BSI Birmingham, March 1995
The following text and graphics provides information based on my presentation entitled "Clinical use and
in-vitro evaluation of humanised CD52 antibodies" within the session chaired by Prof. R. Jefferis on
"Protective effector functions mediated by humoral factors". You are welcome to use and to refer to the
images for teaching and research but please remember that they remain copyright of Mike Clark. If you do
find the information of use or if you would like to make comments I would be happy to hear from you by
A major question concerning the in-vivo effectiveness of antibodies directed to cell surface antigens is why
some appear to be able to deplete the target cells whereas others are not. Antibodies to the CD52 antigen
present on human lymphocytes have been shown to be highly effective both in-vivo and in-vitro at killing
human targets and this therefor provides a good system for study (Reviewed Greenwood & Clark
1993). However for some applications it may be desirable to have antibodies which bind but do not activate
effector functions eg in the blockade of T-cell receptor molecules for immunosuppression or tolerance
induction (Bolt et al 1993). Studies carried out with conventional and recombinant monoclonal
antibodies have provided some but not all of the answers.
Studies with chimaeric antibodies to the hapten NP (Bruggemann et al 1987) and also to the CD52
antigen (Riechmann et al 1987) have shown that the human IgG1 (allotype G1m(1,17)) is very
effective in killing human targets in-vitro by complement lysis or by ADCC through activated K-cell
Using chimaeric rat antibodies to the hapten NP (Bruggemann et al 1989) it was shown that the rat
IgG2b was also effective in both complement lysis and ADCC but that the rat IgG2a was only effective in
complement lysis. In-vivo in the treatment of lymphoma using antibodies directed to CD52 the rat IgG2a
was found to be much less effective than the rat IgG2b (Dyer et al 1989).
Humanisation of the rat IgG2a specific for CD52 was carried out and the four human isotypes IgG1, IgG2,
IgG3 and IgG4 were made (Riechmann et al 1988). The IgG1 antibody called CAMPATH-1H
proved most effective in-vitro in complement mediated lysis and ADCC and was chosen for therapy (Hale et al 1988). Therapy with CAMPATH-1H has shown it to be effective at depleting
lymphocytes in a number of situations (Hale et al 1988, Dyer et al 1989, Mathieson et al 1990, Isaacs
et al 1992b). Whereas the rat IgG2b antibody CAMPATH-1G was frequently immunogenic on a first course
of treatment, the humanised antibody CAMPATH-1H has been found to be much less immunogenic
although repeated administration can lead to anti-idiotypic responses in some patients (Isaacs et al
The availability of good in-vitro assays for effector functions means that the CAMPATH-1 (CD52)
antibodies provide a good system for studying factors to do with IgG effector functions (Greenwood, Clark & Waldmann 1993, Greenwood & Clark 1993). Human IgG1 and IgG4 are very
homologous and differ in only a few residues in the constant region domains (see models)
and they also have different hinge region sequences. In previous studies on mouse IgG, Duncan and Winter,
identified a motif Glu 318, Lys 320, Lys 322 as being important for complement lysis, however all four
human IgG antibodies are identical in sequence at this point. Using domain swap mutants of the CD52
antibodies we managed to identify that the residues responsible for the differences in function between IgG1
and IgG4 in complement lysis and ADCC were in the C-terminal half of the CH2 domain (Greenwood, Clark & Waldmann 1993). The residues concerned are Ala 330 Pro 331 in IgG1 and
Ser 330 Ser 331 in IgG4 (see models) .
However during these studies we discovered a polymorphism in ADCC with human isotypes (Greenwood, Clark & Waldmann 1993). Some individual donors of effector cells mediate ADCC
with all four human IgG subclasses whilst others only mediate ADCC with human IgG1 CD52 antibodies.
This polymorphism remains to be resolved but is obviously of importance to those who have implied that
IgG4 would be a good choice for a non-depleting use of a recombinant antibody. In a separate series of
experiments in collaboration with Inez-Anne Haagen and Jan van de Winkel we discovered that the
polymorphism in human Fc gamma RIIa which affects human IgG2 binding and mouse IgG1 binding, also
effects rat IgG2b binding (Haagen et al 1995).
We have also studied IgG1 allotypes and aglycosyl allotypes for their effector functions. In human IgG1
there are allotypic residues in the CH1 and the CH3 domains (see models) . In comparisons
in complement mediated lysis with CD52 antibodies of different IgG1 allotypes there is very little
difference between them. We then compared antibodies in which the conserved N-linked glycosylation site
Asn 297 was mutated to Ala. We expected the mutation to knock out all activity in complement lysis. To our
surprise the allotypes seemed to differ in their residual activity (Greenwood & Clark 1993). The
allotype G1m(3) has more activity than G1m(1,17) and domain swap mutants giving allotypes G1m(1,3)
and G1m(17) showed that it was the Arg 214 G1m(3) for Lys 214 G1m(17) in the CH1 domain which was
more important ie a residue outside of the CH2 domain.
In-vivo models using recombinant antibodies to murine CD8 have shown that depletion occurs with wild
type antibodies but that aglycosyl mutants fail to deplete. The depletion is unaffected by cobra venom factor
treatment and thus presumably does not depend on complement lysis (Isaacs et al 1992).
Thus it is still not clear which effector functions are absolutely required for in-vivo depletion. FcR binding
rather than complement would seem to be most important but we don't as yet know which FcR or which
cells which express them are most important. The results are likely to be complicated by polymorphisms in
the antibodies and in the FcR.
Williams,G.T., Bindon,C.I., Clark,M.R., Walker,M.R., Jefferis,R.,
Waldmann,H., and Neuberger,M.S. (1987), J. Exp. Med. 166, 1351-1361 Comparison of the effector
functions of human immunoglobulins using a matched set of chimeric antibodies.
Dyer,M.J.S., Clark,M.R., Phillips,J.M., Marcus,R., Riechmann,L.,
Winter,G., and Waldmann,H. (1988), Lancet 2, 1394-1399 Remission induction in non-Hodgkin lymphoma
with reshaped human monoclonal antibody CAMPATH-1H.
Clark,M.R., Waldmann,H., and Winter,G. (1988), Nature 332, 323-
327 Reshaping human antibodies for therapy.
Teale,C., Bindon,C., Clark,M., and Waldmann,H. (1989), J.
Immunol. 142, 3145-3150 A matched set of rat/mouse chimeric antibodies.
Hale,G., Hayhoe,F.G.J., and Waldmann,H. (1989) Blood 73, 1431-
1439 Effects of CAMPATH-1 antibodies in vivo in patients with lymphoid malignancies: influence of
Cobbold,S.P., Hale,G., Clark,M.R., Oliveira,D.B.G.,
Lockwood,C.M. and Waldmann,H. (1990), New England Journal of Medicine 323, 250-254 Monoclonal
antibody therapy in systemic vasculitis.
- Isaacs, J.D.,
Clark, M.R., Greenwood, J. and Waldmann, H. (1992), J. Immunol
148, 3062-3071 Therapy with monoclonal antibodies - an in vivo model for the assessment of therapeutic
- Isaacs, J.D,
Watts, R.A., Hazleman, B.L., Hale, G., Keogan, M.T., Cobbold, S.P.,
Waldmann, H. (1992b) Lancet 340, 748-752 Humanized monoclonal-antibody therapy for rheumatoid-
Routledge,E., Lloyd,I., Chatenoud,L., Pope,H., Gorman,S.D., Clark,M. and
Waldmann,H. (1993), Eur. J. Immunol. 23, 403-411 The generation of a humanised, non-mitogenic CD3
monoclonal antibody which retains in vitro immunosuppressive properties
Clark,M. and Waldmann,H. (1993), Eur. J. Immunol. 23, 1098-1104
Structural motifs involved in human IgG antibody effector functions
and Clark,M. (1993) In: Clark, M. (Ed). Protein engineering of
antibody molecules for prophylactic and therapeutic applications in man. Academic Titles, Nottingham, UK
(Pub). Effector functions of matched sets of recombinant human IgG subclass antibodies. pp. 85-100
Geerars,A.J.G., Clark,M.R., and van de Winkel,J.G.J. (1995), Journal
of Immunology 154, 1852-1860 Interaction of human monocyte Fc gamma receptors with rat IgG2b: A new
indicator for the Fc gamma RIIA (R-H131) polymorphism.
This page is from Mike Clark
"An antibody engineer who also enjoys the mountains."
11th June 1997