Bacterial secretion of soluble and functional trivalent scFv-based N-terminal trimerbodies.

Recombinant antibodies are used with great success in many different diagnostic and therapeutic applications. A variety of protein expression systems are available, but nowadays almost all therapeutic antibodies are produced in mammalian cell lines due to their complex structure and glycosylation requirements. However, production of clinical-grade antibodies in mammalian cells is very expensive and time-consuming. On the other hand, Escherichia coli (E. coli) is known to be the simplest, fastest and most cost-effective recombinant expression system, which usually achieves higher protein yields than mammalian cells. Indeed, it is one of the most popular host in the industry for the expression of recombinant proteins. In this work, a trivalent single-chain fragment variable (scFv)-based N-terminal trimerbody, specific for native laminin-111, was expressed in human embryonic kidney 293 cells and in E. coli. Mammalian and bacterially produced anti-laminin trimerbody molecules display comparable functional and structural properties, although importantly the yield of trimerbody expressed in E. coli was considerably higher than in human cells. These results demonstrated that E. coli is a versatile and efficient expression system for multivalent trimerbody-based molecules that is suitable for their industrial production.

Is there a deficit in recognition in old age? / ¿El reconocimiento empeora al envejecer?

BACKGROUND: There is some debate over of the effect of aging on the ability to recognize previously processed information. The aim of the present study is to analyze, by means of different measurements, whether aging has differential effects on recall and recognition of visual and verbal materials. METHOD: A within-subject design was used to compare two groups of different age (younger, older) in tasks of recall and recognition of images and of the verbal descriptors exchanged in a conversation. RESULTS: The results indicated that, unlike the recall and recognition of words, better in younger participants, the recall and recognition of images was equal in both groups, or even better in older participants when assessed by means of d'. Nevertheless, a more strict recently proposed measurement, the conditional probability for recall given recognition, yielded significant age differences in all instances. Besides, the conditional probability shows the aging changes usually found in the serial position curve: decline of the primacy effect, while maintenance of the recency effect. CONCLUSIONS: Results are explained according to the theories that postulate two components in the process of recognition (familiarity and recollection), which are independently affected by aging

ANTECEDENTES: existe controversia acerca del efecto del envejecimiento sobre la habilidad para reconocer información previamente procesada. El principal objetivo del estudio es analizar, mediante distintas medidas, si al envejecer se produce un declive diferencial del recuerdo y el reconocimiento de materiales visuales y verbales. MÉTODO: se comparó con un diseño intrasujetos el rendimiento de dos grupos de distinta edad (joven, mayor) en tareas de recuerdo y reconocimiento de imágenes y de los descriptores verbales intercambiados en una conversación. RESULTADOS: los resultados indicaron que, a diferencia del recuerdo y reconocimiento de palabras, mejor en jóvenes, el recuerdo y reconocimiento de imágenes es igual en ambos grupos, o incluso mejor en los mayores mediante estimaciones como d'. Sin embargo, una medida más estricta recientemente propuesta, la probabilidad condicionada de recordar la información reconocida, muestra diferencias significativas en función de la edad en todos los casos. Además, dibuja el cambio habitual de la curva de posición serial al envejecer: declive del efecto de primacía y mantenimiento del efecto de recencia. CONCLUSIONES: los resultados se explican en el marco de las teorías que postulan dos componentes en el proceso de reconocimiento (familiaridad y recuperación), sobre los que el envejecimiento tiene distintos efectos

Is there a deficit in recognition in old age?

BACKGROUND: There is some debate over of the effect of aging on the ability to recognize previously processed information. The aim of the present study is to analyze, by means of different measurements, whether aging has differential effects on recall and recognition of visual and verbal materials. METHOD: A within-subject design was used to compare two groups of different age (younger, older) in tasks of recall and recognition of images and of the verbal descriptors exchanged in a conversation. RESULTS: The results indicated that, unlike the recall and recognition of words, better in younger participants, the recall and recognition of images was equal in both groups, or even better in older participants when assessed by means of d´. Nevertheless, a more strict recently proposed measurement, the conditional probability for recall given recognition, yielded significant age differences in all instances. Besides, the conditional probability shows the aging changes usually found in the serial position curve: decline of the primacy effect, while maintenance of the recency effect. CONCLUSIONS: RESULTS are explained according to the theories that postulate two components in the process of recognition (familiarity and recollection), which are independently affected by aging.

Functional comparison of single-chain and two-chain anti-CD3-based bispecific antibodies in gene immunotherapy applications.

Gene therapy to achieve in vivo secretion of recombinant anti-CD3 x anti-tumor bispecific antibodies in cancer patients is being explored as a strategy to counterbalance rapid renal elimination, thereby sustaining levels of bispecific antibodies in the therapeutic range. Here, we performed a comparative analysis between single- and two-chain configurations for anti-CD3 x anti-CEA (carcinoembryonic antigen) bispecific antibodies secreted by genetically-modified human cells. We demonstrate that tandem single-chain variable fragment (scFv) antibodies and two-chain diabodies are expressed as soluble secreted proteins with similar yields. However, we found significant differences in their biological functionality (i.e., antigen binding) and in their ability to induce non-specific T cell activation. Whereas single-chain tandem scFvs induced human T cell activation and proliferation in an antigen-independent manner, secreted two-chain diabodies exerted almost no proliferative stimulus when human T cells were cultured alone or in co-cultures with CEA negative cells. Thus, our data suggest that two-chain diabodies are preferable to single-chain tandem scFvs for immunotherapeutic strategies comprising in vivo secretion of bispecific antibodies aiming to recruit and activate anticancer specific lymphocytic effector T cells.

Generation and characterization of monospecific and bispecific hexavalent trimerbodies.

Here, we describe a new class of multivalent and multispecific antibody-based reagents for therapy. The molecules, termed "trimerbodies," use a modified version of the N-terminal trimerization region of human collagen XVIII noncollagenous 1 domain flanked by two flexible linkers as trimerizing scaffold. By fusing single-chain variable fragments (scFv) with the same or different specificity to both N- and C-terminus of the trimerizing scaffold domain, we produced monospecific or bispecific hexavalent molecules that were efficiently secreted as soluble proteins by transfected mammalian cells. A bispecific anti-laminin x anti-CD3 N-/C-trimerbody was found to be trimeric in solution, very efficient at recognizing purified plastic-immobilized laminin and CD3 expressed at the surface of T cells, and remarkably stable in human serum. The bispecificity was further demonstrated in T cell activation studies. In the presence of laminin-rich substrate, the bispecific anti-laminin x anti-CD3 N-/C-trimerbody stimulated a high percentage of human T cells to express surface activation markers. These results suggest that the trimerbody platform offers promising opportunities for the development of the next-generation therapeutic antibodies, i.e., multivalent and bispecific molecules with a format optimized for the desired pharmacokinetics and adapted to the pathological context.

Epstein-Barr virus infection of naïve B cells in vitro frequently selects clones with mutated immunoglobulin genotypes: implications for virus biology.

Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growth-transforming infections yet establishes persistence only in IgD⁺ CD27⁺ non-switched memory (NSM) and IgD⁻ CD27⁺ switched memory (SM) B cells, not in IgD⁺ CD27⁻ naïve (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBV-driven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectively-harvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naïve B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCL's evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes.

Improved stability of multivalent antibodies containing the human collagen XV trimerization domain.

We recently described the in vitro and in vivo properties of an engineered homotrimeric antibody made by fusing the N-terminal trimerization region of collagen XVIII NC1 domain to the C-terminus of a scFv fragment [trimerbody (scFv-NC1) 3; 110 kDa]. Here, we demonstrated the utility of the N-terminal trimerization region of collagen XV NC1 domain in the engineering of trivalent antibodies. We constructed several scFv-based trimerbodies containing the human type XV trimerization domain and demonstrated that all the purified trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Importantly, type XV trimerbodies demonstrated substantially greater thermal and serum stability and resistance to protease digestion than type XVIII trimerbodies. In summary, the small size, high expression level, solubility and stability of the trimerization domain of type XV collagen make it the ideal choice for engineering homotrimeric antibodies for cancer detection and therapy.

Multivalent antibodies: when design surpasses evolution.

Evolutionary pressure has selected antibodies as key immune molecules acting against foreign pathogens. The development of monoclonal antibody technology has allowed their widespread use in research, real-time diagnosis and treatment of multiple diseases, including cancer. However, compared with hematologic malignancies, solid tumors have often proven to be relatively resistant to antibody-based therapies. In an attempt to improve the tumor-targeting efficacy of antibodies, new formats with modified, multivalent properties have been generated. Initially, these formats imitated the structure of native IgG, creating mostly monospecific, bivalent antibodies. Recently, novel trivalent antibodies have been developed to maximize tumor targeting capabilities through enhanced biodistribution and functional affinity. We review recent advances in the engineering of multivalent antibodies and further discuss their promise as agents for in vivo diagnostics and therapy.

Deglycosylation to obtain stable and homogeneous Pichia pastoris-expressed N-A1 domains of carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is a seven domain membrane glycoprotein widely used as a tumour marker for adenocarcinomas and as a target for antibody-directed therapies. Structural models have proposed that the first two domains of CEA (the N terminal and adjoining A1 domains) bind MFE-23, a single chain Fv antibody in experimental clinical use. We aimed to produce recombinant N-A1 to test this hypothesis. The N-A1 domains were expressed as soluble protein with a C-terminal hexahistidine tag (His6-tag) in the yeast Pichia pastoris. His6-tagged N-A1 was captured from the supernatant by batch purification with copper-loaded Streamline Chelating, an immobilised metal affinity chromatography (IMAC) matrix usually utilised in expanded bed techniques. Purified N-A1 was heterogeneous with a molecular weight range from 38 to 188 kDa. Deglycosylation with endoglycosidase H (Endo H) resulted in three discrete molecular weight forms of N-A1, one partially mannosylated, one fully Endo H-digested and one fully Endo H-digested but lacking the His6-tag. These were separated by concanavalin A chromatography followed by HiTrap IMAC. The procedure resulted in single-band-purity, mannose-free N-A1. The binding interaction of MFE-23 to N-A1 was analysed by surface plasmon resonance. The affinity constants retrieved were KD = 4.49 x 10(-9)M for the P. pastoris expressed, native N-A1, and 5.33 x 10(-9) M for the Endo H-treated N-A1. To our knowledge this is the first time that two consecutive domains of CEA have been stably expressed and purified from P. pastoris. This work confirms that the CEA epitope recognised by MFE-23 resides in N-A1.

Epstein-Barr virus infection in vitro can rescue germinal center B cells with inactivated immunoglobulin genes.

Immunoglobulin genotyping of Epstein-Barr virus (EBV)-positive posttransplantation lymphoproliferative disease has suggested that such lesions often arise from atypical post-germinal center B cells, in some cases carrying functionally inactivated immunoglobulin genes. To investigate whether EBV can rescue cells that are failed products of the somatic hypermutation process occurring in germinal centers (GCs), we isolated GC cells from tonsillar cell suspensions and exposed them to EBV in vitro. Screening more than 100 EBV-transformed cell lines of GC origin identified 6 lines lacking surface immunoglobulin, a phenotype never seen among lines derived from circulating naive or memory B cells. Furthermore, 3 of the 6 surface immunoglobulin-negative GC lines carried inactivating mutations in the immunoglobulin H (IgH) variable gene sequence. The ability of EBV to rescue aberrant products of the germinal center reaction in vitro strengthens the probability that a parallel activity contributes to EBV's lymphomagenic potential in vivo.