novel antibody reagents: production and potential

T I B T E C H - FEBRUARY 1988 [Vol. 6]

Novel antibody reagents: production and potential

Gareth Williams

Immunologists have had l imited control over the composit ions of monoclonal antibodies. They could choose the cell l ines to be fused and screen hybridomas for the production of antibodies with an appropriate specificity. Recently, however, the degree of control has been extended by advances in cell fusion and genetic engineering. In particular, monoclonal antibodies with dual specificities, pre- determined specificities or addit ional functional moieties can be

produced.

In 1975, K6hler and Milstein produced a murine hybridoma by the fusion of a culture of mouse myeloma cells with spleen lymphocytes from an immunized mouse 1. The development of this technology was responsible for an enormous increase in the potential for the use of antibodies as reagents in medicine and biology. Before 1975, the sole source of monoclonal antibodies was a number of myeloma cell lines. Today, hybridomas producing antibodies of a variety of specificities exist in almost limitless quantities. In recent years however, advances in hybridoma production techniques, recombinant DNA manipulation and gene transfection technology have made possible the production of entirely novel antibody reagents.

Bispecific antibodies Naturally occurring antibodies

have two antigen combining sites which both recognize the same antigenic determinant. It is possible, however, to produce antibodies with dual specificity by a number of methods. These bispecific immunoglobulin molecules can react with and link two distinct antigens.

Chemical reassociation Bispecific antibodies have been

Gareth Williams is at the MRC Laboratory of Molecular Biology, University Post- graduate Medical School, Hills Road, Cambridge CB2 2QH, UK.

produced by the chemical reassociation of monovalent fragments derived from two monoclonal antibodies [Box 1, Fig. la and centrefold, part (C)] 2-3. This method suffers from a number of technical disadvantages. For instance, it may be difficult to dis- sociate the immunoglobulin chains without some protein denaturation and subsequent loss of antibody activity; or unorthodox disulphide bonds within chains may be created when the antibody heavy chains are allowed to reassociate.

Heterogeneous aggregation An alternative method of prepara-

tion involves the covalent attach- ment of whole monoclonal antibodies of different specificities using a heterobifunctional cross-linker [Box 1, Fig. la and centrefold, part (b)]. Heterogeneous aggregates have been produced using the chemical cross-linker N-succinimidyl-3-(2-py- ridyldithiol)propionate (SPDP) 4. The potential use of these reagents in the targeting of cytotoxic T cells has been recently demonstrated 5'6 and is discussed in Box 1.

Cell fusion A third method for the preparation

of bispecific monoclonal antibodies, cell fusion, was described in 19837 . A hypoxanthine-aminopterin-thymidine (HAT) sensitive hybridoma producing an anti-peroxidase monoclonal antibody was fused with cells

© 1988, Elsevier Publications, Cambridge 0166- 9430/88/$02.00

from the spleen of a rat that had been immunized with a somatostatin-thyroglobin conjugate. The resultant culture yielded resistant hybrid clones for assay when incu- bated with medium supplemented with HAT. All the hybrids produced anti-peroxidase monoclonal antibodies and a proportion were manufacturing bispecific hybrid molecules [Box 1, Fig. la and centrefold, part (a)] positive for both anti- somatostatin and anti-peroxidase activity.

The hybrid hybridoma expresses light and heavy chains from both antibody types. These protein chains recombine in the cisternal space to produce parental and hybrid antibody molecules. Although only a small proportion of light and heavy chain associations yield bispecific molecules, in practice different combinations are not produced with equal probability. Some chain associations are particularly favoured. The bispecific reagent produced in this experiment was used in a one step immunochemica] detection assay of somatostatin, demonstrating one area of application for dual specificity monoclonal antibodies (For others, see Box 1).

The production of novel antibodies by gene transfection

Recent innovations in DNA manipulation and transfection procedures and in gene expression techniques have opened the way to make new antibodies and antibody-related proteins. The process begins with the construction of bacterial plasmids containing the immunoglobulin genes of interest together with a selectable marker. The different domains of immunoglobulins are encoded by separate exons making assembly of the plasmid reasonably easy. Rodent variable regions of desired antigen-specificity can be obtained from DNA libraries made from hybridomas prepared by conventional methods. The human constant regions have already been cloned.

Expression systems Although experiments have been

carried out using bacteria, yeast and non-lymphoid cell lines as expres-

T I B T E C H - FEBRUARY 1988 [Vol. 6]

sion systems for antibody molecules 14-17, myeloma cell lines are currently widely used. Myelomas are plasma cell tumours and are, therefore, equipped for antibody production and secretion. Many myeloma cell lines can express transfected antibody genes and secrete the products.

The demonstration of techniques for introducing foreign DNA encoding antibody molecules into the germline of higher animals 18 has led to interest in the use of transgenic animals as antibody expression systems. This is an attractive prop- osition since the production of large quantities of monoclonal antibodies from mammalian tissue culture is expensive.

Selectable markers Only a small proportion of cells

become stably transfected whilst using standard transfection techniques for the introduction of foreign DNA into myeloma cell lines. There- fore a selectable marker is needed as part of the bacterial plasmid. The thymidine kinase gene has been used although this requires the recipient cells to be tk- (Ref. 19). Two widely used alternative markers are gpt (Ref. 20) and neo (Ref. 21). Both are bacterial genes coupled to SV40 virus transcription signals and both produce a selectable change in the phenotype of normal cells. The gpt gene encodes a guanine-xanthine phosphoribosyltransferase activity that confers resistance to myco- phenolic acid. Neo allows transfected cells to survive in the presence of the antibiotic G418.

Both of these markers rely on different selection mechanisms and so they can be used to introduce two plasmids either simultaneously or sequentially into a single myeloma host. A gene conferring resistance to hygramycin is also available as a selectable marker in lymphocyte transfection 22.

Gene transfection Antibody genes have been intro-

duced into lymphoid cells by a number of methods. These include cal- cium phosphate co-precipitation 23, spheroplast f u s i o n 24 and electroporation 25. The advantages oftransfection

TIBTECH- FEBRUARY 1988 [Vol. 6]

by electroporation include the speed of operation, reproducibility and efficiency in producing good yields of stable transfectants for a variety of cell lines. In electroporation, the membranes of recipient cells are reversibly permeabilized during the application of a high intensity electric field. When this is done in a solution containing a high concentra- tion of DNA, a small proportion of the cells will become stably transfected.

Different cell lines vary in trans- fectability, for unknown reasons. This appears to hold true for all the gene transfer methods mentioned over a variety of cell lines.

Chimaeric antibodies The major impact on therapy

promised by the advent of hybridoma technology has not yet been felt. Although rodent monoclonal antibodies are used in a number of clinical situations, the prospect of allergic reactions and anti-antibody responses is of real concern. This is especially true when immunization protocols necessitate multiple injec- tions of antibody reagents. Clearly the use of human monoclonal antibodies would be best suited to immunotherapy. However, the ethical restraints on in-vivo immunization and the technical problems of an alternative technique in vitro pre- clude, in the near future, the isolation of cell lines secreting a wide range of useful human monoclonal antibodies.

Variable region grafting To address this problem, several

laboratories have produced chimaeric monoclonal antibodies in which the antigen-binding variable region from a rodent source is genetically combined to a human constant region [Box 2, Fig. 2a and centrefold, part (d)] 26-~:~. These antibodies, when used in vivo, might be less antigenic than their pure rodent counterparts because the constant domains of mouse immunoglobulins are probably the most antigenic.

Apart from their potential use in immunotherapy, the availability of chimaeric monoclonal antibodies of known antigen specificity has allowed a controlled comparison of

the effector functions of human immunoglobulins using a set of chimaeric antibodies of different heavy chain class and subclass 33.

Complem en rarity-determining regions (CDR) grafting

The presence of a rodent variable region might still be sufficient to elicit an immune response. Existing technology has made it possible to produce antibodies in which human constant regions are attached to variable regions which are partially human in composition [Box 2, Fig. 2b and centrefold, part (e)]. These antibodies may be more suitable for application in vivo.

The variable domains of an antibody comprise complementarity- determining regions (CDRs) and framework regions. The CDRs are largely responsible for antigen binding and are positioned adjacent to the framework regions whose archi- tecture is conserved in rodents and humans. Recombinant DNA technology has been used recently to graft the CDRs, and hence the antigen specificity, from the heavy chain

variable domain (VH) of a mouse monoclonal antibody into the VH domain of a human myeloma antibody 34. The antibody produced which contained a 'humanized' VH region retained a similar binding affinity to an analogous monoclonal antibody with a solely murine VH region.

Thus, a foreign specificity can be conferred upon a human antibody by grafting on the CDRs of an antibody from an appropriate hybridoma. Apart from the implications of this technique to modify antibodies for use in therapy it also demonstrates a technically convenient method for the in-vitro manipulation of antibody binding affinities.

Antibodies with novel effector functions

Immunotoxins and enzyme-linked immunosorbent assays (ELISA) are two applications for antibodies that could be expected to benefit from recombinant DNA technology directed at antibody design. In model systems, antibodies have been chemically conjugated to the A chains of both

TIBTECH - FEBRUARY 1988 [Vol. 6]

diptheria and ricin toxins to produce antibody reagents capable of targeting to and killing specific cells 35-37. The production of immunotoxins might be simplified if immunoglobulin and toxin genes were ligated together via DNA splicing methods and introduced into an expression system host cell.

Similarly, problems of batch reproducibility are inherent in the production of ant ibody-enzyme conjugates required for the ELISA assays already available. These problems might be minimized if myeloma cell lines could be persuaded to manufacture and secrete monoclonal antibodies with both antigen-binding specificity and enzyme activity [Fig. 3 and centrefold, part (f)].

The feasibility of this approach has already been demonstrated 38-39. A myeloma cell line was constructed that secretes a hapten-specific F(ab')2 genetically linked to an enzyme moiety. The enzyme was the nuclease of Staphylococcus aureus. The resultant recombinant antibody could be shown to exhibit Ca 2+- dependent activity for degrading nucleic acid as well as hapten specificity. It could also be easily purified to homogeneity in a single step on an antigen column.

The nuclease from S. aureus is viewed as an ideal candidate for fusion to an antibody heavy chain because (a) it can easily refold after denaturation, (b) it contains no thiol residues and so would not be glycosylated in a myeloma host and (c) is a secreted enzyme active as a monomer. Therefore, as a further example, a hapten-specific Fab fragment, similar to that used above, was genetically linked to the Klenow fragment of DNA polymerase I, derived from Escherichia coli, and introduced into a myeloma host cell line 4°. This recombinant antibody was demonstrated to have retained enzyme activity by its subsequent use in dideoxy nucleotide sequencing.

A similar approach might be usefully applied to specific enzymes that cannot be easily purified by conventional methods. An Fab fragment antibody tag not only ensures the export of an enzyme from a cell but facilitates its purification on an antigen column.

- -Fig. 3-

Genetically engineered antibody with novel effector function. The Fc region of the antibody is replaced by another active protein (enzyme, toxin).

Single chain antibodies The production of single chain

antibodies [Fig. 4 and centrefold, part (g)] might be feasible by genetically linking the genes for variable regions of immunoglobulin heavy and light chains by a DNA segment encoding a synthetic peptide linker. The binding sites of antibodies are to a large extent dependent on the accurate positioning of the heavy and light chain variable domains with respect to each other. For this reason it is essential for a peptide linker that holds together a putative single chain antibody to be carefully designed to ensure it does not interfere with the correct folding of the molecule.

The prospect of single chain antibodies is encouraging for a number of reasons. If it is possible to produce them in bacteria then this might lead to lower costs. They may be less immunogenic in vivo and their small size might give them the

- -Fig. 4

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ability to penetrate to tissues in the body normally restrictive to larger molecules.

When the production problems of these truncated proteins are over- come they may have practical applications as biosensor probes, im- munotoxin reagents and diagnostic aids.

Widening the scope The technical innovation of hy-

bridoma production has already led to the wide use of monoclonal antibodies in biological research and medicine. These uses include affinity purification procedures, immunoassays and therapy. The application of DNA manipulation techniques to antibody design and improvements in expression systems will further widen the scope for the practical applications of this family of proteins. Furthermore, the enormous natural diversity of antibody specificity provides a tempting range of protein-binding moieties that may, in the future, represent the building blocks of entirely new proteins.

Acknowledgements I would like to thank Michael

Neuberger for invaluable encourage-

genes coding for the histocompati- bil ity antigens which are important

gens cause, amongst other things, graft rejection.

Advisor: Keith James

February1988

Second generation monoclor

Cell fusion and antibody combinations Hybridoma cell production - Hybridoma cells are produced by fusing myeloma cells (malig- nant plasma cells) with lymphocytes (usually spleen cells) from immunized animals. Fusing myeloma cells with activated B lymphocytes produces long-lived hybridoma cells which syn- thesize uniform antibodies with a single specificity. Large amounts of monoclonal antibodies can be obtained by growing the hybridoma cells in bulk culture in vitro or as ascites in mice. Hybrid cells acquire 'immortality' from the myeloma cell and HAT resistance (i.e. their ability to survive hypoxanthine, aminopterin, thymine selection) from the immune normal B cell.

B lymphocyte Short-lived, Myeloma cell

HAT resistant Immortal, HAT sensitive

Hybridoma cell Immortal. HAT resistant

Hybrid hybridoma ce!

Secretion, recovery a

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Cross-linking

antibody A

I ~ Reassoctation

Dissociation

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M o n o v a l e n t ~ ~ ' fragment A

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Bi-specific monoclonal antibodies- Antibodies in which the combining sites on the immune- globulin molecule react with two distinct antigenic determinants. The antibodies can simultaneously interact with different antigens. (a) Cell fusion: a hybridoma cell synthesizing antibodies of one specificity is fused either with a second hybridoma cell (giving a 'quadroma') or with B lymphocytes. The bi-specific antibody shown is just one of the molecular species produced by the hybridoma cell. (b) Heterogeneous aggregation: monoclonal antibodies of different specificity synthesized by two separate hybridoma cells are joined using a chemical cross-linker. (c) Antibody fragment amalgamaUon: antibodies are dissociated into monovalent fragments by reducing the disulphide bonds which join the two heavy chains. The fragments are then allowed to reassociate With fragments of a different specificity. Denaturation of the chains and incorrect reformation of disulphide bridges may cause a loss of antibody activity.

Natural gene rearrangement ant expression ol antibodies

Germ line cell DNA contains many regions coding for parts of anti ecules (see Antibody diversity). Tl'ese coding regions arq together by a combination of DNA recombination and RNA spl During B cell development, one V (variable) and one J (join) gen~ recombine to produce a functional VJ ~.ne. (For heavy chains, sity) segment gene is also incorporate:L forming a VDJ gene). RNA transcript produced from the fur~ctional gene includes s coding for the constant region. Introns separating the various s are spliced out as RNA is processed il~to mRNA. Heavy and light chains are produced in the same cell from gene,, ent chromosomes and, after translation, combine to produce a i molecule. Before the molecule is excreted it undergoes glycos

V gone Dgene J gene constant region segments segments segments gene segments

m r~.~1 HN~,~ HNP'~ tlnuau itnnnn ,J ~,'~ t/~7"/'~ it ~'17"~ .... ~ J . , d / I ~I~G(Z~I,' ;a~,'l llllt' LlUUtJ ~ z l ~ I f ¢/.Z*~Z£,l

Recombination during B cell matuiation

..... w #/ . / .d i i w u.cdmJ u u r~ r~

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Antibody diversity-The diversity of inmunoglobulin specificit multiple germ line V genes (hundreds), recomb nat on between genes during B cell maturation, inaccuracies in recombination, I ations in immunoglobulin genes (particularly in those coding f( and hypervariable regions) and the cor#3ination of different lighb chains. There is also diversity in the antibody c:~astant region. The lighh of either kappa or lambda type. There! are four lambda const~ genes but only one kappa C gone. The vi~rious heavy chain const genes;determine the immunoglobuli~J class and sub-class i IgG, IgD, IgE and IgA).

/Antigen binding

/~,!Binding complement C4f Binding complement Clq

| Regulation of catabolism Protein A binding

J RF antigenic sites Binding to monocytes, K c B cells, neutrophils, heter( mast cells, macrophages RF antigenic sites

Structure and function of immunoglobulin G-Immun oglubuli r composed of two distinct types of polypeptides, the light (L) and chains. The chains are held together ~v covalent disulphide br non-covalent forces. Regions of each chain are associated with functions. The variable (V) regions ar~ responsible for antige Within the variable regions are relatively constant framework s which seem to act as a scaffold for the hypervariable regions known as complementarity-determinin!] regions (CDR)) which responsible for antibody specificity. F~e heavy chain constaf interact with receptors on various cells involved in the immune and interact with other proteins. ThesetJnctions are associated cular domains of the constant region.

monoclonal antibodies

le rearrangement and sion ot antibodies many regio ns coding for parts of antibody mol- ersity). Tl~ese coding regions are brought )f DNA recumbination and RNA splicing. )no V (variable) and one J (join) gene segment -tional VJ ~ne. (For heavy chains, a D (diver- ~corporate:t, forming a VDJ gene). A primary 'om the fur~ctional gene includes sequences on. Introns separating the various sequences rocessed into mRNA. roduced in the same cell from genes on differ- "translatiori, combine to produce a multichain ule is excreted it undergoes glycosylation.

le J gene constant region ~nts segments gene segments

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versity of immunoglobulin specificity is due to hundreds), recombination between V, D and J on, inaccuracies in recombination, point mut- .rues (particularly in those coding for variable Lnd the cor#3ination of different light and heavy

Peninsula Laboratories inc., 611 Taylor Way, Bolmont, CA 94O02, USA. Tel: (415) 592-5392 Telex: 172511 PEN LABS BLMT. Peninsula Laboratories Europe Ltd., P.O Box 62, 17K Westside, Jackson Street, St. Heens WA9 3A J, UK. Tel: (0744) 612108/30064 Fax: (0744) 30064 Telex: 0297O5 PENEUR G

Peninsula Laboratories provide a wide range of monoclonal ar]tibodies te complement the companies 'second to no¢/e' lange of peptldes and antibodies. El

Monoclonal antibodies produced by genetic engineering r DNA libraries

Sequence CH1 comparisor, constant

I DNA region synthesizer gone Effector

Hybrid protein variable gene

region gene

Expression, assembly, modification, secretion

d

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Synthetic linker peptide

DNA sequence

Expression and secretion(?j

antibody c~nstant region. The light chains are lype. There! are four lambda constant region ~lene. The vi~rious heavy chain constant region Jnoglobuli~J class and sub-class (e.g. IgM,

/Antigen binding

;H1 H/ / /H/Binding complement C4f (CH1)

-~AF"/L~" , m "l Binding complement Clq I • / Regulation of catabolism I • ~ Protein A binding L I j RF antigenic sites I 1 ~ Binding to monocytes, K cells, I I B cells, neutrophils, heterologous I • mast cells, macrophages 1 RF antigenic sites

lmunoglobulin G-Immunoglubulin G (IgG)is ,es of polypeptides, the light (L) and heavy (H) together ~v covalent disulphide bridges and

~s of each chain are associated with particular regions at!? responsible for antigen binding. are relativel'y constant framework sequences Lffold for the hypervariable regions (HV, also determinin!t regions (CDR)) which are largely )ecificity. ~ e heavy chain constant regions arious cells involved in the immune response ns. These'~"dnctions are associated with parti- nt region.

e

Rodent-human chimaeric molecules - Antibodies of this type have advantages for in vivo clinical use. Their reduced antigenicity means they are better tolerated. Their constant regions may interact more effictively with the receptors on cells involved in the immune response. (d) Variable region grafting: rodent DNA sequences coding for antigen-binding variable regions were combined with human sequences coding for the constant regions of both light and heavy chains. The chimaeric gene is expressed in a myeloma cell line.

(e) Complementarity-determining region (CDR) grafting: comparison of rodent and human sequences led to the construction of a chimaeric synthetic gene coding for rodent complementarity-determining regions on a human variable region framework. The synthetic variable region gene combined with a human constant region gene produced chi-

g

maeric antibodies when expressed in a mye- Ioma cell line.

Other constructs (f) Antibodies with novel effector functions: genes for proteins other than antibodies can be spliced to genes for antibody fragments and expressed as a conjugate molecule. For instance, the gene for nuclease from Staphyl- ococcus aureus and that for the Klenow fragment of DNA polymerase I from E. coil have been combined with genes for antibody fragments and expressed in myelorna cell lines.

(g) Single chain antibodies: these may be produced by genetically linking heavy and light chain variable region genes with a DNA sequence encoding a synthetic peptide linker. Avoiding the need for disulphide bridges may enable single chain antibodies to be produced in bacteria.

T IBTECH - FEBRUARY 1988 [VoI. 6]

m e n t and he lpfu l cr i t icism; and Kathy Wes ton for ass is t ing m e in the p repa ra t ion of this manusc r ip t .

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novel antibody reagents: production and potential

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