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Cancer is the second cause of mortality worldwide. Early diagnosis of this multifactorial disease is challenging, especially in populations with limited access to healthcare services. A vast repertoire of cancer biomarkers has been studied to facilitate early diagnosis; particularly, the use of antibodies against these biomarkers has been of interest to detect them through biorecognition. However, there are certain limitations to this approach. Emerging biorecognition engineering technologies are alternative methods to generate molecules and molecule-based scaffolds with similar properties to those presented by antibodies. Molecularly imprinted polymers, recombinant antibodies, and antibody mimetic molecules are three novel technologies commonly used in scientific studies. This review aimed to present the fundamentals of these technologies and address questions about how they are implemented for cancer detection in recent scientific studies. A systematic analysis of the scientific peer-reviewed literature regarding the use of these technologies on cancer detection was carried out starting from the year 2000 up to 2021 to answer these questions. In total, 131 scientific articles indexed in the Web of Science from the last three years were included in this analysis. The results showed that antibody mimetic molecules technology was the biorecognition technology with the highest number of reports. The most studied cancer types were: multiple, breast, leukemia, colorectal, and lung. Electrochemical and optical detection methods were the most frequently used. Finally, the most analyzed biomarkers and cancer entities in the studies were carcinoembryonic antigen, MCF-7 cells, and exosomes. These technologies are emerging tools with adequate performance for developing biosensors useful in cancer detection, which can be used to improve cancer diagnosis in developing countries.

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Activation of the immune system using antigen targeting to the dendritic cell receptor DEC205 presents great potential in the field of vaccination. The objective of this work was to evaluate the immunogenicity and protectiveness of a recombinant mouse x pig chimeric antibody fused with peptides of structural and nonstructural proteins of porcine respiratory and reproductive syndrome virus (PRRSV) directed to DEC205+ cells. Priming and booster immunizations were performed three weeks apart and administered intradermally in the neck area. All pigs were challenged with PRRSV two weeks after the booster immunization. Immunogenicity was evaluated by assessing the presence of antibodies anti-PRRSV, the response of IFN-γ-producing CD4+ cells, and the proliferation of cells. Protection was determined by assessing the viral load in the blood, lungs, and tonsils using qRT-PCR. The results showed that the vaccine exhibited immunogenicity but conferred limited protection. The vaccine group had a lower viral load in the tonsils and a significantly higher production of antibodies anti-PRRSV than the control group (p < 0.05); the vaccine group also produced more CD4+IFN-γ+ cells in response to peptides from the M and Nsp2 proteins. In conclusion, this antigenized recombinant mouse x pig chimeric antibody had immunogenic properties that could be enhanced to improve the level of protection and vaccine efficiency.

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The production of recombinant antibodies has had a tremendous impact on several research fields, most prominently in biotechnology, immunology and medicine, enabling enormous advances in each. Thus far, a broad diversity of recombinant antibody (rAb) forms have been designed and expressed using different expression systems. Even though the majority of rAbs approved for clinical use are targeted to humans, advances in veterinary medicine seem promising. The aim of this mini-review is to present an update regarding the rAbs in veterinary medicine reported to date, as well as their potential use in diagnostics, prophylaxis and therapeutics. Full- and single-chain fragment variables are the most common forms of rAbs developed for the detection, prevention and control of parasitic, bacterial and viral diseases, as well as pain and cancer treatment. Nonetheless, advances in research seem to be skewed toward economically important animals, such as pigs, cows, poultry and dogs. Although significant results have been obtained from the rAbs reported here, most have not been developed enough to be approved. Further research and clinical trials should be encouraged to enable important findings to fulfill their intended potential to improve animal well-being.

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Background: Gardnerella vaginalis is a bacterial vaginosis (BV)-associated vaginal bacterium that produces the toxin vaginolysin (VLY). VLY is a pore-forming toxin that is suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and the emergence of antibiotic-resistant bacterial species demonstrate the need for the development of recombinant antibodies as novel therapeutic agents for disease treatment. Single-chain variable fragments (scFvs) generated against VLY exhibited reduced efficacy to neutralize VLY activity compared to the respective full-length antibodies. To improve the properties of scFvs, monospecific dimeric scFvs were generated by the genetic fusion of two anti-VLY scFv molecules connected by an alpha-helix-forming peptide linker. Results: N-terminal hexahistidine-tagged dimeric scFvs were constructed and produced in Escherichia coli and purified using metal chelate affinity chromatography. Inhibition of VLY-mediated human erythrocyte lysis by dimeric and monomeric scFvs was detected by in vitro hemolytic assay. The circulating half-life of purified scFvs in the blood plasma of mice was determined by ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life than parental monomeric scFv. Conclusions: The protein obtained by the genetic fusion of two anti-VLY scFvs into a dimeric molecule exhibited improved properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by the bacteria G. vaginalis.

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Shiga toxin-producing Escherichia coli (STEC) causes hemorrhagic colitis and hemolytic uremic syndrome by producing Shiga toxin (Stx), a AB5 type toxin. The B subunit binds to ganglioside receptors, and the active A subunit is translocated into the cell, where it inhibits protein synthesis by acting on rRNA, leading to cell death. Diagnosis of these strains is not commonly done in the routine laboratory of low-income countries. Besides, therapy for intoxication is based on minimizing the symptoms, since no toxin antidote is available. Thus, the development of effective tools for toxin detection and therapy is highly relevant. Antibodies exhibit excellent high affinity for their specific antigens. To maintain the homogeneity and specificity of monoclonal antibodies, together with largescale production and low cost, genetic engineering has been used to develop recombinant antibodies. These include single chain variable fragments and antigenbinding fragments, which consist of antibody fragments that have antigen recognition regions. In the present study we obtained and characterized two different recombinant antibody fragments (Fab and scFv), produced in bacteria, against Stx toxins from STEC isolates. Furthermore, for the first time a human monovalent fragment was constructed (Fab), which was able to recognize and neutralize Stx toxins. These fragments obtained are promising tools for its use in the diagnosis and therapy of intoxication by STEC.

Linhagens de Escherichia coli produtoras da toxina de Shiga (STEC) causam colite hemorrágica e síndrome hemolítica urêmica pela produção da citotoxina de Shiga (Stx), uma toxina do tipo AB5, a subunidade B se liga ao receptor, e transloca a subunidade A ativa que inibe a síntese proteica por agir no rRNA levando à morte celular. O diagnóstico dessas linhagens não é realizado na rotina laboratorial de países em desenvolvimento e a terapia da intoxicação se baseia em tratar os sintomas, pois não há antídoto para a toxina. A obtenção de ferramentas para a detecção e terapia destas toxinas são de grande importância. Os anticorpos tem se mostrado moléculas excelentes como reagentes ligantes de alta afinidade à proteínas. Com o intuito de manter a homogeneidade e a especificidade dos anticorpos monoclonais, aliados ainda à produção em larga escala com baixo custo, a engenharia genética tem sido utilizada para obtenção de anticorpos recombinantes como os fragmentos variáveis de cadeia única e os fragmentos de ligação ao antígeno, que são fragmentos de anticorpos que possuem as regiões de reconhecimento ao antígeno. No presente trabalho foram obtidos e caracterizados dois tipos diferentes de fragmentos de anticorpos recombinantes (scFv e Fab), produzidos em bactérias, contra as toxinas Stx produzidas por isolados de STEC. Além disso, pela primeira vez, foi construído um fragmento monovalente humano (Fab), capaz de reconhecer e neutralizar as toxinas Stx. Estes fragmentos obtidos são promissores para seu uso como ferramentas tanto no diagnóstico como na terapia das intoxicações por STEC.

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Accidents with snakes are a problem of public health. It is estimated that about 1.8 million poisonings by snakes occur every year worldwide, resulting in at least 94,000 deaths. This issue has been included in the list of neglected tropical diseases by the World Health Organization. Among the causative genera of snakebites in Brazil, the genus Crotalus has the highest coefficient of lethality. The venom of the rattlesnake Crotalus durissus terrificus (Cdt) is composed of a mixture of substances including toxins. The majority toxin and main toxic component of the venom is called crotoxin. It is highly toxic and is formed by subunits CA and CB. The administration of heterologous antibodies has been the treatment of choice for snake bite accidents. But its administration may cause hypersensitivity reactions. Furthermore, Crotalic antivenom production by horses is slow and hampered by the action of immunosuppressive components present in the venom of Cdt. Currently, recombinant antibody fragments are becoming popular alternative therapies for whole antibodies. Single chain fragment variable (scFv) comprise the VH and VL domains connected by a short linker flexible and may be useful as treatment for poisoning by snakes. Recombinant human antibodies anti-crotoxin have been previously isolated from a naive library of over 1010scFv by phage display technology. The aim of this study was the expression of original scFvs anti-crotoxin and mutants suggested by modeling molecular. ScFv6 3D model was constructed in silico. Docking and energy minimization calculations of CTX-antibody complex were also performed. From these simulations, three mutations were chosen. The S30A and Y31F mutants have mutation in CDR H2 and CDR H3 in the mutant R103H. The first mutant (S30A) was obtained by site-directed mutagenesis, while the other two (Y31F and R103H) were obtained from synthetic genes. ScFv original and mutants were cloned in pET20b + vector and expressed in E. coli C43 (DE3), resulting in soluble protein of approximately 30 kDa. Expression was induced with IPTG. After lysis, the bacterial content was purified by IMAC. The presence of the desired mutations was confirmed by sequencing. The secondary structure of scFvs was evaluated by circular dichroism and showed to be preserved. We conclude that original scFv and its mutants were clone and expressed successfully in soluble form. All scFv presented similar yield after purification. Moreover, they presented preserved secondary structure, as evaluated by circular dichroism.

Acidentes com serpentes são um problema de saúde pública. Estima-se que ocorram cerca de 1,8 milhões de envenenamentos por serpentes a cada ano no mundo, resultando em pelo menos 94 mil mortes. Tal problema foi incluído na lista de doenças tropicais negligenciadas da Organização Mundial da Saúde. Dentre o gêneros causadores de acidentes ofídicos no Brasil, o gênero Crotalus apresenta o maior coeficiente de letalidade. O veneno da cascavel Crotalus durissus terrificus (Cdt) é composto por uma mistura de substâncias, dentre elas as toxinas. A toxina majoritária e principal componente tóxico do veneno é denominada crotoxina. É altamente tóxica e é formada por duas subunidades CA e CB. A administração de anticorpos heterólogos tem sido o tratamento de escolha para indivíduos que sofreram acidentes ofídicos. Porém, sua administração pode causar reações de hipersensibilidade, além da sua produção ser lenta e dificultada pela ação de componentes imunossupressores presentes no veneno de Cdt. Atualmente, fragmentos de anticorpos recombinantes estão se tornando alternativas terapêuticas populares para substituição de anticorpos íntegros. Fragmentos variáveis de cadeia única (scFv, do inglês, single chain fragmente variable) são compostos dos domínios VH e VL unidos por um pequeno linker flexível e podem ser úteis como terapia para o envenenamento por serpentes. Anticorpos recombinantes humanos anti-crotoxina foram isolados previamente de uma biblioteca naive de mais 1010scFvs pela tecnologia de phage display. O objetivo desse estudo foi a expressão de scFvs anticrotoxina original e mutantes sugeridos por modelagem molecular. Um modelo 3D do scFv6 foi construído por modelagem in silico. Docking e cálculos de minimização de energia do complexo anticorpo-CTX também foram realizados. A partir dessas simulações, três mutações foram escolhidas. Os mutantes S30A e Y31F apresentam mutação no CDR H2 e o mutante R103H no CDR H3. O primeiro mutante (S30A) foi obtido por mutagênese sitio dirigida, enquanto os outros dois (Y31Fe R103H) foram obtidos através de genes sintéticos. O scFv original e os mutantes foram clonados em vetor pET20b+ e expressos em E.coli C43(DE3), resultando em proteínas solúveis de aproximadamente 30 kDa. A indução foi feita com IPTG. Após a lise bacteriana, o conteúdo foi purificado por IMAC. A presença das mutações desejadas foi confirmada por sequenciamento. A estrutura secundária dos scFvs foi avaliada por dicroísmo circular e se mostrou preservada. Concluímos que tanto o scFv original como os mutantes foram clonados e expressos com sucesso na forma solúvel. Todos os scFvs apresentaram rendimentos similares após a etapa de purificação. Além disto, os scFvs apresentaram estrutura secundária preservada, conforme avaliado por dicroísmo circular.

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Monoclonal antibodies represent the fastest growing class of biopharmaceutical products and have a host of applications in medical research, diagnosis, therapy, and basic science. The production of recombinant monoclonal antibodies has revolutionized the generation of immunoglobulins, and their use represents a strategic breakthrough, affecting the global pharmaceutical market for therapeutic proteins. In the present work, a review of scFv, and the number of related patents, has been carried out. The results show that several countries have scFv patents, most notably the United States, China and United Kingdom. The target of these scFv antibodies was also assessed and the results demonstrate that most are directed toward cancer therapy.

Anticorpos monoclonais representam a classe de maior crescimento em produtos de biofármacos e possuem várias aplicações em pesquisa médica, diagnóstico, terapias e ciência básica. A produção de anticorpos monoclonais recombinantes revolucionou a geração de imunoglobulinas e sua utilização implica em avanço estratégico, afetando o mercado farmacêutico global de proteínas terapêuticas. No presente trabalho, uma revisão sobre scFv e a relação do seu número de patentes foi analisada. Os resultados mostram que vários países apresentam patentes de scFv com destaque para os Estados Unidos, China e Reino Unido. Os alvos desses anticorpos também foram avaliados e as análises revelaram que a maioria é destinado a terapias contra o câncer.

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Los anticuerpos surgieron en los organismos en respuesta a las necesidades imperantes de neutralizar y destruir los embates de agentes externos nocivos para los mismos. Los anticuerpos son macromoléculas que por sus propiedades de especificidad y afinidad a sus antígenos, han sido utilizados para toda una gama de estudios en la medicina, su manipulación fuera de los sistemas vivientes ha permitido su aplicación en la terapéutica y el diagnóstico oportuno de varias enfermedades. El presente trabajo muestra una sinopsis de las propiedades bioquímicas de los anticuerpos y de las estrategias más recientes que han permitido la manipulación de estas moléculas, con la finalidad de mejorar su afinidad y avidez, así como en los métodos de producción para incrementar su potencial de aplicación en la investigación biológica y médica.

Antibodies appeared in the organisms in response to the needs of neutralizing and destroying the attacks of external agents injurious to themselves. Antibodies are macromolecules that because of their properties of specificity and affinity to their antigens, have been used in a great variety of studies in medicine. Moreover, their manipulation out of living systems has permitted their application in the treatment and opportune diagnosis of several diseases. The present work shows a synopsis of antibodies’ biochemical properties and the most recent strategies that have allowed the manipulation of these molecules in order to improve their affinity and avidity. This work will also present the methodological advances that can increase antibodies application potential in biology and in medical research.

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Nicotiana tabacum hairy roots that express the antibody 14D9 were established. The 14D9 antibody yield obtained after 20 days of culture was 5.95 μg 14D9ml-1. The addition of the reticulum endoplasmic retention sequence KDEL demonstrated a positive effect over the intracellular 14D9 amounts with a yield increase up to 20.82 µg ml-1. DMSO increased the antibody amount in the biomass from 20.00 to 64.03 µg ml-1 while PVP (at 1.5 gl-1) and gelatine (at 5.0 gl-1) increased total 14D9 amounts in the culture medium to 25 µg and 14 µg respectively.