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Intrabodies are antibodies that are not secreted but bind to their antigens inside the cell producing them. Intrabodies targeting antigens in the endoplasmatic reticulum were successfully used in vitro and in vivo. However, many target antigens interesting for research or therapy are located in the reducing environment of the cytosol, where correct folding and formation of disulfide bonds cannot be ensured. The majority of different scFv fragments, when expressed in the cytosol of the cell, do not fold correctly, are not stable or cannot bind their antigen. Such scFv antibodies are therefore not suited as intrabodies.In this study, we evaluated fast and simple screening methods to identify scFv fragments that are stable and functional in the cytosol. We analyzed various phage display derived human scFv antibodies recognizing extracellular signal-regulated kinase 2 (Erk2) for stability and antigen binding under reducing and non-reducing conditions. Further, we developed an assay allowing to measure the interaction of the scFv intrabodies with their antigen in the cytosol of in living cells, by using a Split-Luciferase (Split-Luc) assay. ScFv fragments showing antigen binding in the cytosol could successfully be identified.

The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed a sub nM IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody was demonstrated in the Syrian hamster and in the hACE2 mice model using a silenced human IgG1 Fc part. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD was solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibtion of STE90-C11 is not blocked by many known RBD mutations including N439K, L452R, E484K or L452R+E484Q (emerging B.1.617). STE90-C11 derived human IgG1 with FcγR silenced Fc (COR-101) is currently undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.In BriefHuman antibodies were selected from convalescent COVID-19 patients using antibody phage display. The antibody STE90-C11 is neutralizing authentic SARS-CoV-2 virus in vitro and in vivo and the crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD revealed that this antibody is binding in the RBD-ACE2 interface. S1 binding of STE90-C11 and inhibition of ACE2 binding is not blocked by many known RBD mutations.

The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed an IC50 of 0.56 nM in a plaque-based live SARS-CoV-2 neutralization assay. The crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD was solved at 2.0 A resolution showing that the antibody binds at the same region as ACE2 to RBD. In contrast to other published anti-SARS-CoV-2 antibodies, the binding of STE90-C11 is not blocked by known RBD mutations, endowing our antibody with higher intrinsic resistance to those possible escape mutants. Funding: We kindly acknowledge the financial support of MWK Niedersachsen (14-76103-184CORONA-2/20) for antibody generation. We also acknowledge support of the European Union for the ATAC (“antibody therapy against corona”, Horizon 2020 number 101003650) consortium. Conflict of Interest: The authors declare a conflict of interest. F.B., D.M.,N.L., S.S., P.A.H., R.B., M.R., K.T.S.,K.D.R.R., S.Z.-E., M.B., V.F., S.T.,M.S. and M.H. are inventors on a patent application on blocking antibodies against SARS-CoV-2. A.H., A.F. and T.S. are officers of CORAT Therapeutics GmbH, a company founded by YUMAB GmbH for the clinical and regulatory development of STE90-C11 (COR-101). A.F., T.S., S.D. and M.H. are shareholders of YUMAB GmbH. Ethical Approval: Approval number, FV-2020-02. Approved by the TU Braunschweig Institute for Psychology.

The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF-β signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF-β signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF-β signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA.Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.

Durch die Moglichkeit, rekombinante Antikorper herzustellen, anstatt auf die Produktion von Antikorpern in einem Versuchstier oder in Hybridomen angewiesen zu sein, wird der molekulare Aufbau von Antikorpern steuerbar. Das kann beispielsweise genutzt werden, um Antikorper mit verschiedenen Molekulgrosen und Formaten herzustellen (Frenzel et al. 2013).

Der Begriff „rekombinante Antikorper“ bezeichnet Immunglobuline oder antigenbindende Fragmente, welche in heterologen Produktionssystemen hergestellt werden. Da die Voraussetzung der heterologen Produktion eine Transformation mit bekannter DNA ist, sind rekombinante Antikorper stets durch ihre bekannte Sequenz – dem Bauplan – definiert, und unterscheiden sich so grundsatzlich von den polyklonalen Antiseren und den monoklonalen Antikorpern aus Hybridomen. Rekombinante Antikorper sind also auch stets gentechnologische Produkte und in aller Regel durch „ antibody engineering “ (eine sinnvolle deutsche Ubersetzung gibt es nicht) hergestellt.

Antikorper werden seit Jahrzenten in zahlreichen unterschiedlichen Assays eingesetzt. Der wohl meistverbreitete Assay in der Forschung und Diagnostik ist der Immunblot, auch haufig als Westernblot bezeichnet. Hier werden zuerst Proteine mittels SDS-PAGE aufgetrennt, dann auf eine Nitrocellulose- oder PVDF-Membran ubertragen und die nachzuweisenden Proteine mit Hilfe eines Detektionsantikorpers gefarbt.

Ihren Durchbruch verdankte die Technologie rekombinanter Antikorper vor allem dem Bedarf an menschlichen Antikorpern fur die Therapie – durch den Bedarf fur Klonierung und genetische Modifikation von Maus-Hybridom-Antikorpern. Es folgten bald darauf revolutionare neue Selektionsmoglichkeiten fur komplett menschliche Antikorper durch Phagendisplay oder transgene Mausstamme. In den 1990er Jahren zeigte sich sehr schnell, dass E. coli fur die Produktion groserer Mengen nicht immer der geeignete Organismus ist, insbesondere nicht fur die groseren Immunglobulinformate wie komplette IgG.

Emil von Behring und Shibasaburō Kitasato haben als erstes nachgewiesen, dass eine Substanz im Blut existiert, die Toxine neutralisieren kann (von Behring und Kitasato von Behring and Kitasato 1890). Diese Substanz wurde damals als „Antitoxin“ bezeichnet. Durch die aus dieser Erkenntnis abgeleitete Serumtherapie konnte erstmals die Diphtherie (Krupphusten) behandelt werden.

ImmunoRNases combine tumor targeting by antibodies with the cytotoxic action of ribonucleases from the RNase A superfamily. This study investigated for the first time all catalytic active human RNase A family members (1 to 8) as effector components of antibody fusion proteins. ImmunoRNase fusion proteins were constructed using the CD30-specific bivalent recombinant scFv-Fc antibody SH313-B5. Production of the resulting entirely human immunoRNases 1 to 8 was done in mammalian cells by secretion of active forms. The immunoRNases mediated CD30-specific cell binding and showed ribonucleolytic activity. Interestingly, immunoRNases 1 and 2 were active in the presence of up to 5-/20-fold molar excess of the pancreatic RNase inhibitor (RI), which is supposed to efficiently inhibit all human RNase A activity. ImmunoRNases 3, 4, 6 and 7 were only inhibited by several fold molar excess of RI, whereas immunoRNases 5 and 8 were already completely inactive at equimolar RI concentrations. Compared to free RNases, activity and RI sensitivity were not significantly changed by antibody fusion or dimerisation. ImmunoRNase3 and 5 mediated tumor growth inhibition at low nanomolar concentrations. Anti-tumor activity was antigen-specific and did not show any correlation with ribonucleolytic activity or RI sensitivity.

The use of antibodies to target their antigens in living cells is a powerful analytical tool for cell biology research. Not only can molecules be localized and visualized in living cells, but interference with cellular processes by antibodies may allow functional analysis down to the level of individual post-translational modifications and splice variants, which is not possible with genetic or RNA-based methods. To utilize the vast resource of available antibodies, an efficient system to deliver them into the cytosol from the outside is needed. Numerous strategies have been proposed, but the most robust and widely applicable procedure still remains to be identified, since a quantitative ranking of the efficiencies has not yet been done. To achieve this, we developed a novel efficiency evaluation method for antibody delivery based on a fusion protein consisting of a human IgG 1 Fc and the recombination enzyme Cre (Fc-Cre). Applied to suitable GFP reporter cells, it allows the important distinction between proteins trapped in endosomes and those delivered to the cytosol. Further, it ensures viability of positive cells and is unsusceptible to fixation artifacts and misinterpretation of cellular localization in microscopy and flow cytometry. Very low cytoplasmic delivery efficiencies were found for various profection reagents and membrane penetrating peptides, leaving electroporation as the only practically useful delivery method for antibodies. This was further verified by the successful application of this method to bind antibodies to cytosolic components in living cells.

This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69-3A2) neutralized 50% of 5x10(4) TCID 50/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69-3A2 antibody is a promising candidate for future passive vaccine development.

Hodgkin's lymphoma (HL) and ALK(+) anaplastic large-cell lymphoma (ALCL) have become highly curable due to the success of modern regimens of chemotherapy and radiotherapy. However, up to one-third of the patients experience relapse or do not respond to first-line therapy, and half of them relapse again after secondary therapy with limited options for further treatment. In the last 15 years, monoclonal antibodies (mAbs) directed to surface receptors became a new and valuable therapeutic option in many hematologic malignancies. Due to its restricted expression on normal activated lymphocytes and its high expression on malignant cells, CD30 represents an attractive target molecule for HL and ALCL therapy. However, unconjugated CD30 mAbs have demonstrated a lack of objective clinical responses in patients with recurrent HL. CD30 exhibits complex signaling pathways, and binding of its natural ligand or anti-CD30 mAbs can induce apoptosis but may also promote proliferation and activation depending on the cellular context. Moreover, CD30 rapidly internalizes after crosslinking, which counteracts efficient recruitment of immunologic effectors but also provides the opportunity to transfer cytotoxic payloads coupled to CD30-specific mAbs into the tumor cells. Several tumor targeting approaches have been studied, including radio-immunoconjugates, immunotoxins, immunoRNases, immunokinases, and antibody drug conjugates (ADCs). In 2011, the ADC brentuximab-vedotin, consisting of the CD30-specific chimeric mAb cAC10 and the potent tubulin toxin monomethyl auristatin E, gained regulatory approval as a well tolerated and highly active drug in patients with refractory and relapsed HL and ALCL. SGN-35 is on the way to being incorporated in the standard management of CD30(+) lymphoma with significant therapeutic impact. This review gives a critical overview about anti-CD30 therapies with unconjugated, engineered, and conjugated mAbs and the therapeutic challenges of treatment of CD30(+) lymphoma.

C-reactive protein (CRP) is a serum marker highly upregulated in inflammation after bacterial infection. Robust, reliable and quick quantification of CRP would be a substitute for erythrocyte sedimentation rate (ESR) with superior diagnostic value. Quartz crystal microbalance (QCM) based sensors coated with specific antibodies and integrated into lab-on-chip systems are in development for rapid point of care quantification. In this study, we isolated three CRP specific single chain (sc)Fv antibody fragments using phage display from an antibody gene library. Their affinities ranged from 2.7 × 10(-8) to 1.0 × 10(-8) M when measured by surface plasmon resonance. ScFv antibody fragment LA13-IIE3 showed best affinity, high long-term stability and remarkable resistance to denaturation. This scFv antibody fragment was coupled to a QCM sensor. CRP quantification in up to 15 samples sequentially measured on the same sensor with intermitting regeneration by buffer was demonstrated.

For proteome research, antibodies against a growing number of antigens must be generated and characterized. The high throughput generation of antibody fragments, using in vitro selection, requires bacterial expression of antibody fragments. This created a need to establish an expression method to improve the parallel production of many antibody fragments. In this study, we describe the development of a high throughput bacterial production method for single chain fragment variables (scFvs) using shaking flasks or the LEX™ bioreactor. We compared the influence of a set of production parameters on Escherichia coli production of four different scFv. The results led to an optimized protocol for the parallel production of multiple antibody fragments.

Over the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of “fully” human antibodies with potentially superior clinical efficacy and lowest immunogenicity. Antibody phage display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by phage display were approved for therapy. In 2002, adalimumab (Humira®) became the first phage display-derived antibody granted a marketing approval. Humira® was also the first approved human antibody, and it is currently the best-selling antibody drug on the market. Numerous phage display-derived antibodies are currently under advanced clinical investigation, and, despite the availability of other technologies such as human antibody-producing transgenic mice, phage display has not lost its importance for the discovery and engineering of therapeutic antibodies. Here, we provide a comprehensive overview about phage display-derived antibodies that are approved for therapy or in clinical development. A selection of these antibodies is described in more detail to demonstrate different aspects of the phage display technology and its development over the last 25 years.

Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies.

The functional decryption of the human proteome is the challenge which follows the sequencing of the human genome. Specific binders to every human protein are key reagents for this purpose. In vitro antibody selection using phage display offers one possible solution that can meet the demand for 25,000 or more antibodies, but needs substantial standardisation and minimalisation. To evaluate this potential, three human, naive antibody gene libraries (HAL4/7/8) were constructed and a standardised antibody selection pipeline was set up. The quality of the libraries and the selection pipeline was validated with 110 antigens, including human, other mammalian, fungal or bacterial proteins, viruses or haptens. Furthermore, the abundance of VH, kappa and lambda subfamilies during library cloning and the E. coli based phage display system on library packaging and the selection of scFvs was evaluated from the analysis of 435 individual antibodies, resulting in the first comprehensive comparison of V gene subfamily use for all steps of an antibody phage display pipeline. Further, a compatible cassette vector set for E. coli and mammalian expression of antibody fragments is described, allowing in vivo biotinylation, enzyme fusion and Fc fusion.

Pork meat is one of the major sources for human infections with Salmonella enterica subspecies enterica serovars. Further, zoonoses caused by S. enterica subspecies enterica serovars are responsible for substantial economical losses in industrial countries. Quick and reliable detection of this infection is urgently needed to improve consumer security. Due to its capability to identify infections independent of the species, a competitive ELISA is the preferable method for the detection of anti-Salmonella antibodies in serum. Recombinant antibody fragments (scFvs) were isolated from the naive human antibody gene library HAL7 by phage display. Recombinant produced outer membrane protein D (OmpD) of Salmonella Typhimurium was used as antigen. The characterization of the isolated single chain Fv (scFv) antibodies was done by enzyme-linked immunosorbent assay (ELISA), immunoblot, sequencing, epitope mapping and size exclusion chromatography (SEC). The detection of anti-OmpD IgGs in swine sera by competitive ELISA was shown in a proof of principle concept. Furthermore, the developed competitive ELISA would be compatible to a recently published DIVA vaccine, allow to distinguish between infected and vaccinated pigs.

The new era of functional genomics demands several antibodies as specific detection reagents for proteins, their complexes and post-translational modifications. Only in vitro antibody selection technologies are able to provide the required throughput to generate these large numbers. Phage display is the most widely used technology for in vitro selection of antibodies. The major bottleneck of a phage display selection pipeline is the production of monoclonal antibody fragments for screening and further analysis. In this study, we describe the development of improved protocols for the production of single chain Fv (scFv) antibody fragments in 96-well microtitre plates (MTPs) in Escherichia coli. Four scFvs were expressed using the antibody expression vector pOPE101-XP to analyse the influence of a set of different parameters on their production. Further, six scFvs were expressed using the phage display vector pHAL14 to investigate the effect on the production of functional scFvs using those parameters that improved production from pOPE101-XP. Yield in MTPs was influenced by a variety of conditions and was also strongly dependent on the individual scFv clone. Although it was not possible to deduce a single set of optimal parameters applicable to all the tested scFvs, a combined protocol was developed which improved the expression of scFv fragments over standard methods.

Branched beta-1,3/1,6-glucans (scleroglucan) were produced by cultivation of Sclerotium rolfsii ATCC 15205. Regioselective hydrolysis at the beta-1,3-linkage of the cell-free and purified polysaccharide was performed in borosilicate glass bottles at pH 5, 121 degrees C, and 1 bar for 72 h. The mixture was divided into four molar mass fractions by stepwise cross-flow filtration using different cutoffs. In vitro studies revealed that scleroglucan hydrolysates with a low molar mass of less than 5 kDa significantly stimulated the activation and maturation of porcine monocyte derived dendritic cells (MoDC) by upregulation of CD40 and CD80/86 as well as by reduction of antigen uptake. MoDC treated with low molar mass scleroglucan showed a considerable increase in the amounts of secreted proinflammatory cytokine tumor necrosis factor alpha and stimulated the proliferation of lymphocytes. Therefore, scleroglucan molecules of low molecular weight are able to induce activation and maturation of porcine DC, which are key initiators of inflammatory and adaptive immune responses, and could provide improved protection against infectious diseases.

An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a –mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies.

Antibody phage display is an in vitro technology to generate recombinant antibodies. In particular for pathogens like viruses or toxins, antibody phage display is an alternative to hybridoma technology, since it circumvents the limitations of the immune system. Phage display allows the generation of human antibodies from naive antibody gene libraries when either immunized patients are not available or immunization is not ethically feasible. This technology also allows the construction of immune libraries to select in vivo affinity matured antibodies if immunized patients or animals are available.In this review, we describe the generation of human and human-like antibodies from naive antibody gene libraries and antibodies from immune antibody gene libraries. Furthermore, we give an overview about phage display derived recombinant antibodies against viruses and toxins for diagnostics and therapy.

Antikorper sind unersetzliche Werkzeuge fur die Analyse von Proteinen und damit Schlusselreagenzien fur eine Hauptaufgabe der Genomforschung – der Zuordnung von Funktionen zu der noch riesigen Zahl von uncharakterisierten Gensequenzen und den dazu gehorigen Genprodukten. Zur Bewaltigung dieser Aufgabe entwickelt die Antikorperfabrik (Antibody Factory) Verfahren, mit denen spezifische Antikorper in einem weit hoherem Durchsatz als bisher generiert werden konnen. Als zentrale Technologie wird hierfur das Antikorper-Phagen-Display eingesetzt, mit dem Antikorper vollstandig ohne Versuchstiere aus menschlichen Antikorper-Genbibliotheken isoliert werden konnen.

T cells are a new and promising antigen-specific therapeutic option for the treatment of malignant diseases. To achieve antigen specificity against tumor antigens, T cells can be manipulated by gene transfer to express chimeric antigen receptors (CARs). CAR-expressing T cells are called redirected T cells. CARs are composed of an extracellular antibody-derived antigen recognition domain, a transmembrane domain and a cytoplasmatic signal domain. Therefore, redirected T cells combine the exchangeable specificity of an antibody with the cytotoxic machinery of a T cell. Early clinical trials with redirected T cells targeting cluster of differentiation (CD) 19 have shown impressive results in CD19-positive hematological cancers. However, for solid cancers only limited clinical experience exists and new and innovative concepts have to be developed to overcome tumor-mediated immune suppression. Herein, we describe the general design of a CAR, the function of the different domains and the different strategies to produce redirected T cells. Furthermore, we summarize and discuss the preclinical and clinical data indicating the tremendous potential of redirected T cells to become a mainstay of cancer immunotherapy.

Background Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. Results The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×1010 independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. Conclusion The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

T cells are a new and promising antigen-specific therapeutic option for the treatment of malignant diseases. To achieve antigen specificity against tumor antigens, T cells can be manipulated by gene transfer to express chimeric antigen receptors (CARs). CAR-expressing T cells are called redirected T cells. CARs are composed of an extracellular antibody-derived antigen recognition domain, a transmembrane domain and a cytoplasmatic signal domain. Therefore, redirected T cells combine the exchangeable specificity of an antibody with the cytotoxic machinery of a T cell. Early clinical trials with redirected T cells targeting cluster of differentiation (CD) 19 have shown impressive results in CD19-positive hematological cancers. However, for solid cancers only limited clinical experience exists and new and innovative concepts have to be developed to overcome tumor-mediated immune suppression. Herein, we describe the general design of a CAR, the function of the different domains and the different strategies to produce redirected T cells. Furthermore, we summarize and discuss the preclinical and clinical data indicating the tremendous potential of redirected T cells to become a mainstay of cancer immunotherapy.

Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies.

The gene transfer of tumor-specific chimeric immunoglobulin T-cell receptors (cIgTCRs) combining antibody-like specificity with the effector cell function could be an attractive tool in immunotherapy. In this study, we directed the human natural killer (NK) cell line YT to tumor cells by gene transfer of a cIgTCR with specificity against the human carcinoembryonic antigen (CEA). The cIgTCR was constructed of a CEA-specific humanized single-chain Fv antibody fragment fused to the IgG1 Fc domain and the CD3 zeta chain. YT cells were transfected with the cIgTCR gene by electroporation and cIgTCR-expressing cells were enriched by immunoaffinity purification. cIgTCR-expressing YT cells specifically lysed CEA(+) colon carcinoma cell lines, which were resistant to the parental YT cell line. The lysis was not inhibited in the presence of soluble CEA. Receptor gene-modified YT cells retained their CEA-specific cytolytic activity after gamma-irradiation in vitro and inhibited the tumor growth in vivo after adoptive transfer into NOD/SCID mice. This gene-modified NK cell line available in unlimited source might be useful in clinical immunotherapy of CEA(+) cancer.

Abs bind to unprocessed Ags, whereas cytotoxic CD8+ T cells recognize peptides derived from endogenously processed Ags presented in the context of class I MHC complexes. We screened, by ELISA, human sera for Abs reacting specifically with the influenza matrix protein (IMP)–derived peptide58–66 displayed by HLA-A*0201 complexes. Among 653 healthy volunteers, blood donors, and women on delivery, high-titered HLA-A*0201/IMP58–66 complex–specific IgG Abs were detected in 11 females with a history of pregnancies and in 1 male, all HLA-A*0201−. These Abs had the same specificity as HLA-A*0201/IMP58–66–specific cytotoxic T cells and bound neither to HLA-A*0201 nor the peptide alone. No such Abs were detected in HLA-A*0201+ volunteers. These Abs were not cross-reactive to other self–MHC class I alleles displaying IMP58–66, but bound to MHC class I complexes of an HLA nonidentical offspring. HLA-A*0201/IMP58–66 Abs were also detected in the cord blood of newborns, indicating that HLA-A*0201/IMP58–66 Abs are produced in HLA-A*0201− mothers and enter the fetal blood system. That Abs can bind to peptides derived from endogenous Ags presented by MHC complexes opens new perspectives on interactions between the cellular and humoral immune system.

Transient gene expression has evolved into an attractive technology for the rapid production of milligram to gram amounts of recombinant proteins. This review describes the different methods for introducing foreign DNA into suitable mammalian cells with either viral or non-viral vectors. Particular emphasis is given to non-viral transient transfection which represents meanwhile the most prominent variant due to recent progress in the resulting protein productivity. Non-viral transient transfection protocols are always based on the use of specific transfection reagents or the application of an electroporation device. The corresponding methods are compared with regard to their scale-up potential, also in consideration of potential production costs. The underlying cellular pathways of plasmid DNA incorporation, cytoplasmic release and translocation into the nucleus are important details to understand the transfection principle and further improve the technology. Problems associated with the application of transient gene expression at a larger scale are also addressed. In particular, the requirement of different cell culture media conditions for plasmid DNA complex preparation (if necessary), the transfection process itself and a high titer recombinant production need to be harmonized. Strategies to improve recombinant protein productivity by increasing the cell-specific output and/or sustaining the production phase are itemized as well. This can be accomplished by enabling cells to perform episomal plasmid replication, co-transfection with other plasmids, altering the cellular metabolism, temperature reduction, supplementation of specific production enhancers or combinations thereof. A number of examples for successful applications at pilot scale are also provided.

Targeting of immunological effector cells to tumor cells could be an efficient strategy of adoptive immunotherapy. The success of this strategy depends on the specificity of the effector cells and their availability in sufficient numbers. The aim of this study was to target the human natural killer cell line YT specifically to tumor cells. The cell line was modified by transfection with the cDNA of the human asialoglycoprotein receptor (ASGPR). This C-type lectin recognizes carbohydrates containing terminal galactosyl (Gal) residues, including the beta1-Gal bearing Thomsen-Friedenreich (TF) antigen, which is found on tumor cells. Binding assays revealed that the ASGPR-gene-transfected YT cell line binds significantly higher to tested target tumor cell lines than the mock-transfected control cells. Cytolytic activity against the tumor cell lines Raji, Jurkat and the TF-positive KG1 subline was increased. Genetic modification of YT cells could provide a useful tool for tumor targeting in immunotherapy.

International audience; Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease.

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG. In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.