the changing balance of NK receptor expression may influence susceptibility to infectious, inflammatory, and neoplastic diseases.

A relevância das células natural killer (NK) e killer immunoglobulin-like receptors (KIR) no transplante de células-tronco hematopoéticas (TCTH): [revisão] / The relevance of natural killer (NK) cells and killer immunoglobulin-like receptors (KIR) in hematopoietic stem cell transplantation (HSCT): [review]

As células natural killer (NK) foram identificadas há mais de 30 anos por sua capacidade de matar células tumorais e infectadas por vírus sem precisar de sensibilização prévia. No entanto, a forma como as células NK matam seus alvos ficou desconhecida por muito tempo. Na década de 90, a partir de várias observações, foi proposto que as células NK matariam células com a expressão diminuída de antígeno leucocitário humano (HLA), protegendo as células autólogas normais, o que ficou conhecido como hipótese do missing-self. Esta teoria foi confirmada através da descoberta de vários receptores, principalmente os da família killer immunoglobulin-like receptors (KIR), que reconhecem moléculas de HLA de classe I. Estes novos conceitos levaram à busca da importância dos receptores KIR no transplante de células-tronco hematopoéticas (TCTH). Foi sugerido que as disparidades de HLA entre o doador e o paciente poderiam ser reconhecidas por células NK levando à aloreatividade, o que ajudaria no efeito enxerto contra leucemia. No entanto, apesar de alguns resultados promissores, até hoje, os diferentes estudos sobre o assunto não chegaram a um consenso. Nesta revisão, será abordada a relevância das células NK e dos receptores KIR nos diferentes tipos de TCTH.

Natural killer (NK) cells were identified over 30 years ago by their ability to kill cancer and virally infected cells without prior sensitization. For years the recognition mechanisms of target cells were unknown, until the 1990s when the "missing-self" hypothesis was proposed. According to this theory, although tolerant to normal autologous cells, NK cells can recognize and attack cells that have down-regulated human leukocyte antigen (HLA) class I molecules. The discovery of killer immunoglobulin-like receptors (KIR) that specifically recognize HLA class I molecules corroborated this hypothesis. These new concepts point to the importance of studying KIR in hematopoietic stem cell transplantation (HSCT). HLA disparities between the donor and patient in HSCT may be distinguished by NK cells leading to alloreactivity. Even though there are some promising results, until now existing studies have not reached any consensus. Here, we will review the relevance of NK cells and KIR in the different types of HSC.

Rhodamine 123 efflux in human subpopulations of hematopoietic stem cells: comparison between bone marrow, umbilical cord blood and mobilized peripheral blood CD34+ cells.

Hematopoietic stem cells (HSC) can be identified by the expression of the CD34 molecule. CD34+ cells are found in bone marrow (BM), umbilical cord blood (UCB) and in mobilized peripheral blood (PB). CD34+ cells express P-glycoprotein (Pgp), a product of the multidrug resistance (MDR) gene. Pgp activity can be measured by the efflux of the dye Rhodamine 123 (Rho 123) and can be blocked by verapamil. Transport activity in HSC suggests that Pgp could have a functional role in stem cell differentiation. This study compared the number of CD34+ cells with Pgp activity measured by efflux of Rho 123 in the hematopoietic population obtained from different sources. Samples were analysed for their content of CD34+ cells, and BM had a significantly higher amount of CD34+ cells compared to UCB, mobilized PB and normal PB. When the frequency of Rholow cells was studied among the CD34+ population, an enrichment of cells with Pgp activity was observed. The frequency in BM was significantly lower than that in UCB and mobilized PB. The low retention of Rho 123 could be modified by verapamil, indicating that the measurements reflected dye efflux due to Pgp activity. Although UCB and mobilized PB had a lower number of CD34+ cells compared to BM, the total number of CD34+ cells with Pgp activity was similar in the three tissues. The different profiles may indicate the existence of subpopulations of stem cells or different stages of cellular differentiation detected by the extrusion of the dye Rho 123.