WO1990011090A1

WO1990011090A1 - Pharmaceutical compositions

Info

Publication number: WO1990011090A1
Application number: PCT/GB1990/000405
Authority: WO
Inventors: Andrew Gordon Hadley; Belinda Mary Kumpel
Original assignee: Central Blood Laboratories Authority
Priority date: 1989-03-17
Filing date: 1990-03-16
Publication date: 1990-10-04
Also published as: GB8906129D0; AU5276690A

Abstract

The present invention provides a mixture comprising at least one monoclonal anti-Rh(D) antibody of the IgG1 subclass and at least one monoclonal anti-Rh(D) antibody of the IgG3 subclass, the ratio of said antibodies in said mixture being selected to provide in use a synergistic enhancement of the metabolic rate of monocytes, which may be employed as a pharmaceutical composition for passive immunisation to prevent haemolytic disease of the newborn.

Description

Pharmaceutical Compositions

This invention relates to novel pharmaceutical compositions for use in prophylaxis against haemolytic disease of the newborn (HDN) and related conditions. When an Rh(D-) mother gives birth to an Rh(D+) child, the mother frequently becomes sensitive to the RhD antigen and develops anti-Rh(D) antibodies which give rise to HDN when a second Rh(D+) child is born. Rh(D-) mothers carrying Rh(D+) foetuses are therefore commonly treated prophylactically by passive immunisation with anti-Rh(D) antibodies to destroy Rh(D+) cells which may transfer to the mother's vascular system from the foetus before such cells stimulate an immune response.

Thus, hyperimmune anti-Rh(D) immunoglobulin is widely used in such i munoprophylaxis against HDN and may be obtained from sera of sensitised Rh (D-) individuals. In general, it has been found that the anti-Rh(D) antibodies are restricted to the IgGl and IgG3 subclasses. The mechanism of red cell destruction in HDN has not been elucidated. The studies of Urbaniak and Greiss (1980) suggest that severe HDN can occur with a predominantly IgGl response and the study of Parinaud et al (1985) suggested that the severity of HDN was correlated with the Gm allotype of IgGl antibody. In contrast however, another study by Ouwehand et al (1984) showed that high activity of a monocyte ADCC assay (due to IgG3 anti-Rh(D)) correlated with the severity of HDN. These inconsistent findings suggest that the anti-Rh(D) response is complex. Our studies suggest that the IgGl and IgG3 antibodies function differently in red cell destruction by antibody dependent cellular cytotoxicity (ADCC) . Both the red cells and the mononuclear phagocytes (monocytes) which destroy them are negatively charged and are mutually repelled when in close proximity. Although both IgGl and IgG3 antibodies react with D-antigens on the red cell surface, the IgG3 antibodies are far more effective in binding the red cells to monocytes via their FcR receptors. It seems possible that the longer hinge region of the IgG3 antibodies permit multiple binding between cells spaced at greater distances, where repulsion between the cells is lower, thereby enabling fewer antibody molecules to effect a stable interaction. In monocyte-mediated ADCC, red cell destruction is largely extracellular, and IgG3 anti-Rh(D) is more effective than IgGl. On the other hand, we have observed that under certain conditions IgGl antibodies appear to activate phagocytosis more effectively than IgG3, perhaps due to their short hinge region resulting in more stable immobilisation of membrane receptors. We have now found that IgGl and IgG3 antibodies simultaneously attached to red cells show a surprising and therapeutically valuable synergistic enhancement of the metabolic rate of the monocytes.

In its broadest aspect our invention provides a mixture of at least one monoclonal anti-Rh.D) antibody of the IgGl subclass and at least one monoclonal anti-Rh(D) antibody of the IgG3 subclass.

In one aspect our invention provides a mixture suitable for use in prophylaxis against haemolytic disease of the newborn comprising at least one monoclonal anti-Rh(D) antibody of the IgGl subclass and at least one monoclonal anti-Rh(D) antibody of the IgG3 subclass, the ratio of said antibodies in said mixture being selected to provide in use a synergistic enhancement of the metabolic rate of monocytes.

In a further aspect of the invention we provide a pharmaceutical composition comprising a mixture of a) at least one monoclonal anti-Rh(D) antibody of the IgGl subclass for simultaneous or sequential administration with b) at least one monoclonal anti- Rh(D) antibody of the IgG3 subclass. Although not wanting to be limited by any theory of the invention, which is founded on empirical evidence, we suggest that the presence of both IgGl and IgG3 anti-Rh(D) on the same red cell may result both in efficien red cell adherence and Fc receptor crosslinking with increased metabolism as a consequence. Thus we suggest that IgG3 anti-Rh(D) promotes rapid clearance of foetal cells to the maternal spleen, and IgGl is necessary for efficient phagocytosis.

A pharmaceutical composition according to the invention is substantially more effective than using either the IgGl or IgG3 alone. Although the IgGl and IgG3 components may be administered sequentially, it is important that they are available substantially simultaneously at the site of action, and simultaneous administration is preferred. The use of monoclonal antibodies has significant advantages over the use of anti-Rh(D) preparations from pooled blood: substantiall pure compositions can be made which are free of contaminant such as viruses and antibodies of other specificities, and are consistent in strength and reliability.

The mixture of monoclonal antibodies may be provided in a lyophilised form for dissolution in a pharmaceutically acceptable excipient, e.g. saline, when required. Alternatively the pharmaceutical composition may comprise the antibody mixture dissolved in a carrier, e.g. any physiologically acceptable aqueous medium for example isotonic phosphate buffered saline or normal human immunoglobulin. In the latter instance it would usually be necessary to store the composition under cold conditions.

The invention includes within its scope not only mixtures of specific monoclonal IgGl anti-Rh(D) antibodies with specific monoclonal IgG3 anti-Rh(D) anti¬ bodies but also compositions comprising a plurality of sub-types of monoclonal IgGl and/or IgG3 anti- Rh(D) antibodies. The synergistic effect only occurs when a mixture of monoclonal IgGl and IgG3 is used. We prefer to use the two subclasses of antibody in a ratio in the range 3:1 to 1:3, preferably 2:1 to 1:2. It appears that synergism is highest when the IgGl and IgG3 are present on the red cells in about equal amounts. Moreover, it should be noted that the synergistic effect is at least predominantly reflected in metabolic activity (as assessed by CL activity) , rather than in enhanced adherence, phagocytosis or ADCC. Suitable monoclonal IgGl antibodies for use in pharmaceutical compositions according to the invention include e.g. those disclosed in our co-pending applications PCT/GB88/00755 and 757. Suitable monoclonal IgG3 antibodies for use in pharmaceutical compositions according to the invention include e.g. those disclosed in our co-pending application PCT/GB88/00756.

The amount of antibody which will be effective is well documented (Bowman, 1988) . Recommended doses of the currently used polyclonal anti-Rh(D) vary depending on country (300 microgrammes typically being given intramuscularly (IM) in Canada compared with 100 microgrammes IM in Great Britain) and Bowman recommends doses ranging from 50 microgrammes for every Rh negative woman who had had a spontaneous or induced abortion to 300 microgrammes for every 25 ml of foetal blood which may have entered her circulation via a trans- placental haemorrhage. Thus a typical dosage unit for human use may contain e.g. 20-1000 meg, generally 50-500 meg of antibody mixture. A suitable formulation for prophylaxis may comprise e.g. 20-150, preferably about 50 meg of IgGl anti-Rh(D) designated "Bll" in our earlier application PCT/GB88/00755 together with e.g. 20-150, preferably about 50 eg of IgG3 anti-Rh(D) designated "A8" in our earlier application PCT/GB88/00756. Of course other IgGl or IgG3 anti-Rh(D) monoclonal antibodies may be used. For example, FOG-1 (an IgGl anti-Rh(D) monoclonal antibody) described by Wiener et al (1987) and in GB-A-2189506, Table II on page 6 (number 4) may be used alone as the IgGl component or in combination with one or more other IgGls. Further examples include other IgGl monoclonal antibodies disclosed in GB-A- 2189506, in Table II on page 6.

The invention will now be described by way of illustration in the following non-limiting Examples. Results obtained in these Examples are shown graphically in the accompanying drawings wherein:

Figure 1, shows the interactions between human monocytes and red cells sensitized with IgGl and IgG3 anti-Rh (D) antibodies in various proportions; Figure 2 shows the level of chemiluminescence (CL) produced by various IgGl/IgG3 mixtures at different concentrations per red blood cell; and

Figure 3, shows the increase in CL of monocytes incubated with red cells sensitized with both IgGl and IgG3 monoclonal antibodies against Rh(D).

Example 1

Materials and Methods

Binding of sensitized red cells to adherent human monocytes was determined as described previously (Hadley et al 1989) . The results are given as the percentage of monocytes with one or more peripherally- bound red cells. Phagocytosis of red cells was determined by incubating adherent monocytes with sensitized red cells (30 red cells per monocyte) for 4 hours at 37°C in 24-well tissue culture plates (Costar) . Unbound red cells were removed by rinsing the wells with phosphate-buffered saline (PBS) . Peripherally- bound (but not internalized) red cells were then lysed by addition of 1.0% w/v ammonium oxalate containg 2mM Na EDTA for 2 in. Thereafter cells were restored to isotonicity with PBS for 5 min, then dried, fixed, stained and assessed microscopically. The results are given as the percentage of monocytes with one or more internalized red cells. Generation of luminol- enhanced chemiluminescence (CL) by monocytes during erythrophagocytosis was measured as described previously (Hadley, Kumpel & Merry, 1988) . Changes in CL are directly proportional to changes in metabolic rate of the cells under test. The CL response of monocytes incubated with sensitized cells was recorded for Ih, then compared with the CL response to unsensitized cells, and expressed as the ratio or opsonic index. Monocyte-mediated lysis of Cr labelled red cells

(Hadley et al. 1989) is expressed as a percentage of the 51Cr released from red cells by 1% v/v Triton X100. Lymphocyte-mediated lysis of red cells (Urbaniak, 1976) was measured using red cells presensitized at 20,000 IgG molecules per red cell, and at a lymphocyte to target cell ratio of 15:1.

The IgGl and IgG3 anti-Rh(D) monoclonal antibodies used below in relation to Figures 1 to 3 were Bll (disclosed in PCT/GB88/0755) and A8 (disclosed in PCT/GB88/00756) respectively.

Figure 1 shows that the adherence and lysis of sensitized red cells by human monocytes increased with increasing proportions of IgG3 in the sensitizing mixture. Conversely, phagocytosis of red cells was greater with increasing proportions of IgGl. However, the metabolic response of monocytes was greatest when both subclasses were present in the opsonizing mixture in approximately equal proportions. Quantitation of cell-bound IgG showed that this increase was not due to raised levels of bound anti-Rh(D) when red cells were incubated in mixtures of antibodies. This syner¬ gistic increase in the ability of red cell-bound IgG to elicit a CL response from monocytes was observed only when IgGl-containing supernatants were blended with IgG3-containing supernatants. Increased CL responses were not observed in four IgGl/IgGl mixtures, and two IgG3/IgG3 mixtures tested but were observed for all eight IgGl/IgG3 mixtures tested. The additional IgGl and IgG3 anti-Rh(D) monoclonals used in these mixtures were selected from Al, A2 and Bll disclosed in PCT/GB88/ 00755, RID7 (an IgGl) disclosed by Ku pel et al (1988) and A4 and A5 disclosed in PCT/GB88/00756. The synergistic increase in CL response was detected at levels of sensitization from 40,000 IgG molecules per cell (corresponding to the approximate number of D antigen sites) to 1,000 IgG molecules per cell (limit of assay sensitivity - see figure 2). Further, increased CL responses were seen only when both subclasses were present on the same red cells^', as shown in Figure 3. Figure 3 shows the increase in CL response of monocytes incubated with red cells sensitized with both IgGl and IgG3 anti-Rh(D). The results are from a single representative experiment. The solid lines show monocyte responses to red cells sensitized with IgGl anti-Rh(D) , IgG3 anti-Rh(D), or a 50/50 blend of IgGl and IgG3 anti-Rh(D) . For comparison, the broken line shows monocyte response to a mixed population of IgGl-sensistized and IgG3-sensitized red cells. Thus, mixing of red cells sensitized with only IgGl anti-Rh(D) or only IgG3 anti-Rh(D) prior to their addition to monocytes did not result in increased CL generation. The synergistic effect only occurs when a mixture of monoclonal IgGl and IgG3 is used to sensitise the red cells. Example 2

Pharmaceutical compositions

a) A mixture of 50 meg of each of monoclonal antibodies produced by heterohybridomas of numbers 4(IgGl) and 5(IgG3) in Table II on page 6 of GB-A-2189506 in 1ml of sterile isotonic saline.

b) A mixture of 50 meg of each of monoclonal antibodies produced by heterohybridomas of numbers 11(IgGl) and 3(IgG3) in Table II on page 6 of GB-A-2189506 in 1ml of sterile isotonic saline.

REFERENCES

BOWMAN J.M. (1988) The Prevention of Rh immunization. Trans. Med. Rev. 2 , 129.

HADLEY A.G., KUMPEL B.M. & MERRY A.H. (1988) The Chemiluminescent response of human monocytes to red cells sensitized with monoclonal anti-Rh(D) antibodies. Clin. Lab. Haemat. ^, 377.

HADLEY A.G., KUMPEL B.M., LEADER K., MERRY A.H. BROJER E. & ZUPANSKA B. (1989) An in vitro assessment of the functional acitivity of monoclonal anti-D. Clin. Lab. Haemat. (In Press) .

KUMPEL B.M., LEADER K.A. AND BRADLEY B.A. (1988) The relationship between IgG subclass and Gm allotype of monoclonal anti-Rh(D) antibodies and their functional activity. Biochem. Soc. Trans. 16:733-734.

OUWEHAND W.H., MALLENS T.E.J.M., HUISKES E., WEBER A., JAGER K. , BENNEBROEK GRAVENHORST J. & ENGELFRIET C.P. (1984) Predictive value of a monocyte-driven eytotoxicity assay for the severity of rhesus (D) haemolytic disease of the newborn; a comparison with other techniques. In Ouwehand, W.H.: The activity of IgGl and IgG3 antibodies in immune-mediated destruction of red cells. Thesis, Amsterdam, pp 88-114.

PARINAUD J., et al (1985) IgG subclass and Gm allo types of anti-D antibodies during pregnancy. AM. J. Obstet, Gynec. 151, 1111-1115.

URBANIAK S.J. (1976) Lymphoid cell dependent (K-cell) lysis of human erythrocytes sensitized with rhesus alloantibodies. Br. J. Haemat. 33, 409. URBANIAK S.J., Greiss M.A. , (1980) ADCC (K-cell) lysis of human erythrocytes sensitized with rhesus alloantibodies III. Br. J. Haemat. 4_6, 447-453.

WIENER E., ATWAL A., THOMPSON K.M. , MELAMED M.D., GORICK B., and HUGHES-JONES N.C. (1987). Difference between the acitivities of human monoclonal IgGl and lgG3 subclasses of anti-D(Rh) antibody in their ability to mediate red cell-binding to macrophages. Immunology, 62:401-404.

Claims

CL IMS :

1. A mixture suitable for use in prophylaxis against haemolytic disease of the newborn comprising at least one monoclonal anti-Rh(D) antibody of the IgGl subclass and at least one monoclonal anti-Rh(D) antibody of the IgG3 subclass, the ratio of said antibodies in said mixture being selected to provide in use a synergistic enhancement of the metabolic rate of monocytes.

2. A mixture as claimed in claim 1 comprising IgGl and IgG3 antibodies in a ratio in the range

3:1 to 1:3.

3. A mixture as claimed in claim 1 wherein the ratio of IgGl to IgG3 antibodies is 1:1.

4. A pharmaceutical composition comprising a mixture of a) at least one monoclonal anti-Rh(D) antibody of the IgGl subclass for simultaneous or sequential administration with b) at least one monoclonal anti- Rh(D) antibody of the IgG3 subclasses.

5. A pharmaceutical composition as claimed in claim 4 wherein the antibody mixture is provided in a sterile solution in a physiologically acceptable aqueous medium.

6.. A pharmaceutical composition as claimed in claim 4 for passive immunisation of an Rh (D)-negative mother after the birth of an Rh(D)-positive child to prevent sensitization of the mother to Rh(D) antigen.