Novel genetically glycoengineered human dendritic cell model reveals regulatory roles of α2,6-linked sialic acids in DC activation of CD4+ T cells and response to TNFα.

Dendritic cells (DCs) are central for the initiation and regulation of appropriate immune responses. While several studies suggest important regulatory roles of sialoglycans in DC biology, our understanding is still inadequate primarily due to a lack of appropriate models. Previous approaches based on enzymatic- or metabolic-glycoengineering and primary cell isolation from genetically modified mice have limitations related to specificity, stability, and species differences. This study addresses these challenges by introducing a workflow to genetically glycoengineer the human DC precursor cell line MUTZ-3, described to differentiate and maturate into fully functional dendritic cells, using CRISPR-Cas9, thereby providing and validating the first isogenic cell model for investigating glycan alteration on human DC differentiation, maturation, and activity. By knocking out (KO) the ST6GAL1 gene, we generated isogenic cells devoid of ST6GAL1-mediated α(2,6)-linked sialylation, allowing for a comprehensive investigation into its impact on DC function. Glycan profiling using lectin binding assay and functional studies revealed that ST6GAL1 KO increased the expression of important antigen presenting and co-stimulatory surface receptors and a specifically increased activation of allogenic human CD4 + T cells. Additionally, ST6GAL1 KO induces significant changes in surface marker expression and cytokine response to TNFα-induced maturation, and it affects migration and the endocytic capacity. These results indicate that genetic glycoengineering of the isogenic MUTZ-3 cellular model offers a valuable tool to study how specific glycan structures influence human DC biology, contributing to our understanding of glycoimmunology.

Expanding the CRISPR toolbox for Chinese hamster ovary cells with comprehensive tools for Mad7 genome editing.

The production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.

Immobilization-Free Binding and Affinity Characterization of Higher Order Bispecific Antibody Complexes Using Size-Based Microfluidics.

Simultaneous targeting of different antigens by bispecific antibodies (bsAbs) is permitting synergistic binding functionalities with high therapeutic potential, but is also rendering their analysis challenging. We introduce flow-induced dispersion analysis (FIDA) for the in-depth characterization of bsAbs with diverse molecular architectures and valencies under near-native conditions without potentially obstructive surface immobilization. Individual equilibrium dissociation constants are determined in solution, even in higher-order complexes with both antigens involved, hereby allowing the analysis of binding cooperativity and elucidation of a potential interference between the interactions. We further illustrate bispecific binding functionality as incremental increases in complex sizes when the bsAbs are exposed to one or two antigens. The possibility for comprehensive binding analysis with low material consumption and high matrix tolerability irrespective of molecular format and with little optimization renders FIDA a versatile tool for format selection and characterization of complex bi/multispecific protein therapeutics throughout the drug development and biomanufacturing pipeline.

Fully human glyco-optimized recombinant FSH (follitropin epsilon) - a randomized, comparator-controlled phase II clinical trial.

RESEARCH QUESTION: The study aimed to determine the standard treatment dose of follitropin epsilon for ovarian stimulation in the context of IVF treatment. DESIGN: A total of 247 women aged 18-37 years were treated with either 52.5, 75, 112.5 or 150 IU follitropin epsilon daily, or 150 IU every other day, or 150 IU follitropin alfa daily in a long gonadotrophin-releasing hormone agonist protocol. The study was performed as a randomized, assessor-blinded, comparator-controlled, six-armed phase II trial in eight fertility clinics in two European countries. RESULTS: The primary results were as follow. First, none of the doses of follitropin epsilon showed superiority for the main outcome measure, i.e. number of follicles ≥12 mm in size. Follitropin epsilon 75 IU produced results most similar to those of follitropin alfa 150 IU. In terms of secondary results, stronger effects of follitropin epsilon 112.5 IU compared with follitropin alfa 150 IU were seen for secondary outcome measures such as hormone concentrations (oestradiol, inhibin B and progesterone) and oocyte number. CONCLUSIONS: Follitropin epsilon 75 IU daily results in a similar ovarian response to a standard dose of 150 IU follitropin alfa. This dose could be tested in a phase III trial.

A new fully human recombinant FSH (follitropin epsilon): two phase I randomized placebo and comparator-controlled pharmacokinetic and pharmacodynamic trials.

STUDY QUESTION: What are the differences and similarities of pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of the novel recombinant human FSH follitropin epsilon expressed in the human cell line GlycoExpress compared with a Chinese hamster ovary (CHO) derived compound and a urinary derived product? SUMMARY ANSWER: Overall follitropin epsilon, with a fully human glycosylation, shows a comparable PK profile at single-dose as well as multiple-dose administration compared to recombinant CHO-derived FSH as well as urinary derived FSH, whereas the PD properties differ from product to product with follitropin epsilon being most active in PD parameters. WHAT IS KNOWN ALREADY: Recombinant FSH produced in CHO and FSH obtained from the urine of postmenopausal women show comparable PK and PD properties. However, more recently a comparative study of a recombinant FSH produced in the human cell line PerC6 and a CHO-derived FSH preparation revealed differences in PK and PD properties of the molecule. STUDY DESIGN, SIZE, DURATION: Both studies were randomized, placebo- and comparator-controlled, single-blind phase I studies in healthy pituitary-suppressed female volunteers aged 18 and 40 years. The single-dose, dose escalation study included 19 women (April 2011 to September 2011) with three ascending dose levels per subject or placebo/comparators with a 14-day washout phase between dosings. The multiple-dose study included 57 women (October 2011 to April 2012) in five cohorts with three dose levels versus placebo and two comparators. Randomization to the respective treatment was performed after successful downregulation of the pituitary gland prior to Investigational Medicinal Product dosing. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the single-dose study, 12 subjects received follitropin epsilon (25, 75, 150 and 300 IU) in three of four possible ascending doses and seven subjects received one dose of two comparators (150 IU Bravelle and 150 IU Gonal-f) and placebo in random order in each treatment period. In the multiple-dose study, 30 subjects received follitropin epsilon (75 IU or 150 IU once daily [QD], or 150 IU every other day [QAD], 10 subjects each) and 27 subjects received 150 IU Gonal-f, 150 IU Bravelle, or placebo for 7 days (11/10/6 subjects). Blood samples for measuring PK as well as PD parameters were collected systematically before, during and after dosing. Adverse events (AEs) and other relevant safety parameters were recorded. Data were summarized using descriptive statistics. MAIN RESULTS AND THE ROLE OF CHANCE: The single- and multiple-dose PK parameters maximum concentration (Cmax) and area under the concentration-time curve (AUC0-last) increased in a linear fashion with increasing dose levels of follitropin epsilon. Follitropin epsilon showed PK characteristics comparable to the comparators indicating that well established treatment schemes could be applied. There was a dose-response effect of single and multiple doses of follitropin epsilon on follicular growth, which was shown for the biomarker inhibin B as well as for the mean number and size of follicles. Multiple doses of 75 IU follitropin epsilon given daily, as well as 150 IU follitropin epsilon every second day, showed a follicle growth comparable with 150 IU Gonal-f given daily, while in case of daily administration of 150 IU Bravelle only weak follicle stimulation was observed. Multiple doses of 150 IU follitropin epsilon induced a much higher follicle growth compared to the same dose of Gonal-f. All single and multiple follitropin epsilon doses tested were safe and well tolerated, and overall there were no relevant differences between follitropin epsilon and the comparators in terms of safety. The average number of AEs increased with increasing dose levels. No clinically relevant abnormalities were reported for any of the other safety parameters assessed. No follitropin epsilon anti-drug antibodies were observed. LIMITATIONS, REASONS FOR CAUTION: The studies were conducted as a single-blind design. Hormone levels or other parameters assessed in serum are generally not considered as being subject to bias. Other assessments directly performed by the investigators, such as transvaginal ultrasound assessments, may have been subject to personal bias. No prospective calculations of statistical power had been made, as is common practice for first in human and early phase I studies in healthy volunteers. WIDER IMPLICATIONS OF THE FINDINGS: These early development studies showed that follitropin epsilon exhibits comparable PK characteristics, as well as inducing stronger PD effects in terms of follicle growth and serum inhibin B, than the comparators. Follitropin epsilon induced a dose-dependent increase in follicular growth. The results warrant further studies with this new fully human recombinant FSH. STUDY FUNDING/COMPETING INTEREST(S): The studies were sponsored by GLYCOTOPE GmbH, Berlin, Germany. K.A-E. is an employee of QPS-Netherlands, B.V., which received funding for the studies from Glycotope GmbH; I.D. and C.K. are employees of Dinox B.V., which received funding for the studies from Glycotope GmbH; L.S. and S.G. are employees and shareholders of Glycotope GmbH; B.D. and K.E. are employees of Glycotope GmbH. TRIAL REGISTRATION NUMBER: www.clinicaltrials.gov: NCT01354886 (single-dose); NCT01477073 (multiple-dose). TRIAL REGISTRATION DATE: The single-dose trial was registered on 11 May 2011 while the multiple-dose trial was registered on 09 November 2011. DATE OF FIRST SUBJECT'S ENROLMENT: First subject was enroled in the single-dose trial in 27 April 2011 and in the multiple-dose trial in 02 October 2011.

Design and engineering of bispecific antibodies: insights and practical considerations.

Bispecific antibodies (bsAbs) have attracted significant attention due to their dual binding activity, which permits simultaneous targeting of antigens and synergistic binding effects beyond what can be obtained even with combinations of conventional monospecific antibodies. Despite the tremendous therapeutic potential, the design and construction of bsAbs are often hampered by practical issues arising from the increased structural complexity as compared to conventional monospecific antibodies. The issues are diverse in nature, spanning from decreased biophysical stability from fusion of exogenous antigen-binding domains to antibody chain mispairing leading to formation of antibody-related impurities that are very difficult to remove. The added complexity requires judicious design considerations as well as extensive molecular engineering to ensure formation of high quality bsAbs with the intended mode of action and favorable drug-like qualities. In this review, we highlight and summarize some of the key considerations in design of bsAbs as well as state-of-the-art engineering principles that can be applied in efficient construction of bsAbs with diverse molecular formats.

Deep mining of antibody phage-display selections using Oxford Nanopore Technologies and Dual Unique Molecular Identifiers.

Antibody phage-display technology identifies antibody-antigen interactions through multiple panning rounds, but traditional screening gives no information on enrichment or diversity throughout the process. This results in the loss of valuable binders. Next Generation Sequencing can overcome this problem. We introduce a high accuracy long-read sequencing method based on the recent Oxford Nanopore Technologies (ONT) Q20 + chemistry in combination with dual unique molecular identifiers (UMIs) and an optimized bioinformatic analysis pipeline to monitor the selections. We identified binders from two single-domain antibody libraries selected against a model protein. Traditional colony-picking was compared with our ONT-UMI method. ONT-UMI enabled monitoring of diversity and enrichment before and after each selection round. By combining phage antibody selections with ONT-UMIs, deep mining of output selections is possible. The approach provides an alternative to traditional screening, enabling diversity quantification after each selection round and rare binder recovery, even when the dominating binder was > 99% abundant. Moreover, it can give insights on binding motifs for further affinity maturation and specificity optimizations. Our results demonstrate a platform for future data guided selection strategies.

Structural trends in antibody-antigen binding interfaces: a computational analysis of 1833 experimentally determined 3D structures.

Antibodies are attractive therapeutic candidates due to their ability to bind cognate antigens with high affinity and specificity. Still, the underlying molecular rules governing the antibody-antigen interface remain poorly understood, making in silico antibody design inherently difficult and keeping the discovery and design of novel antibodies a costly and laborious process. This study investigates the characteristics of antibody-antigen binding interfaces through a computational analysis of more than 850,000 atom-atom contacts from the largest reported set of antibody-antigen complexes with 1833 nonredundant, experimentally determined structures. The analysis compares binding characteristics of conventional antibodies and single-domain antibodies (sdAbs) targeting both protein- and peptide antigens. We find clear patterns in the number antibody-antigen contacts and amino acid frequencies in the paratope. The direct comparison of sdAbs and conventional antibodies helps elucidate the mechanisms employed by sdAbs to compensate for their smaller size and the fact that they harbor only half the number of complementarity-determining regions compared to conventional antibodies. Furthermore, we pinpoint antibody interface hotspot residues that are often found at the binding interface and the amino acid frequencies at these positions. These findings have direct potential applications in antibody engineering and the design of improved antibody libraries.

Specific humoral immune response to the Thomsen-Friedenreich tumor antigen (CD176) in mice after vaccination with the commensal bacterium Bacteroides ovatus D-6.

The tumor-specific Thomsen-Friedenreich antigen (TFα, CD176) is an attractive target for a cancer vaccine, especially as TF-directed antibodies play an important role in cancer immunosurveillance. However, synthetic TF vaccines have not overcome the low intrinsic immunogenicity of TF. Since natural TF-directed antibodies present in human sera are generated in response to microbes found in the gastrointestinal tract, microbial TF structures are obviously more immunogenic than synthetic TF. We recently isolated a new strain (D-6) of the human gut bacterium Bacteroides ovatus, which carries the true TFα antigen. Here, we present experimental data on the immunogenicity of this strain. Mice immunized with B. ovatus D-6 in the absence of adjuvants developed specific anti-TFα IgM and IgG antibodies which also bound to human cancer cells carrying TFα. Our data suggest that B. ovatus D-6 presents a unique TFα-specific immunogenicity based on a combination of several inherent properties including: expression of the true TFα antigen, clustering and accessible presentation of TFα as repetitive side chains on a capsular polysaccharide, and intrinsic adjuvant properties. Therefore, B. ovatus strain D-6 is an almost perfect candidate for the development of the first adjuvant-free TFα-specific anti-tumor vaccine.

Generation of robust bispecific antibodies through fusion of single-domain antibodies on IgG scaffolds: a comprehensive comparison of formats.

Bispecific antibodies (bsAbs) enable dual binding of different antigens with potential synergistic targeting effects and innovative therapeutic possibilities. The formation of bsAbs is, however, often dependent on complex engineering strategies with a high risk of antibody chain mispairing leading to contamination of the final product with incorrectly assembled antibody species. This study demonstrates formation of bsAbs in a generic and conceptually easy manner through fusion of single-domain antibodies (sdAbs) onto IgG scaffolds through flexible 10 amino acid linkers to form high-quality bsAbs with both binding functionalities intact and minimal product-related impurities. SdAbs are attractive fusion partners due to their small and monomeric nature combined with antigen-binding capabilities comparable to conventional human antibodies. By systematically comparing a comprehensive panel of symmetric αPD-L1×αHER2 antibodies, including reversely mirrored antigen specificities, we investigate how the molecular geometry affects production, stability, antigen binding and CD16a binding. SdAb fusion of the heavy chain was generally preferred over light chain fusion for promoting good expression and high biophysical stability as well as maintaining efficient binding to both antigens. We find that N-terminal sdAb fusion might sterically hinder antigen-binding to the Fv region of the IgG scaffold, whereas C-terminal fusion might disturb antigen-binding to the fused sdAb. Our work demonstrates a toolbox of complementary methods for in-depth analysis of key features, such as in-solution dual antigen binding, thermal stability, and aggregation propensity, to ensure high bsAb quality. These techniques can be executed at high-throughput and/or with very low material consumption and thus represent valuable tools for bsAb screening and development.

Eliminating OFF-frame clones in randomized gene libraries: An improved split ß-lactamase enrichment system.

Large, randomized libraries are a key technology for many biotechnological applications. While genetic diversity is the main parameter most libraries direct their resources on, less focus is devoted to ensuring functional IN-frame expression. This study describes a faster and more efficient system based on a split ß-lactamase complementation for removal of OFF-frame clones and increase of functional diversity, suitable for construction of randomized libraries. The gene of interest is inserted between two fragments of the ß-lactamase gene, conferring resistance to ß-lactam drugs only upon expression of an inserted IN-frame gene without stop codons or frameshifts. The preinduction-free system was capable of eliminating OFF-frame clones in starting mixtures of as little as 1% IN-frame clones and enriching to about 70% IN-frame clones, even when their starting rate was as low as 0.001%. The curation system was verified by constructing a single-domain antibody phage display library using trinucleotide phosphoramidites for randomizing a complementary determining region, while eliminating OFF-frame clones and maximizing functional diversity.

A window into the human immune system: comprehensive characterization of the complexity of antibody complementary-determining regions in functional antibodies.

The human immune system uses antibodies to neutralize foreign antigens. They are composed of heavy and light chains, both with constant and variable regions. The variable region has six hypervariable loops, also known as complementary-determining regions (CDRs) that determine antibody diversity and antigen specificity. Knowledge of their significance, and certain residues present in these areas, is vital for antibody therapeutics development. This study includes an analysis of more than 11,000 human antibody sequences from the International Immunogenetics information system (IMGT). The analysis included parameters such as length distribution, overall amino acid diversity, amino acid frequency per CDR and residue position within antibody chains. Overall, our findings confirm existing knowledge, such as CDRH3's high length diversity and amino acid variability, increased aromatic residue usage, particularly tyrosine, charged and polar residues like aspartic acid, serine, and the flexible residue glycine. Specific residue positions within each CDR influence these occurrences, implying a unique amino acid type distribution pattern. We compared amino acid type usage in CDRs and non-CDR regions, both in globular and transmembrane proteins, which revealed distinguishing features, such as increased frequency of tyrosine, serine, aspartic acid, and arginine. These findings should prove useful for future optimization, improvement of affinity, synthetic antibody library design, or the creation of antibodies de-novo in silico.

Exploration of cell state heterogeneity using single-cell proteomics through sensitivity-tailored data-independent acquisition.

Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, single-cell proteomics by Mass Spectrometry (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential. To this end, we carry out comprehensive analysis of orbitrap-based data-independent acquisition (DIA) for limited material proteomics. Notably, we find a fundamental difference between optimal DIA methods for high- and low-load samples. We further improve our low-input DIA method by relying on high-resolution MS1 quantification, thus enhancing sensitivity by more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we are able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we demonstrate the capability of our approach by profiling mouse embryonic stem cell culture conditions, showcasing heterogeneity in global proteomes and highlighting distinct differences in key metabolic enzyme expression in distinct cell subclusters.

Clinical Staphylococcus aureus inhibits human T-cell activity through interaction with the PD-1 receptor.

IMPORTANCE: Therapies that target and aid the host immune defense to repel cancer cells or invading pathogens are rapidly emerging. Antibiotic resistance is among the largest threats to human health globally. Staphylococcus aureus (S. aureus) is the most common bacterial infection, and it poses a challenge to the healthcare system due to its significant ability to develop resistance toward current available therapies. In long-term infections, S. aureus further adapt to avoid clearance by the host immune defense. In this study, we discover a new interaction that allows S. aureus to avoid elimination by the immune system, which likely supports its persistence in the host. Moreover, we find that blocking the specific receptor (PD-1) using antibodies significantly relieves the S. aureus-imposed inhibition. Our findings suggest that therapeutically targeting PD-1 is a possible future strategy for treating certain antibiotic-resistant staphylococcal infections.

Preliminary safety evaluation of a new Bacteroides xylanisolvens isolate.

Besides conferring some health benefit to the host, a bacterial strain must present an unambiguous safety status to be considered a probiotic. We here present the preliminary safety evaluation of a new Bacteroides xylanisolvens strain (DSM 23964) isolated from human feces. First results suggest that it may be able to provide probiotic health benefits. Its identity was confirmed by biochemical analysis, by sequencing of its 16S rRNA genes, and by DNA-DNA hybridization. Virulence determinants known to occur in the genus Bacteroides, such the bft enterotoxin and other enzymatic activities involved in the degradation of the extracellular matrix and the capsular polysaccharide PS A, were absent in this strain. The investigation of the antibiotic susceptibility indicated that strain DSM 23964 was sensitive to metronidazole, meropenem agents, and clindamycin. Resistance to penicillin and ampicillin was identified to be conferred by the ß-lactamase cepA gene and could therefore be restored by adding ß-lactamase inhibitors. The localization of the cepA gene in the genome of strain DSM 23964 and the absence of detectable plasmids further suggest that a transfer of ß-lactamase activity or the acquisition of other antibiotic resistances are highly improbable. Taken together, the presented data indicate that the strain B. xylanisolvens DSM 23964 has no virulence potential. Since it also resists the action of gastric enzymes and low-pH conditions, this strain is an interesting candidate for further investigation of its safety and potential health-promoting properties.

Safety assessment of the commensal strain Bacteroides xylanisolvens DSM 23964.

We recently isolated and characterized the new strain Bacteroides xylanisolvens DSM 23964 and presented it as potential candidate for the first natural probiotic strain of the genus Bacteroides. In order to evaluate the safety of this strain for use in food, the following standard toxicity assays were conducted with this strain in both viable and pasteurized form: in vitro bacterial reverse mutation assay, in vitro chromosomal aberration assay, and 90day subchronic repeated oral toxicity studies in mice. No mutagenic, clastogenic, or toxic effects were detected even at extremely high doses. In addition, no clinical, hematological, ophthalmological, or histopathological abnormality could be observed after necropsy at any of the doses tested. Hence, the NOAEL could be estimated to be greater than 2.3×10(11) CFUs, and 2.3×10(14) for pasteurized bacteria calculated as equivalent for an average 70kg human being. In addition, the absence of any in vivo pathogenic properties of viable B. xylanisolvens DSM 23964 cells was confirmed by means of an intraperitoneal abscess formation model in mice which also demonstrated that the bacteria are easily eradicated by the host's immune system. The obtained results support the assumed safety of B. xylanisolvens DSM 23964 for use in food.

Occurrence of the human tumor-specific antigen structure Galß1-3GalNAcα- (Thomsen-Friedenreich) and related structures on gut bacteria: prevalence, immunochemical analysis and structural confirmation.

The Thomsen-Friedenreich antigen (TF; CD176, Galß1-3GalNAcα-) is a tumor-specific carbohydrate antigen and a promising therapeutic target. Antibodies that react with this antigen are frequently found in the sera of healthy adults and are assumed to play a role in cancer immunosurveillance. In this study, we examined the occurrence of α-anomeric TF (TFα) on a large variety of gastrointestinal bacteria using a novel panel of well-characterized monoclonal antibodies. Reactivity with at least one anti-TF antibody was found in 13% (16 of 122) of strains analyzed. A more in-depth analysis, using monoclonal antibodies specific for α- and ß-anomeric TF in combination with periodate oxidation, revealed that only two novel Bacteroides ovatus strains (D-6 and F-1), isolated from the faeces of healthy persons by TF-immunoaffinity enrichment, possessed structures that are immunochemically identical to the true TFα antigen. The TF-positive capsular polysaccharide structure of strain D-6 was characterized by mass spectrometry, monosaccharide composition analysis, glycosidase treatments and immunoblot staining with TFα- and TFß-specific antibodies. The active antigen was identified as Galß1-3GalNAc-, which was α-anomerically linked as a branching structure within a heptasaccharide repeating unit. We conclude that structures immunochemically identical to TFα are extremely rare on the surface of human intestinal bacteria and may only be identifiable by binding of both antibodies, NM-TF1 and NM-TF2, which recognize a complete immunomolecular imprint of the TFα structure. The two novel B. ovatus strains isolated in this study may provide a basis for the development of TF-based anti-tumor vaccines.

Expression of CD176 (Thomsen-Friedenreich antigen) on lung, breast and liver cancer-initiating cells.

The cancer-initiating capacity of most malignant tumours is considered to reside in a small subpopulation of cells. Therapeutical interventions should target these cells rather than the tumour mass. Numerous studies have shown that the carbohydrate antigen structure CD176 (Thomsen-Friedenreich antigen, core-1) is present in many types of cancer and absent in normal adult human tissues. In this study, we assessed whether CD176 is co-expressed with CD44 or CD133 [markers of cancer-initiating cells (CIC)] in human lung, breast and liver carcinoma. A variety of human cancer cell lines and surgical specimens of these malignancies were examined. It was found that in most cases the majority of tumour cells stained strongly for CD44 by immunohistochemistry and flow cytometry, whereas CD133 expression was found on a smaller, but varying proportion of cells. Co-expression of CD176 with CD44 was found at a surprisingly high percentage of cancer cells in vitro and in vivo. Co-expression of CD176 with CD133 was also detected, although at a lower rate. Tamoxifen treatment of MDA-435 breast cancer cells enhanced the CD44(+) /CD176(+) phenotype. Evidence is provided through a new sandwich solid-phase enzyme-linked immunosorbent assay (ELISA) suggesting that CD44 is a carrier molecule for CD176 not only in colorectal cancer as previously reported, but also in lung, breast and liver cancer. The expression of CD176 in CIC suggests that it may represent an effective target for tumour therapies.

A novel scale-down mimic of perfusion cell culture using sedimentation in an automated microbioreactor (SAM).

Continuous upstream processing in mammalian cell culture for recombinant protein production holds promise to increase product yield and quality. To facilitate the design and optimization of large-scale perfusion cultures, suitable scale-down mimics are needed which allow high-throughput experiments to be performed with minimal raw material requirements. Automated microbioreactors are available that mimic batch and fed-batch processes effectively but these have not yet been adapted for perfusion cell culture. This article describes how an automated microbioreactor system (ambr15) can be used to scale-down perfusion cell cultures using cell sedimentation as the method for cell retention. The approach accurately predicts the viable cell concentration, in the range of about 1 × 107 cells/mL for a human cell line, and cell viability of larger scale cultures using a hollow fiber based cell retention system. While it was found to underpredict cell line productivity, the method accurately predicts product quality attributes, including glycosylation profiles, from cultures performed in bioreactors with working volumes between 1 L and 1,000 L. The spent media exchange method using the ambr15 was found to predict the influence of different media formulations on large-scale perfusion cultures in contrast to batch and chemostat experiments performed in the microbioreactor system. The described experimental setup in the microbioreactor allowed an 80-fold reduction in cell culture media requirements, half the daily operator time, which can translate into a cost reduction of approximately 2.5-fold compared to a similar experimental setup at bench scale.