Effector functions of matched sets of recobinant ...

Final version edited 11th February 1993

Effector functions of matched sets of recombinant human IgG subclass antibodies

Judith Greenwood & Mike Clark Running title: Effector functions of human IgG

Cambridge University Department of Pathology Immunology Division Box 238 Addenbrooke’s Hospital Hills Road Cambridge CB2 2QQ Tel 0223 336900 Fax 0223 336901

Judith Greenwood & Mike Clark

Effector Functions of Human IgG

Introduction When monoclonal antibodies were first described by Kohler and Milstein in1975 [1] it was immediately recognised that they had great potential as powerful and specific therapeutic agents. In attempts to exploit that potential, large numbers of clinically-relevant monoclonal antibodies have been derived and many of these have been tested in-vivo. However, with a few notable exceptions such as the licensing of OKT3 for use in immunosuppression [2,3] the results of the great majority of clinical trials have been disappointing, and data on the effectiveness of the antibodies have been collected on a largely empirical basis. Because this initial "suck it and see" approach has been generally unsuccessful, a more considered and rational approach to the application of monoclonal antibodies in therapy is now required. The design of a therapeutic strategy requires consideration of a number of different factors including short- and long-term side-effects as well as benefits. Particular attention needs to be paid to deciding which therapeutic effect is most desirable and how it is expected that the antibody may produce that effect (reviewed by Waldmann [4]). For many forms of therapy such as in the treatment of malignancies, the requirement is that the abnormal target cells should be destroyed. Similarly, in the cases of immunosuppression to prevent organ graft rejection or to treat autoimmune disease, or of prevention of graft-versus-host disease in bone marrow transplantation one could envisage using antibodies to destroy the normal haemopoietic cells responsible for the damage [4]. Alternatively, in such cases, antibodies may be used to block the activity of cells temporarily, allowing useful cellular functions to return once tolerance has become established [4]. In other examples such as in the use of monoclonal antibodies to provide passive immunity to toxins, drugs and pathogens it is necessary that the antibody activates the appropriate effector mechanisms. In order to make better predictions of the therapeutic value of monoclonal

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antibodies it is necessary to understand how different antibodies interact with Fc receptors and complement to activate the cellular and humoral effector systems. Initial studies involved analysing the interactions of rodent monoclonal antibodies with human effector systems. Clearly rat immunoglobulin has evolved to work in-vivo in rats, and mouse immunoglobulin has evolved to work in-vivo in mice, and it should be remembered that the ability of these antibodies to activate human effector mechanisms is a result of the conservation of some of the key features. Conservation of effector functions between species is not absolute however, and this has muddied the interpretation of many of the studies in this area. Thus, traditionally, workers studying complement activation have often used heterologous systems, for example human target cells, rat or mouse monoclonal antibodies and rabbit or guinea pig complement. In such classical studies certain combinations of complement and target cells were found to be very effective with a large range of antibodies. In contrast, when experiments were carried out with human complement and human targets, mimicking more closely the therapeutic scenario, far fewer of the monoclonal antibodies gave good lysis [5]. This is now known to be due to the presence of a number of homologous complement restriction factors. These are cell surface molecules which act to restrict the toxicity of the animals own complement to the animals own cells and they include such molecules as "homologous restriction factor", 'decay accelerating factor, CD59 and 'membrane cofactor protein' (reviewed by Lachmann [6]). Hence, many monoclonal antibodies which appear to be potent mediators of complement lysis when measured in heterologous, in-vitro systems are completely ineffective in-vivo. Experiments using complement and target cells derived from the same donor revealed that some antigens were consistently poor targets for activation of complement regardless of the isotype of the antibody, whilst others were consistently good targets providing that the antibody had even a modest ability to bind complement [7]. An exceptionally good target for cell lysis is the human antigen CDw52 [8], recognised by the CAMPATH-1 family of

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antibodies. Experiments with this system will be described later in this Chapter. The Kohler and Milstein hybridoma technology facilitated the preparation of panels of monoclonal antibodies, mainly of rodent origin, for use in comparative studies [7,9]. However, the antibodies in such panels differed in terms of their fine specificities and avidities so it was difficult to reach absolute conclusions about their properties. In addition, it has always proved difficult to make large numbers of human antibodies by this route (in most early studies the human immunoglobulins were myeloma proteins). Advances in recombinant DNA technology were necessary before further progress could be made. The cloning of immunoglobulin genes was a particular turning point and the ability to manipulate the genes and then express them in transfected cells, has made a very valuable contribution to the field [10, 11, 12, 13, 14]. Antibody V-genes can be expressed with different constant region genes, permitting production of matched sets of antibodies of different isotypes but identical specificity and single-site affinity, allowing a direct comparison of properties [15, 16, 17]. There is no restriction on the species from which the V-regions and C-regions can be derived, so that antibodies may be chimeric (eg. mouse or rat variable regions with human constant regions) or purely from one species (eg. fully reshaped antibodies as described in greater detail in other chapters of this book). In this chapter we describe some of the information learned from studying two matched panels of recombinant antibody molecules [15, 18]. In the first panel all of the antibodies have specificity for a hapten molecule, 4-hydroxy3-nitrophenylacetyl (NP) [15]. This hapten can conveniently be coupled to carriers such as soluble proteins, cell membrane proteins or to membrane soluble lipid anchors. The antibodies can therefore be purified on affinity columns made from the antigen, they can be quantified and compared in enzyme linked immunoadsorption assays and they can be targeted to hapten modified cells. Reviewed elsewhere [19] and this volume (Morrison et al., Chapter 6) are studies using a different matched set of antibodies to the

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hapten dansyl. The second matched set of antibodies discussed here [18] is to a human cell surface antigen, CAMPATH-1 (CDw52) which is expressed on human lymphocytes and monocytes [8, 20]. The CAMPATH-1 antigen was first investigated because rat monoclonal antibodies of this specificity were extremely cytotoxic to human lymphocytes in the presence of autologous complement [21]. It has become a very important specificity for a number of clinical investigations [22, 23, 24, 25, 26, 27]. It is hoped that much of the information gained from these two panels of antibodies will be widely applicable and will help in directing the general use of monoclonal antibodies in therapy as well as extending our understanding of the normal functions of antibodies.

Effector functions of anti-NP antibodies A complete matched set of monoclonal antibodies consisting of the human isotypes IgM, IgG1(allotype G1m(za), IgG2, IgG3 (allotypes G3m(b) and G3m(g)), IgG4, IgA2 and IgE was constructed with specificity for the hapten NP and its derivative NIP (5-iodo-4-hydroxy-3-nitrophenylacetyl) [15]. The antibodies were affinity purified on hapten columns. The hapten, available as either a lipid soluble molecule NIP-kephalin, or as a protein modifier NIPsuccinimide ester, was used to label cell surfaces. In this way the antibodies could be assayed for activation of effector mechanisms leading to cell killing. The antibodies were tested for their abilities to mediate autologous complement-dependent lysis of human red blood cells labelled with the NIPkephalin derivative and for antibody dependent cell-mediated cytotoxicity (ADCC) by activated human mononuclear cells of a human lymphoblastoid cell line coupled with NIP-succinimide ester. The results from these experiments are summarised in Figure 1, shown as the results relative to the IgG1 isotype, and have been adapted from Bruggemann et al [15]. The IgG1 antibody proved to be the most effective in both complement-dependent

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and cell-mediated cytotoxicity. The concentration of IgG1 antibody needed for 50% of maximal killing was approximately 0.1µg/ml for complement lysis and for ADCC. The effector cells in ADCC were inhibited by a CD16 (FcgRIII) monoclonal antibody and had the phenotype of killer-cells (K-cells). As expected, the IgM antibody proved to be good at complement activation but poor in ADCC. Surprisingly the two IgG3 antibodies were not quite as good as the IgG1 antibody in either assay. For complement, this was despite the fact that IgG3 was shown to be fixing many more molecules of C1q. Bindon et al [28] later went on to show that although human IgG1 bound less C1 than human IgG3 there was a much more efficient deposition of C4b on the cell surface which accounted for the more effective cell lysis by IgG1. None of the other isotypes IgG2, IgG4, IgA2 and IgE showed any significant functional activity in these assays. The cell lines making this matched panel of antibodies have been widely distributed and are available from the European Collection of Animal Cell Cultures (Division of Biologics, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, SP4 0JG,UK), and purified antibody is commercially available. Other groups making use of this set of antibodies have extended the original observations. Two groups have investigated the effect of varying the antigen density as well as the epitope patchiness for complement lysis triggered by the NP chimaerics [29, 30]. The IgG1 antibody was most effective when the antigen concentration was higher, and whereas the IgG3 antibody was relatively better at lower concentrations. The IgG2 antibody gave good lysis at very high concentrations of antigen but the IgG4 antibody did not give lysis under any conditions [29]. It was also shown that IgG1 and IgG3, along with IgM activated the classical pathway of complement but not the alternative pathway [30]. At high antigen concentration the IgG2 antibody could also activate the classical pathway but the IgG4 and IgA2 antibodies could not [30]. However the IgA2 antibody, and to a lesser extent the IgG2 antibody, did activate the alternative pathway of complement [30].

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Effector functions of IgG antibodies to CAMPATH-1 (CDw52) antigen The matched sets of antibodies to CAMPATH-1 (CDw52) antigen fall into two subgroups, a subgroup of chimaerics with the original rat VH region and and the rat k-1b light chain and a second set which uses a reshaped fullyhumanised VH region and a reshaped fully-humanised VL region on a human k light chain [18 , 31]. The chimeric antibodies were essentially the human correlates of the original rat IgG2b antibody CAMPATH-1G [32] and were used to decide which human isotype seemed the most suitable for therapy. This could be assessed by comparing the performance of the chimaerics in in-vitro assays with that of CAMPATH-1G, the specificity and affinity of the antibodies being identical in all cases [18]. For in-vivo use it was necessary to minimise the possibilities of an antiglobulin response to the rodent V-region, therefore the complimentarity regions were grafted onto human frameworks (see chapter 2 this volume). The antibodies with the fully reshaped V-regions have a slightly lower affinity than the original chimeric antibodies [18] and so this needs to be considered with the proper controls when interpreting the experimental data. The results from the comparison of the chimeric antibodies are given in Figure 2. Targets for these experiments were human peripheral blood mononuclear cells. The ADCC effector cells were these same mononuclear cells but after activation with a mitogenic CD3 antibody and expansion in 40 units per ml of recombinant human Interleukin-2. These expanded cells contain a low percentage of CD16 positive cells and are extremely potent as effectors [15, 18, 31]. The IgG1 antibody was found to be the most effective antibody in both complement-mediated lysis and ADCC. The IgG3 antibody was also active in both assays whilst the IgG2 antibody had a lower titre in complementmediated lysis and gave very little ADCC. The IgG4 antibody did not work in

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either system. Comparing these results with the NIP chimaerics reveals a large degree of similarity except that the IgG2 antibody to CAMPATH-1 is more active in complement lysis. Following the studies of others using the NIP system [29, 30] this observation may be due to different antigen densities, or clustering of antigen, the CAMPATH-1 antigen being equivalent to the NIP antigen at high concentrations. The human IgG1 isotype appeared to be the most similar to rat IgG2b and the reshaped version of that isotype, CAMPATH-1H [18, 32], was chosen for therapeutic evaluation and comparison to the previously used CAMPATH-1G [24, 25]. The antibody has now been used in a number of clinical settings and has proved to be very effective at depleting cells [22, 26, 27]. The antibody is currently being evaluated in therapeutic trials in the USA and Europe by the Wellcome Foundation. More recent experiments have made the interpretation of the ADCC experiments more complex [31]. Repeating the experiments with a larger panel of donors of effectors and targets demonstrated that there were differences in the pattern of ADCC seen with different individuals. Thus some people (3 out of 8) showed the originally described pattern where only the IgG1 gave good levels of ADCC. For other individuals there were significant levels of ADCC seen with all four isotypes of IgG, including IgG4. This variation has been found to be consistent with the source of effectors and is not a difference in target susceptibility [31]. This represents an intriguing polymorphism and also predicts that unexpected results might be obtained with some human isotypes such as IgG2, and particularly IgG4, when used therapeutically in some individuals. It has often been assumed that IgG4 binds only poorly to FcgRI and does not mediate ADCC and, therefore, that it would be a good choice for non-depleting antibodies used, for example, in imaging with radioisotopes (Reviewed by Adair [33]).

Functional epitopes of human IgG1 and IgG4 The four human IgG subclasses are remarkably similar in sequence and differ

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significantly only in the hinge exons ( Reviewed by Burton et al [34] and by Jefferis [35]). However, they exhibit major differences in their effector functions (as described above). By comparing effector functions and sequences it is possible to speculate as to which of the sequence differences are responsible for the observed functional differences [34, 35]. Using recombinant DNA techniques, including site-directed mutagenesis and exon shuffling, these speculations may be tested experimentally. Several groups have carried out such experiments using different combinations of antibodies with either the anti-dansyl specificity (Reviewed by Shin et al [19] and also by Morrison et al Chapter 6) or the anti-NP specificity described above and also elsewhere in this volume (Lund & Jefferis Chapter 7). These studies have identified some of the structural features which determine the different antibody effector functions. Although the hinges of the four human IgG subclasses are quite different in sequence, early speculation that this might account for the large functional differences was not upheld experimentally. Thus in experiments with antibodies specific for dansyl, the hinge regions of IgG3 and IgG4 could be exchanged without significantly affecting complement activation, i.e. the activity of IgG3 with an IgG4 hinge was only slightly reduced, and IgG4 with an IgG3 hinge still did not activate complement [36]. In another series of experiments with antibodies specific for NP, the long internally repeated hinge of IgG3 was reduced in length by sequential deletion of the repeated exons. In this case the activation of complement increased at shorter hinge lengths [37]. Further studies have indicated that the CH2 domain, including the lower hinge region, contains the sequences responsible for differences in binding to Fc receptors [38, 39, 40, 41, 42], and to complement [43, 44]. These studies are reviewed in Chapters 6 and 7 this volume. The CAMPATH-1 (CDw52) specificity represents an ideal natural target antigen for investigating the structural requirements within antibodies for recruitment and activation of autologous effector functions (in this case for the destruction of human lymphocytes and lymphoid tumours). The

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activation of such autologous effector functions can be conveniently measured in-vitro [18] and at least for the rat IgG isotypes there has been a correlation between the ability to activate in-vitro effector functions such as ADCC and the in-vivo effectiveness of these antibodies [ 24, 25, 32]. Thus results from studies in this system are of direct relevance to an understanding of how antibodies work in-vivo. Wild-type IgG1 with CAMPATH-1 specificity works well in both complement mediated lysis and ADCC [18,31] whereas IgG4 does not work in complement mediated lysis and only works in ADCC with some donors [31]. By exploiting a combination of naturally occurring and specially engineered restriction sites a series of domain swap mutants between these antibodies was constructed and tested [31]. The results are summarised in Figure 3. Initial swapping of the exons indicated that despite earlier speculation on segmental flexibility, the hinge region was not a major contributory factor to the observed functional differences in IgG1 and IgG4. The major differences seemed to correlate with the CH2 domain of the antibody. A convenient conserved restriction site within the CH2 domains of g1 and g4 allowed the Nterminal and C-terminal halves of the domain to be swapped independently. The functional differences in both complement and ADCC correlated with the C-terminal half of the domain. There are only four residues which differ in this region between IgG1 (Tyr 296; Ala 327; Ala 330; Pro 331) and IgG4 (Phe 296; Gly 327; Ser 330; Ser 331). Of significance perhaps is the Pro/Ser 331 change which, in the X-ray crystallographic structures of IgG1 Fc, occurs at the elbow of a loop just adjacent to the hinge region [45]. Perhaps there is an overall distortion of the structure as a result of this change which affects other epitopes involved in interactions with complement and with Fcg receptors. Unfortunately there are only a limited number of solved structures for human IgG and they are all of the IgG1 subclass. Models of IgG2, 3 or 4 would, of course, be of tremendous value in predicting how the amino acid differences might affect interactions with other molecules.

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Immunoglobulin Allotypes So far considerable attention has been paid to the engineering of the immunoglobulin isotype but very little to problems of immunoglobulin allotype. A number of allelic forms of human immunoglobulins have been identified [46] (reviewed by van Loghem [47]). The epitopes have been mapped to amino acid substitutions for some but not all of these differences. Of interest is the classification of markers into 'allotypes' and 'isoallotypes' [46, 47]. These are distinguished on different serological bases dependent upon the strong sequence homologies between isotypes. Allotypes are sequence differences between alleles of a subclass whereby the antisera recognises only the allelic differences. An isoallotype is an allele in one isotype which produces an epitope which is shared with a non-polymorphic homologous region of one or more other isotypes and because of this the antisera will react with both the relevant allotypes and the relevant homologous isotypes. There are several reasons to be concerned about immunoglobulin allotypes with regard to the engineering of antibodies for therapy. Firstly, when a patient has a different immunoglobulin allotype from the therapeutic monoclonal then there is an increased potential for an antiglobulin response, directed to both the constant region and the variable region (discussed in more detail in chapter 2). The antiglobulin response to allotypes is the major concern here - an anti-isoallotype response is less likely to occur because of the cross reactivity with the patients own immunoglobulins of different isotype. One way to avoid this problem would be to create a panel of matched therapeutic antibodies with the different allotypes and then to match the therapeutic antibody to the patient. Commercially this approach is unfeasable because of the complications in manufacture and testing of several similar products each with a restricted application. An alternative approach is to select the most common allotype for general use. Unfortunately, the frequency of different alleles varies between different racial groups [46, 47]. A possible solution to this is to exploit the idea of isoallotypes by artificially creating a new allele, consisting entirely of

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isoallotypes [48]. An example of such an allele is described in Figure 4. This antibody has been constructed with the CAMPATH-1 specificity and does not react with the tested anti-G1m allotypic antisera whilst still retaining its effector function activity in-vitro. A clinical trial of this antibody will be necessary to establish whether most patients will fail to a make an antiglobulin response to the constant region. Another question concerning allotypes relates to polymorphisms affecting the function of the antibody. At present there is very little evidence of gross differences in effector functions of human IgG allotypes when compared to the big differences seen between subclasses, for example IgG1 versus IgG4. However a detailed comparison between matched sets of allotypes has not yet been carried out. In the NP specific series of antibodies described above, two different alleles of IgG3 were compared and there were differences in titres in complement activation and lysis [15, 28], see Figure 1. In a recent set of experiments a matched set of IgG1 allotype antibodies with specificity for CAMPATH-1H have been compared in complement mediated lysis and no significant differences were seen for the "wild type" forms [48] (see Figures 4 and 5). However when the allotype differences were combined with a Asn/ Ala 297 mutation which results in an aglycosyl form of the antibody then a difference was observed (see Figures 4 & 5). The carbohydrate on antibodies has been shown to be important for most effector functions [34, 35, 49, 50] and aglycosyl antibodies may have a therapeutic use in situations where activation of effector functions need to be minimised [51]. It is interesting to speculate on the functional differences seen between these two aglycosyl mutants in Figure 5 since the only structural differences are in the allotypic residues in the CH1 and CH3 region (see Figure 4) and do not involve any of the residues from the CH2 and hinge region discussed above. Studies on an aglycosyl mutant of IgG3 specific for NP have also been described and in this case there was also some residual activity in complement activation observed [50]. The IgG3 isotype has an Arg at position 214 which is the same as the allotype G1m(3) and different to the Lys found at this position in the allotype G1m(17).

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Allotype differences between individuals have also been implicated in disease associations [52] although the linkage is often very weak, suggesting either that the allotype is only a minor contributing factor or that it is in linkage disequilibrium with a much more important locus, possibly even the immunoglobulin V-gene segments. Polymorphisms of the immunoglobulin genes are further complicated by polymorphisms in the Fc receptors (reviewed by Ravetch & Anderson [53]) and complement components (reviewed by Campbell et al [54]) with which they interact and so it is not yet possible to make clear statements about the role of any given antibody.

Animal models Whilst it is possible to analyse the interaction of antibodies with effector functions in in-vitro systems it is still very difficult to understand why some antibodies are effective in-vivo and others are not. Unfortunately, in many circumstances attempts to correlate the human in-vitro and in-vivo data are neither practical nor ethical. As a result the information gained about a set of antibodies, such as the CAMPATH-1 antibodies, is often incomplete or contradictory. For example, the original rat IgM antibody CAMPATH-1M (clone YTH66.9) [21] was exceptionally good at lysing cells with autologous complement in-vitro and found use in the removal of lymphocytes from bone marrow prior to allografting [23]. However despite being very efficient in activating complement in-vivo, the antibody proved very poor in lysing lymphoid leukaemia cells [24, 25]. Similarly a rat IgG2a antibody with similar specificity (Clone YTH34.5) was good at fixing complement in-vivo but failed to clear significant numbers of cells [24, 25]. However, a class-switch variant of YTH34.5 from IgG2a to rat IgG2b (CAMPATH-1G)[32] proved very effective in the in-vivo treatment of lymphoid leukaemia [24, 25]. The in-vitro correlate is that rat IgG2b seems to be better than rat IgM or IgG2a at interacting with Fc receptors and triggering ADCC [9 ,32], and that complement activation alone cannot account for in-vivo effectiveness. This does not help in our understanding of what facet of ADCC, or which Fc receptors on which cells,

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or what other effector mechanisms, including complement might contribute to cell killing by antibody in-vivo [4, 33, 34, 35, 53, 54]. In fact it is not even certain that in-vitro assays such as ADCC represent any in-vivo mechanism for cell-killing. Animal models might help in our understanding of the requirements for cell killing through antibody providing that the data is interpreted cautiously. For example it has been observed that some (but not all) monoclonal antibodies to some mouse cell surface antigens are able to effectively deplete those cell populations in-vivo. The depletion appears to be dependant upon both the antibody specificity and on the antibody isotype and therefore this closely resembles the human situation. Similarly the mouse possesses a complement system and a series of Fc receptors expressed on homologous populations of effector cells. To a certain degree these effector systems and immunoglobulin molecules interact with and share conserved structural features with the human systems [53, 54]. That the conservation is not complete is supported by comparisons of the known sequences and by the functional differences which account for phenomena such as homologous restriction of complement activation [6] and for the isotype specificity of binding to Fcg receptors [53]. The important correlation is that different immunoglobulin isotypes interact with different effector mechanisms in a given species [34, 35, 53, 54]. For example mouse IgG3 binds very well to human FcgRI but only poorly to the mouse equivalent whereas mouse IgG2b binds poorly to human FcgRI and well to the mouse equivalent [53]. By observing which effector mechanisms operate in-vivo in the mouse it should be possible to derive a set of rules applicable to most species. In a recent series of experiments a matched set of recombinant antibodies to the mouse CD8 antigen was constructed [55]. All four human IgG subclasses as well as rat IgG2b but not other human isotypes seemed to work effectively in depleting cells in-vivo [55]. The next step was to introduce mutations into the antibodies which would interfere with their abilities to activate the murine effector systems. Thus an aglycosyl mutant of the IgG1 antibody was no

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longer able to deplete cells even though it coated cells in-vivo and remained for a reasonable period of time. As discussed above aglycosyl IgG is unable to interact effectively with several effector systems [34, 35, 49, 50]. Further work in this model using mutations which only interfere with one effector system should help resolve which functions are critical.

Conclusions The studies outlined above show that the choice of isotype is critical to the effectiveness of the antibody at recruiting natural effector functions. However the importance of this should not be over estimated as the nature of the target antigen seems to play an equally important role [7]. Thus when designing a therapeutic strategy it seems likely that the correct combination of target specificity and isotype will produce the optimum results. For depletion of cells in-vivo in man it would seem that the IgG1 isotype is suitable, particularly when combined with specificities such as CAMPATH-1 [15, 18]. However, the in-vitro observation that the other IgG isotypes are also effective with some individuals [31], leads one to speculate that variable results will be seen in-vivo with many antibodies. It may turn out that artificial mutants containing the best features of several isotypes, for example the shortened hinge variants of IgG3 [37], will be the best choice for a range of antigens. For some applications it may be necessary to use a non-depleting isotype which merely targets the antigen or blocks its function [4, 33] . Whilst at one time IgG4 might have been considered for this role, the observations that, at least in some individuals, IgG4 can be as effective as IgG1 in ADCC may preclude its use [31]. An alternative is to use mutants which have had their functions destroyed and perhaps a suitable example is the aglycosyl variant. The aglycosyl CAMPATH-1 IgG1 allotype G1m(1,17) shown in Figure 5 does not cause complement mediated lysis and in the in-vivo model system in the mouse the aglycosyl IgG1 did not deplete the cells [55]. Similarly an aglycosyl variant of a humanised CD3 antibody no longer induced mitogenic responses from human T-cells in-vitro [51].

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What is clear from these studies is that although we have identified some of the important features of antibodies there is still a great deal upon which we have to speculate. However, advances in current technology are allowing a new approach to many questions and we should hopefully find out much more in the near future.

Acknowledgements We would like to thank our colleagues Herman Waldmann, John Isaacs and Steve Cobbold for helpful discussions. This work was supported by grants from the UK Medical Research Council and the Wellcome Trust and also with additional support from the Wellcome Foundation. CAMPATH is a registered trademark of the Wellcome Foundation.

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References 1. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-497. 2. Ortho multicentre transplant study group. A randomized clinical trial of OKT3 monoclonal antibody for acute rejection of cadaveric renal transplants. New Engl J Med 1985; 313: 337-342. 3. Goldstein G. An overview of Orthoclone OKT3. Transplant Proc 1986; 18: 927-930. 4. Waldmann H. Manipulation of T-cell responses with monoclonal antibodies. Ann Rev Immunol 1989; 7: 407-444 5. Clark M, Cobbold S, Hale G, Waldmann H. Advantages of rat monoclonal antibodies. Immunol Today 1983; 4: 100-101. 6. Lachmann PJ. The control of homologous lysis. Immunol Today 1991; 12: 312-315. 7. Bindon CI, Hale G, Waldmann H. Importance of antigen specificity for complement mediated lysis by monoclonal antibodies. Eur J Immunol 1988; 18: 1507-1514. 8. Hale G, Xia M, Tighe HP, et al. The CAMPATH-1 antigen (CDw52). Tissue Antigens 1990; 35: 118-127. 9. Hale G, Clark M, Waldmann H. Therapeutic potential of rat monoclonal antibodies: Isotype specificity of antibody-dependant cell-mediated cytotoxicity with human lymphocytes. J Immunol 1985; 134: 3056-3061. 10. Boulianne GL, Hozumi N, Schulman MJ. Production of functional chimaeric mouse/human antibody region domains. Nature 1984; 312: 643-

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646. 11. Morrison SL, Johnson MJ, Herzenberg LA, et al. Chimeric human antibody molecules: Mouse antigen-binding domains with human constant Proc Natl Acad Sci USA 1984; 81:6851-6855. 12. Morrison SL, Oi VT. Transfer and expression of immunoglobulin genes. Ann Rev Immunol 1984; 2: 239-256. 13. Neuberger MS, Williams GT, Fox RO. Recombinant antibodies possessing novel effector functions. Nature 1984; 312: 604-608. 14. Neuberger MS, Williams GT, Mitchell EB, et al. A hapten-specific chimeric immunoglobulin E antibody which exhibits human physiological effector function. Nature 1985; 314: 268-271. 15. Bruggemann M, Williams GT, Bindon CI, et al. Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies. J Exp Med 1987; 166: 1351-136. 16. Shaw DR, Khazaeli MB, LoBuglio AF Mouse/human chimeric antibodies to a tumour-associated antigen: biological activity of the four human IgG subclasses. J Natl Cancer Inst 1988; 80: 1553-1559. 17. Steplewski Z, Sun LK, Shearman CW, et al. Biological activity of humanmouse IgG1, IgG2, IgG3, and IgG4 chimeric monoclonal antibodies with antitumor specificity. Proc Natl Acad Sci USA 1988; 85: 4852-4856. 18. Riechmann L, Clark MR, Waldmann H, Winter G. Reshaping human antibodies for therapy. Nature 1988; 332: 323-327. 19. Shin S, Wright A, Bonagura V, et al. Genetically-engineered antibodies: tools for the study of diverse properties of the antibody molecule. Immunol Rev 1992; 130: 87-107.

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20. Xia M, Tone M, Packman L, et al. Characterization of the CAMPATH-1 (CDw52) antigen: biochemical analysis and cDNA cloning reveal an unusually small peptide backbone. Eur J Immunol 1991; 21: 1677-1684. 21. Hale G, Bright S, Chumbley G, et al. Removal of T cells from bone marrow for transplantation: a monoclonal antilymphocyte antibody that fixes human complement. Blood 1983; 62: 873-882. 22. Hale G, Dyer MJS, Clark MR, et al. Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H. Lancet 1988; 2: 1394-1399. 23. Hale G, Waldmann H, for CAMPATH users. CAMPATH-1 for prevention of graft-versus-host disease and graft rejection. Summary of results from a multicentre study. Bone Marrow Transplantation 1988; 3 (suppl 1): 11-14. 24. Dyer MJS, Hale G, Hayhoe FGJ, Waldmann H. Effects of CAMPATH-1 antibodies in-vivo in patients with lymphoid malignancies: influence of antibody isotype. Blood 1989; 73: 1431-1439. 25. Dyer MJS, Hale G, Marcus R, et al. Remission induction in patients with lymphoid malignancies using unconjugated CAMPATH-1 monoclonal antibodies. Leukaemia and Lymphoma 1990; 2: 179-193 26. Mathieson PW, Cobbold SP, Hale G, et al. Monoclonal antibody therapy in systemic vasculitis. New Engl J Med 1990; 323: 250-254. 27. Isaacs JD, Watts RA, Hazleman BL, et al. Humanised monoclonal antibody therapy for rheumatoid arthritis. The Lancet 1992; 340: 748-752. 28. Bindon CI, Hale G, Bruggemann M, Waldmann H. Human monoclonal IgG isotypes differ in complement activating function at the level of C4 as well as C1q. J Exp Med 1988; 168: 127-42.

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29. Michaelsen TE, Garred P, Aase A. Human IgG subclass pattern of inducing complement-mediated cytolysis depends on antigen concentration and to a lesser extent on epitope patchiness, antibody affinity and complement concentration. Eur J Immunol 1991; 21: 11-16. 30. Valim YML, Lachmann PJ. The effect of antibody isotype and antigenic epitope density on the complement-fixing activity of immune complexes: a systematic study using chimaeric anti-NIP antibodies with human Fc regions. Clin Exp Immunol 1991; 84: 1-8. 31. Greenwood J, Clark M, Waldmann H. Structural motifs involved in human IgG antibody effector functions Eur J Immunol 1993; in press. 32. Hale G, Cobbold SP, Waldmann H, et al. Isolation of low-frequency classswitch variants from rat hybrid myelomas. J Immunol Meth 1987; 103: 59-67. 33. Adair JR. Engineering antibodies for therapy. Immunol rev 1992; 130: 5-40. 34. Burton DR, Gregory L, Jefferis R. Aspects of the molecular Structure of IgG Subclasses. Monogr Allergy 1986; 19: 7-35 35. Jefferis R. Molecular structure of human IgG subclasses. In: Shakib F, ed. The Human IgG subclasses, Pergamon press. 1990: 15-30. 36. Tan LK, Shopes RJ, Oi VT, et al. Influence of the hinge region on complement activation , C1q binding, and segmental flexibility in human immunoglobulins. Proc Natl Acad Sci USA 1990; 87: 162-166. 37. Michaelsen TE, Aase A, Westby C, Sandlie I. Enhancement of complement activation and cytolysis of human IgG3 by deletion of hinge exons. Scand J Immunol 1990; 32: 517-528. 38. Duncan AR, Woof JM, Partridge LJ, et al. Localization of the binding site for the human high-affinity Fc receptor on IgG. Nature 1988; 332: 563-564.

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Effector Functions of Human IgG

39. Jefferis R, Lund J, Pound J. Molecular definition of interaction sites on human IgG for Fc receptors (huFcgR). Molec Immunol 1990; 27: 1237-1240 40. Canfield SM, Morrison SL. The binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated by the hinge region. J Exp Med 1991; 173: 1483-1491. 41. Chappel MS, Isenman DE, Everett M, et al. Identification of the Fcg receptor class I binding site in human IgG through the use of recombinant IgG1/IgG2 hybrid and point mutated antibodies. Proc Natl Acad USA 1991; 88: 9036-9040. 42. Sarmay G, Lund J, Rozsnyay Z, et al. Mapping and comparison of the interaction sites on the Fc region of IgG responsible for the triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fcg receptor Molec Immunol. 1992; 29: 633-639. 43. Duncan AR, Winter G. The binding site for C1q on antibodies. Nature 1988; 332: 738-740. 44. Tao M, Canfield SM, Morrison SL.The differential ability of human IgG1 and IgG4 to activate complement is determined by the COOH-terminal sequence of the the CH2 domain J Exp Med 1991;173: 1025-1028. 45. Diesenhofer J. Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from staphylococcus aureas at 2.9 and 2.8 angstroms resolution. Biochem 1981; 20 : 2361-2370. 46. WHO Review of the notation for the allotypic and related markers of human immunoglobulins. J Immunogen 1976; 3: 357-362. 47. Loghem E van. Allotypic markers. Monogr Allergy 1986; 19: 40-51.

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48. Gorman SD, Clark MR. Humanisation of monoclonal antibodies for therapy. Sem Immunol 1990; 2: 457-466. 49. Tao M, Morrison SL. Studies of aglycoyslated chimeric mouse human IgG: Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region J Immunol 1989; 143: 2595-2601. 50. Lund J, Tanaka T, Takahashi N, et al. A protein structural change in aglycosylated IgG3 correlates with loss of huFcgRI and huFcgRIII and/or activation. Mol Immunol 1990; 27: 1145-1153. 51. Bolt S, Routledge E, Lloyd I, et al. The generation of a humanised, nonmitogenic CD3 monoclonal antibody which retains in-vitro immunosuppressive properties. Eur J Immunol 1993; 23: 403-411. 52. Whittingham S, Propert DN. Gm and Km Allotypes, Immune response and disease susceptibility. Monogr Allergy 1986; 19: 52-70. 53. Ravetch JV, Anderson CL. FcgR Family: Proteins, Transcripts, and Genes. In: Metzler H, ed. Fc Receptors and the action of antibodies, American Society for Microbiology, Washington, USA 1991: 211-235. 54. Campbell RD, Law SKA, Reid KBM, et al. Structure, organization, and regulation of the complement genes. Ann Rev Immunol 1988; 6: 161-195. 55. Isaacs JD, Clark MR, Greenwood J Waldmann, H. Therapy with monoclonal antibodies - an in-vivo model for the assessment of therapeutic potential. J Immunol 1992; 148: 3062-3071.

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Effector Functions of Human IgG

Figure Legends Figure 1. Effector functions of anti-NP chimaeric antibodies. A matched set of human chimaeric antibodies specific for the hapten NP was constructed and assayed for their ability to lyse haptenated target cells. The results summarise the findings for autologous complement mediated lysis of human group O red blood cells (left panel) and for lysis of haptenated lymphoblastoid cells by activated human peripheral blood mononuclear cells containing CD16 positive Killer cells (right panel). A consistent finding of these studies is the observation that the IgG1 isotype has been the most active of the IgG subclasses in the effector functions measured. The results presented in this chapter have therefore been summarised by giving them relative to the activity of IgG1, where the activity of IgG1 equals 1. Data adapted from Bruggemann et al [15].

Figure 2. Effector functions of CAMPATH-1 (CDw52) chimaeric antibodies. CAMPATH-1(CDw52) chimaeric antibodies were constructed from the four human IgG subclasses and were assayed for their abilities to lyse human lymphocyte targets by autologous complement or by antibody dependent cell-mediated cytotoxicity using activated autologous effector cells. All results are shown relative to the best IgG isotype human IgG1. Data adapted from Riechmann et al [18].

Figure 3. Identification of functional features of IgG by domain swapping. Starting from "wild type" IgG1 and IgG4 constant region genes various "swap" mutants were constructed using natural and introduced restrictions sites within the two highly conserved genes. The constructs are indicated schematically in the right panel of the figure using black to denote g1 derived and grey g4 derived gene segments. These constructs were then expressed as

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Effector Functions of Human IgG

CAMPATH-1 (CDw52) antibodies using the fully reshaped V-genes [18, 31]. These antibodies were then compared in complement mediated lysis and ADCC. The results are shown relative to the "wild type" IgG1 antibody (Data Greenwood et al [31]).

Figure 4. IgG1 allotypes Starting from a gene for the human IgG1 allotype G1m(1,17) site directed mutagenesis was used to create alternative allotypes by changing the key residues indicated [48]. Thus a change of Lys (214) to Arg (214) changes allotype 17 to 3 and a change of Asp (356), Leu (358) to Glu (356), Met (358) changes the allotype 1 to the isoallotype non-1 which is homologous to IgG2 in this region. Using the isoallotype logic a novel "null allotype" was constructed by mutating residue 214 to a Thr which is again homologous to IgG2 [48]. Also indicated is the conserved N-linked glycosylation site Asn (297) which was mutated to an Ala to produce aglycosyl variants of the antibodies (M.R. Clark unpublished).

Figure 5 Comparison of IgG1 allotypes. The allotypes described in Figure 4, G1m(1,17), G1m(3) and the "null mutant", along with the the aglycosyl mutants of the G1m(1,17) and G1m(3) were expressed along with the CAMPATH-1 (CDw52) fully reshaped variable regions [18, 48] and were then compared for their abilities to lyse human lymphocytes with autologous complement. The results are given relative to the "wild-type" IgG1 G1m(1,17). (Data M.R. Clark unpublished).

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