Publications by authors named "Gertrudis Rojas"

29 Publications

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Baculovirus-free insect cell expression system for high yield antibody and antigen production.

Sci Rep 2020 12 7;10(1):21393. Epub 2020 Dec 7.

Department of Biotechnology, Technische Universität Braunschweig, Spielmannstraße 7, 38106, Braunschweig, Germany.

Antibodies are essential tools for therapy and diagnostics. Yet, production remains expensive as it is mostly done in mammalian expression systems. As most therapeutic IgG require mammalian glycosylation to interact with the human immune system, other expression systems are rarely used for production. However, for neutralizing antibodies that are not required to activate the human immune system as well as antibodies used in diagnostics, a cheaper production system would be advantageous. In our study, we show cost-efficient, easy and high yield production of antibodies as well as various secreted antigens including Interleukins and SARS-CoV-2 related proteins in a baculovirus-free insect cell expression system. To improve yields, we optimized the expression vector, media and feeding strategies. In addition, we showed the feasibility of lyophilization of the insect cell produced antibodies. Furthermore, stability and activity of the antibodies was compared to antibodies produced by Expi293F cells revealing a lower aggregation of antibodies originating from High Five cell production. Finally, the newly established High Five expression system was compared to the Expi293F mammalian expression system in regard of yield and costs. Most interestingly, all tested proteins were producible in our High Five cell expression system what was not the case in the Expi293F system, hinting that the High Five cell system is especially suited to produce difficult-to-express target proteins.
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http://dx.doi.org/10.1038/s41598-020-78425-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721901PMC
December 2020

Affinity-matured variants derived from nimotuzumab keep the original fine specificity and exhibit superior biological activity.

Sci Rep 2020 Jan 27;10(1):1194. Epub 2020 Jan 27.

Center of Molecular Immunology, calle 216 esq 15, PO Box 16040, Atabey, Playa, CP, 11300, La Habana, Cuba.

Nimotuzumab is a humanized monoclonal antibody against the Epidermal Growth Factor Receptor with a long history of therapeutic use, recognizing an epitope different from the ones targeted by other antibodies against the same antigen. It is also distinguished by much less toxicity resulting in a better safety profile, which has been attributed to its lower affinity compared to these other antibodies. Nevertheless, the ideal affinity window for optimizing the balance between anti-tumor activity and toxic effects has not been determined. In the current work, the paratope of the phage-displayed nimotuzumab Fab fragment was evolved in vitro to obtain affinity-matured variants. Soft-randomization of heavy chain variable region CDRs and phage selection resulted in mutated variants with improved binding ability. Two recombinant antibodies were constructed using these variable regions, which kept the original fine epitope specificity and showed moderate affinity increases against the target (3-4-fold). Such differences were translated into a greatly enhanced inhibitory capacity upon ligand-induced receptor phosphorylation on tumor cells. The new antibodies, named K4 and K5, are valuable tools to explore the role of affinity in nimotuzumab biological properties, and could be used for applications requiring a fine-tuning of the balance between binding to tumor cells and healthy tissues.
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http://dx.doi.org/10.1038/s41598-019-57279-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985160PMC
January 2020

Directed evolution of super-secreted variants from phage-displayed human Interleukin-2.

Sci Rep 2019 01 28;9(1):800. Epub 2019 Jan 28.

Center of Molecular Immunology, calle 216 esq 15, apartado 16040, Atabey, Playa, CP 11300, La Habana, Cuba.

Selection from a phage display library derived from human Interleukin-2 (IL-2) yielded mutated variants with greatly enhanced display levels of the functional cytokine on filamentous phages. Introduction of a single amino acid replacement selected that way (K35E) increased the secretion levels of IL-2-containing fusion proteins from human transfected host cells up to 20-fold. Super-secreted (K35E) IL-2/Fc is biologically active in vitro and in vivo, has anti-tumor activity and exhibits a remarkable reduction in its aggregation propensity- the major manufacturability issue limiting IL-2 usefulness up to now. Improvement of secretion was also shown for a panel of IL-2-engineered variants with altered receptor binding properties, including a selective agonist and a super agonist that kept their unique properties. Our findings will improve developability of the growing family of IL-2-derived immunotherapeutic agents and could have a broader impact on the engineering of structurally related four-alpha-helix bundle cytokines.
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http://dx.doi.org/10.1038/s41598-018-37280-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349883PMC
January 2019

Combining computational and experimental biology to develop therapeutically valuable IL2 muteins.

Semin Oncol 2018 01 1;45(1-2):95-104. Epub 2018 May 1.

Center of Molecular Immunology (CIM), Havana, Cuba.

High-dose IL2, first approved in 1992, has been used in the treatment of advanced renal cell carcinoma and melanoma. In these indications, IL2 induces long lasting objective responses in 5% to 20% of patients. However, toxicity and the unexpected expansion of regulatory T cells (Tregs) have limited its practical use and therapeutic impact, respectively. At the Center of Molecular Immunology in Havana, Cuba, a project was launched in 2005 to rationally design IL2 muteins that could be deployed in the therapy of cancer. The basic goal was to uncouple the pleiotropic effect of IL2 on different immune T cells, to obtain a mutein with a therapeutic index that was better than that achieved with wild type (wt) IL2. Using a combination of computational and experimental biology approaches, we predicted and developed two novel IL2 muteins with therapeutic potential. The first, designated no-alpha mutein, is an agonist of IL2R signaling with a reduced ability to expand Treg in vivo. In mice, the no-alpha mutein IL2 has higher antitumor activity and lower toxicity than wt IL2. It represents a potential best-in-class drug that has begun phase I/II clinical trials in solid tumors. The second, designated no-gamma mutein, is an antagonist of IL2R signaling, with some preferential affinity for Tregs. This mutein has antitumor activity in mice that likely derives from its ability to reduce Treg accumulation in vivo. It represents a first-in-class drug that offers a novel strategy to inhibit Treg activity in vivo.
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http://dx.doi.org/10.1053/j.seminoncol.2018.04.001DOI Listing
January 2018

Crystal structure of an L chain optimised 14F7 anti-ganglioside Fv suggests a unique tumour-specificity through an unusual H-chain CDR3 architecture.

Sci Rep 2018 Jul 18;8(1):10836. Epub 2018 Jul 18.

Department of Chemistry, University of Oslo, NO-0315 Oslo, Norway.

Targeted cancer immunotherapy offers increased efficacy concomitantly with reduced side effects. One antibody with promising clinical potential is 14F7, which specifically recognises the NeuGc GM3 ganglioside. This antigen is found in the plasma membrane of a range of tumours, but is essentially absent from healthy human cells. 14F7 can discriminate NeuGc GM3 from the very similar NeuAc GM3, a common component of cell membranes. The molecular basis for this unique specificity is poorly understood. Here we designed and expressed 14F7-derived single-chain Fvs (scFvs), which retained the specificity of the parent antibody. Detailed expression and purification protocols are described as well as the synthesis of the NeuGc GM3 trisaccharide. The most successful scFv construct, which comprises an alternative variable light chain (V), allowed structure determination to 2.2 Å resolution. The structure gives insights into the conformation of the important CDR H3 loop and the suspected antigen binding site. Furthermore, the presence of V instead of the original V elucidates how this subdomain indirectly stabilises the CDR H3 loop. The current work may serve as a guideline for the efficient production of scFvs for structure determination.
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http://dx.doi.org/10.1038/s41598-018-28918-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052152PMC
July 2018

Phagekines: Screening Binding Properties and Biological Activity of Functional Cytokines Displayed on Phages.

Methods Mol Biol 2018 ;1701:535-560

Center of Molecular Immunology, Calle 216 esq 15, Atabey, Playa, La Habana, CP, 11300, Cuba.

The current chapter focuses on the use of filamentous phages to display, modify, and characterize cytokines, which are proteins belonging to a versatile group of essential mediators involved in cell-cell communication. Cytokines exhibit a considerable diversity, both in functions and in structural features underlying their biological effects. A broad variety of cytokines have been successfully displayed on phages, allowing the high-throughput study of their binding properties and biological activities and the discovery of novel therapeutics through directed evolution. The technical singularities and some potential applications of cytokine phage display are illustrated here with the case of Interleukin-2, a prototypic member of the four-alpha-helix bundle cytokine family playing a pivotal role in the immune response and having a long history of therapeutic use.
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http://dx.doi.org/10.1007/978-1-4939-7447-4_30DOI Listing
July 2018

Molecular dissection of the interactions of an antitumor interleukin-2-derived mutein on a phage display-based platform.

J Mol Recognit 2015 Apr 16;28(4):261-8. Epub 2015 Feb 16.

Systems Biology Department, Center of Molecular Immunology, Calle 216 esq 15, PO Box 16040, Atabey, Playa, La Habana, CP, 11600, Cuba.

A mutein with stronger antitumor activity and lower toxicity than wild-type human interleukin-2 (IL-2) has been recently described. The rationale behind its design was to reinforce the immunostimulatory potential through the introduction of four mutations that would selectively disrupt the interaction with the IL-2 receptor alpha chain (thought to be critical for both IL-2-driven expansion of T regulatory cells and IL-2-mediated toxic effects). Despite the successful results of the mutein in several tumor models, characterization of its interactions was still to be performed. The current work, based on phage display of IL-2-derived variants, showed the individual contribution of each mutation to the impairment of alpha chain binding. A more sensitive assay, based on the ability of phage-displayed IL-2 variants to induce proliferation of the IL-2-dependent CTLL-2 cell line, revealed differences between the mutated variants. The results validated the mutein design, highlighting the importance of the combined effects of the four mutations. The developed phage display-based platform is robust and sensitive, allows a fast comparative evaluation of multiple variants, and could be broadly used to engineer IL-2 and related cytokines, accelerating the development of cytokine-derived therapeutics.
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http://dx.doi.org/10.1002/jmr.2440DOI Listing
April 2015

High throughput functional epitope mapping: revisiting phage display platform to scan target antigen surface.

MAbs 2014 ;6(6):1368-76

a Systems Biology Department ; Center of Molecular Immunology ; La Habana , Cuba.

Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.
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http://dx.doi.org/10.4161/mabs.36144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622681PMC
September 2015

Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor.

MAbs 2014 Jul-Aug;6(4):1013-25. Epub 2014 Apr 23.

Systems Biology Department; Center of Molecular Immunology; Habana, Cuba.

Molecular details of epidermal growth factor receptor (EGFR) targeting by nimotuzumab, a therapeutic anti-cancer antibody, have been largely unknown. The current study delineated a functional map of their interface, based on phage display and extensive mutagenesis of both the target antigen and the Fv antibody fragment. Five residues in EGFR domain III (R353, S356, F357, T358, and H359T) and the third hypervariable region of nimotuzumab heavy chain were shown to be major functional contributors to the interaction. Fine specificity differences between nimotuzumab and other anti-EGFR antibodies were revealed. Mapping information guided the generation of a plausible in silico binding model. Knowledge about the epitope/paratope interface opens new avenues for the study of tumor sensitivity/resistance to nimotuzumab and for further engineering of its binding site. The developed mapping platform, also validated with the well-known cetuximab epitope, allows a comprehensive exploration of antigenic regions and could be expanded to map other anti-EGFR antibodies.
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http://dx.doi.org/10.4161/mabs.28915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4171005PMC
February 2015

A combinatorial mutagenesis approach for functional epitope mapping on phage-displayed target antigen: application to antibodies against epidermal growth factor.

MAbs 2014 May-Jun;6(3):637-48. Epub 2014 Mar 3.

Systems Biology Department; Center of Molecular Immunology; La Habana, Cuba.

Although multiple different procedures to characterize the epitopes recognized by antibodies have been developed, site-directed mutagenesis remains the method of choice to define the energetic contribution of antigen residues to binding. These studies are useful to identify critical residues and to delineate functional maps of the epitopes. However, they tend to underestimate the roles of residues that are not critical for binding on their own, but contribute to the formation of the target epitope in an additive, or even cooperative, way. Mapping antigenic determinants with a diffuse energetic landscape, which establish multiple individually weak interactions with the antibody paratope, resulting in high affinity and specificity recognition of the epitope as a whole, is thus technically challenging. The current work was aimed at developing a combinatorial strategy to overcome the limitations of site-directed mutagenesis, relying on comprehensive randomization of discrete antigenic regions within phage-displayed antigen libraries. Two model antibodies recognizing epidermal growth factor were used to validate the mapping platform. Abrogation of antibody recognition due to the introduction of simultaneous replacements was able to show the involvement of particular amino acid clusters in epitope formation. The abundance of some of the original residues (or functionally equivalent amino acids sharing their physicochemical properties) among the set of mutated antigen variants selected on a given antibody highlighted their contributions and allowed delineation of a detailed functional map of the corresponding epitope. The use of the combinatorial approach could be expanded to map the interactions between other antigens/antibodies.
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http://dx.doi.org/10.4161/mabs.28395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4011908PMC
May 2015

Fine epitope mapping based on phage display and extensive mutagenesis of the target antigen.

Authors:
Gertrudis Rojas

Methods Mol Biol 2014 ;1131:447-76

Systems Biology Department, Center of Molecular Immunology, La Habana, Cuba.

The residues contributing to the formation of the epitope recognized by a monoclonal antibody can be defined in several ways. Mutagenesis on the target antigen, followed by screening of the reactivity of the new variants with the antibody, is particularly powerful to reveal the functional contribution of each amino acid in the context of the native antigen. The current protocol provides a relatively simple procedure to study the surface of the target antigen in the search for residues involved in recognition. If the antigen is successfully displayed on the surface of filamentous bacteriophages, it can be quickly scanned by simultaneous mutagenesis of multiple solvent-exposed residues and high-throughput screening of the new variants with the antibody to be mapped. Once a few amino acids critically involved in recognition are defined, they can be used as starting points for a comprehensive exploration of the antigenic region by randomization of their whole neighborhood. The analysis of binding and sequence data allows delineating a detailed picture of the epitope recognized by the antibody under investigation.
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http://dx.doi.org/10.1007/978-1-62703-992-5_27DOI Listing
October 2014

Fine epitope specificity of antibodies against interleukin-2 explains their paradoxical immunomodulatory effects.

MAbs 2014 Jan 19;6(1):273-85. Epub 2013 Nov 19.

Systems Biology Department; Center of Molecular Immunology; La Habana, Cuba.

The functional dichotomy of antibodies against interleukin-2 (IL-2) is thought to depend upon recognition of different cytokine epitopes. Beyond functional studies, the only molecular evidence obtained so far located the epitopes recognized by the immunoenhancing antibodies S4B6 and JES6-5H4 within the predicted interface of IL-2 with the α receptor subunit, explaining the preferential stimulation of effector cells displaying only β and γ receptor chains. A consistent functional map of the epitope bound by the immunoregulatory antibody JES6-1A12 has now been delineated by screening the interactions of phage-displayed antigen variants (with single and multiple mutations) and antigen mimotopes. The target determinant resides in a region between the predicted interfaces with α and β/γ receptor subunits, supporting the dual inhibitory role of the antibody on both interactions. Binding by JES6-1A12 would thus convert complexed IL-2 into a very weak agonist, reinforcing the advantage of T regulatory cells (displaying the high affinity αβγ heterotrimeric receptor) to capture the cytokine by competition and expand over effector cells, ultimately resulting in the observed strong tolerogenic effect of this antibody. Detailed knowledge of the epitopes recognized by anti-IL-2 antibodies with either immunoenhancing or immunoregulatory properties completes the molecular scenario underlying their use to boost or inhibit immune responses in multiple experimental systems. The expanded functional mapping platform now available could be exploited to study other interactions involving related molecular pairs with the final goal of optimizing cytokine and anti-cytokine therapies.
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http://dx.doi.org/10.4161/mabs.27224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3929449PMC
January 2014

Affinity maturation and fine functional mapping of an antibody fragment against a novel neutralizing epitope on human vascular endothelial growth factor.

Mol Biosyst 2013 Aug 24;9(8):2097-106. Epub 2013 May 24.

Recombinant Antibodies Laboratory, Cancer Research Department, Center for Genetic Engineering and Biotechnology, PO Box 6162, Ave. 31 e/ 158 y 190, Cubanacan, Playa, La Habana, CP 10600, Cuba.

We have previously reported the isolation of a novel single-chain variable fragment (scFv) against vascular endothelial growth factor (VEGF), from a phage-displayed human antibody repertoire. This scFv, denominated 2H1, was shown to block the binding of VEGF to its receptor but exhibited a moderate binding affinity. Here, we describe the affinity maturation of the 2H1 scFv. Two phage-displayed libraries were constructed by diversification of the third complementarity-determining regions (CDRs) of the light (VL) and heavy (VH) chain variable domains of 2H1 using parsimonious mutagenesis. A competitive phage-selection strategy in the presence of the parental scFv as a competitor was used to eliminate low affinity binders. High affinity variants were retrieved from both libraries. An optimized VL variant was designed and constructed by combining recurrent replacements found among selected variants in a single molecule, resulting in an additional affinity increase. Further affinity improvements were achieved by combining this optimized VL with the best VH variants. The final variant obtained here, L3H6, showed an overall affinity improvement of 18-fold over the parental scFv and exhibited an enhanced potency to block the binding of VEGF to its receptor. Using phage display and extensive mutagenesis of VEGF, we determined the fine specificity of L3H6. This functional mapping revealed a novel neutralizing epitope on human VEGF defined by the residues Y25, T71, E72, N100, K101, E103 and R105. The conformational epitope recognized by L3H6 was recapitulated by grafting human VEGF residues into the mouse molecule, providing further confirmation of the nature of the identified epitope.
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http://dx.doi.org/10.1039/c3mb70136kDOI Listing
August 2013

Engineering the binding site of an antibody against N-glycolyl GM3: from functional mapping to novel anti-ganglioside specificities.

ACS Chem Biol 2013 Feb 21;8(2):376-86. Epub 2012 Nov 21.

Center of Molecular Immunology, Calle 216 esq 15, Atabey, Playa, PO Box 16040, La Habana CP 11600, Cuba.

The structurally related gangliosides N-glycolyl GM3 and N-acetyl GM3 are potential targets for tumor immunotherapy. 14F7 is a monoclonal antibody able to discriminate the tumor-specific antigen N-glycolyl GM3 from the closely related N-acetyl GM3 on the basis of the presence of a single additional hydroxyl group in the former. A combinatorial phage display strategy, based on the screening of a large library followed by refined mutagenesis, allowed a thorough exploration of the binding chemistry of this unique antibody. Three essential features of the heavy chain variable region were identified: two aromatic rings (in positions 33 and 100D) contributing to the binding site architecture and an arginine residue (position 98) critical for recognition. Directed evolution of 14F7 resulted in novel variants that cross-react with the tumor-associated antigen N-acetyl GM3 and display recurrent replacements: the substitution W33Q and the appearance of additional arginine residues at several positions of CDR H1. Successful conversion of such engineered variable regions into whole cross-reactive anti-GM3 immunoglobulins validated our phage-based approach to study and modify the lead antibody 14F7. The resulting family of closely related antibodies offers new tools to study the mechanisms of cell death induced by antibodies targeting gangliosides. In vitro directed evolution was useful to overcome the technical limitations to obtain anti-ganglioside antibodies. The case of 14F7 illustrates the power of combining library screening with focused site-directed randomization for a comprehensive scanning of protein interactions.
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http://dx.doi.org/10.1021/cb3003754DOI Listing
February 2013

Deciphering the molecular bases of the biological effects of antibodies against Interleukin-2: a versatile platform for fine epitope mapping.

Immunobiology 2013 Jan 16;218(1):105-13. Epub 2012 Feb 16.

Systems Biology Department, Center of Molecular Immunology, Calle 216 esq 15, Atabey, Playa, PO Box 16040, La Habana, CP 11600, Cuba.

Elucidating the network of interactions established by Interleukin-2 is a key step to understanding its role as a master regulator of the immune system. Binding of this cytokine by specific antibodies gives rise to different classes of immune complexes that boost or inhibit immune responses. The molecular bases of such functional dichotomy are likely related to the nature of the recognized epitopes, making it necessary to perform fine epitope mapping studies. The current work was aimed at developing a versatile platform to do so. This was accomplished by display of human and mouse Interleukin-2 on filamentous phages, together with extensive mutagenesis of both antigens and high throughput screening of binding properties of more than 200 variants. Detailed molecular pictures of the epitopes were thus delineated for four antibodies against either human or mouse Interleukin-2, which refined and, in some cases, modified the conclusions derived from previous mapping studies with peptide libraries. Overlapping surface patches on mouse Interleukin-2 that also coincide with the predicted interface between the cytokine and its receptor alpha chain were shown to be recognized by two monoclonal antibodies that promote enhancement of immune responses, shedding new light on the structural bases of their biological activity. Our strategy was powerful enough to reveal multiple binding details and could be used to map the epitopes recognized by other antibodies and to explore additional interactions involving Interleukin-2 and related cytokines, thus contributing to our understanding of the complex structure-function relationships within the immune system.
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http://dx.doi.org/10.1016/j.imbio.2012.02.009DOI Listing
January 2013

Isolation of a novel neutralizing antibody fragment against human vascular endothelial growth factor from a phage-displayed human antibody repertoire using an epitope disturbing strategy.

J Biotechnol 2011 Jan 15;151(2):166-74. Epub 2010 Dec 15.

Recombinant Antibodies Laboratory, Cancer Research Department, Center for Genetic Engineering and Biotechnology, La Habana, CP 10600, Cuba.

Following the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between vascular endothelial growth factor (VEGF) and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments with diverse binding properties in terms of inter-species cross-reactivity and sensitivity to chemical modifications of the antigen were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Purified soluble 2H1 inhibited in a dose dependent manner the ligand-receptor interaction and abolished VEGF-dependent proliferation of human umbilical vein endothelial cells. Our epitope disturbing strategy based on a triple mutant target antigen was successful to focus selection on epitopes different from a known one. Similar approaches could be used to direct phage isolation towards the desired specificity in other antigenic systems.
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http://dx.doi.org/10.1016/j.jbiotec.2010.12.007DOI Listing
January 2011

Phage-displayed antibody fragments recognizing dengue 3 and dengue 4 viruses as tools for viral serotyping in sera from infected individuals.

Arch Virol 2009 6;154(7):1035-45. Epub 2009 Jun 6.

Department of Virology, PAHO/WHO Collaborating Center for the Study of Dengue and its Vector, "Pedro Kourí" Tropical Medicine Institute, Marianao, Havana, Cuba.

The current study shows the usefulness of dengue-3- and dengue-4-specific phage-displayed antibody fragments as tools for viral detection and serotyping in sera from infected individuals. C6/36 HT cells were inoculated with acute-phase sera from patients, and supernatants were collected daily and analyzed by ELISA using phage-displayed antibody fragments as serotype-specific detector reagents. Serotyping of most samples was possible as early as two to three days postinoculation. Results were comparable with those obtained by indirect immunofluorescence assay but were obtained in a shorter period of time (<1 week). Phage-displayed antibody fragments were better tools for diagnosis and serotyping than their soluble counterparts. Our approach combines the advantages of viral isolation and ELISA techniques. These results could be the basis for the development of a high-throughput method for identifying dengue virus serotypes, which is crucial for the management and control of the disease.
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http://dx.doi.org/10.1007/s00705-009-0401-1DOI Listing
July 2009

Preferential selection of Cys-constrained peptides from a random phage-displayed library by anti-glucitollysine antibodies.

J Pept Sci 2008 Nov;14(11):1216-21

Center for Genetic Engineering and Biotechnology, La Habana, CP 10600, Cuba.

Phage-displayed peptides recognized by two monoclonal antibodies against glucitollysine were selected. The most prominent feature of the peptide panel was the presence of paired Cys in most of them (21/24 peptides). The availability of a wide variety of peptides having differently spaced paired Cys, as well as truly linear Cys-free peptides, gave the opportunity to explore the role of disulfide bridges in phage selection. Some Cys-containing peptides came from a Cys-flanked cyclic 9-mer library, but most of them (18/21) were derived from a totally random 12-mer library, and hence the presence of Cys was dictated by the selector antibodies. Motifs shared by several peptides (potentially involved in binding) often contained or were flanked by Cys residues. Binding of all Cys-containing phage-displayed peptides was abolished/decreased after a reducing treatment. Screening a random peptide library (without invariant Cys residues) is powerful enough to clearly reveal the need, preferences, and diversity of Cys-mediated structural constraints for recognition.
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http://dx.doi.org/10.1002/psc.1061DOI Listing
November 2008

Selection of phage-displayed human antibody fragments on Dengue virus particles captured by a monoclonal antibody: application to the four serotypes.

J Virol Methods 2008 Feb 22;147(2):235-43. Epub 2007 Oct 22.

Department of Virology, PAHO/WHO Collaborating Center for the Study of Dengue and its Vector, Pedro Kourí Tropical Medicine Institute, Autopista Novia del Mediodía, km 6 5, Marianao 13, Habana, Cuba.

Antibody fragments to the four Dengue virus serotypes were isolated from a human universal naïve library using phage display technology. Phage-displayed antibody fragments were selected on Dengue virus particles directly captured from infected Vero cells supernatant by an anti-dengue monoclonal antibody, in order to avoid laborious virus concentration/purification procedures. A total of nine phage-displayed antibody fragments were obtained. Seven of them were highly specific for three of the selector serotypes (two for Dengue 1, four for Dengue 3 and one for Dengue 4). One clone (Dengue 3-selected) cross-reacted with Dengue 1, whereas another (selected with Dengue 2) cross-reacted with the three remaining serotypes. The soluble variants of six antibody fragments recognized their target viruses when used at nanomolar and even subnanomolar concentrations. All phage-displayed antibody fragments were cross-reactive against several strains of distinct genotypes within the corresponding serotype(s). These antibody fragments are potentially useful for the future development of tools for viral diagnosis and serotype identification. The simple phage selection method on captured virus could be applied in a high throughput way to obtain larger panels of antibody fragments to Dengue virus for multiple applications.
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http://dx.doi.org/10.1016/j.jviromet.2007.09.001DOI Listing
February 2008

A simple vector system to improve performance and utilisation of recombinant antibodies.

BMC Biotechnol 2006 Dec 7;6:46. Epub 2006 Dec 7.

Atlas of Protein Expression, Wellcome Trust Sanger Institute, Genome Campus, Morgan Building, Hinxton, Cambridgeshire, CB10 1HH, UK.

Background: Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression.

Results: We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs). Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3). The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2 (DE3)) was investigated and found to be inferior to periplasmic expression in BL21 (DE3) cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner), bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule), or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and immunohistochemistry.

Conclusion: Nine scFv expression vectors have been generated and tested. Three vectors showed utility for expression of functional scFv fragments. One vector, pSANG14-3F, produces scFv-alkaline phosphatase fusion molecules which offers a simple, convenient and sensitive way of determining the reactivity of recombinant antibody fragments in a variety of common assay systems.
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http://dx.doi.org/10.1186/1472-6750-6-46DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1712229PMC
December 2006

Biologically active vascular endothelial growth factor as a bacterial recombinant glutathione S-transferase fusion protein.

Biotechnol Appl Biochem 2006 Apr;44(Pt 1):45-53

Recombinant Antibodies Laboratory, Cancer Research Department, Pharmaceuticals Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba.

Human VEGF121 (vascular endothelial growth factor isoform 121) was produced as a recombinant fusion protein with GST (glutathione S-transferase) in Escherichia coli. After affinity purification with glutathione, the GST-VEGF121 fusion protein preparation was used to obtain antibodies in mice against commercial hrVEGF (human recombinant VEGF) through immunization. It was also employed successfully to select specific antihuman VEGF antibody fragments of human origin employing phage-display technology. The fusion protein preparation was separated in monomeric, dimeric and oligomeric forms using size-exclusion chromatography. The dimers were recognized by a soluble VEGF receptor 2-Fc chimaera, and stimulated the growth of human umbilical-vein endothelial cells in vitro in a similar fashion to a commercial hrVEGF. The presence of GST in the fusion protein apparently did not affect the correct assembly of dimers and display of residues critical for receptor recognition. The two-step purification method reported in the present paper involves no laborious renaturalization methods, yields 10 mg/l of the mixture of different aggregation states after affinity chromatography, and 5 mg/l of the biologically active dimer after gel filtration, thus providing a source of material for the development of new anti-angiogenic therapeutic molecules.
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http://dx.doi.org/10.1042/BA20050169DOI Listing
April 2006

Efficient construction of a highly useful phage-displayed human antibody repertoire.

Biochem Biophys Res Commun 2005 Nov;336(4):1207-13

Recombinant Antibodies Laboratory, Pharmaceuticals Division, Center for Genetic Engineering and Biotechnology, Playa, La Habana, Cuba.

We have constructed a highly useful phage-displayed human antibody repertoire with limited cloning efforts. Our strategy was to maximize diversity during the first steps of library construction through the use of various lymphoid sources from several donors, inclusion of different immunoglobulin isotypes, and performance of multiple separate amplification reactions with all possible combinations within a complex primer set. The resulting variable region collections were cloned to form a moderate size library, composed by 4.25x10(8) single chain antibody fragments. This repertoire was successfully used to retrieve binders to seven model antigens: six proteins and one 12 aa peptide. Binding affinities reached nanomolar and even subnanomolar range. Sequence diversity and V-gene usage variability among binders were proven. Our approach was not focused on absolute library size, but on a high quality sampling of variable regions from the human antibody repertoire.
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http://dx.doi.org/10.1016/j.bbrc.2005.09.005DOI Listing
November 2005

Functional hierarchy of plasminogen kringles 1 and 4 in fibrinolysis and plasmin-induced cell detachment and apoptosis.

FEBS J 2005 Jul;272(13):3387-400

INSERM U698, Centre Hospitalier Universitaire Bichat-Claude Bernard, Paris, France.

Plasmin(ogen) kringles 1 and 4 are involved in anchorage of plasmin(ogen) to fibrin and cells, an essential step in fibrinolysis and pericellular proteolysis. Their contribution to these processes was investigated by selective neutralization of their lysine-binding function. Blocking the kringle 1 lysine-binding site with monoclonal antibody 34D3 fully abolished binding and activation of Glu-plasminogen and prevented both fibrinolysis and plasmin-induced cell detachment-induced apoptosis. In contrast, blocking the kringle 4 lysine-binding site with monoclonal antibody A10.2 did not impair its activation although it partially inhibited plasmin(ogen) binding, fibrinolysis and cell detachment. This remarkable, biologically relevant, distinctive response was not observed for plasmin or Lys-plasminogen; each antibody inhibited their binding and activation of Lys-plasminogen to a limited extent, and full inhibition of fibrinolysis required simultaneous neutralization of both kringles. Thus, in Lys-plasminogen and plasmin, kringles 1 and 4 act as independent and complementary domains, both able to support binding and activation. We conclude that Glu-/Lys-plasminogen and plasmin conformations are associated with transitions in the lysine-binding function of kringles 1 and 4 that modulate fibrinolysis and pericellular proteolysis and may be of biological relevance during athero-thrombosis and inflammatory states. These findings constitute the first biological link between plasmin(ogen) transitions and functions.
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http://dx.doi.org/10.1111/j.1742-4658.2005.04754.xDOI Listing
July 2005

Light-chain shuffling results in successful phage display selection of functional prokaryotic-expressed antibody fragments to N-glycolyl GM3 ganglioside.

J Immunol Methods 2004 Oct;293(1-2):71-83

Recombinant Antibodies Laboratory, Pharmaceuticals Division, Center for Genetic Engineering and Biotechnologym, P.O. Box 6162, Ave 31 e/ 158 y 190, Cubanacán, Playa, La Habana 10600, Cuba.

Phage display technology makes it possible to introduce and rapidly screen diversity in antibody binding sites. Chain shuffling has been successfully used to humanize murine antibody fragments and also to obtain affinity matured variants. Here we report a different application of this method: the use of chain shuffling to overcome improper prokaryotic expression behavior of a hybridoma-derived single-chain antibody fragment. Construction and expression of such recombinant antibody fragments remain as empirical entities, hampered by the inability to express some antibody genes coming from eukaryotic cells in bacterial expression systems. Such problems are different for each combination of variable regions and can be serious enough to preclude the use of some hybridomas as sources of V regions to obtain recombinant antibody fragments. The particular binding properties and potential usefulness of some monoclonal antibodies make it highly desirable to bypass these technical limitations in order to develop smaller size therapeutic agents in the form of antibody fragments. The 14F7 mouse monoclonal antibody is one such attractive candidate due to its high specificity for the N-glycolyl GM3 ganglioside overexpressed in tumor cells and its ability to distinguish this antigen from closely related gangliosides like N-acetyl GM3. Our goal was to construct a phage-displayed single-chain Fv antibody fragment derived from 14F7. After cloning the original variable regions from the 14F7 hybridoma in a phagemid vector, we were unable to detect either binding activity or even expression of antibody fragments in bacteria, despite repetitive efforts. We constructed light-chain shuffling libraries, from which functional antibody fragments were readily selected. These combined the original 14F7 heavy chain variable region with a wide variety of unrelated murine and human light-chain variable regions. New antibody fragments retained the valuable properties of the monoclonal antibody in terms of fine specificity, affinity and tumor recognition. They were readily produced by bacteria, either in phage-displayed form or as soluble molecules, and provided a panel of potentially useful variants for cancer diagnosis and immunotherapy. Chain shuffling and phage display were found to be useful strategies for selecting antibody fragments on the basis of both prokaryotic expression and antigen binding criteria.
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http://dx.doi.org/10.1016/j.jim.2004.07.002DOI Listing
October 2004

Structure and molecular interactions of a unique antitumor antibody specific for N-glycolyl GM3.

J Biol Chem 2004 Feb 19;279(7):5597-603. Epub 2003 Nov 19.

Department of Chemistry and Biosciences, Chalmers University of Technology, P. O. Box 462, SE-40530 Göteborg, Sweden.

N-glycolyl GM3 ganglioside is an attractive target antigen for cancer immunotherapy, because this epitope is a molecular marker of certain tumor cells and not expressed in normal human tissues. The murine monoclonal antibody 14F7 specifically recognizes N-glycolyl GM3 and shows no cross-reactivity with the abundant N-acetyl GM3 ganglioside, a close structural homologue of N-glycolyl GM3. Here, we report the crystal structure of the 14F7 Fab fragment at 2.5 A resolution and its molecular model with the saccharide moiety of N-glycolyl GM3, NeuGcalpha3Galbeta4Glcbeta. Fab 14F7 contains a very long CDR H3 loop, which divides the antigen-binding site of this antibody into two subsites. In the docking model, the saccharide ligand is bound to one of these subsites, formed solely by heavy chain residues. The discriminative feature of N-glycolyl GM3 versus N-acetyl GM3, its hydroxymethyl group, is positioned in a hydrophilic cavity, forming hydrogen bonds with the carboxyl group of Asp H52, the indole NH of Trp H33 and the hydroxyl group of Tyr H50. For the hydrophobic methyl group of N-acetyl GM3, this environment would not be favorable, explaining why the antibody specifically recognizes N-glycolyl GM3, but not N-acetyl GM3. Mutation of Asp H52 to hydrophobic residues of similar size completely abolished binding. Our model of the antibodycarbohydrate complex is consistent with binding data for several tested glycolipids as well as for a variety of 14F7 mutants with replaced VL domains.
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http://dx.doi.org/10.1074/jbc.M311693200DOI Listing
February 2004

TCR-like human antibodies expressed on human CTLs mediate antibody affinity-dependent cytolytic activity.

J Immunol 2002 Jul;169(2):1110-8

Department of Pathology, Maastricht University, Maastricht, The Netherlands.

The permanent genetic programming via gene transfer of autologous T cells with cell surface receptors directed toward tumor-related Ags holds great promise for the development of more-specific tumor therapies. In this study we have explored the use of Abs directed to MHC-peptide complexes (or TCR-like Abs) to engraft CTLs with exquisite specificity for cancer cells. First, we affinity matured in vitro a previously selected TCR-like Ab, Fab-G8, which is highly specific for the peptide melanoma-associated Ag-A1 presented by the HLA-A1 molecule. A combination of L chain shuffling, H chain-targeted mutagenesis, and in vitro selection of phage display libraries yielded a Fab-G8 Ab derivative, Fab-Hyb3, with an 18-fold improved affinity yet identical peptide fine specificity. Fab-G8 and Fab-Hyb3 were expressed on primary human T lymphocytes as cell surface-anchored Fab, demonstrating that T cells expressing the high-affinity Fab-Hyb3 molecule eradicate tumor cells much more effectively. Furthermore, the gain in ligand-binding affinity resulted in a 2-log improvement in the detection of peptide/MHC complexes on melanoma-associated Ag-A1 peptide-loaded cells. In summary, an affinity-matured Ab specifically recognizing a cancer-related peptide/MHC complex was generated and used to improve the tumor cell killing capacity of human T cells. This strategy, based on engraftment of T cells with in vitro engineered Abs, is an attractive alternative to the laborious, and in many cases unsuccessful, generation of highly potent tumor-specific T lymphocytes.
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http://dx.doi.org/10.4049/jimmunol.169.2.1110DOI Listing
July 2002

Apolipoprotein(a): structure-function relationship at the lysine-binding site and plasminogen activator cleavage site.

Biol Chem 2002 Jan;383(1):93-9

Institut National de la Santé et de la Recherche, Médicale, Faculté de Médecine Xavier-Bichat, Paris, France.

Apolipoprotein(a) [apo(a)] is the distinctive glycoprotein of lipoprotein Lp(a), which is disulfide linked to the apo B100 of a low density lipoprotein particle. Apo(a) possesses a high degree of sequence homology with plasminogen, the precursor of plasmin, a fibrinolytic and pericellular proteolytic enzyme. Apo(a) exists in several isoforms defined by a variable number of copies of plasminogen-like kringle 4 and single copies of kringle 5, and the protease region including the backbone positions for the catalytic triad (Ser, His, Asp). A lysine-binding site that is similar to that of plasminogen kringle 4 is present in apo(a) kringle IV type 10. These kringle motifs share some amino acid residues (Asp55, Asp57, Phe64, Tyr62, Trp72, Arg71) that are key components of their lysine-binding site. The spatial conformation and the function of this site in plasminogen kringle 4 and in apo(a) kringle IV-10 seem to be identical as indicated by (i) the ability of apo(a) to compete with plasminogen for binding to fibrin, and (ii) the neutralisation of the lysine-binding function of these kringles by a monoclonal antibody that recognises key components of the lysine-binding site. In contrast, the lysine-binding site of plasminogen kringle 1 contains a Tyr residue at positions 64 and 72 and is not recognised by this antibody. Plasminogen bound to fibrin is specifically recognised and cleaved by the tissue-type plasminogen activator at Arg561-Val562, and is thereby transformed into plasmin. A Ser-Ile substitution at the activation cleavage site is present in apo(a). Reinstallation of the Arg-Val peptide bond does not ensure cleavage of apo(a) by plasminogen activators. These data suggest that the stringent specificity of tissue-type plasminogen activator for plasminogen requires molecular interactions with structures located remotely from the activation disulfide loop. These structures ensure second site interactions that are most probably absent in apo(a).
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http://dx.doi.org/10.1515/BC.2002.009DOI Listing
January 2002

Phage antibody fragments library combining a single human light chain variable region with immune mouse heavy chain variable regions.

J Biotechnol 2002 Apr;94(3):287-98

Recombinant Antibodies Laboratory, Pharmaceuticals Division, P.O. Box 6162, Ave 31 e/158 y 190, Cubanacán, Playa, La Habana 10600, Cuba.

We describe the construction of a phage antibody fragments library which combines, in a single cloning step, a synthetic human light chain variable region (V(L)) with a diverse set of heavy chain variable regions, from a mouse immunized with the prostate specific antigen (PSA). Despite V(L) restriction, selection from this library rendered two different single chain Fv antibody fragments, specifically recognizing PSA. The human V(L), used as a general partner for mouse heavy chains, was constructed by linking the germline A27 gene and the J(K)1 minigene segment, both of which are prominently involved in human antibody responses. Our approach offers a fast and simple way to produce half-human molecules, while keeping the advantage of immunizing animals for high affinity antibodies.
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http://dx.doi.org/10.1016/s0168-1656(01)00432-1DOI Listing
April 2002

Development and validation of a quantitative ELISA for the measurement of PSA concentration.

Clin Chim Acta 2002 Mar;317(1-2):55-63

Recombinant Antibodies Laboratories, Pharmaceutical Division, Center for Genetic Engineering and Biotechnology, Ave 31 and 58, Cubanacán, P.O. Box 6162, 10600, Havana, Cuba.

Background: Prostate-specific antigen (PSA) has been used for the diagnosis and follow up of prostate cancer (PCa).

Methods: Mouse monoclonal antibodies (MAbs) were generated against human prostate-specific antigen (PSA) for the development of a sensitive total PSA (t-PSA) assay. Two MAbs, denoted CB-PSA.4 and CB-PSA.9, with affinities of 3.7 x 10(9) and 4.7 x 10(10) l/mol, respectively, were used to develop an enzyme-linked immunosorbent assay (ELISA) for quantifying serum t-PSA concentration.

Results: The detection limit (DL) of the assay was 0.1 microg/l (n=20, mean of "zero" standard+3S.D.), and the recovery of t-PSA was 96-103%. The within-run and between-day coefficients of variation (CV) ranged from 2.1% to 3.2%, and from 2.8% to 6.3% for PSA concentrations of 10 and 1 microg/l, respectively. The equimolar detection of t-PSA and free-PSA was demonstrated by two different methods, one consisted in the comparative evaluation of a sera panel (n=9) with our enzyme-linked immunosorbent assay (ELISA) and four commercial total PSA assays and the concordance with CIS bio total PSA assay. The assay had a linear range of 0.12 to 25 microg/l.

Conclusions: The analytical performance characteristics of our PSA ELISA suggest that it will provide clinically useful PSA results, particularly when diagnostic algorithms are used.
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http://dx.doi.org/10.1016/s0009-8981(01)00749-5DOI Listing
March 2002