Immunological Techniques

Immunological techniques in biotechnology research R. G. Werner, W. Berthold, H. Hoffmann, J. Walter and W. Werz Dr Karl Thomae GmbH, Department of Biotechnology, D-7950 Biberach an der Riss, Germany

Summary Specific interactions between antigen and antibody provided the basis for a variety of applications of monoclonal antibodies of mouse origin. In enzymelinked immunoassays (e.1.is.a.) they are used for quantification and qualification of recombinant DNA-derived proteins and contaminants of proteinaceous nature. In addition, in epitope mapping they are a useful tool in characterization of the protein structure. For purification of proteins, monoclonal antibodies can be used for immunoaffinity chromatography to gain the desired protein in high purity within a short period of development. In tumour-imaging, monoclonal antibodies provide a high sensitivity and selectivity to tumour markers, and therefore, improve the detection of solid tumours and metastasis. In tumour therapy, they are used for drug targeting or act as a cytotoxic agent themselves. Monoclonal antibodies, which specifically interact with cell-adhesion molecules are currently developed as immunomodulating or immunosuppressive drugs. The opportunity of humanization of such monoclonal antibodies of mouse origin or the production of human monoclonal antibodies after immunization in vitro will provide future perspectives for the application of therapeutic monoclonal antibodies in cases where the human anti-mouse antibody (HAMA) response so far does not allow a long-term therapeutic application.

Introduction The introduction of monoclonal-antibody technology by Kohler & Milstein [ 11 opens a broad field of applications. The initial use of monoclonal antibodies took place in the basic research for identification of tumour markers and thereby lead to applications in diagnosis and therapy of cancers and other diseases. Owing to the specific interaction of the monoclonal antibodies with their antigen, they can be used for analytical purposes, for immunoaffinity chromatography, for diagnosis and therapy and for drug targeting. Thereby analytical methods with improved sensitivity, chromatoAbbreviations used: ICAM- 1, intercellular adhesion molecule- 1; [,FA- 1, leucocyte function associated-1 antigen; ADCC. antibody-dependent cellular cytotoxicity; HAMA, human anti-mouse antibody.

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graphic procedures with higher selectivity, tumour diagnosis with increased efficacy and new tailormade therapies could be developed.

Enzyme-linked immunoassay applications in the quantification and qualification of recombinant DNAderived proteins and monoclonal antibodies Owing to the specificity of monoclonal antibodies and of affinity-purified polyclonal antibodies, selective e.1.i.s.a. tests can be established which are suitable for the quantification of active ingredients and of protein contaminants in pharmaceutical products. These e.1.i.s.a. tests are qualified by their sensitivity, specificity, accuracy and reproducibility as well as by their suitablility in routine test systems. E.1.i.s.a. tests are the method of choice for the quantification of active ingredient in complex protein mixtures, such as the determination of product titres in cell-culture fluids or product concentration in early steps of protein recovery. Another application is the quantification of specific trace-protein contaminants in products derived from biotechnical production. In most cases a sandwich-e.1.i.s.a. arrangement with two different specific monoclonal antibodies is applied. A detection limit of below 1 p.p.m. protein contaminant in the purified product can be reached. This is far beyond the detection limit of other analytical methods such as SDS/ PAGE with sensitive silver staining, where in favourable cases the detection limit reaches 200 p.p.m. for individual protein contaminants. In addition, if two monoclonal antibodies can differentiate between the native and the denatured form of protein these antibodies can be used for qualification of the native product and for quantification of small amounts of denatured protein (Fig. 1a). Using such differentiating antibodies, stabilityindicating assays can be developed, which detect specifically inactive product (Fig. 1b). These antibodies can also be used for the purification of the native molecule by removal of the denatured form. For quantification of host-cell proteins in pharmaceutical products from biotechnical production, a special multi-antigen e.1.i.s.a. can be developed. A so-called ‘blank run’ has to be performed with host cells which lack the genetic information for the product synthesis to isolate

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The detection of small atttounts of denatured protein using an e.1.i.s.a. test based on a monoclonal antibody which specifically binds to denatured protein but not to the native form of the protein Specificity of binding is shown in (a). Key to symbols -, native product: +, native product+ I% denatured pronative product + 0. I% denatured protein. The tein; +, stability indication properties of this e.1.i.s.a. assay are shown in (b). Key to symbols: +, 6 O " C : m , 50"C-, 40°C IZZI,25'CI\, 4°C.

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host-cell-protein contaminants free from the product of interest. The antigen-specific polyclonal antibodies which are gained after immunization are used in all e.l.i.s.a., which detects very low amounts of various host-cell-protein contaminants in the purified product. Again a detection limit in the p.p.m. range can be reached. The advantage is that this type of a host-cell-protein e.1.i.s.a. has the potential to detect trace amounts of a broad spectrum of different contaminant proteins with unknown features.

Epitope mapping for structure analysis

of complex proteins In addition to the quantification of proteins or peptides by e.1.i.s.a. techniques, monoclonal antibodies can be used for the qualification and detailed

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characterization of proteins [2]. Examples for the detection of surface structures of a complex recombinant protein are given in Fig. ~ ( u - c )for the tissue plasminogen activator molecule. In many cases epitope mapping of proteins can be considered as a useful tool for detection of binding sites, determination of surface-oriented amino-acid sequences, measurement of proteolytic degradation, confirmation of the integrity of the N-and C-terminal aminoacid sequence, detection of carbohydrate-binding sites and, in complementation to computer modelling, elucidation of the three-dimensional structure of complex proteins.

Immunoflinity chromatography is the method of choice for the isolation of proteins from natural sources or fermentation processes for research purposes in relatively high purity without an elaborated process development for protein purification. Immunoafinity chromatography is especially designated for quantitative purification of proteins present in small concentrations in large volumes and with a high degree of contaminating proteins. The separating performance and capacity in affinity chromatography correlates with the specificity of the ligand and is of outstanding performance with appropriate antibodies. For its unique specificity, immunoaffinity chromatography would be the sophisticated method of choice, even for a manufacturing process. However, it is very costly and mostly associated with the disadvantage of leakage of the monoclonal antibody during elution or regeneration. . For all reagents which will be in contact with the protein during the manufacturing process, it is required that they have to be produced, analysed and certified according to Good Manufacturing Practice (GMP) Guidelines. In accordance with these guidelines, regulatory agencies require that the production of monoclonal antibodies used as ligands for immunoafinity chromatography is comparable with that of a therapeutic agent according to the corresponding guidelines. This implies an expensive GMP production scheme for the monoclonal antibody itself in order to obtain the ligand. The application of an immunoafinity-chromatography step within the purification process implies additional purification steps behind the immunoaffinity chromatography to remove antibody contaminations derived from column leakage. Corresponding analytical methods for detection of trace amounts of murine antibodies must be established also.

Immunological Techniques

Fig. 2 Epitope characterization of rt-PA using three different monoclonal antibodies 223

Epitope characterization using the monoclonal antibodies 17-I34/20 ( a ) , 103-23 ( b ) and I I 1-321 (c). Peptides representing the overlapping amino-acid sequence were coupled on plastic rods (pins); pin I representing the amino-acid sequence 1-8; pin 2, 2-9, etc. The epitope detected by the monoclonal antibody is determined using an e.1.i.s.a. technique. Positive pins are indicated by their numbers and the binding reaction is expressed as absorbance (Aw5",,,).

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Basic matrices which allow an application in low-pressure chromatography at a technical scale, are preferably highly cross-linked agarose or beads or organic polymers because of their rigidity at high linear flow rates up to 1500 atm/h [3, 41. With respect to adsorption and desorption kinetics silicabased matrices for high-pressure-liquid-afinity chromatography refer more to analytical applications [S]. The antibodies used as immunoafinity ligands are linked to these matrices via appropriate spacers to avoid steric hindrance and to guarantee a stable linkage [6]. Only a few immobilization methods lead to matrices, such as N-hydroxysuccinimide-, triazine- and tresyl-activated matrices, which really exhibit those features absolutely necessary for an immunoafinity matrix value. They pro-

First e e amii e q u e n c e number

vide a gentle and perfectly stable linkage. However, a disadvantage might be the random coupling of the antibodies, thus lowering the acessibility to the immunospecific sites (Fab part of the antibodies)

[71. Hydrazide-activated matrices enable a sitedirected immobilization to the carbohydrate moiety of the Fc part, thereby allowing free immunospecific interactions, but they contain a less stable bond than the other matrices mentioned above. This leads to enhanced bleeding of the antibodies during protein elution for these matrics [8].

Tumour imaging and therapy Tumour cells express unique proteins at their membrane surface. Quite a number of such proteins are already identified by specific monoclonal anti-

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bodies. Radioactive, preferentially technetiumlabelled, antibodies can be used in immuno-szintigraphy for detection of solid tumours and metastasis. Using this methology, a higher sensitivity to tumour imaging can be achieved [9, 101. Based on their specific interaction with protein structures at the membrane surface of tumour cells monoclonal antibodies can also be applied to kill specifically tumour cells, or they can be used for drug targeting of cytotoxic compounds to the tumour cell [ 11, 121. By the application of anti-idiotype monoclonal antibodies it is possible to interact with the network of the immune system and to inactivate B-cell lymphomas which carry corresponding idiotype antibodies on their cell surface [ 131.

Fig. 3

Schematic representation of the molecular interaction between T-cells and monocytes during the early stage of immune responsiveness Abbreviation used: MHC 11, major histocompatibility complex II. Reproduced from [27] with permission.

Immunosuppressive therapy using monoclonal antibodies directed against cellular adhesion molecules Leucocyte adhesion and signalling are important steps in the development of inflammatory and immune responses. Intercellular adhesion molecule-1 (ICAM-1, CD54), a molecule bound to the cell-surface membrane, mediates various cell-cell interactions in immunity and inflammation by binding to the leucocyte integrin adhesion receptor LFA-1 (CDlla/CD18) and partially also to Mac-1 (CDl lb/CD18) [ 14-16] (Fig. 3). ICAM-1 is a sialylated glycoprotein with tissue-specific differential glycosylation resulting in a wide molecular mass range of 70-120 kDa. It belongs to the immunoglobulin supergene family and has a restricted tissue distribution [17]. It is, however, highly inducible on various cells during inflammation. It responds in vitro to pro-inflammatory cytokines, which suggests that ICAM-1 upregulation and cell-surface expression are important in the regulation of immunoresponses [ 181. ICAM- 1 cell-surface expression has been reported in a variety of diseases such as liver and kidney allograft rejection, allergic reactions type IV, psoriasis, hyper-reactive bronchial system, metastatic carcinoma and progression of melanoma [9-241. In addition, ICAM-1 represents the receptor for the main group of rhinoviruses to invade epithelial cells which have been stimulated by inflammatory mediators. Upper-respiratory-tract infection or common cold represent the medicinal indications [25]. This molecule seems also to be an endothelial receptor for Plasmodium falciparum, the causative agent of malaria tropica [26]. Therefore, specific monoclonal antibodies directed against ICAM- 1 have a great potential to be successfully used for diagnosis and

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Monocyte

therapy of a variety of diseases where ICAM1-related adhesion is involved. First successful clinical results have been obtained with a monoclonal antibody to ICAM-1 in the prevention of kidney allograft rejection, where this molecule seems to play a key role during an immunologically mediated graft rejection. The heterodimeric adhesion molecule LFA- 1 (leucocyte function associated- 1 antigen) can be found on cells of the haematopoetic lineage: T- and €3-lymphocytes, monocytedmacrophages, granulocytes, natural killer cells and thymocytes. 15 000-40000 receptor sites are expressed on individual cells. Regarding T-lymphocytes there exists a functional co-operation between target-cell LFA- 1 and the T-cell antigen receptor, whereas during the granulocyte-mediated ADCC-reaction (antibodydependent cellular cytotoxicity) the interaction occurs between LFA-1 and the Fc receptors of granulocytes. Accordingly, LFA-1 plays a key role during cytotoxic T-lymphocyte responses (lysis of allogenetic, xenogenetic, virus-infected or haptenmodified cells), natural killer-cell functions, CD4dependent reactions (including T-cell-dependent activation of B-lymphocytes), phagocytosis of antibody-coated particles and extravasation of activated immunocompetent cells [24, 271. Therefore, this

Immunological Techniques

integrin is involved in antigen-dependent and antigen-independent cell adhesion. Monoclonal antibodies which specifically bind to LFA-1 and thereby inhibit immune-cell adhesion may be used for immunosuppressive or immunomodulating therapy of various diseases. In general, cellular adhesion molecules and receptors are molecular targets for monoclonal antibodies and peptids or peptidomedica as a therapeutic approach to influence cell-cell, receptorligand and receptor-virus interaction. This offers a great potential, both for diagnostic and therapeutic application of drugs derived from biotechnical production.

Humanized monoclonal antibodies Mouse monoclonal antibodies are a common source for imaging and therapeutic applications. A major disadvantage is the potential of these heterologeous proteins to induce HAMAs. This HAMAresponse is undesirable because it may cause adverse effects in the human body and neutralize the therapeutic antibodies. Several approaches to avoid a HAMA-response have been followed: preparation of less antigenic Fab or F(ab),-fragments, polyethylene glycol modification of antibodies, preparation of chimeric or hybrid antibodies, and preparation of humanized monoclonal antibodies. In chimeric or hybrid antibodies the variable regions of the heavy and light chain of the mouse monoclonal antibody are combined with the constant regions of a human antibody by genetic engineering. In humanized antibodies only the gene fragments for the variable-antigen binding sites are integrated in the framework of a human-antibody gene. The advantages of humanized antibodies versus mouse monoclonal antibodies are a lower antigenicity, a longer half-life and stronger support of Fc-mediated immune reactions, such as antibody-dependent cytotoxicity and complementmediated cytotoxicity [ 11,281. The basis for the production of chimeric or humanized monoclonal antibodies is the genetic information of a mouse monoclonal antibody and the expression of the resulting recombinant DNA in appropriate host cells. A different approach is the isolation of native human antibodies after immunization in vdro [29]. Hybridoma cells which are derived from human B-lymphocytes often show a lower stability and productivity than mouse hybridoma cells. In these cases the method of choice is the isolation of the gene for the human antibody, its integration into suitable vector systems and its expression in appropriate host cells. Using these

techniques, a stable production of sufficient amounts of human monoclonal antibodies can be established. Recombinant DNA-technology can also be applied for the production of antibody fragments or of antibodies which are conjugated to toxins [ 121. Thereby, highly active immunotoxins for cancer therapy can be generated with high toxicity to the target cells and low toxicity to non-target cells. As a further advantage, small immunotoxins with a high stability can be produced which show a good penetration into cancer tissues. These molecules represent a new generation of anti-cancer drugs, which owing to their specific mode of action have a lower toxicity than conventional chemotherapeutic drugs, but still have to prove their efficacy for cancer treatment in the clinic.

Conclusion Epitope specificity and availability of monoclonal antibodies made them not only a useful tool in basic research, but provided a broad field of application in diagnosis and therapy. In clinical indications where the HAMA response causes adverse effects, humanization of mouse monoclonal antibodies will be the method of choice and will provide further perspectives in human therapy.

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Dustin, M. L., Springer, T. A., Clark, E. A., Mannoni, P. & Shaw, S. (1988) Nature (London) 331.86-88 Diamond, M. S., Staunton, D. E., de Fougerolles, A. R, Stacker,S. A., Garcia-Aguilar,J., Hibbs, M. L. & Springer, T. A. (1990) J. Cell Biol. 111,3129-3139 Simmons, D., Makgoba, M. W. & Seed, B. (1988) Nature (London) 331,624-627 Wawryk, S. O., Novotny, J. R, Wicks, I. P., Wilkinson, D., Maher, D., Salvaris, E., Welch, K., Fecondo, J. & Boyd, A. W. (1989) Apr. Immunol. Rev. 108,135-161 Adams, D. H., Hubscher, S. C., Shaw, J., Rothlein, R. & Neuberger,J. M. (1989) Lancet ii, 1122-25 Weetman, A. P., Cohen, S., Makgoba, M. W. & Borysiewicz, L. K. (1989) J. Endocrinol. 122, 185-191

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Received 28 August 1991

Peptide antigens G. Brian Wisdom Division of Biochemistry, The Queen’s University, Medical Biology Centre, Belfast BT9 7BL, U.K.

Introduction The advent of rapid, accurate and economic methods of peptide synthesis has allowed the exploitation of peptides both as immunogens and as antigens s m stn’cto. Immunological techniques based on peptides are used for two main purposes in biochemistry. First, anti-peptide antibodies are used to identify and characterize peptides and proteins; the ability of proteins to cross-react with these antibodies results from the immunogenic peptide mimicking a portion of the protein’s structure. This is especially valuable when a protein is rare, or when the only information available is its primary structure derived from the DNA sequence. Secondly, peptides themselves are used to analyse immune responses, particularly the mapping of protein epitopes recognized by antibodies and by T-cell receptors. Peptides can also be used to mimic ‘natural’ epitopes and to fractionate antibodies and lymphocytes. An important feature of the use of peptide antigens is the ability of some peptides to resemble the parts of a protein which react with an antibody or a T-lymphocyte; these parts are called epitopes

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or antigenic determinants. Epitopes for antibodies are classified rather artifically into two types: continuous (or linear or sequential) epitopes and noncontinuous (or assembled or conformational) epitopes. The latter are composed of different parts of the polypeptide chain which are contiguous in space; most epitopes in native proteins are thought to be of this type. Peptides can mimic continuous epitopes and, in some cases, can at least partially mimic discontinuous epitopes. In the few examples of antigen-antibody complexes studied in detail to date about 20 of the antigen’s amino-acid residues are in close proximity to the antibody’s binding site, but synthetic epitopes with as few as six residues can react effectively. The epitopes for T-cells are different in that a digested and relatively linear fragment of polypeptide is presented to the T-cell receptor protein by a major-histocompatabilitycomplex molecule. In this overview I wish to describe recent developments in the techniques involved in the application of peptide antigens, particularly those peculiar to the use of peptides, and provide examples of a few of the applications. Several publica-

Immunological techniques in biotechnology research.

Immunological Techniques Immunological techniques in biotechnology research R. G. Werner, W. Berthold, H. Hoffmann, J. Walter and W. Werz Dr Karl Tho...
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