Advantages of rat monoclonal antibodies

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Immunology Today, vol. 4, No. 4, 1983

Advantages of rat monoclonal antibodies

making more time available, it is possible to approach this ideal more closely.

from Mike Clark, Steve Cobbold, Geoff Hale and Herman Waldmann During the past three years the pages of this journal have witnessed the explosion of interest in monoclonal antibodies and particularly in their applications for diagnosis and therapy of disease. For reasons of history and, we suspect, of tradition, virtually all investigators now use one of the various mouse myeloma lines to construct their hybrid myelomas. We are often tantalized by reports of h u m a n myeloma lines which are said to be suitable for fusions to produce h u m a n monoclonal antibodies, but so far none of them has been particularly fruitful. During this time we have been using the rat myeloma line Y3/Ag 1.2.3 (Ref. 1) in rat x rat fusions to make antibodies against cell-surface antigens of mouse and h u m a n leukocytes and have noticed several advantages of this system, some of which may not be widely appreciated. One of the main reasons for the development of a rat myeloma line was to enable larger amounts of antibody to be made as ascitic fluid in vivo and this remains a technical advantage, especially when gram quantities of antibody are required, for example for clinical trials. It is unlikely that most large-scale production of antibody will ultimately be by culture of cells in vitro but even then rat hybrids may offer advantages of more favourable growth requirements. An immediate application of the rat system was for production of antibodies against monomorphic differentiation antigens of the mouse 2'3 or polymorphic antigens in the rat 4. Previously, these types of specificity were available only from fusions of rat spleen cells with mouse myeloma lines with their attendant disadvantages.

Stabilityofrat × rathybridmyelomas The points made above have longbeen obvious to many, and it is to other advantages that we wish to draw particular attention. In our opinion the most important distinguishing feature of the Y3/Ag 1.2.3 line and hybrids derived from it is their marked stability against loss of secretion of immunoglobulin chains. It has been estimated that the rate of loss of immunoglobulin expression by the M O P C line (from which most mouse lines were derived) is about 10-3/cell/ generation (Ref. 5) whereas chain-loss variants of the Y3 line have never been detected and the loss rate is therefore less 4 than 10-/cell/generation (Ref. 6). This difference is not so important for established hybrid myelomas which usually are reasonably stable and can

easily be maintained by regular recloning, but it does have a significant effect during the early stages of a fusion. In mouse x mouse fusions (using a variety of myeloma lines) only about 60% of the hybrids which are growing after 2-3 weeks secrete intact spleen-cell-derived immunoglobulin 7. The corresponding frequency for fusions between rat spleen cells and mouse myeloma lines is, if anything, somewhat lower. It is supposed that the non-secretors arise by rapid loss of chromosomes and it is commonly observed that they will rapidly overgrow the antibody-producing hybrids if they are not quickly cloned6's'9. This has led to the appearance of techniques for immediate cloning of hybrids 1° which require large numbers of quick assays in order to decide which clones to keep. In contrast, rat x rat fusions result in 90-99 % of hybrids secreting spleen-cellderived antibody 7and this has enabled us to rethink the strategy for cloning and assays. Fusions are distributed amongst 96 wells and almost invariably growth of hybrids occurs in every one. Cells from each well (probably containing four or more clones) are frozen and culture supernatant is stored. Relatively few supernatants can then be assayed at leisure, which is an advantage if complex biological assays are necessary and especially if the assays are not available at the time of the fusion. When the original immunogen consists of a complex mixture of molecules (for example cell membranes) it is possible to return repeatedly to the same fusion and isolate clones with new specificities, and we have done this successfully up to three years after the fusion. O f course, when assaying supernatants which probably contain a mixture of antibodies it is necessary to be aware that subtle specificities can be obscured. This could be a problem when screening antibodies against cell surfaces because purified antigens are usually not available for the assays. One way to overcome it is to use a variety of detection systems and reagents which are specific for particular subsets of the rat antibodies and will, on average, detect only about one antibody per well. Examples of suitable systems we have used are: complement fixation, protein-A binding, and second antibodies specific for particular rat isotypes. The ideal assay is always the one most closely related to the ultimate, purpose for which the antibody is required. By cutting down the initial screening to a manageable size and

High frequency of rat monoclonal antibodies which fix human complement The other major reason why we prefer rat x rat fusion is the relatively large n u m b e r of antibodies obtained which fix h u m a n complement, and this may be of great importance for their therapeutic use. Several mouse monoclonal antibodies have been tried as potential immunosuppressive or anti-leukaemic agents 11, but few, if any, fix h u m a n complement. It remains to be seen whether that is a property which would have made them more effective at clearing ceils in vivo. However, there can be little doubt that for in-vitro applications such as removing cells from bone marrow, whether T cells (to prevent graftversus-host disease), or leukaemic cells (for autologous transplants), lysis with antibody and h u m a n complement would be preferable to other procedures such as mere opsonization with antibody or treatment with heterologous complement. The disadvantage of opsonization alone is that its effectiveness may be low when the patient's reticuloendothelial system is compromised by pre-transplant conditioning. When heterologous complement is used, lysis of cells can easily be monitored but is not always reproducible because different batches can vary greatly in potency and non-specific toxicity. In three rat × rat fusions using different h u m a n cells as immunogens we found 25/73, 35/84 and 61/96 wells positive for killing the immunizing cells with h u m a n complement (S. Cobbold, J. Martin and H. Waldmann, unpublished observations). One of the antibodies ( C A M P A T H 1) was found to react with T and B cells in humans and other primates. Injections of this antibody into monkeys caused rapid decreases in the n u m b e r of circulating lymphocytes and also in the level of total haemolytic complement. However, no acute toxic or anaphylactic reactions resulted even after repeated infusions (G. Hale and H. Waldrnann, unpublished observations). This demonstrates that there is no reason, in principle, why humancomplement-fixing monoclonal antibodies should not be considered for therapeutic use in vivo. The new hybrid myeloma technology is clearly having a profound effect in immunology and beyond, but is not without its own particular problems and limitations. We hope to have shown that some of the difficulties can be overcome by making rat hybrids rather than the

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Immunology Today, vol. 4, No. 4, 1983

traditional mouse ones. The myeloma line Y3/Ag 1.2.3 is now readily available to bona fide investigators, from Cesar Milstein's laboratory. References 1 Galfrd, G., Milstein, C. and Wright, B. (1979) Nature (London) 277, 131-133 2 Hoang, T., Gilmore, D., Metcalf, D., Cobbold, S., Watt, S., Clark, M., Furth, M. and Waldmann, H. Blood (in press) 3 Watt, S., Gilmore, D., Metcalf, D., Hoang,

Another look at chronic lymphocytic leukaemia in the context of B-cell differentiation

1

SIR, In many tumours there is a bulk population of cells which has apparently suffered maturation arrest. In the common form of chronic lymphocytic leukaemia (CLL) the major component of the neoplastic clone consists of cells which have the morphology, surface phenotype, enzyme profile and migratory properties characteristic of small B lymphocytes. Are C L L cells at a stage of development intermediate between pre-B cells and mature B lymphocytes, as is suggested by Dr Johnstone (Immunology Today, 1982, V01. 3, pp. 343-348)? It is doubtful whether we know enough about the details of normal B-cell differentiation o r about how closely C L L cells resemble members of the normal B-cell series to provide any certain answer to this question. We propose to re-examine some of the evidence which Johnstone considers to favour his hypothesis. O n the comparison of immunoglobulin production and expression by CLL and normal cells, which forms the basis of much of Johnstone's discussion, we should like to make three comments: (1) The association of IgM (to the exclusion of other classes of immunoglobulin) with B-cell immaturity can be over-emphasized, particularly if cellsurface immunoglobulin (slg) is the sole criterion. The immaturity of cord blood lymphocytes is not reflected in the slg isotype profile. In our experience and that of others 1, there is approximately the same proportion of B cells expressing IgG and IgA on their surface in the blood of the newborn as of adults. Furthermore, in tests on spleens from foetuses 16-18 weeks old we have found 5-12% and 1-2% of cells bearing IgG and IgA, re-

T. and Waldmann, H . J . Cell. PhysioL (in press) 4 Butcher, G. W., Diamond, A. G., Clark, M.

9 Coding, J. w. (1980)J. Immunol. Methods 39, 285-308 10 Davis,J. M., Pennington,J. E., Kubler, A.M. and Conscience,J.-F. (1982)d~ Immunol Methods 50, 161-172 11 Ritz, J. and Schlossman, S. F. (1982) Blood 59, 1-11

and Howard, J. C. Transplant. Proc. (in press) 5 Cotton, R. G. H., Secher, D. S. and Milstein, C. (1973) Eur. J. lmmunoL 3, 136-140 6 Galfr~, G., Butcher, G. W., Howard, J. C., Wilde, C. D. and Milstein, C. (1980) Transplant. Proc. 12, 371-375 7 Clarke, M. and Milstein, C. (1981) Somatic Cell Genet. 7, 657-666 8 Fazekasde St. Groth, S. and Scheidegger, D. (1980)J. Immunol. Methods 35, 1-21

Mike Clark, Steve Cobbold, GeoffHale and Herman Waldmann are in the Division of Immunology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 2 Q ~ UK.

spectively, compared with 25-34% and 19-21% bearing IgM and IgD. (2) The significance of small amounts of IgG on C L L cells of IgM and IgD type is certainly controversial and should be reinvestigated now that monoclonal antibodies reacting with epitopes in the three constant-region domains of y chains are available2. There appear to be antigenic differences between sIgG and secreted IgG (Ref. 3). Dhaliwal et al. ~ found that the proportion of IgG-positive cases depended on the choice of anti-y reagent. Stevenson et at. 5 also found sIgG on CLL cells in some IgM, IgD-positive cases~ but, as Johnstone pointed out, the sIgG lacked the idiotype of the leukaemic clone in the case studied 7. However, one cannot ignore the fact that there are some CLLs where IgG is the only sIg. In these cases the sIgG is well expressed and is of one light-chain type. Furthermore, the IgG found in 8 cases examined (L. Partridge, unpublished observations) has been of one subclass. (3) We have classified our B-cell leukaemias, according to heavy chain detected on the surface, into: M only; high M, low D; roughly equal M and D; low M, high D; D only; M D G ; G only; MA; MDA; A only; GA and light chain only. No differences in other markers were associated with a particular sIg phenotype. In particular, C L L cells of all sIg phenotypes formed strong rosettes with mouse erythrocytes (sometimes regarded as a selective marker for 'early' B cells). Normal B cells do not form rosettes with mouse erythrocytes under the conditions used in our laboratory. There is some degree of negative correlation with the quantity of surface immunoglobulin in that cells from cases ofprolymphocytic leukaemia fail to form mouse red-cell rosettes. Johnstone brings together two propositions which have achieved popularity: (1) the stage of differentiation of a B cell is closely related to the class of immunoglobulin it expresses on its surface; (2) C L L cells have the properties of 'early' B cells. In view of the range of sIg profiles encountered in CLL, and particularly the existence of IgG CLLs,

these propositions are not jointly tenable. Even the low level of surface immunoglobulin on C L L cells (10% of that on normal B cells) does not necessarily place them at an early stage of differentiation since similar low levels are found on B cells in germinal centress. Passage from surface expression of immunoglobulin to secretion is also an accepted indicator of progression along the differentiation pathway. Here again the matter can be oversimplified. It has become dogma that antigen-independent B-cell differentiation stops abruptly with the formation oflymphocytes expressing surface IgM and IgD and that, in CLL, the cells are 'frozen' at this stage or earlier. Neither of these statements is quite true. Recent studies on mice kept under germ-free conditions suggest that some progression to a stage of antibody secretion occurs independently of antigenic stimulation9. Other studies on C LL cases of 'non-secreting' type using antiidiotype reagents have shown that IgM bearing the clonal marker is frequently present in the serum and, further, that this IgM is of the 19s secretory type 6'7. It may not be produced by the CLL cells present in the peripheral blood but it must be produced by cells of what has been presumed to be a single clone 'frozen' at the B-cell stage. How is the pattern ofimmunoglobulin production after in-vitro stimulation related to maturity? Cells from cord blood, foetal liver and adult bone marrow secrete exclusively IgM after activation by Epstein-Barr virus (EBV) 5. However, even peripheral lymphocytes from adults produce immunoglobulin of predominantly the IgM class in response to this stimulant 10. Cord blood lyrnphocytes do not secrete any immunoglobulin alter stimulation with P W M , LPS or PHA (Refs 11, 12), but the interpretation of this finding is not simple since it has been shown that both T and B cells are incriminated 11'13. Some IgA, a lot of IgM but no IgG is secreted by cord blood lymphocytes which have been stimulated by staphylococci of the Cowan I strain, which is regarded as a T-independent B-cell stimulant 12. C L L ceils have, in