Combinatorial Biology Research Articles

Understanding and Modulating Antibody Fine Specificity: Lessons from Combinatorial Biology

Combinatorial biology methods such as phage and yeast display, suitable for the generation and screening of huge numbers of protein fragments and mutated variants, have been useful when dissecting the molecular details of the interactions between antibodies and their target antigens (mainly those of protein nature). The relevance of these studies goes far beyond the mere description of binding interfaces, as the information obtained has implications for the understanding of the chemistry of antibody–antigen binding reactions and the biological effects of antibodies. Further modification of the interactions through combinatorial methods to manipulate the key properties of antibodies (affinity and fine specificity) can result in the emergence of novel research tools and optimized therapeutics.

Comprehensive Genomic Analysis of Marine Strain Streptomyces sp. 891, an Excellent Producer of Chrysomycin A with Therapeutic Potential.

Chrysomycin A is one of the most promising therapeutic candidates for treating infections caused by multidrug-resistant Gram-positive bacteria. By hybridizing next-step generation (Illumina) and third-generation (PacBio) sequencing technologies, a high-quality chromosome-level genome together with a plasmid was firstly assembled for chrysomycin A-producing marine strain 891. Phylogenetic analysis of the 16S rRNA gene and genome sequences revealed that this strain unambiguously belonged to the genus Streptomyces, and its genomic features and functional genes were comprehensively analyzed and annotated. AntiSMASH analysis of this strain unveiled one key biosynthetic gene cluster, T2PKS, responsible for the biosynthesis of chrysomycin, the biosynthesis pathway of which was putatively proposed. These findings definitely shed light on further investigation for construction of a robust industrial strain with high-yield chrysomycin A production using genetic engineering techniques and combinatorial biology approaches.

Phage display as a tool for identifying HIV-1 broadly neutralizing antibodies.

Combinatorial biology methods offer a good solution for targeting interactions of specif ic molecules by a high-throughput screening and are widely used for drug development, diagnostics, identif ication of novel monoclonal antibodies, search for linear peptide mimetics of discontinuous epitopes for the development of immunogens or vaccine components. Among all currently available techniques, phage display remains one of the most popular approaches. Despite being a fairly old method, phage display is still widely used for studying protein-protein, peptide-protein and DNA-protein interactions due to its relative simplicity and versatility. Phage display allows highly representative libraries of peptides, proteins or their fragments to be created. Each phage particle in a library displays peptides or proteins fused to its coat protein and simultaneously carries the DNA sequence encoding the displayed peptide/protein in its genome. The biopanning procedure allows isolation of specif ic clones for almost any target, and due to the physical link between the genotype and the phenotype of recombinant phage particles it is possible to determine the structure of selected molecules. Phage display technology continues to play an important role in HIV research. A major obstacle to the development of an effective HIV vaccine is an extensive genetic and antigenic variability of the virus. According to recent data, in order to provide protection against HIV infection, the so-called broadly neutralizing antibodies that are cross-reactive against multiple viral strains of HIV must be induced, which makes the identif ication of such antibodies a key area of HIV vaccinology. In this review, we discuss the use of phage display as a tool for identif ication of HIV-specif ic antibodies with broad neutralizing activity. We provide an outline of phage display technology, brief ly describe the design of antibody phage libraries and the affinity selection procedure, and discuss the biology of HIV-1-specif ic broadly neutralizing antibodies. Finally, we summarize the studies aimed at identif ication of broadly neutralizing antibodies using various types of phage libraries.

Biallelic PI4KA variants cause neurological, intestinal and immunological disease

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα/PI4KA/OMIM:600286) is a lipid kinase generating phosphatidylinositol 4-phosphate (PI4P), a membrane phospholipid with critical roles in the physiology of multiple cell types. PI4KIIIα’s role in PI4P generation requires its assembly into a heterotetrameric complex with EFR3, TTC7 and FAM126. Sequence alterations in two of these molecular partners, TTC7 (encoded by TTC7A or TCC7B) and FAM126, have been associated with a heterogeneous group of either neurological (FAM126A) or intestinal and immunological (TTC7A) conditions.Here we show that biallelic PI4KA sequence alterations in humans are associated with neurological disease, in particular hypomyelinating leukodystrophy. In addition, affected individuals may present with inflammatory bowel disease, multiple intestinal atresia and combined immunodeficiency. Our cellular, biochemical and structural modelling studies indicate that PI4KA-associated phenotypical outcomes probably stem from impairment of PI4KIIIα-TTC7-FAM126's organ-specific functions, due to defective catalytic activity or altered intra-complex functional interactions.Together, these data define PI4KA gene alteration as a cause of a variable phenotypical spectrum and provide fundamental new insight into the combinatorial biology of the PI4KIIIα-FAM126-TTC7-EFR3 molecular complex.

Abstract 5079: Immuno-competent 3d in vitro tumor models as novel platform to assess combinatorial biologics therapy

Abstract Background: Immunotherapy has revolutionized cancer treatment, but clinical success is still limited to a subset of patients, underlining the need for novel therapeutic targets. Available ex vivo animal platforms for drug screening show limitations due to only partial recapitulation of tumor-intrinsic features and of the interactions of tumors with stromal/immune cells. Here, we review our 3D InSightTM tumor models, a complex in-vitro platform allowing to study the efficacy of immune-targeting agents on tumor infiltration and killing by peripheral blood mononuclear cells (PBMCs) and to acquire insights on the modulatory capacity of immune checkpoint inhibitors. Aim: In the present study we have developed a 3D tumor - immune cell coculture model and evaluated its application to study the effect of immune checkpoint inhibitors on tumor viability, cytokine secretion, and immune cell infiltration. Material and Methods: Lung carcinoma GFP-A549 cells were cultured with dermal fibroblasts and PBMCs in Akura 96/384-well plates. Immune cells were pre-treated using different activation protocols to induce priming and pT-cell exhaustion. We assessed the effect of PD-1 inhibitors in combination with cytokines for potential synergistic anti-cancer effects. Killing activity and effector function of T cells were evaluated by measuring tumor microtissue size by automatic stage fluorescence microscopy and measuring release of IL-6, TNF, IFNγ and GM-CSF with a MAGPIXTM Luminex system over time. Immune tumor infiltration was quantified using fluorescent staining of CD3+ cells and the effect of the immune cell attack on the tumor cells was assessed by IHC staining. Results: Combination treatment with cytokines and anti-PD-1 led to higher release of IL-6/TNF/IFNγ/GM-CSF and increased tumor microtissue infiltration by CD3+ lymphocytes, stronger expression of Granzyme B, resulting in enhanced tumor killing and apoptosis. Conclusion: Our 3D InSightTM tumor models can be successfully employed for optimal high throughput screening of biologics to unveil the efficacy and anti-tumor efficacy of immune-targeting therapeutic agents. Citation Format: Irina Agarkova, Nicole Buschmann, Silvan Strebel, Armin Wolf, Francesca Chiovaro. Immuno-competent 3d in vitro tumor models as novel platform to assess combinatorial biologics therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5079.

Immune-competent 3D InSightTM tumour models as novel platform to assess combinatorial biologics therapy

Abstract Background Immunotherapy has revolutionized cancer treatment, but clinical success is still limited to a subset of patients, underlining the need for novel therapeutic targets. Available ex vivo platforms for drug screening show limitations due to only partial recapitulation of tumor-intrinsic features and of the interactions of tumours with stromal/immune cells. Here, we review our 3D InSightTM tumour models, a complex platform allowing us to study the efficacy of immune-targeting agents on tumour infiltration and killing by peripheral blood mononuclear cells (PBMCs) using clinically relevant models of 3D tumour microtissues from patient-derived xenografts (PDX) with high translational potential. Methods Lung carcinoma GFP-A549 cells were cultured with dermal fibroblasts and PBMCs in Akura 96/384-well plates. To generate a pro-inflammatory tumour microenvironment, PBMCs were activated with cytokines, anti-CD3/CD28 or their combination. We assessed the effect of PD-1 inhibitors in combination with cytokines for potential synergistic anti-cancer effects. 3D InSight™ tumour Microtissue from Melanoma PDX were labeled with CellTracker to monitor for tumour cell viability once exposed to PBMCs. Killing activity and effector function of T cells were evaluated by measuring tumour microtissue size by automatic stage fluorescence microscopy and measuring release of IL-6, TNF, IFNγ and GM-CSF with a MAGPIXTM Luminex system over time. Immune tumour infiltration was quantified and the impact on survival was assessed histologically. Results Combination treatment with cytokines and anti-PD-1 led to higher release of IL-6/TNF/IFNγ/GM-CSF and increased tumour microtissue infiltration by CD8+ T cells, resulting in enhanced tumour killing. Conclusion Our 3D InSightTM tumour models can be successfully employed for optimal high throughput screening of biologics to unveil the efficacy and anti-tumour properties of immune-targeting therapeutic agents. Legal entity responsible for the study InSphero. Funding InSphero AG. Disclosure All authors have declared no conflicts of interest.

Neuroinflammation as Part of a Possible Neuregeneration on a Patient with ASIA-A Scale Using Combinatorial Biologics: Bioquantine® and Allogeneic Mesenchymal Stem Cells

Neuroinflammation as Part of a Possible Neuregeneration on a Patient with ASIA-A Scale Using Combinatorial Biologics: Bioquantine® and Allogeneic Mesenchymal Stem Cells

Neuroinflammation as Part of a Possible Neuro-regeneration in a Patient with ASIA-A Scale Using Combinatorial Biologics: Bioquantine® and Allogeneic Mesenchymal Stem Cells

A complete spinal cord injury (SCI) is the complete sensory and motor loss below the site of spinal cord injury following acute or chronic destruction, compression, or ischemia of the spinal cord. Initially, this may present as spinal shock, which is an acute physiological loss or depression of spinal cord function. It presents as a flaccid are flexic paralysis below the level of the injury with autonomic features (e.g., hypotension and bradycardia). After some days to weeks the spinal shock wears off and a complete spinal cord injury may remain. It presents with spastic paresis, hyperreflexia, and continued sensory loss.

Neuroinflammation as part of a possible neuro-regeneration in a patient with ASIA-A scale using combinatorial biologics: Bioquantine® and allogeneic mesenchymal stem cells

Neuroinflammation as part of a possible neuro-regeneration in a patient with ASIA-A scale using combinatorial biologics: Bioquantine® and allogeneic mesenchymal stem cells

RNA, local moves on plane trees, and transpositions on tableaux

We define a collection of functions $s_i$ on the set of plane trees (or standard Young tableaux). The functions are adapted from transpositions in the representation theory of the symmetric group and almost form a group action. They were motivated by $\textit{local moves}$ in combinatorial biology, which are maps that represent a certain unfolding and refolding of RNA strands. One main result of this study identifies a subset of local moves that we call $s_i$-local moves, and proves that $s_i$-local moves correspond to the maps $s_i$ acting on standard Young tableaux. We also prove that the graph of $s_i$-local moves is a connected, graded poset with unique minimal and maximal elements. We then extend this discussion to functions $s_i^C$ that mimic reflections in the Weyl group of type $C$. The corresponding graph is no longer connected, but we prove it has two connected components, one of symmetric and the other of asymmetric plane trees. We give open questions and possible biological interpretations.

RegenerAge system: Therapeutic effects of combinatorial biologics (mRNA and Allogenic MSCs) with a spinal cord stimulation system on a patient with spinal cord section

RegenerAge system: Therapeutic effects of combinatorial biologics (mRNA and Allogenic MSCs) with a spinal cord stimulation system on a patient with spinal cord section

RegenerAge System: Therapeutic effects of combinatorial biologics (mRNA and allogenic MSCs) with a spinal cord stimulation system on a patient with spinal cord section

RegenerAge System: Therapeutic effects of combinatorial biologics (mRNA and allogenic MSCs) with a spinal cord stimulation system on a patient with spinal cord section

Clinical Pharmacology and Translational Aspects of Bispecific Antibodies.

Clinical Pharmacology and Translational Aspects of Bispecific Antibodies.

"Dipstick" Colorimetric Detection of Metal Ions Based on Immobilization of DNAzyme and Gold Nanoparticles onto a Lateral Flow Device.

Real-time, on-site detection and quantification of different trace analytes is a challenge that requires both searching a general class of molecules to recognize a broad range of contaminants and translating this recognition to easily detectable signals. Functional nucleic acids, which include DNAzymes (DNA with catalytic activity) and aptamers (nucleic acids that bind an analyte), are ideal candidates for the target recognition. These nucleic acids can be selected by a combinatorial biology method called in vitro selection to interact with a particular analyte with high specificity and sensitivity. Furthermore, they can be incorporated into sensors by attaching signaling molecules. Due to the high extinction coefficients and distance-dependent optical properties, metallic nanoparticles such as the commonly used gold nanoparticles have been shown to be very attractive in converting analyte-specific functional DNA into colorimetric sensors. DNAzyme directed assembly of gold nanoparticles has been used to make colorimetric sensors for metal ions such as lead, uranium, and copper. To make the operation even easier and less vulnerable to operator's errors, dipstick tests have been constructed. Here, we describe protocols for the preparation of DNAzyme-linked gold nanoparticles (AuNP) that are then immobilized on to lateral flow devices to make easy-to-use dipstick tests for metal ions.

Biomedical Innovation: Lessons From the Past and Perspectives for the Future

Back around the turn of the millennium, the future of the pharmaceutical industry was bright. Amazing technologies were converging to enable a top-to-bottom reengineering of drug research and development (R&D). The "omics," combinatorial chemistry, high-throughput screening, robotic automation, and systems biology, promised to bring order and method to drug research's bewildering complexity. Pharmaceutical executives-many of whom were ill at ease with their scientists' freewheeling ways-were excited. Gushing with an enthusiasm that was typical of the times, a former industry Chief Executive Officer spoke glowingly of the launch of "two to three new blockbusters… each year" driving a quadrupling of revenues.

Q-Polynomial Invariant of Rooted Trees

We describe in this note a new invariant of rooted trees. We argue that the invariant is interesting on it own, and that it has connections to knot theory and homological algebra. However, the real reason that we propose this invariant to readers of Arnold Journal of Mathematics is that we deal here with an elementary, interesting, new mathematics, and after reading this essay readers can take part in developing the topic, inventing new results and connections to other disciplines of mathematics, and likely, statistical mechanics, and combinatorial biology.

Recombinant antibodies and recombinant proteins: Prolonged-action drugs

An intensively developing trend in modern biotechnology is the production of recombinant antibodies and proteins. Recombinant antibodies have immunogenic properties and can stay in the blood for a long time; neutralize pathogens, toxins, and viruses; and destroy tumor cells. Recombinant proteins are widely used to cure, prevent, and diagnose diseases. A topical trend in modern medical biotechnology is the creation of prolonged-action drugs based on proteins obtained by genetic engineering methods. The issue of improving the specificity and selectivity of preparations based on enzymes and antibodies awaits solution. All this can be implemented using the approaches of modern combinatorial chemistry and biology, as well as quantum-mechanical calculations.

Aptamer-based biosensors for biomedical diagnostics.

Aptamers are single-stranded nucleic acids that selectively bind to target molecules. Most aptamers are obtained through a combinatorial biology technique called SELEX. Since aptamers can be isolated to bind to almost any molecule of choice, can be readily modified at arbitrary positions and they possess predictable secondary structures, this platform technology shows great promise in biosensor development. Over the past two decades, more than one thousand papers have been published on aptamer-based biosensors. Given this progress, the application of aptamer technology in biomedical diagnosis is still in a quite preliminary stage. Most previous work involves only a few model aptamers to demonstrate the sensing concept with limited biomedical impact. This Critical Review aims to summarize progress that might enable practical applications of aptamers for biological samples. First, general sensing strategies based on the unique properties of aptamers are summarized. Each strategy can be coupled to various signaling methods. Among these, a few detection methods including fluorescence lifetime, flow cytometry, upconverting nanoparticles, nanoflare technology, magnetic resonance imaging, electronic aptamer-based sensors, and lateral flow devices have been discussed in more detail since they are more likely to work in a complex sample matrix. The current limitations of this field include the lack of high quality aptamers for clinically important targets. In addition, the aptamer technology has to be extensively tested in a clinical sample matrix to establish reliability and accuracy. Future directions are also speculated to overcome these challenges.

Functional nucleic acids for detecting bacteria

Abstract Bacterial infection represents one of the leading causes of disease and death, and as such, bacterial detection is an important step in managing infectious diseases. The current protocol requires growing cell cultures, which can take several days. Fast detection of low copies of bacterial cells has thus posed an analytical challenge. Among the new strategies developed to achieve this goal, functional nucleic acids (FNAs) have emerged to be a promising platform. FNAs include DNAzymes, aptamers, and aptazymes, all of which can recognize analytes other than complementary nucleic acids. FNAs are obtained using a combinatorial biology technique called systematic evolution of ligands by exponential enrichment (SELEX). FNAs have been isolated against not only purified proteins and surface markers from bacterial cells but also whole cells. A diverse range of signaling mechanisms including fluorescence, color, and electrochemistry-based detection has been reported. Although the majority of current sensors cannot achieve single-cell sensitivity, with improved combinatorial selection techniques and the incorporation of nanomaterials to realize multivalent binding and signal amplification, FNAs represent a feasible solution for bacterial detection.

Concepts in biotechnology: history, science and business

Preface. Abbreviations and Glossary. Part One. History. 1. Introduction. 2. The Early Period to 1850. 2.1 Introduction. 2.2 Experimental Scientific Findings. 2.3 Application. 2.4 Theoretical Approaches. References. 3. The Period from 1850 to 1890. 3.1 Introduction. 3.2 Experimental Findings. 3.3 Practical Application, Technical Progress and Institutional Development. 3.4 Theoretical Approaches. References. 4. The Period from 1890 to 1950. 4.1 Introduction. 4.2 Research Advances in the Basics of Biotechnology: Experimental Findings. 4.3 Technological Development, Progress and Application. 4.4 Theoretical Approaches. References. 5. Outlook, from 1950 Onwards: Biotechnology Science or What? 5.1 Introduction. 5.2 Traditional Biotechnology and the Decehma Report. 5.3 The Changing Focus in BT in the USA in the Early 1980s. 5.4 Conclusions. References. Part Two The New Paradigm Based on Molecular Biology and Genetics. 6. Broadening of Biotechnology through Understanding Life, Genetics and Evolution. References. 7. The Beginning of the New Biotechnology. 7.1 Introduction. 7.2 The Beginnings of Evolution Theory and Genetics. 7.3 The Origin of Recombinant DNA Technology. 7.4 Oligonucleotide Synthesis Leads to Protein Engineering. 7.5 Synthetic DNA, Reverse Transciptase: Isolating Genes. 7.6 Biodiversity and Gene Mining. 7.7 Creating New Diversity by Design or Empirically. 7.8 Genetic Fingerprinting . 7.9 Inheritable Predisposition to Disease. References. 8. Ethical Aspects Related to Genome Research, and Reproductive Medicine. 8.1 Negative Public Reaction to Gene Technology. 8.2 Ethical Aspects: Animal Cloning and Fertility Research. References. 9. Elucidating Protein Structure: The Beginnings of Rational Protein Design. 9.1 Cambridge England, the Cradle of Structural Analysis of Macromolecules. 9.2 Redesigning the Protein Core. 9.3 Redesigning the Protein by Altering Primary Sequence. 9.4 Post Translational Modifications. 9.5 Total Chemical Synthesis. 9.6 Validation of Drug Design Based on the known Structure of the Target. 9.7 General Considerations in Drug Development. References. 10. The Development of Antibodies as Pharmaceutical Products. 10.1 An Introduction to the Immune System. 10.2 The Beginnings of Applied Immunology. 10.3 Monoclonal Antibodies. 10.4 Producing Antibodies via rDNA and Combinatorial Biology. 10.5 Affinity Enrichment on Surface of Immobilized Target Molecules. 10.6 Mice with Human Antibody Gene Repertoires. 10.7 Two Severe Setbacks during Clinical Testing. 10.8 A Survey of Therapeutic Antibodies. References. 11. Hereditary Disease and Human Genome Analysis. 11.1 Introduction. 11.2 Heredity Studies and Family Counselling . 11.3 Early Attempts to Analyse the Human Genome. 11.4 The Personalized Genome and Personal Medicine. 11.5 Analysing the Effect of the Environment on the Human Genome: Epigenetics. References. 12. Transgenic Animals and Plants. 12.1 Introduction. 12.2 Stem Cells and Gene Targeting. References. 13. Extrapolating to the Future. 13.1 Summary of the Status Quo. 13.2 Insect Control Through Sterile Males (SIT). 13.3 The Future of Gene Therapy. 13.4 Stem Cell Therapy. 13.5 Flash Sequencing DNA: A Human Genome Sequence in Minutes? 13.6 Systems Biology and Looking for Druggable Targets. 13.7 Synthetic Biology. References. 14. Biotechnology and Intellectual Property. 14.1 Introduction. 14.2 Patents Ensure Growth and Rapid Dissemination of Knowledge. 14.3 Owning a Patent does not simply Mean that it can be Implemented: Freedom to Operate (FTO). 14.4 Life-forms as Novel Subject Matter for Patents? 14.5 Technology State of the Art : Precedence/Directives, not Fixed by Law. 14.6 Who can make Decisions about Public Morality? 14.7 Biotechnology-oriented Directives Guide Patenting Decisions. References. Part Three. Application. 15. Bioprocess Engineering. 15.1 Introduction. 15.2 Aspects of Applied Microbiology. 15.3 Biocatalysis. 15.4 Biochemical Engineering. 15.5 Process Sustainability and Ecological Considerations. 15.6 Biosystems Engineering, including Omics Technologies. 15.7 Outlook and Perspectives. References. 16. Industrial Biotechnology. 16.1 Introduction. 16.2 General Aspects. 16.3 Commodities. 16.4 Chemicals. 16.5 Food Processing and Products. 16.6 Environmental Processes. 16.7 Summary, Trends and Perspectives. References. 17. Pharmaceutical Biotechnology. 17.1 Introduction. 17.2 Drug Targeting, Discovery Strategies and Development. 17.3 Pharmaceuticals Production. 17.4 Products, Pharmaceuticals Made by Biotechnology. 17.5 Medicinal Techniques, Diagnostics. 17.6 Business: Companies and Economic Aspects. References. 18. Plant Biotechnology. 18.1 Introduction. 18.2 Political, Ethical and Biosafety Aspects. 18.3 Research and Development. 18.4 Application of Modified Plants and Products. 18.5 Economic Aspects. 18.6 Summary and Outlook. References. Index.