It has been more than three decades since the first monoclonal antibody was approved by the United States Food and Drug Administration (US FDA) in 1986, and during this time, antibody engineering has dramatically evolved. Current antibody drugs have increasingly fewer adverse effects due to their high specificity. As a result, therapeutic antibodies have become the predominant class of new drugs developed in recent years. Over the past five years, antibodies have become the best-selling drugs in the pharmaceutical market, and in 2018, eight of the top ten bestselling drugs worldwide were biologics. The global therapeutic monoclonal antibody market was valued at approximately US$115.2 billion in 2018 and is expected to generate revenue of $150 billion by the end of 2019 and $300 billion by 2025. Thus, the market for therapeutic antibody drugs has experienced explosive growth as new drugs have been approved for treating various human diseases, including many cancers, autoimmune, metabolic and infectious diseases. As of December 2019, 79 therapeutic mAbs have been approved by the US FDA, but there is still significant growth potential. This review summarizes the latest market trends and outlines the preeminent antibody engineering technologies used in the development of therapeutic antibody drugs, such as humanization of monoclonal antibodies, phage display, the human antibody mouse, single B cell antibody technology, and affinity maturation. Finally, future applications and perspectives are also discussed.

Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.

Human embryonic stem cells (hESCs) are unique pluripotent cells capable of self-renewal and differentiation into all three germ layers. To date, more cell surface markers capable of reliably identifying hESCs are needed. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein expressed in several progenitor cell populations and cancers. It has been used to enrich cells with tumor-initiating activity in xenograft transplantation studies. Here, we comprehensively profile the expression of EpCAM by immunofluorescence microscopy, Western blotting, and flow cytometry using an anti-EpCAM monoclonal antibody (mAb) OC98-1. We found EpCAM to be highly and selectively expressed by undifferentiated rather than differentiated hESCs. The protein and transcript level of EpCAM rapidly diminished as soon as hESC had differentiated. This silencing was closely and exclusively associated with the radical transformation of histone modification at the EpCAM promoter. Moreover, we demonstrated that the dynamic pattern of lysine 27 trimethylation of histone 3 was conferred by the interplay of SUZ12 and JMJD3, both of which were involved in maintaining hESC pluripotency. In addition, we used chromatin immunoprecipitation analysis to elucidate the direct regulation by EpCAM of several reprogramming genes, including c-MYC, OCT-4, NANOG, SOX2, and KLF4, to help maintain the undifferentiation of hESCs. Collectively, our results suggest that EpCAM might be used as a surface marker for hESC. The expression of EpCAM may be regulated by epigenetic mechanisms, and it is strongly associated with the maintenance of the undifferentiated state of hESCs.

The coronavirus disease 2019 (COVID-19) pandemic is an exceptional public health crisis that demands the timely creation of new therapeutics and viral detection. Owing to their high specificity and reliability, monoclonal antibodies (mAbs) have emerged as powerful tools to treat and detect numerous diseases. Hence, many researchers have begun to urgently develop Ab-based kits for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ab drugs for use as COVID-19 therapeutic agents. The detailed structure of the SARS-CoV-2 spike protein is known, and since this protein is key for viral infection, its receptor-binding domain (RBD) has become a major target for therapeutic Ab development. Because SARS-CoV-2 is an RNA virus with a high mutation rate, especially under the selective pressure of aggressively deployed prophylactic vaccines and neutralizing Abs, the use of Ab cocktails is expected to be an important strategy for effective COVID-19 treatment. Moreover, SARS-CoV-2 infection may stimulate an overactive immune response, resulting in a cytokine storm that drives severe disease progression. Abs to combat cytokine storms have also been under intense development as treatments for COVID-19. In addition to their use as drugs, Abs are currently being utilized in SARS-CoV-2 detection tests, including antigen and immunoglobulin tests. Such Ab-based detection tests are crucial surveillance tools that can be used to prevent the spread of COVID-19. Herein, we highlight some key points regarding mAb-based detection tests and treatments for the COVID-19 pandemic.

A growing body of evidence supports the coordination of mRNA synthesis and its subsequent processing events. Nuclear proteins harboring both WW and FF protein interaction modules bind to splicing factors as well as RNA polymerase II and may serve to link transcription with splicing. To understand how WW domains coordinate the assembly of splicing complexes, we used glutathione S-transferase fusions containing WW domains from CA150 or FBP11 in pull-down experiments with HeLa cell nuclear extract. The WW domains associate preferentially with the U2 small nuclear ribonucleoprotein and with splicing factors SF1, U2AF, and components of the SF3 complex. Accordingly, WW domain-associating factors bind to the 3 part of a pre-mRNA to form a pre-spliceosome-like complex. We performed both in vitro and in vivo splicing assays to explore the role of WW/FF domain-containing proteins in this process. However, although CA150 is associated with the spliceosome, it appears to be dispensable for splicing in vitro. Nevertheless, in vivo depletion of CA150 substantially reduced splicing efficiency of a reporter pre-mRNA. Moreover, overexpression of CA150 fragments containing both WW and FF domains activated splicing and modulated alternative exon selection, probably by facilitating 3 splice site recognition. Our results suggest an essential role of WW/FF domaincontaining factors in pre-mRNA splicing that likely occurs in concert with transcription in vivo.Gene expression in eukaryotic cells involves several steps, including transcription, mRNA processing, and export. Synthesis of mRNA by RNA polymerase II (Pol II) is coordinated with subsequent RNA processing events such as capping, splicing, and cleavage and polyadenylation (4,12,20,23,37). The largest subunit of RNA Pol II recruits mRNA processing activities during transcriptional elongation via its heptapeptide repeat-containing C-terminal domain (CTD) (4, 23). Truncation of the Pol II CTD results in reduced splicing in vivo (34). Under some circumstances, in vitro splicing of pre-mRNA can be stimulated by phosphorylated Pol II or CTD (22, 52). Several transcriptional activators or coactivators associate physically with small nuclear ribonucleoproteins (snRNPs) or serine/arginine-rich splicing factors (SR proteins) and thereby may modulate pre-mRNA splicing (18,29,35). Moreover, promoter structure affects alternative splice site selection during pre-mRNA splicing (11), and further analyses reveal that transcriptional activators differentially modulate the rate of transcriptional elongation, which in turn determines the outcome of two competing alternative splicing reactions (15,26,38). Nevertheless, several lines of evidence support the concept of a physical and functional coupling between transcription and splicing (12,20).Since the RNA Pol II CTD is implicated in promoting premRNA splicing, proteins that associate with the CTD may mediate this function. A two-hybrid screen previously revealed a set of RS domain-containing proteins via their interaction with the CTD (51). Amon...

Spinal muscular atrophy (SMA) is a recessive neuromuscular disorder caused by the homozygous loss of the SMN1 gene. The human SMN2 gene has a C-to-T transition at position ؉6 of exon 7 and thus produces exon 7-skipping mRNAs. However, we observed an unexpectedly high level of exon 7-containing SMN2 transcripts as well as SMN protein in testis of smn ؊/؊ SMN2 transgenic mice. Using affinity chromatography, we identified several SMN RNA-associating proteins in mouse testis and human HeLa cells, including hnRNP Q. The major hnRNP Q isoform, Q1, directly bound SMN exon 7 in the vicinity of nucleotide ؉6. Overexpression of hnRNP Q1 promoted the inclusion of exon 7 in SMN2, probably by activating the use of its upstream 3 splice site. However, the minor isoforms Q2/Q3 could antagonize the activity of hnRNP Q1 and induced exon 7 exclusion. Intriguingly, enhanced exon 7 inclusion was also observed upon concomitant depletion of three hnRNP Q isoforms. Thus, differential expression of hnRNP Q isoforms may result in intricate control of SMN precursor mRNA splicing. Here, we demonstrate that hnRNP Q is a splicing modulator of SMN, further underscoring the potential of hnRNP Q as a therapeutic target for SMA.Spinal muscular atrophy (SMA) is an autosomal recessive disorder that results in degeneration of motor neurons in the spinal cord, and it is one of the most common genetic diseases causing infant mortality (6, 33). SMA is primarily caused by homologous deletion or mutations of the survival motor neuron 1 (SMN1) gene (22). The duplicate gene, SMN2, in the human genome differs from SMN1 by a few nucleotides. The only difference in the coding region is the C-to-T transition at position ϩ6 of exon 7. This difference is translationally silent but results in exon 7 skipping in the SMN2 mRNA (27, 31). Thus, SMN2 transcripts lacking exon 7, which are abundant in cells, encode a C-terminally truncated SMN protein. The SMN protein is primarily involved in assembly of snRNPs (small nuclear ribonucleoproteins) and may also participate in precursor mRNA (pre-mRNA) splicing and in neuronal mRNA transport (35, 37). The SMN protein with a deletion of exon 7 is less stable and has lower self-oligomerization activity (26, 28). Moreover, this protein is unable to interact with the snRNP Sm proteins and fails to promote splicing in vitro (34, 35). Therefore, SMN2 is insufficient to fully compensate for SMN1 loss in SMA.Regulation of the SMN pre-mRNA splicing involves several cis elements around exon 7 that either serve as recognition sites for splicing regulatory factors or form secondary structures (26, 39-41). At least three splicing enhancers (SE) are located within exon 7. The C-to-T change at nucleotide ϩ6 of SMN2 likely disrupts the enhancer activity of SE1 and may also create a splicing silencer (3, 16). ASF/SF2 has been predicted to bind a sequence encompassing nucleotides ϩ6 to ϩ11 of exon 7 according to a sequence motif matrices analysis (3). An in vitro splicing assay has shown that excess ASF/SF2 could activate exon 7 inclusion ...

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Targeted delivery of drugs to tumors represents a significant advance in cancer diagnosis and therapy. Therefore, development of novel tumor-specific ligands or pharmaceutical nanocarriers is highly desirable. In this study, we utilized phage display to identify a new targeting peptide, SP90, which specifically binds to breast cancer cells, and recognizes tumor tissues from breast cancer patients. We used confocal and electron microscopy to reveal that conjugation of SP90 with liposomes enables efficient delivery of drugs into cancer cells through endocytosis. Furthermore, in vivo fluorescent imaging demonstrated that SP90-conjugated quantum dots possess tumor-targeting properties. In tumor xenograft and orthotopic models, SP90-conjugated liposomal doxorubicin was found to improve the therapeutic index of the chemotherapeutic drug by selectively increasing its accumulation in tumors. We conclude that the targeting peptide SP90 has significant potential in improving the clinical benefits of chemotherapy in the treatment and the diagnosis of breast cancer.