Antibody-like Molecules Research Articles

Surrogate antibodies

Researchers at Scripps and Sea Lane have developed antibody-like molecules with high antigen-binding affinities, called Surrobodies, which they say could generate molecules with greater diversity and more functionality than antibodies. To be validated as a new platform, antibody companies want evidence that the underlying natural structure of Surrobodies can be modified to create improved and stable binding properties.

Immunoassays: Biological Tools for High Throughput Screening and Characterisation of Combinatorial Libraries

In the demanding field of proteomics, there is an urgent need for affinity-catcher molecules to implement effective and high throughput methods for analysing the human proteome or parts of it. Antibodies have an essential role in this endeavour, and selection, isolation and characterisation of specific antibodies represent a key issue to meet success. Alternatively, it is expected that new, well-characterised affinity reagents generated in rapid and cost-effective manners will also be used to facilitate the deciphering of the function, location and interactions of the high number of encoded protein products. Combinatorial approaches combined with high throughput screening (HTS) technologies have become essential for the generation and identification of robust affinity reagents from biological combinatorial libraries and the lead discovery of active/mimic molecules in large chemical libraries. Phage and yeast display provide the means for engineering a multitude of antibody-like molecules against any desired antigen. The construction of peptide libraries is commonly used for the identification and characterisation of ligand-receptor specific interactions, and the search for novel ligands for protein purification. Further improvement of chemical and biological resistance of affinity ligands encouraged the "intelligent" design and synthesis of chemical libraries of low-molecular-weight bio-inspired mimic compounds. No matter what the ligand source, selection and characterisation of leads is a most relevant task. Immunological assays, in microtiter plates, biosensors or microarrays, are a biological tool of inestimable value for the iterative screening of combinatorial ligand libraries for tailored specificities, and improved affinities. Particularly, enzyme-linked immunosorbent assays are frequently the method of choice in a large number of screening strategies, for both biological and chemical libraries.

Targeting Transmembrane Domains

Reagents such as antibodies that target specific proteins are useful in research and medicine. There are methods to engineer antibody-like molecules that target the soluble regions of proteins, but targeting transmembrane regions remains a challenge. Now Yin et al . describe a computational method to design peptides that target specific transmembrane helices. Peptides were designed that were specific for each of two closely related integrins involved in cell adhesion. H. Yin, J. S. Slusky, B. W. Berger, R. S. Walters, G. Vilaire, R. I. Litvinov, J. D. Lear, G. A. Caputo, J. S. Bennett, W. F. DeGrado, Computational design of peptides that target transmembrane helices. Science 315 , 1817-1822 (2007). [Abstract] [Full Text]

Crossing the Threshold of Lymphocyte Activation

In 1972, Neils K. Jerne stated in the First Annual Report of the Basel Institute for Immunology, “For bursa cells and B lymphocytes, it is relatively easy to demonstrate that receptors are antibody-like molecules which enable the cell to recognize antigens. Membrane fluidity permits the receptors

Combinatorial methods for discovery of peptide ligands which bind to antibody-like molecules.

In the 1980s, new methods of parallel peptide synthesis were used to make large libraries of peptides, which were then screened for binding to Bence-Jones dimers. Subsequent X-ray crystallography of the Bence-Jones proteins, which had been infiltrated with peptide ligand, was used to determine the structural correlate of the binding data. The mode of binding was found to be not predictable and insight was gained into the forces determining how the so-called mimotopes interacted with binding sites.

Coupling complement regulators to immunoglobulin domains generates effective anti-complement reagents with extended half-life in vivo.

Complement activation and subsequent generation of inflammatory molecules and membrane attack complex contributes to the pathology of a number of inflammatory and degenerative diseases, including arthritis, glomerulonephritis and demyelination. Agents that specifically inhibit complement activation might prove beneficial in the treatment of these diseases. Soluble recombinant forms of the naturally occurring membrane complement regulatory proteins (CRP) have been exploited for this purpose. We have undertaken to design better therapeutics based on CRP. Here we describe the generation of soluble, recombinant CRP comprising rat decay accelerating factor (DAF) or rat CD59 expressed as Fc fusion proteins, antibody-like molecules comprising two CRP moieties in place of the antibody Fab arms (CRP-Ig). Reagents bearing DAF on each arm (DAF-Ig), CD59 on each arm (CD59-Ig) and a hybrid reagent containing both DAF and CD59 were generated. All three reagents inhibited C activation in vitro. Compared with soluble CRP lacking Fc domains, activity was reduced, but was fully restored by enzymatic release of the regulator from the Ig moiety, implicating steric constraints in reducing functional activity. In vivo studies showed that DAF-Ig, when compared to soluble DAF, had a much extended half-life in the circulation in rats and concomitantly caused a sustained reduction in plasma complement activity. When given intra-articularly to rats in a model of arthritis, DAF-Ig significantly reduced severity of disease. The data demonstrate the potential of CRP-Ig as reagents for sustained therapy of inflammatory disorders, including arthritis, but emphasize the need for careful design of fusion proteins to retain function.

TEM-1 beta-lactamase as a scaffold for protein recognition and assay.

A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules. We have previously shown that the TEM-1 beta-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to beta-lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that beta-lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to beta-lactamases binding to streptavidin, beta-lactamase clones binding to horse spleen ferritin and beta-galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining beta-lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for beta-lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme-conjugated antibodies in enzyme-linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme.

Antibody Modeling: Implications for Engineering and Design

Our understanding of the rules relating sequence to structure in antibodies has led to the development of accurate knowledge-based procedures for antibody modeling. Information gained from the analysis of antibody structures has been successfully exploited to engineer antibody-like molecules endowed with prescribed properties, such as increased stability or different specificity, many of which have a broad spectrum of applications both in therapy and in research. Here we describe a knowledge-based procedure for the prediction of the antibody-variable domains, based on the canonical structures method for the antigen-binding site, and discuss its expected accuracy and limitations. The rational design of antibody-based molecules is illustrated using as an example one of the most widely employed modifications of antibody structures: the humanization of animal-derived antibodies to reduce their immunogenicity for serotherapy in humans.

Progress in immunoassay technology.

Immunoassay is now the most widely used analytical technique in laboratory medicine embracing a vast repertoire of analytes and delivered through an increasingly diverse range of devices. This explosion of analytical techniques is complemented by an increasing knowledge base on the antigen antibody reaction that is being used to explore new and improved methodologies. Our knowledge of the molecular chemistry of the antigen antibody reaction coupled with the molecular biology of immunoglobulin expression has led to the development of antibody and antibody-like molecules with enhanced specificity, and reaction kinetics. These developments include novel fusion proteins, antibody mimics and antibodies that recognise antigen antibody complexes. The advances in the chemistry of the analytical systems are matched by the design of delivery vehicles: on the one hand the automated analyser that facilitates the analysis of large numbers of samples and on the other, the encapsulated, miniaturised device that enables an immunoassay to be performed at the point of care.

Immunoadhesins: An Alternative to Human Monoclonal Antibodies

Immunoadhesins are chimeric, antibody-like molecules that combine the functional domain of a binding protein, usually a receptor, ligand, or cell-adhesion molecule, with immunoglobulin constant domains, usually including the hinge and Fc regions. Immunoadhesins have many applications as research tools, for example, in studies of receptor–ligand interactions. In addition, human immunoadhesins have potential applications in the clinic and may provide an alternative to human monoclonal antibodies as agents that recognize and neutralize foreign or human proteins that possess pathologic functions.

Immunoassay: recent developments and future directions

All analytical techniques employed in the biological sciences rely on recognition of the shape and structure of molecules of the substance of interest (the analyte). Such molecular recognition and sensing usually relies on the use other molecules possessing a complementary structure, implying a specific lock and key relationship between the two. Antibodies comprise a class of recognition molecules evolved by nature for the purpose of bodily defence, and are clearly of particular utility in this context. However techniques of increasing sophistication (including the techniques of molecular biology) are currently being developed which enable the artificial construction of antibody-like molecules possessing improved molecular recognition properties which can be harnessed for microanalytical purposes. Oligonucleotide probes likewise exhibit the property of binding to complementary nucleotide sequences, and the techniques of, for example, in situ hybridisation therefore share many features with immunoassay techniques. Microanalytical techniques relying on binding reactions between substances possessing complementary lock and key molecular structures are unlikely to be superseded within the foreseeable future, only the labels used to monitor suche reactions, and the means of production of “recognition molecules”, being subject to further development. Such techniques already enter into all areas of life, including medicine, agriculture, etc, and are likely to increase further in importance with increasing concern regarding chemically complex contaminants in food, the environment, etc. Developments in this field are clearly directed to slightly differing objectives as indicated in this presentation. These include methodological simplification (making the techniques cheaper and more widely available), improvements in sensitivity (to enable the detection and measurement of substances beyond the reach of current methods) and the construction of transducer-based sensor methods (permitting, inter alia, the monitoring of changing analyte concentrations). However the combination of the “ultrasensitivity” of current single analyte assay methods with the ability simultaneously to determine multiple analytes in the same sample represents, in my view, the next major methodological challenge in this field, and -if successfully addressed- will constitute a quantum advance on present analytical methodes. Indeed the development of miniaturised multianalyte binding assay techniques may ultimately come to be seen as analagous to, for example, the introduction of the word processor, and other similar major technological advances of the past decade.

Antiviral effects of different CD4-immunoglobulin constructs against HIV-1 and SIV: immunological characterization, pharmacokinetic data and in vivo experiments.

The CD4 cell surface antigen belongs to the immunoglobulin superfamily and is the primary receptor for the human immunodeficiency virus 1 (HIV-1). The high affinity interaction between HIV-1 and CD4 is mediated by the viral envelope glycoprotein gp120. Recombinant soluble CD4 (rsCD4) has been shown in vitro to be an effective inhibitor of HIV-1 and HIV-2 propagation in lymphoid cells. A variety of antibody-like molecules were constructed, consisting of different parts of the extracellular domain of CD4 fused to immunoglobulin constant regions. The fusion proteins were expressed in mammalian cell lines and purified via affinity chromatography. The specificity and anti-viral effects of the different CD4-immunoglobulin constructs against HIV were analysed by different immunological tests, i.e., immunofluorescence, neutralisation and in vitro assays. In pharmacokinetic studies, differences were found in serum half-life between the four- and two-domain CD4 constructs in cynomolgus monkeys and between glycosylated and deglycosylated CD4-Fc constructs in rabbits. In two in vivo experiments using the four-domain CD4-Fc in SIV-infected macaques, no beneficial effects were observed.

Protein Engineering of Antibodies

This article reviews the technical advances in antibody engineering and the clinical applications of these molecules. Recombinant DNA technology facilitates the construction and expression of engineered antibodies. These novel molecules are designed to meet specific applications. Although genomic and cDNA cloning have been used widely in the past to isolate the relevant antibody V domains, at present, the PCR-based cloning is the preferred system. Bacterial and mammalian expression systems are used commonly for the production of antibodies, antibody fragments, and antibody fusion proteins. A range of chimeric antibodies with murine V domains joined to C regions from human and other species have been produced and found to exhibit the expected binding characteristics and effector functions. Humanized antibodies have been developed to minimize the HAMA response, and bifunctional immunoglobulins are being used in tumor therapy and diagnosis. Single chain antibodies and fusion proteins with antibody specificities jointed to nonimmunoglobulin sequences provide a source of antibody-like molecules with novel properties. The potential applications of minimal recognition units and antigenized antibodies are described. Combinatorial libraries produced in bacteriophage present an alternative to hybridomas for the production of antibodies with the desired antigen binding specificities. Future developments in this field are discussed also.

Expression and Characterization of Human CD4: Immunoglobulin Fusion Proteins

Different chimeric antibody-like molecules consisting of the four human CD4 extracellular domains (amino acids 1-369) fused to different parts of human IgG1 and IgM heavy-chain constant regions have been created and expressed in mammalian cells. For both IgG1 and IgM fusion proteins, the best expression in COS cells was observed for molecules lacking the CH1 domain of the heavy-chain constant region. The chimeric molecules are potent inhibitors of human immunodeficiency virus (HIV) infection and HIV-mediated cytotoxicity. A CD4:IgG1 hinge fusion protein, which was analyzed in more detail, binds efficiently to HIV gp160 and human Fc receptors and shows complement-assisted inhibition of viral propagation in culture. Half-life studies after intravenous application of the latter human fusion protein into mice and monkeys showed significant prolongation of serum survival compared to soluble CD4. An IgG2b murine homolog of the human CD4:IgG1 hinge fusion protein was prepared and evaluated in mice, where it was found to be nontoxic and to have no detectable effect on the humoral response to soluble antigen.

A CD 4: immunoglobulin fusion protein with antiviral effects against HIV

Antibody-like molecules consisting of the human CD 4 extracellular domain fused to human IgG1 heavy chain constant regions were genetically constructed and expressed in a BHK cell stable transfectant. Purified chimeric antibodies bound to HIV particles as it was shown by immuno electron microscopy, inhibited fusions of HIV-1-infected cells with uninfected cells, neutralized HIV-1, and were able to inhibit the spread of a cellular HIV-1 infection in CD 4+ cells. Plaque reduction assays with CD 4(+)-transfected Hela-cells showed a comparable inhibition of HIV-1 and HIV-2. Inhibitory functions were enhanced in the presence of complement. HIV-1- and HIV-2-infected CD 4+ cells were efficiently lysed by a slow, complement-dependent mechanism, whereas uninfected CD 4+ cells and HLA-DR+ cells were not affected.

Genetically Engineered Antibody Molecules

Publisher Summary The availability of monoclonally derived hybridoma antibodies provided an infinite supply of homogeneous reagents so that laboratories around the world could use standardized procedures. However, some limitations persisted. One approach to producing hybridoma antibodies with more desirable biological effector functions or antigen-binding specificities has been to take advantage of the high rate of somatic mutation which occurs in cultured myeloma and hybridoma cell lines. The limitation inherent in this approach is that it is possible only to switch to isotypes that lie downstream of the expressed heavy chain gene isotype in the heavy chain gene complex. An alternative approach to producing improved monoclonal antibodies is to use recombinant deoxyribonucleic acid (DNA) techniques and eukaryotic gene expression methods, the objective being to produce antibodies with improved antigen specificities and effector functions. An advantage of this approach is that producing antibodies is not a limitation as they exist in nature. Instead, design and production of antibody molecules can be done with optimized specificities and effector functions. Antigen-binding specificities can be joined to both immunoglobulin and nonimmunoglobulin sequences to provide antibody and antibody-like molecules for use in diagnosis and immunotherapy. Standard techniques of genetic engineering and gene transfection make it feasible to produce antigen-binding molecules with widely varying structures. The challenge is to define the structure that is optimal for a desired function.

The hemolytic activity of (Fab-Fc) recombinant immunoglobulins with specificity for the sheep red blood cells

Antibody-like molecules were formed by artificial recombination of proteolytic IgG fragments (Fab′ with anti-SRBC activity, and Fc) and used for studies concerning the complement fixation. Such molecules, when composed of single Fab′ bound to Fc fragment appeared inactive, while species containing two Fab′ fragments revealed the hemolytic activity. The results were discussed and interpreted, assuming that the interaction of Fab domains with C H2 domains in the Fc fragment is a main structural effect influencing the binding of the complement.

A bicellular mechanism in the immune response to chemically defined antigens: I. Antibody formation in vitro

Studies were made on the in vitro initiation of primary and secondary antibody responses to DNP-protein or DNP-poly- l-amino acid conjugates using the Millipore filter well technique for spleen organ cultures. Antibodies to DNP were assayed by inactivation of DNP-conjugated T4 bacteriophages. The production of a primary response to DNP after immunization with α-DNP-poly- l-lysine was inhibited by either free DNP-lysine or free poly- l-lysine. Hence, specific cell receptors recognizing both the hapten and the carrier interact with the immunogen in the primary response. The inhibition of the response by the affinity labeling reagent to DNP, BADE, suggests that the cell receptors are antibody-like molecules which thus exist prior to the experimental induction of antibody response. Spleens from mice immunized against RSA, then stimulated in vitro with DNP-RSA manifested a higher response to DNP than spleen cultures from animals which had not been primed with the carrier. Free carrier molecules prevented this anti-DNP response. The effect of preimmunization against the carrier is specific: Spleen cultures from donors primed against RSA did not manifest increased response to DNP after in vitro stimulation with DNP-Hcy, DNP-HSA, or DNP-GPA. To study the cellular basis of the carrier effect, the effect of suppressing cell replication was tested. Vinblastine given simultaneously with RSA inhibited the response to the subsequent in vitro stimulation with DNP-RSA. When given just 24 hr after the carrier, however, it did not inhibit this response. This is interpreted as an indication that the crucial event in the carrier effect is the stimulation of just one cycle of DNA replication, which might be necessary for more carrier receptors to be produced per cell. The results are discussed in relation to the bicellular interaction in antibody production.

Specific blocking in vitro of antibody synthesis by affinity labelling reagents.

INDUCTION of an antibody response is thought to involve an initial phase of antigen recognition by cells, but it is not yet known whether the recognizing cell is the precursor of the antibody producing cell or a distinct antigen sensitive cell1. In any case, recognition is believed to take place through a cell-surface receptor. This was suggested from experiments in which free haptens inhibited the immunogenic effect of hapten–protein conjugates. Furthermore, closely related haptens interfered with the response induced by the hapten–protein conjugate; the antibodies inhibited were those which cross-reacted with the related haptenic determinant2. Studies to characterize such a cell receptor indicated that antibodies inhibit induction of a response, possibly by competing with the receptor for the antigen2, which suggested that the receptor is an antibody-like entity. The idea that antibody-like molecules are present on the membrane of the lymphoid cell is supported by observations that antibodies to immunoglobulins induce transformation in lymphoid cells3.