Monoclonal Antibodies-Principles and Practice (original) (raw)
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Modification of Antibody Function by Mutagenesis
Cold Spring Harbor Protocols
The ability to “fine-tune” recombinant antibodies by mutagenesis separates recombinant antibodies from hybridoma-derived antibodies because the latter are locked with respect to their properties. Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available. After immunoglobulin heavy and light chains for a particular antibody have been cloned, the binding site—namely, the complementarity determining regions (CDR)—can be manipulated by mutagenesis to obtain antibody variants with improved properties. The method described here is relatively simple, uses commercially available reagents, and is effective. Using the pComb3H vector, a commercial mutagenesis kit, PfuTurbo polymerase (Agilent), and two mutagenic primers, a library of phage with mutagenized heavy and light CDR3 can be obtained.
Antibodies and Genetically Engineered Related Molecules: Production and Purification
Biotechnology Progress, 2004
Antibodies and antibody derivatives constitute 20 % of biopharmaceutical products currently in development, and despite early failures of murine products, chimeric and humanized monoclonal antibodies are now viable therapeutics. A number of genetically engineered antibody constructions have emerged, including molecular hybrids or chimeras that can deliver a powerful toxin to a target such as a tumor cell. However, the general use in clinical practice of antibody therapeutics is dependent not only on the availability of products with required efficacy but also on the costs of therapy. As a rule, a significant percentage (50-80%) of the total manufacturing cost of a therapeutic antibody is incurred during downstream processing. The critical challenges posed by the production of novel antibody therapeutics include improving process economics and efficiency, to reduce costs, and fulfilling increasingly demanding quality criteria for Food and Drug Administration (FDA) approval. It is anticipated that novel affinity-based separations will emerge from the development of synthetic ligands tailored to specific biotechnological needs. These synthetic affinity ligands include peptides obtained by synthesis and screening of peptide combinatorial libraries and artificial non-peptidic ligands generated by a de novo process design and synthesis. The exceptional stability, improved selectivity, and low cost of these ligands can lead to more efficient, less expensive, and safer procedures for antibody purification at manufacturing scales. This review aims to highlight the current trends in the design and construction of genetically engineered antibodies and related molecules, the recombinant systems used for their production, and the development of novel affinitybased strategies for antibody recovery and purification. Contents 1. Introduction 639 2. Antibody Engineering: Tools and Products 641 2.1. Humanized Antibodies 642 2.1.1.Chimeric Antibodies 642 2.
Strategies in the development of human monoclonal antibodies
Developments in biological standardization, 1990
A high-efficiency, HAT-sensitive heteromyeloma fusion line CB-F7 has been developed from an 8-Azaguanin-treated Ig-non-secreting human X mouse heterohybridoma. The use of this line allowed us to produce human hybridomas more successfully by fusion of cell material from blood, lymph node or spleen. A polyspecific repertoire of IgM isotype was detected among the hybridomas obtained from the spleen. These IgM antibodies reacted with autoantigens as well as with foreign material. This naturally occurring repertoire may be of interest since it has anti-bacterial activity. The frequency of the occurrence of polyspecific antibody-producing hybridomas was high in the spleen. Apart from the detection of polyspecific IgM antibodies we did not find IgG-secreting hybridomas with anti-bacterial reactivity among thousands of initial lines derived from non-immunized persons. We therefore tried to fuse lymphocytes from donors, who were boosted with Tetanus Toxoid (TTd). A short and limited optimum ...
Characterization of the antibody response
Journal of cellular physiology. Supplement, 1957
By way of characterizing the antibody response I would like to bring together some of the available information on the disposition of antigen and the synthesis of antibody in a single antigen-host system. The correlation of the data on these two basic processes in a given antibody response may afford a general picture of the antibody response and serve as a frame of reference for some of the more specific topics. The immunologic system I have chosen as a basis f o r this discussion is the rabbit's response to heterologous serum proteins. The fate of these antigens has been extensively studied by isotopic and immunologic techniques and the antibody response has been investigated from both metabolic and morphologic points of view.
The American Biology Teacher, 1984
As a graduate student 15 years ago, I worked in a lab with a student whose research involved purifying an antibody. At the time, pure antibody was difficult to come by. Immunizing an animal against a particular antigen could, of course, induce production of antibodies, but they were a heterogeneous collection with such similar properties that they were almost impossible to purify to homogeneity. Even if purification were achieved, the yield was so small as to be almost useless. Researchers tried to get around these problems by purifying antibodies from individuals with diseases in which large quantities of a single antibody were produced. Many used myelomas, which are tumors composed of B cells, the lymphocytes that make antibodies. Since the tumor was derived from a single cell, it secreted a single type of antibody. In our lab, antibody was purified from the psoriasis scales of a patient with a severe form of the disease. This antibody was more basic than most, which helped in purification, but it was still a difficult job (Lawrence, Tye, and Liss, Immunochemistry, January 1972). These strategies did succeed in providing researchers with relatively homogeneous antibody, and in making it possible to work out antibody structure. But since it was impossible to know the antigen that the antibody was directed against, the antibody's value as a research tool was limited. This situation changed in 1975 when George Kohler and Cesar Milstein developed the first antibodyproducing hybridoma. They began by immunizing a mouse against a specific antigen. The mouse's spleen cells then made antibody against that antigen. Since they were normal B cells, they had limited lifespans in culture, and so produced limited amounts of antibody. To overcome this problem, Kohler and Milstein
A novel method for making human monoclonal antibodies
Journal of Autoimmunity, 2010
We have developed a B cell immortalization method for low B cell numbers per well using simultaneous B cell stimulation by CpG2006 and B cell infection by Epstein-Barr virus (EBV), followed by an additional CpG2006 and interleukin-2 (IL-2) stimulus. Using this method, immunoglobulin G (IgG)-producing immortalized B cell lines were generated from peripheral blood IgG þ CD22 þ B cells with an efficiency of up to 83%. Antibody can already be obtained from the culture supernatant after 3e4 weeks. Moreover, clonality analysis demonstrated monoclonality in 87% of the resulting immortalized B cell lines. Given the high immortalization efficiency and monoclonality rate, evidence is provided that no further subcloning is necessary. An important application of this B cell immortalization method is the characterization of (autoreactive) antibodies from patients with autoimmune disease. This could eventually lead to the identification of new autoantigens, disease markers or targets for therapy.