Global Analysis of Aggregation Profiles of Three Kinds of Immuno-Oncology mAb Drug Products Using Flow Cytometry.

Accurately quantifying the protein particles in both subvisible (1-100 µm) and submicron (≤1 µm) ranges remains a prominent challenge in the development and manufacturing of protein drugs. Due to the limitation of the sensitivity, resolution, or quantification level of various measurement systems, some instruments may not provide count information, while others can only count particles in a limited size range. Moreover, the reported concentrations of protein particles commonly have significant discrepancies owing to different methodological dynamic ranges and the detection efficiency of these analytical tools. Therefore, it is extremely difficult to accurately and comparably quantify protein particles within the desired size range at one time. To develop an efficient protein aggregation measurement method that can span the entire range of interest, we established, in this study, a single particle-sizing/counting method based on our highly sensitive lab-built flow cytometry (FCM) system. The performance of this method was assessed, and its capability of identifying and counting microspheres between 0.2 and 25 µm was demonstrated. It was also used to characterize and quantify both subvisible and submicron particles in three kinds of top-selling immuno-oncology antibody drugs and their lab-produced counterparts. These assessment and measurement results suggest that there may be a role for an enhanced FCM system as an efficient investigative tool for characterizing and learning the molecular aggregation behavior, stability, or safety risk of protein products.

The application of adenine deaminase in antibody affinity maturation.

Previously, we established a platform for antibody/protein affinity maturation based on CHO cell display. The gene of interest was mutated by activation-induced cytidine deaminase (AID), and then, a mutation library mainly containing G/C to A/T conversion was obtained by simply proliferating cells. However, the AID-induced G/C to A/T conversion limits the diversity space of the mutation library. In contrast to AID, adenine deaminase (ADA) can convert A/T to G/C. In this study, we demonstrated that ADA could efficiently induce random A/T to G/C mutations on the target gene in the CHO cell display and could be applied in affinity maturation. Our data also showed that more mutant types were obtained through the combined use of AID and ADA, thus offering an opportunity to acquire new mutants offering higher affinities than those obtained by only using AID. Examples presented in this study showed that ADA contributed to the improvement of antibody affinity either with or without AID in CHO display. KEY POINTS: • ADA is able to induce random mutations on antibody gene in mammalian cells. • ADA induces mutations on A/T bases to compensate AID which can induce mutation on G/C. • Combination of AID and ADA can increase mutation types and maturation efficiencies.

Synthesis and purification of biotinylated oligodeoxynucleotides containing single TpT dimeric pyrimidine (6-4) pyrimidone lesion.

Ultraviolet (UV) radiation could induce pyrimidine-related dimeric lesions in genomic DNA. Though the cyclobutane pyrimidine dimers (CPDs) are the most abundant UV-induced lesions, the pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) may have more serious, potentially lethal, and mutagenic effects. It is important to have 6-4PP-containing oligodeoxynucleotides to be prepared for studying their adverse biological effects. Here, we developed a UV-irradiated water droplet method for the preparation of a biotinylated, 6-4PP-containing 10-mer oligodeoxynucleotide. By the use of HPLC purification and enrichment twice, the final yield is estimated to be about 8.1%. In contrast, without applying droplet technique, the direct UV irradiation against oligonucleotide-containing aqueous solution, the product yield is very low. The enzymatic hydrolyzation of the obtained product shows a 6-4PP characteristic ion transition of 545.12 → 432.13 in negative ion mode UHPLC-Q-TOF/MS. The established procedure for the preparation of 6-4PP-containing oligonucleotides is convenient with an improved yield. Graphical abstract ᅟ.

In vitro affinity maturation of antibody against membrane-bound GPCR molecules.

G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, are among the most important targets against which many small molecule drugs have been developed. However, only two antibody drugs targeting GPCRs have been approved for clinical use although many antibody drugs against non-GPCR protein targets have been successfully developed for various disease indications. One of the challenges for developing anti-GPCR drugs is the high difficulty to perform affinity maturation due to their insolubility in aqueous solutions. To address this issue, CHO cell display libraries of single-chain variable fragments (scFvs) and full-length antibodies were maturated directly against vesicle probes prepared from CHO cells displaying the endothelin A receptor (ETaR) GPCR. The probe in the vesicle form ensures the physiological conformation and functional activity of the protein and avoids issues with membrane protein insolubility. The size of the vesicle had a clear effect on protein-ligand interaction; we used small-sized vesicles with low expression levels of GPCRs for the affinity maturation. Four rounds of affinity maturation combining vesicles as probes with the CHO cell display platform improved affinity by 13.58-fold for scFvs and 5.05-fold for full-length antibodies. We expect that this method will not only be used for the affinity maturation of antibodies against GPCRs but will also be used to mature antibodies for other types of proteins where the conformation/activity of which depends on the proper membrane environment.

Affinity maturation of an antibody for the UV-induced DNA lesions 6,4 pyrimidine-pyrimidones.

DNA lesions, associated mostly with minor changes in DNA structure, may induce permanent change in heritable coding information. Biochemically, these minor structural changes are difficult to be explored for generating high-affinity antibodies to detect specific DNA lesions in varying sequence contexts. Herein, we established a platform of bacterial display to facilitate antibodies to be matured with high affinity and high specificity against DNA lesions. To achieve this goal, we, for the first time, developed a two-round mutation/screening strategy: (1) using multiple lesion-containing DNA probes for primary maturation and (2) using single lesion-containing DNA probes for second maturation. Specifically, we capitalized on 64M-2 as a parental template to improve affinity for 6-4PP by 710-fold, compared with the model one. In addition, the matured antibody (9c3) is found to be much less dependent on the bases surrounding 6-4PPs than the model one. The mechanistic study from both computational simulation and reverse mutations revealed the critical roles of the two-round mutations in the enhanced binding affinity and independence of surrounding bases. This selection strategy opens a new way to improve affinity and specificity of antibodies for other DNA lesions.

Coupling recombinase-mediated cassette exchange with somatic hypermutation for antibody affinity maturation in CHO cells.

Heterologous expression of activation-induced cytidine deaminase (AID) can induce somatic hypermutation (SHM) for genes of interest in various cells, and several research groups (including ours) have successfully improved antibody affinity in mammalian or chicken cells using AID-induced SHM. These affinity maturation systems are time-consuming and inefficient. In this study, we developed an antibody affinity maturation platform in Chinese hamster ovary (CHO) cells by coupling recombinase-mediated cassette exchange (RMCE) with SHM. Stable CHO cell clones containing a single copy puromycin resistance gene (PuroR) expression cassette flanked by recombination target sequences (FRT and loxP) being able to highly express a gene of interest placed in the cassette were developed. The PuroR gene was replaced with an antibody gene by RMCE, and the antibody was displayed on the cell surface. Cells displaying antibodies on their membrane were transfected with the AID gene, and mutations of the antibody gene were accumulated by AID-mediated hypermutation during cell proliferation followed by flow cytometric cell sorting for cells bearing antibody mutants with improved affinity. Affinity improvements were detected after only one round of cell sorting and proliferation, mutant clones with 15-fold affinity improvement were isolated within five rounds of maturation (within 2 months). CHO cells are fast growing, stress-resistant and produce antibody with glycosylations suitable for therapy. Our antibody-evolution platform based on CHO cells makes antibody-affinity maturation more efficient and is especially convenient for therapeutic antibody affinity improvement.

Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting.

Recombination of antibody light and heavy chain libraries greatly increases the size of a two-chain paired antibody library, thus easing the construction of large antibody libraries. Here, light and heavy chain variable domains paired by a coiled coil were applied to a bacterial inner membrane display system. However, the probability of the correct pairing of light and heavy chains through random recombination after each round of flow cytometric sorting and cloning was very low in the presence of mostly unmatched light and heavy chain genes, resulting in inefficient enrichment; a target antibody clone in the ratio of 1:100,000 negative control spheroplasts was unable to be enriched by six rounds of sorting and cloning by a conventional sorting strategy (sorting the top 1 %). By just sorting the top 0.000025 % of spheroplasts, we succeeded in enriching the target antibody clone mixed with negative control spheroplasts in a ratio of 1:10(8) by just one round of sorting and cloning. Furthermore, using this gating strategy, we efficiently enriched for an antibody clone with an affinity slightly better than the parent antibody clone from mixed spheroplasts which were present in the ratio of 1 better affinity clone to 10 parent clones to 10(6) negative control clones after just two rounds of sorting and cloning, suggesting that this gating strategy is highly sensitive in distinguishing between clones with a small difference in affinity and also enriching for clones with a higher affinity. Taken together, the combination of the display of a two-chain paired antibody library and the use of stringent gating has significantly increased the efficiency of the antibody maturation system.

Evolved bacterial biosensor for arsenite detection in environmental water.

Arsenic, a ubiquitous presence in the biosphere, often occurs from both natural and anthropogenic sources. Bacterial biosensors based on genetically engineered bacteria have promising applications in detecting the chemical compound and its toxicity. However, most of the bacteria biosensors take advantage of the existing wild-type substrate-induced promoters, which are often low in specificity, affinity and sensitivity, and thus limiting their applications in commercial or field use. In this study, we developed an in vivo evolution procedure with a bidirectional selection scheme for improving the sensitivity of an arsenite-responsive bacterial biosensor through optimization of the inducible operon. As a proof of concept, we evolved the arsenite-induced arsR operon for both low background and high expression through three successive rounds of fluorescence activated cell sorting (FACS) with bidirectional strategy. An arsR operon variant with 12-fold higher activity over the control was isolated, confirming multiple rounds of construction and screening of mutation library, as described here, can be efficiently applied to bacterial biosensor optimization. The evolved arsenite-responsive biosensor demonstrated an excellent performance in the detection of low trace arsenite in environmental water. These results indicate that the technologies of directed evolution could be used to improve the performance of bacterial biosensors, which will be helpful in promoting the practical application of bacterial biosensors.

In vitro evolution of diagnostic antibodies targeting native antigens in plasma by sandwich flow cytometry.

Monoclonal antibodies (mAbs) that recognize and bind to specific antigens (Ags) have a wide range of applications in research, therapy, and diagnostics. However, many of these antibodies cannot bind well to the native Ags. In this study, based on the Chinese hamster ovary (CHO) cell display platform developed previously in our lab, we reported a novel artificial evolution procedure to improve the affinity of mAb against the native Ag directly using the plasma samples without purification of the native Ag. In this procedure, a pair of antibodies able to bind the Ag in sandwich manner are first confirmed (Ab1/Ab2) and the antibody (Ab) to be affinity-improved (Ab1) is displayed on CHO cells for Ab mutation. Then the cells were detected and sorted with flow cytometry in the form of Ab1-Ag-fluorescence labeled Ab2, which we named sandwich flow cytometry. Here, we used soluble isoform of suppression of tumorigenicity 2 (sST2) protein as model Ag, carried out "sandwich" maturation directly using the plasma samples containing the native sST2 protein and optimized a pair of antibodies with significantly improved sensitivity in the detection of the native sST2 in plasma. This method could be very useful in optimization of the diagnostic Ab pairs working in a "sandwich" manner if more antibodies were also successfully affinity-matured with this method.

The Humanization and Maturation of an Anti-PrPc Antibody.

The cellular prion protein (PrPc) is a cell surface glycoprotein that is highly expressed in a variety of cancer tissues in addition to the nervous system, and its elevated expression is correlated to poor prognosis in many cancer patients. Our team previously found that patients with colorectal cancer (CRC) with high-level PrPc expression had significantly poorer survival than those with no or low-level PrPc expression. Mouse antibodies for PrPc inhibited tumor initiation and liver metastasis of PrPc-positive human CRC cells in mouse model experiments. PrPc is a candidate target for CRC therapy. In this study, we newly cloned a mouse anti-PrPc antibody (Clone 6) and humanized it, then affinity-matured this antibody using a CHO cell display with a peptide antigen and full-length PrPc, respectively. We obtained two humanized antibody clones with affinities toward a full-length PrPc of about 10- and 100-fold of that of the original antibody. The two humanized antibodies bound to the PrPc displayed significantly better on the cell surface than Clone 6. Used for Western blotting and immunohistochemistry, the humanized antibody with the highest affinity is superior to the two most frequently used commercial antibodies (8H4 and 3F4). The two new antibodies have the potential to be developed as useful reagents for PrPc detection and even therapeutic antibodies targeting PrPc-positive cancers.

Chronic high glucose induced INS-1ß cell mitochondrial dysfunction: a comparative mitochondrial proteome with SILAC.

As glucose-stimulated insulin secretion of pancreatic ß cell is triggered and promoted by the metabolic messengers derived from mitochondria, mitochondria take a central stage in the normal function of ß cells. ß cells in diabetics were chronically exposed to hyperglycemia stimulation, which have been reported to exert deleterious effects on ß-cell mitochondria. However, the mechanism of the toxic effects of hyperglycemia on ß-cell mitochondria was not clear. In this study, we characterized the biological functional changes of rat INS-1ß cells and their mitochondria with chronic exposure to hyperglycemia and created a research model of chronic hyperglycemia-induced dysfunctional ß cells with damaged mitochondria. Then, SILAC-based quantitative proteomic approach was used to compare the mitochondrial protein expression from high glucose treated INS-1ß cells and control cells. The expression of some mitochondrial proteins was found with significant changes. Functional classification revealed most of these proteins were related with oxidative phosphorylation, mitochondrial protein biosynthesis, substances metabolism, transport, and cell death. These results presented some useful information about the effect of glucotoxicity on the ß-cell mitochondria.

Two distinct states of Escherichia coli cells that overexpress recombinant heterogeneous ß-galactosidase.

The mechanism by which inclusion bodies form is still not well understood, partly because the dynamic processes of the inclusion body formation and its solubilization have hardly been investigated at an individual cell level, and so the important detailed information has not been acquired for the mechanism. In this study, we investigated the in vivo folding and aggregation of Aspergillus phoenicis ß-D-galactosidase fused to a red fluorescence protein in individual Escherichia coli cells. The folding status and expression level of the recombinant ß-D-galactosidase at an individual cell level was analyzed by flow cytometry in combination with transmission electron microscopy and Western blotting. We found that individual E. coli cells fell into two distinct states, one containing only inclusion bodies accompanied with low galactosidase activity and the other containing the recombinant soluble galactosidase accompanied with high galactosidase activity. The majority of the E. coli cells in the later state possessed no inclusion bodies. The two states of the cells were shifted to a cell state with high enzyme activity by culturing the cells in isopropyl 1-thio-ß-D-galactopyranoside-free medium after an initial protein expression induction in isopropyl 1-thio-ß-D-galactopyranoside-containing medium. This shift of the cell population status took place without the level change of the ß-D-galactosidase protein in individual cells, indicating that the factor(s) besides the crowdedness of the recombinant protein play a major role in the cell state transition. These results shed new light on the mechanism of inclusion body formation and will facilitate the development of new strategies in improving recombinant protein quality.

A role for the arginine methylation of Rad9 in checkpoint control and cellular sensitivity to DNA damage.

The genome stability is maintained by coordinated action of DNA repairs and checkpoints, which delay progression through the cell cycle in response to DNA damage. Rad9 is conserved from yeast to human and functions in cell cycle checkpoint controls. Here, a regulatory mechanism for Rad9 function is reported. In this study Rad9 has been found to interact with and be methylated by protein arginine methyltransferase 5 (PRMT5). Arginine methylation of Rad9 plays a critical role in S/M and G2/M cell cycle checkpoints. The activation of the Rad9 downstream checkpoint effector Chk1 is impaired in cells only expressing a mutant Rad9 that cannot be methylated. Additionally, Rad9 methylation is also required for cellular resistance to DNA damaging stresses. In summary, we uncovered that arginine methylation is important for regulation of Rad9 function, and thus is a major element for maintaining genome integrity.

MDM2 Antagonist Nutlin-3 Stimulates Global DNA Hydroxymethylation by Enhancing p53-TET1 Signaling Axis.

DNA hydroxymethylation is involved in many biological processes, including nuclear reprogramming, embryonic development, and tumor suppression. In this study, we report that an anticancer agent, nutlin-3, selectively stimulates global DNA hydroxymethylation in TP53 wild-type cancer cells as manifested by the elevation of 5-hydroxymethylcytosine (5hmC) in genomic DNA. In contrast, nutlin 3 fails to enhance DNA hydroxymethylation in TP53-mutated cancer cells. Consistently, nutlin-3 as a MDM2 antagonist only activates wild-type but not mutated TP53. Furthermore, nutlin-3 does not alter the expression of TET1 but slightly reduces the expression of TET2 and TET3 proteins. These TET family proteins are responsible for converting 5-methylcytosine (5mC) to 5hmC. Interestingly, TET1 knockdown could significantly block the nutlin-3-induced DNA hydroxymethylation as well as TP53 and P21 activation. Immunoprecipitation analysis supports that p53 strongly interacts with TET1 proteins. These results suggest that nutlin-3 activates TP53 and promotes p53-TET1 interaction. As positive feedback, the p53-TET1 interaction further enhances p53 activation and promotes apoptosis. Collectively, we demonstrate that nutlin-3 stimulates DNA hydroxymethylation and apoptosis via a positive feedback mechanism.

Identification of the major functional proteins of prokaryotic lipid droplets.

Storage of cellular triacylglycerols (TAGs) in lipid droplets (LDs) has been linked to the progression of many metabolic diseases in humans, and to the development of biofuels from plants and microorganisms. However, the biogenesis and dynamics of LDs are poorly understood. Compared with other organisms, bacteria seem to be a better model system for studying LD biology, because they are relatively simple and are highly efficient in converting biomass to TAG. We obtained highly purified LDs from Rhodococcus sp. RHA1, a bacterium that can produce TAG from many carbon sources, and then comprehensively characterized the LD proteome. Of the 228 LD-associated proteins identified, two major proteins, ro02104 and PspA, constituted about 15% of the total LD protein. The structure predicted for ro02104 resembles that of apolipoproteins, the structural proteins of plasma lipoproteins in mammals. Deletion of ro02104 resulted in the formation of supersized LDs, indicating that ro02104 plays a critical role in cellular LD dynamics. The putative α helix of the ro02104 LD-targeting domain (amino acids 83-146) is also similar to that of apolipoproteins. We report the identification of 228 proteins in the proteome of prokaryotic LDs, identify a putative structural protein of this organelle, and suggest that apolipoproteins may have an evolutionarily conserved role in the storage and trafficking of neutral lipids.

Simultaneous Maturation of Single Chain Antibody Stability and Affinity by CHO Cell Display.

Antibody stability and affinity are two important features of its applications in therapy and diagnosis. Antibody display technologies such as yeast and bacterial displays have been successfully used for improving both affinity and stability. Although mammalian cell display has also been utilized for maturing antibody affinity, it has not been applied for improving antibody stability. Previously, we developed a Chinese hamster ovary (CHO) cell display platform in which activation-induced cytidine deaminase (AID) was used to induce antibody mutation, and antibody affinity was successfully matured using the platform. In the current study, we developed thermo-resistant (TR) CHO cells for the purpose of maturing both antibody stability and affinity. We cultured TR CHO cells displaying an antibody mutant library and labeled them at temperatures above 41 °C, enriching cells that displayed antibody mutants with both the highest affinities and the highest display levels. To evaluate our system, we chose three antibodies to improve their affinities and stabilities. We succeeded in simultaneously improving both affinities and stabilities of all three antibodies. Of note, we obtained an anti-TNFα antibody mutant with a Tm (dissolution temperature) value 12 °C higher and affinity 160-fold greater than the parent antibody after two rounds of cell proliferation and flow cytometric sorting. By using CHO cells with its advantages in protein folding, post-translational modifications, and code usage, this procedure is likely to be widely used in maturing antibodies and other proteins in the future.

Rad9 is required for B cell proliferation and immunoglobulin class switch recombination.

B cell maturation and B cell-mediated antibody response require programmed DNA modifications such as the V(D)J recombination, the immunoglobulin (Ig) class switch recombination, and the somatic hypermutation to generate functional Igs. Many protein factors involved in DNA damage repair have been shown to be critical for the maturation and activation of B cells. Rad9 plays an important role in both DNA repair and cell cycle checkpoint control. However, its role in Ig generation has not been reported. In this study, we generated a conditional knock-out mouse line in which Rad9 is deleted specifically in B cells and investigated the function of Rad9 in B cells. The Rad9(-/-) B cells isolated from the conditional knock-out mice displayed impaired growth response and enhanced DNA lesions. Impaired Ig production in response to immunization in Rad9(-/-) mice was also detected. In addition, the Ig class switch recombination is deficient in Rad9(-/-) B cells. Taken together, Rad9 plays dual roles in generating functional antibodies and in maintaining the integrity of the whole genome in B cells.

Toxicity of carbon nanotubes to p21 and hus1 gene deficient mammalian cells.

Carbon nanotubes including single wall and multi wall carbon nanotubes (SWNT and MWNT) are attractive nanomaterials with great promise in industrial and medical applications. However, little is known about the role of p21 and hus1 gene in the toxic response of SWNT and MWNT to mammalian cells. The aim of this study is to investigate the role of the p21 and hus1 genes in the toxicity of carbon nanotubes. Comparison of micronucleus fraction between the wild type and p21 -/- , hus1 +/+ mouse embryo fibroblast (MEF) cells was performed experimentally. Our results show that the yield of the micronucleus ratio in p21 gene knock-out MEF cells is lower than that in the wild type counterpart, indicating that p21 may play as anti-apoptosis factor during the signal transduction of DNA damage caused by carbon nanotubes in mammalian cells.

Targeting IL-21 to tumor-reactive T cells enhances memory T cell responses and anti-PD-1 antibody therapy.

T cell rejuvenation by PD-1/PD-L1 blockade, despite emerging as a highly promising therapy for advanced cancers, is only beneficial for a minority of treated patients. There is evidence that a lack of efficient T cell activation may be responsible for the failure. Here, we demonstrate that IL-21 can be targeted to tumor-reactive T cells by fusion of IL-21 to anti-PD-1 antibody. To our surprise, the fusion protein PD-1Ab21 promotes the generation of memory stem T cells (TSCM) with enhanced cell proliferation. PD-1Ab21 treatment show potent antitumor effects in established tumor-bearing mice accompanied with an increased frequency of TSCM and robust expansion of tumor-specific CD8+ T cells with a memory phenotype, and is superior to a combination of PD-1 blockade and IL-21 infusion. Therefore, we have developed a potential strategy to improve the therapeutic effects of immune checkpoint blockade by simultaneously targeting cytokines to tumor-reactive T cells.

A pathogen-like antigen-based vaccine confers immune protection against SARS-CoV-2 in non-human primates.

Activation of nucleic acid sensing Toll-like receptors (TLRs) in B cells is involved in antiviral responses by promoting B cell activation and germinal center responses. In order to take advantage of this natural pathway for vaccine development, synthetic pathogen-like antigens (PLAs) constructed of multivalent antigens with encapsulated TLR ligands can be used to activate B cell antigen receptors and TLRs in a synergistic manner. Here we report a PLA-based coronavirus disease 2019 (COVID-19) vaccine candidate designed by combining a phage-derived virus-like particle carrying bacterial RNA as TLR ligands with the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein as the target antigen. This PLA-based vaccine candidate induces robust neutralizing antibodies in both mice and non-human primates (NHPs). Using a NHP infection model, we demonstrate that the viral clearance is accelerated in vaccinated animals. In addition, the PLA-based vaccine induces a T helper 1 (Th1)-oriented response and a durable memory, supporting its potential for further clinical development.