immunology loday, September 1.980
56
also referred to previous studies in rodents by others in which there was activation of immune and reticuloendothelial systems during pregnancy. Such stimulation has, however, not been reported in man. It is i m p o r t a n t to note that migration is particularly vulnerable to unspecific stimulation during laboratory processing. In this study the cells were washed three times and the concentration of cells was low while in the previously cited studies, cells were washed only once1, 2 or not at alP. The reported enhancement of monocyte r a n d o m migration during pregnancy should preferably be confirmed by further investigation. In summary, data obtained from studies of humans imply that normal pregnancy is indeed associated with depressed inflammatory cell function and specific
K]
immune responsiveness. It is suggested that these effects are mediated by humoral factors which remain to be identified with certainty. BENGT BJ()P.KSTEN
Department of Biomedical Research, Pharmacia AB, Box 181, S-175 04 Uppsala 1, Sweden. References 1 Takeguchi, A. and Persellin, R.H. (1980)J. Clin. Lab. lmmunol. 3, 121-124 2 Persellin, R.H. and Thoi, L.L. (1979) Am. J. Obstet. Gynecol. 134, 250-254 3 Bj6rkst~n, B., S6derstr6m, T., Damber, M.-G., yon Schouhz, B. and Stigbrand, T. (1978) Seand.J. lmmunol. 8, 257 262 4 Hawes, C.S., Kemp, A.S. and Jones, W.R. (1980)J. Reprod. Immunol. 2, 37-44
1 Clinical applications of monoclonal antibodies Andrew J. McMichael and Judy M. Bastin Nuffield Department of Medicine, John Radclille Hospital, Headington, Oxford OX3 9DU, U . K
Monoclonal antibodies offer many distinct advantages over conventional antisera, the most obvious being their precise specificity for a single epitope on a single antigen and their potentially unlimited supply. The quantity and purity of available antibodies facilitates antigen purification by affinity chromatography. Monodonal antibodies can also be used to eharaclerize different parts of a macromolecule with regard to anligenicily, functional activity or genetic variability. IIere Andrew McMichael and Judy Bastin concentrate on lhe potential value of monoclonal antibodies in clinical medicine, surveying papers published up to June 1980, and a number of preprints and personal communications, kindly made available to them by colleagues. We hope in this brief review to demonstrate the range of antibodies becoming available tor clinical studies but we should add the caution that the number of papers describing monoclonal antibodies that are in preparation or in press possibly exceeds the number cited here. This field is moving very rapidly. The principle behind the generation of monoclonal antibodies is well known I and it is described here only in outline. Spleen cells from immune mice or rats are fused, using polyethylene glycol, with cells of a cultured myeloma cell line, such as P3-NS1/1Ag 4.1, which is resistant to 8-azaguanine. The hybrid cells are grown in selective medium containing h y p o x a n thine, aminopterin and thymidine (HAT). This part of the procedure presents little difficulty. Normally the cells are cultured in multiple wells of © Elsevier/North-Holland BiomedicalPiess 1980
plastic tissue culture plates. After 10-14 days the supernatants of each culture are assayed for antibody activity and those of interest identified. A rapid and effective means of screening large numbers of culture supernatants is essential at this stage. Culture wells are then selected for further study and these cells are cloned by limiting dilution on to a feeder layer of lymphoid cells, or in soft agar. Clones that secrete the antibody of interest are then selected and grown, either in bulk culture or as tumours in mice. Most of the difficulty arises just before the cloning stage where problems of overgrowth by 'uninteresting' clones and instability may arise. It is sometimes necessary to clone repeatedly and survey hundreds of culture wells to obtain one stable antibody-secreting clone. Monoctonal antibodies of potential clinical value
57
immunology to@, September 1980
are listed in Table I. W e have somewhat arbitrarily divided them into six categories, according to the antigen recognized. There are two main groups of interest to clinicians: antibodies to h u m a n antigens, an immunological dissection of the body, and antibodies to pathogens. In the first group are: differentiation antigens of the lymphohaemopoietic system; organ differentiation antigens; ubiquitous cell-surface antigens, which m a y be polymorphic; onco/foetal antigens; and antigenic determinants on secreted cell products and their receptors. The anti-pathogen antibodies recognize (so far) various virus or parasite antigens. Each type of antibody will be discussed separately with special reference to their possible use.
Differentiation antigens of the lympho-haemopoeitic system A large number of anti-lymphocyte monoclonal antibodies have been described. Subpopulations of h u m a n tymphocytes have been defined by the recognition of differentiation antigens that are expressed only on certain types of cell. Good examples are the monoclonal antibodies that are specific for thymocytes 2-4, peripheral T cells 5-7, T inducer cells 8-1~ and T suppressor/cytotoxic cells ~. The last two may recognize the h u m a n equivalents of the Lyt 1, 2 and 3 antigens which have proved so useful in defining mouse lymphocyte function. Not all antigens have equivalent distribution in different species, however. Thy-1, found on all mouse T lymphocytes, is confined to only a minor subpopulation of T cells found at the periphery of the thymus, the marginal zone of the spleen and post-capillary venules of the lymph node ~2. Certain antigens such as the 'leucocyte c o m m o n ' antigen ~3 may be quantitatively or even qualitatively different in their expression on different cell populations H. Thus, differentiation antigens do not have to be expressed in an all-or-none fashion. Quantification of indirect, fluorescent staining patterns on single cells by these antibodies, with the fluorescence-activated cell sorter (FACS), has been extremely useful in these analyses. The antibodies that define subpopulations of h u m a n lymphocytes have important clinical applications Is. One major use is the characterization of leukaemias 4,~,~7. At least seven diflerent patterns of reactivity with the anti-T-cell antibodies have been found for T-cell acute lymphatic leukaemias 4. Most of these have the surface antigen phenotypes of the more primitive thymocytes. A monoclonal antibody has been described ~8 which recognizes the antigen characteristic of the non-T, non-B blast cells of the common type of acute lymphatic leukaemia (and on a nqinor population of normal bone marrow cells). Classification of leukaemias with these reagents should help in planning more rational therapeutic strategies because different types of cell may respond to different drugs. In addition, antigens not found on normal
peripheral blood cells may be valuable in early detection of recurrence of leukaemia after induction of remission. Besides these diagnostic uses, the antibodies might be useful in treatment, it has been reported that growth in vitro of a mouse T-cell lymphoma could be inhibited by a monoclonal anti-Thy-1 antibody 19. Use of this type of antibody to treat a transplantable leukaemia in mice in vivo was successful if combined with administration of complement and surgical excision 2°. There have, to date, been no reports of the use of monoclonal antibodies to treat h u m a n leukaemia. Expected problems might be modulation of the antigen on the leukaemic cells, which would be associated with resistance, and immune reactions to the mouse immunoglobulin. The possibility of coupling drugs or toxins to the antibodies has not yet been explored, nor has the use of the antibody to destroy tumour cells before transplantation of autologous remission bone marrow. Some of the anti-T-cell antibodies could also be used to remove the cells responsible for the graftversus-host reaction from normal bone marrow before its transplantation to patients with leukaemia or aplastic anaemia 1°,~5. The anti-T-cell antibodies and those that recognize other leucocyte and monocyte antigens 7,1~,16,21 will be useful in determining whether perturbations of the relative concentrations of each population occur in certain disease states or during therapy. As T~/ cells have now been shown not to be T cells 22, the O K T 5 suppressor T-cell marker wilt be particularly usefu111,15. Similarly, the identification of the cell types in inflammatory lesions in tissue section has obvious potential. Two antibodies specific for platelets have been described. One recognizes glycoprotein I z3 and the other glycoprotein II/II124. These antibodies specifically fail to bind to the platelets of patients with Bernard Soulier syndrome or Glanzmanns thrombasthenia, which lack glycoprotein I and I I / I I I respectively. Thus monoclonal antibodies can identify polypeptides absent in rare genetic disorders. Furthermore, the antibodies block certain platelet functions and will allow a fuller understanding of the role of platelets in haemostasis and thrombosis. The gpI antigen recognized by monoctonal antibody is also present on megakaryocytes and on the blast cells of some chronic myeloid leukaemia patients in blast crisis 2~.
Differentiation antigens on other tissues Differentiation antigens on other organs are beginning to be recognized. Neurone-specific antibodies 7,12,26 should lead to greater understanding of neurone development and anatomy. Intracellular antigens can also be recognized. Morton et al. 27 raised monoclonal antibodies to Mallory bodies, cytoplasmic inclusions characteristic of alcoholic and other liver disease. One antibody is specific for, and therefore
58
diagnostic of, these inclusions. Another binds to Mallory bodies and to intermediate filaments of epithelial cells. This study illustrates the power of the technique in recognizing heterogeneity of intermediate filaments and their relationship with Mallory bodies.
Ubiquitous antigens Most of the antibodies generated when a mouse is immunized with human cells are directed towards 'species' determinants. Some of these may be useful. Thus anti-ABO reagents 28 and, more importantly, antibodies to the rarer blood groups may ultimately be used for blood grouping, although IgG monoclonal antibodies will not agglutinate red cells. Monoclonal antibodies that recognize common, monomorphic determinants on H L A antigens have been described28 32. These have been useful for purifying H L A antigens 33. Antibodies have also been found that detect the polymorphism of H L A A, B and C 3
56
also referred to previous studies in rodents by others in which there was activation of immune and reticuloendothelial systems during pregnancy. Such stimulation has, however, not been reported in man. It is i m p o r t a n t to note that migration is particularly vulnerable to unspecific stimulation during laboratory processing. In this study the cells were washed three times and the concentration of cells was low while in the previously cited studies, cells were washed only once1, 2 or not at alP. The reported enhancement of monocyte r a n d o m migration during pregnancy should preferably be confirmed by further investigation. In summary, data obtained from studies of humans imply that normal pregnancy is indeed associated with depressed inflammatory cell function and specific
K]
immune responsiveness. It is suggested that these effects are mediated by humoral factors which remain to be identified with certainty. BENGT BJ()P.KSTEN
Department of Biomedical Research, Pharmacia AB, Box 181, S-175 04 Uppsala 1, Sweden. References 1 Takeguchi, A. and Persellin, R.H. (1980)J. Clin. Lab. lmmunol. 3, 121-124 2 Persellin, R.H. and Thoi, L.L. (1979) Am. J. Obstet. Gynecol. 134, 250-254 3 Bj6rkst~n, B., S6derstr6m, T., Damber, M.-G., yon Schouhz, B. and Stigbrand, T. (1978) Seand.J. lmmunol. 8, 257 262 4 Hawes, C.S., Kemp, A.S. and Jones, W.R. (1980)J. Reprod. Immunol. 2, 37-44
1 Clinical applications of monoclonal antibodies Andrew J. McMichael and Judy M. Bastin Nuffield Department of Medicine, John Radclille Hospital, Headington, Oxford OX3 9DU, U . K
Monoclonal antibodies offer many distinct advantages over conventional antisera, the most obvious being their precise specificity for a single epitope on a single antigen and their potentially unlimited supply. The quantity and purity of available antibodies facilitates antigen purification by affinity chromatography. Monodonal antibodies can also be used to eharaclerize different parts of a macromolecule with regard to anligenicily, functional activity or genetic variability. IIere Andrew McMichael and Judy Bastin concentrate on lhe potential value of monoclonal antibodies in clinical medicine, surveying papers published up to June 1980, and a number of preprints and personal communications, kindly made available to them by colleagues. We hope in this brief review to demonstrate the range of antibodies becoming available tor clinical studies but we should add the caution that the number of papers describing monoclonal antibodies that are in preparation or in press possibly exceeds the number cited here. This field is moving very rapidly. The principle behind the generation of monoclonal antibodies is well known I and it is described here only in outline. Spleen cells from immune mice or rats are fused, using polyethylene glycol, with cells of a cultured myeloma cell line, such as P3-NS1/1Ag 4.1, which is resistant to 8-azaguanine. The hybrid cells are grown in selective medium containing h y p o x a n thine, aminopterin and thymidine (HAT). This part of the procedure presents little difficulty. Normally the cells are cultured in multiple wells of © Elsevier/North-Holland BiomedicalPiess 1980
plastic tissue culture plates. After 10-14 days the supernatants of each culture are assayed for antibody activity and those of interest identified. A rapid and effective means of screening large numbers of culture supernatants is essential at this stage. Culture wells are then selected for further study and these cells are cloned by limiting dilution on to a feeder layer of lymphoid cells, or in soft agar. Clones that secrete the antibody of interest are then selected and grown, either in bulk culture or as tumours in mice. Most of the difficulty arises just before the cloning stage where problems of overgrowth by 'uninteresting' clones and instability may arise. It is sometimes necessary to clone repeatedly and survey hundreds of culture wells to obtain one stable antibody-secreting clone. Monoctonal antibodies of potential clinical value
57
immunology to@, September 1980
are listed in Table I. W e have somewhat arbitrarily divided them into six categories, according to the antigen recognized. There are two main groups of interest to clinicians: antibodies to h u m a n antigens, an immunological dissection of the body, and antibodies to pathogens. In the first group are: differentiation antigens of the lymphohaemopoietic system; organ differentiation antigens; ubiquitous cell-surface antigens, which m a y be polymorphic; onco/foetal antigens; and antigenic determinants on secreted cell products and their receptors. The anti-pathogen antibodies recognize (so far) various virus or parasite antigens. Each type of antibody will be discussed separately with special reference to their possible use.
Differentiation antigens of the lympho-haemopoeitic system A large number of anti-lymphocyte monoclonal antibodies have been described. Subpopulations of h u m a n tymphocytes have been defined by the recognition of differentiation antigens that are expressed only on certain types of cell. Good examples are the monoclonal antibodies that are specific for thymocytes 2-4, peripheral T cells 5-7, T inducer cells 8-1~ and T suppressor/cytotoxic cells ~. The last two may recognize the h u m a n equivalents of the Lyt 1, 2 and 3 antigens which have proved so useful in defining mouse lymphocyte function. Not all antigens have equivalent distribution in different species, however. Thy-1, found on all mouse T lymphocytes, is confined to only a minor subpopulation of T cells found at the periphery of the thymus, the marginal zone of the spleen and post-capillary venules of the lymph node ~2. Certain antigens such as the 'leucocyte c o m m o n ' antigen ~3 may be quantitatively or even qualitatively different in their expression on different cell populations H. Thus, differentiation antigens do not have to be expressed in an all-or-none fashion. Quantification of indirect, fluorescent staining patterns on single cells by these antibodies, with the fluorescence-activated cell sorter (FACS), has been extremely useful in these analyses. The antibodies that define subpopulations of h u m a n lymphocytes have important clinical applications Is. One major use is the characterization of leukaemias 4,~,~7. At least seven diflerent patterns of reactivity with the anti-T-cell antibodies have been found for T-cell acute lymphatic leukaemias 4. Most of these have the surface antigen phenotypes of the more primitive thymocytes. A monoclonal antibody has been described ~8 which recognizes the antigen characteristic of the non-T, non-B blast cells of the common type of acute lymphatic leukaemia (and on a nqinor population of normal bone marrow cells). Classification of leukaemias with these reagents should help in planning more rational therapeutic strategies because different types of cell may respond to different drugs. In addition, antigens not found on normal
peripheral blood cells may be valuable in early detection of recurrence of leukaemia after induction of remission. Besides these diagnostic uses, the antibodies might be useful in treatment, it has been reported that growth in vitro of a mouse T-cell lymphoma could be inhibited by a monoclonal anti-Thy-1 antibody 19. Use of this type of antibody to treat a transplantable leukaemia in mice in vivo was successful if combined with administration of complement and surgical excision 2°. There have, to date, been no reports of the use of monoclonal antibodies to treat h u m a n leukaemia. Expected problems might be modulation of the antigen on the leukaemic cells, which would be associated with resistance, and immune reactions to the mouse immunoglobulin. The possibility of coupling drugs or toxins to the antibodies has not yet been explored, nor has the use of the antibody to destroy tumour cells before transplantation of autologous remission bone marrow. Some of the anti-T-cell antibodies could also be used to remove the cells responsible for the graftversus-host reaction from normal bone marrow before its transplantation to patients with leukaemia or aplastic anaemia 1°,~5. The anti-T-cell antibodies and those that recognize other leucocyte and monocyte antigens 7,1~,16,21 will be useful in determining whether perturbations of the relative concentrations of each population occur in certain disease states or during therapy. As T~/ cells have now been shown not to be T cells 22, the O K T 5 suppressor T-cell marker wilt be particularly usefu111,15. Similarly, the identification of the cell types in inflammatory lesions in tissue section has obvious potential. Two antibodies specific for platelets have been described. One recognizes glycoprotein I z3 and the other glycoprotein II/II124. These antibodies specifically fail to bind to the platelets of patients with Bernard Soulier syndrome or Glanzmanns thrombasthenia, which lack glycoprotein I and I I / I I I respectively. Thus monoclonal antibodies can identify polypeptides absent in rare genetic disorders. Furthermore, the antibodies block certain platelet functions and will allow a fuller understanding of the role of platelets in haemostasis and thrombosis. The gpI antigen recognized by monoctonal antibody is also present on megakaryocytes and on the blast cells of some chronic myeloid leukaemia patients in blast crisis 2~.
Differentiation antigens on other tissues Differentiation antigens on other organs are beginning to be recognized. Neurone-specific antibodies 7,12,26 should lead to greater understanding of neurone development and anatomy. Intracellular antigens can also be recognized. Morton et al. 27 raised monoclonal antibodies to Mallory bodies, cytoplasmic inclusions characteristic of alcoholic and other liver disease. One antibody is specific for, and therefore
58
diagnostic of, these inclusions. Another binds to Mallory bodies and to intermediate filaments of epithelial cells. This study illustrates the power of the technique in recognizing heterogeneity of intermediate filaments and their relationship with Mallory bodies.
Ubiquitous antigens Most of the antibodies generated when a mouse is immunized with human cells are directed towards 'species' determinants. Some of these may be useful. Thus anti-ABO reagents 28 and, more importantly, antibodies to the rarer blood groups may ultimately be used for blood grouping, although IgG monoclonal antibodies will not agglutinate red cells. Monoclonal antibodies that recognize common, monomorphic determinants on H L A antigens have been described28 32. These have been useful for purifying H L A antigens 33. Antibodies have also been found that detect the polymorphism of H L A A, B and C 3
