FR2641468A1 - Chimeric antibodies to human interleukin-2 receptor, their preparation and pharmaceutical compositions containing them - Google Patents

Abstract

Chimeric antibodies, in which the variable portion of an animal monoclonal antibody is grafted onto divalent or monovalent human IgGs or onto Fc fragments of human IgGs or onto the constant portion of human IgGs, wherein the aforementioned variable portion recognises the human interleukin-2 receptor, and wherein the Fab' fragments of the aforementioned animal antibody are capable of inhibiting the primary allogenic response; their preparation and their application in human medicine, in particular in the prevention of graft rejection.

Description

 The subject of the present invention is chimeric anti-human interleukin-2 receptor antibodies, their preparation and their application in human medicine.

 It is known that the human interleukin-2 receptor (RIL2) is present on the surface of activated T cells, and in particular on the surface of T cells that are responsible for the organ transplant rejection phenomenon.

 Human anti-receptor monoclonal antibodies for this reason are, for this reason, used with some success in preventing the rejection of organ transplants in humans.

 However, monoclonal antibodies, and particularly murine monoclonal antibodies, which are used in human therapy, have various disadvantages. They are quickly eliminated by the body, and they induce in the hood the production of anti-isotype and anti-idiotypic antibodies.

 In addition, some monoclonal antibodies, for example rat IgG2a or mouse IgG1 do not bind complement, and therefore have no cytotoxic effect on the target cells. They only allow to block the receiver of the cell concerned. Furthermore, the murine monoclonal antibodies having the isotypes IgG1, IgG2a, IgG2b and IgG3 are capable of binding to the receptor for the Fc fragment of human mononuclear cells, and this binding results in a significant loss of their specific activity.

 It should also be noted that certain monoclonal antibodies, such as for example the OKT3 monoclonal antibody, which is a mouse IgG2a capable of recognizing the T3 marker present on the surface of T lymphocytes, bind to the receptors for the Fc fragment of human monocytes. and induce a proliferative response that causes undesirable side effects.

In order to avoid the disadvantages of monoclonal antibodies, the preparation of chimeric antibodies obtained by grafting Fab 'fragments of a monoclonal antibody onto human polyclonal IgGs, said Fab' fragments originating from a monoclonal antibody has already been described. mouse capable of recognizing an IgM present on the surface of lymphoid tumor cells, but not recognizing normal human I's. The Fab 'fragments are grafted onto polyclonal human IgGs using a coupling agent. The chimeric antibody obtained, of the IgG-Fab 'type, is capable of recognizing the tumor cell, as is the monoclonal antibody from which it is derived. However, this chimeric antibody is incapable of inducing the antibody-dependent cytotoxicity phenomenon (ADCC ) and is not able to activate the complement; see Hamblin and have,
Blood, vol.69, 790-797 (1987).

 Other authors have described monovalent chimeric antibodies obtained by grafting Fab 'fragments of polyclonal antibodies directed against the surface IgM of lymphoid tumor cells on IgG or Fc fragments; see, in particular, G. T. Stevenson et al., Med. Oncol. & Tusor.

 Pharnacother, vol. 1, n04, 275-278 (1984).

 It has now been discovered that it is possible to obtain chimeric antibodies capable of recognizing the human interleukin-2 receptor (RIL2) and which can be used in human therapy in the prevention and / or treatment of transplant rejection. organs.

 The subject of the present invention is chimeric antibodies in which the variable part of an animal monoclonal antibody is grafted on divalent or monovalent human IgGs or on human IgG Fc fragments or on the constant portion of human IgG, characterized in that said variable portion recognizes the human interleukin-2 receptor, and that Fab 'fragments of said monoclonal antibody are capable of inhibiting the primary allogeneic response.

 It has been discovered that it is possible to thereby obtain chimeric antibodies which are capable of recognizing the human interleukin-2 receptor, and which also have the ability to inhibit the primary allogeneic response, such as the monoclonal antibody of which the variable part of these chimeric antibodies is derived, while it is known that this inhibition is not obtained with human IgGs.

 The chimeric antibodies are in particular those which consist of Fab 'fragments grafted on human IgGs or on Fc fragments of human IgG, characterized in that the said Fab' fragments recognize the human interleukin-2 receptor, and said chimeric antibodies are capable of inhibiting the primary allogeneic response. In such chimeric antibodies, it is not only the variable part, but the whole of the Fab 'fragment which is grafted on the IgG or on the Fc fragment. These chimeric antibodies according to the invention may be of the IgGFab ', Fc-Fab' or Fc- (Fab) type. Reverse notation is sometimes used, for example Fab'-IgG.

 The Fab 'fragments can also be grafted analogously to Fablc-type monovalent IgG obtained by selective enzymatic cleavage of only one of the human IgG Fab fragments, according to the method described by Glennie & Stevenson, Nature, Viol. 295, February 25, 1982.

 The resulting chimeras are then of the Fab / c-Fab 'type.

 The Fab 'fragments can be uro-clonal antibody Fab' fragments obtained by immunizing animals against the human interleukin-2 receptor.

 The Fabt fragments can also be obtained by expression of recoibinant DNA in a suitable vector, said recombinant DNA encoding the variable region of an anti-RIL2 monoclonal antibody.

The human IgG used for the preparation of the chimeric antibodies of the invention may be polyclonal IgGs or else
IgG1, IgG2, IgG3 or IgG4, or mixtures of these various isotypes.

 Likewise, the IgG Fc fragments used in the preparation of the chimeric antibodies of the invention may be Fc fragments derived from polyclonal IgGs or Fc fragments of IgG1, IgG2, IgG3 or IgG4. . It can also be Fc fragments obtained by genetic engineering.

 The chimeric anti-RIL2 antibodies of the invention may also be constructed according to the methods of genetic engineering, for example according to the method described by Zenon Steplewski et al., Proc. Natl. Acad. Sci.

USA, vol.85, 4852-4856, July 1988.

 The invention also relates to a process for preparing chimeric antibodies as defined above.

This method is characterized in that on the one hand divalent or monovalent human IgGs or human IgG Fc fragments are subjected to the action of a reducing agent in an amount calculated to cut a portion of the disulfide bridges connecting the two heavy chains of the IgG molecule or Fc fragment, on the other hand fragments are prepared
Fab ', capable of inhibiting the primary allogeneic response, derived from a monoclonal antibody capable of recognizing RIL2 and that said Fab' fragments are grafted on said reduced IgG or said reduced Fc fragments, via a bifunctional coupling agent.

 The process for preparing the chimeric antibodies is known per se.

 In order to prepare reduced IgG or reduced Fc fragments, a reducing agent capable of cleaving the disulfide bridges into free thiols is reacted with IgG or Fc fragments, in solution in a suitable buffer. For example, dithiothreitol with alkaline pH, close to 8, is used in a suitable buffer. The reduction is carried out for example at a temperature of 20-40 C.

 Controlled amounts of reducing agent are used to leave at least one disulfide bond between the two heavy chains of the IgG or Fc fragment.

 Preferably, the minimum amount of reducing agent is used to obtain on average two SH-groups per molecule of IgG or Fc fragment. The minimum amounts of reducing agent can be determined in each case by simple routine experiments.

 The starting IgGs or the IgG1, IgG2, IgG3 or IgG4 isotypes are prepared from plasma or placental blood according to known methods. The Fc fragments are obtained from IgG according to the known methods by digestion with the papain or the plasmimer.

 The Fab 'fragments are prepared from the F (ab') 2 fragments by reduction according to known methods using the minimum amount of a reducing agent such as 2-mercaptoethanol. The F (ab ') 2 fragments are themselves prepared by limited digestion of IgG with pepsin.

 Reduced IgGs, or reduced Fc fragments, or preferably Fab 'fragments, are reacted with a bifunctional coupling agent capable of establishing a covalent bond by reaction with the -SH groups. The conjugate is thus obtained, such as Fab'-Cpl, where Cpl is the remainder of the coupling agent, still containing a functional group capable of reacting with the reduced Fc fragments or reduced IgGs to obtain, as the case may be, an IgG-derivative. Fab ', Fab / c-Fab', Fc-Fab 'and / or Fc- (Fab') 2. Reduced IgG or reduced Fc fragments may also be reacted with the coupling agent and the resultant conjugate reacted with the Fab 'fragments.

 The coupling agent is for example a diimide such as orthophenylenedimaleimide, which reacts with the growths -SE by creating thioether bonds.

 In the case where the conjugate Fab'-coupling agent is reacted with reduced IgG, an excess of IgG is preferably used, for example from 1.1 to 2 moles of IgG per mole of Fab 'for avoid any loss of Fab '.

 In the case of the reaction of the conjugate, the Fab'-coupling agent with a substantially stoichiometric amount of Fc fragment, a Fc-Fab 'chimera (monovalent) is obtained. Using at least 2 moles of Fab' per Fc fragment, we obtain a divalent Fc- (Fab ') 2 chimera, generally in admixture with the Fc-Fab' monovalent chimera. Such divalent Fc- (Fab ') 2 chimeras have not been described so far in the literature.

 The Fab 'fragments used are fragments derived from a monoclonal antibody obtained by immunization of animals with the human interleukin-2 receptor, fusion of the lymphocytes of the immunized animals with a transformed lymphoid cell capable of being cultured in vitro, and cloning according to known methods.

 The starting anti-RIL2 monoclonal antibody is, for example, the 33B3-1 antibody described by OLIVE D. et al., Eur. J. Immunol. 16, 611-620 (1986), marketed by IMMUNOTECH (France) under the name IOT 14a, or else other existing commercial anti-RIL2 antibodies.

 The IgG or Fc fragments supporting the Fab I fragments to form the chimeric antibody are generally polyclonal IgGs.

Of n% n, Fc fragments are polyclonal IgG Fc fragments. However, it is desirable to use in some cases IgG1, IgG2, IgG3 or
IgG4 or Fc fragments of these isotypes.

 As indicated above, the chimeric antibodies of the invention can also be prepared by isolating the genes of the variable portion of a hybridoma secreting an antibody recognizing human RIL2. These genes are inserted into a vector containing DNAs encoding the heavy catkin and the light channe of the constant part of a human IgG. Transfection of these expression vectors into non-excretory myeloma tumor cells results in cell lines producing chimeric antibodies having the constant portion of a human IgG and the variable portion of the anti-RIL2 monoclonal antibody; see Zenon Steplewski et al., cited article.

 The chimeric antibodies of the invention, administered in humans, do not have the known disadvantages of monoclonal antibodies, namely, short life, non-specific receptor binding for the Fc fragment, induction of lymphocyte proliferation and induction of a response. anti-isotype and anti-idiotype antibodies.

 The chimeric antibodies of the invention do not induce in man the side effects observed with monoclonal antibodies and can be used for long periods of time, especially as agents for preventing rejection of organ transplants, whereas monoclonal antibodies , because of their side effects, are currently only used for a few days before and after the transplantation. They can also be used in the therapy of autoimmune diseases, which requires repeated treatments.

 The chimeric antibodies of the invention are preferably administered intravenously, for example at a dose of 10 to 40 mg / day in adult men weighing about 60 kg.

 The invention also relates to pharmaceutical compositions for human use, intended in particular to prevent the rejection of organ transplants, or to treat autoimmune diseases, characterized in that they contain as active ingredient at least one antibody chimeric as defined above.

 The compositions of the invention are either in the form of injectable solutions or in the form of lyophilized compositions for reconstituting an injectable solution at the time of use.

 The following examples illustrate the invention without limiting it.

EXAMPLE 1
a) Obtaining reduced human IgG
The starting material is a 50mg / ml solution of human polyclonal IgG in 0.2M Tris HCl buffer pH 8.0 containing 20mM dithiothreitol and 1mM disodium ethylenediaminetetraacetate (Na2 EDTA). After 30 minutes of incubation at 30 ° C., a solution of reduced human IgG is obtained. The solution obtained was purified by dialysis against 0.5M sodium acetate buffer 10mM disodium ethylenediaminetetraacetate (pH 5.4).

 A purified solution of reduced human IgG in acetate buffer is thus obtained.

 b) Obtaining Fab 'Fragments of a Rat Monoclonal Antibody.

 The starting monoclonal antibody is a rat anti-human RIL2 isotope IgG2a antibody (reference 33B3-1) in the form of a solution in isotonic saline.

 The solution is purified by dialysis for 16 hours against a 0.1M sodium formate solution pH 2.8, then by dialysis for 24 hours against a solution of 0.1M sodium acetate pH 4.5.

 A pepsin solution is then added to a concentration of 2%, pH 4.5, and incubated for 4 hours at 37 ° C. The enzymatic reaction is then stopped by raising the pH to 8.

By permeation chromatography on Sephacryl S 200 (Pharmacia) and elution with 0.28 Tris buffer pH 8.0, a 5 mg / ml solution of F (ab ') 2 fragments is obtained. Beta-mercaptoethanol is added to a concentration of 10 mM and incubated for 30 minutes at 300 ° C.
By chromatography on Sephadex G (Pharmacia) and elution with 50 mM sodium acetate buffer + 2 mM Na 2 EDTA, pH 5.3, a solution of Fab 'fragments free of beta-mercaptoethanol is obtained.

 The starting monoclonal antibody has been described by OLIVE D. et al., Article cited above.

c) Preparation of Fab'-orthophenylenedimaleiride Conjugate
The orthophenylenedimaleimide in 4 mM solution, obtained by dissolving 18.76 mg in 3.5 ml of dimethylformamide, is added to the solution of Fab 'fragments.

 The mixture is incubated for 45 minutes at 4 ° C. with stirring at pH 4.7.

 The solution is then chromatographed on Phospho Ultragel AGR (IBF) equilibrated with 40 mM sodium acetate buffer, 2 XM Na2EDTA, 4.7 fold. It is eluted with 8 mM sodium acetate buffer, 5 mM Na 2 EDTA, 5.7 fold. A solution of Fab 'maleimide fragments is obtained.

 d) Coupling the Fab 'fragment with the reduced IgG.

 The solution obtained in step c) and the solution of reduced human IgG obtained in step a) are mixed; molar ratio 1/1.

 It is incubated for 24 hours at 4 ° C and then dialyzed against PBS buffer for about 16 hours.

 By chromatography on Sephacryl S 200 (Pharmacia), a solution of the obtained Fab'-IgG chimera (hereinafter called Lot 01) is separated.

The product obtained has the following physico-chemical characteristics
In electrophoresis in SDS PAGE, the Fab'-IgG chimera is characterized by a band whose molecular weight is approximately 230 kD.

The product is further characterized by gel filtration (gel
Superose 12, Pharmacia).

 By replacing the final purification step on Sephacryl S 200 by final chromatography on Sephacryl S 300, a purified Fab'-IgG chimera was obtained, hereinafter Lot 02.

 By replacing the final purification step on Sephacryl S 200 by a affinity chromatography operation on a column containing the fixed commercial antibody MARK 1 (monoclonal antibody recognizing the kappa chain of rat IgG), a purified chimera was obtained. Fab'-IgG hereinafter called Lot 03.

EXAMPLE 2 Preparation of chimeric antibodies of the type
Fc-Fab 'and Fc-F (ab') 2
a) In a manner analogous to that described in Example I, step a), by replacing the IgGs with IgG Fc fragments with human IgG Fc fragments, a purified solution of reduced Fc fragments is obtained.

The Fc fragments used as starting material are obtained from human polyclonal IgGs according to the method of Barandun S. et al.
Vox Sang. 28, 157 (1975).

 b) The Fab 'fragments are obtained as in example 1, stage b, and coupled to orthophenylenedimaleimide as in example 1, stage c.

 c) The solution of dimaleimide-coupled Fab 'fragments, obtained in stage b, is mixed with the solution of reduced Fc fragments obtained from stage a (molar ratio: 2: 1).

 It is left in contact for 24 hours at +4 ° C. and then dialysed against PBS at + 40 ° C.

At the exit of dialysis, a chromatography is carried out on
Sephacryl S200 (gel filtration) in column of 1m length.

 Three successive chromatographies are carried out to separate the chimeras and then a reconcentration by ultrafiltration. A chimera of the (Fab) '2-Fc type and a Fab'-Fc type chimera are thus obtained, characterized in electrophoresis and in gel filtration.

 In electrophoresis (SDS-PAGE), the two chimeras have respective molar masses of about 160,000 and 100,000.

 EXAMPLE 3 Biological Activity of Chimeric Antibodies

a) Recognition of antigen
The RIL2 receptor is expressed on activated T cells. A T (1E7) clone described by Moreau J et al., J.Clin, is used as target cells. Invest., 78, 874 (1986).

 The test is carried out by indirect immunofluorescence according to the technique of Preudthomme J.L., Eur.J.Immunol. 7, 191 (1977), using a rat anti-IgG (Biophys supplier) and a fluorescein-labeled human anti-IgG (Bio Merieux).

 106 1E7 cells are incubated with 50 μl of the antibody solution to be tested at 40 ° C. for 1 hour; then the cells were washed 3 times with PBS buffer containing 0.1% sodium azide. 100 μl of fluorescent conjugated antibody diluted 1/100 in PBS buffer containing 1% fetal calf serum are then added. The cells are again washed 3 times. The immunofluorescence is read with a fluorescence microscope.

The results are summarized in Table I below:
TABLE I
Percentage of fluorescent cells

<tb><SEP> Anti-IgG <SEP> rat <SEP> anti <SEP> Human IgG <SEP><SEP>
<tb> Conjugate <SEP> control <SEP> 0 <SEP> Z <SEP> 3 <SEP> Z
<tb> Chimera <SEP>Fab'-IgG * <SEP> 1/5 <SEP> 54 <SEP>% <SEP> 78 <SEP>%
<tb><SEP> 1/50 <SEP> 53 <SEP> Z <SEP> 75 <SE> Z <SEP>
<tb> chimera <SEP>Fab'-IgG ** <SEP> 1/5 <SEP> 57 <SEP> Z <SEP> 54 <SEP> Z
<tb><SEP> 1/50 <SEP> 67 <SEP> Z <SEP> 59 <SEP> Z <SEP>
<tb> Chimera <SEP>Fab'-IgG *** <SEP> 1/5 <SEP> 50 <SEP>% <SEP> 47 <SEP>%
<tb><SEP> 1/50 <SEP> 44 <SEP> Z <SEP> 52 <SEP> Z <SEP>
<tb> Chimera <SEP> (Fab ') 2-Fc <SEP> **** <SEP> 1/5 <SEP> 60 <SEP>% <SEP> 32 <SEP>%
<tb><SEP> 1/50 <SEP> 44 <SEP>% <SEP> 8 <SEP>%
<tb> Control: <SEP> Fc <SEP> of LgG <SEP> human <SEP> 1/5 <SEP> 0 <SEP>% <SEP> 26 <SEP>%
<tb><SEP> 1/50 <SEP> O <SEP> Z <SEP> 7%
<tb> Control: <SEP> IgG <SEP> human <SEP> 1/5 <SEP> 1 <SEP> Z <SEP> 8 <SEP>% <SEP>
<tb><SEP> 1/50 <SEP> 1.5% <SEP> 10 <SEP> Z
<tb> Control: <SEP>Fab'rat<SEP> 33B31 <SEP> 1/5 <SEP> 58 <SEP> Z <SEP> 12 <SEP> Z
<tb><SEP> 1/50 <SEP> 58 <SEP> Z <SEP> 12 <SEP> Z <SEP>
<tb> Control <SEP> IgG2a <SEP> rat <SEP> 33B31 <SEP> 1110 <SEP> 43 <SEP> Z <SEP> 1 <SEP> Z
<tb><SEP> 11100 <SEP> 54 <SEP> Z <SEP> 3 <SEP> Z
<Tb>
Immunofluorescence on 1E7 cells
Positive anti-RIL2 control (Becton Dickinson) - 77% Conjugate control: cells + fluorescent antibody alone.

 The solutions of antibodies and chimeras studied have, before dilution, an optical density of 280 nm equal to 0.5.

* obtained in Example 1 (Lot 02) ** obtained in Example 1 (Lot 03) *** obtained in Example 1 (Lot 01) **** obtained in Example 2
b) Inhibition of Mixed Lymphocyte Reaction (MLR)
It operates according to the conventional technique described for example by
DUPONT B. et al., Adv. Immunol., 23, 107 (1976).

The results, expressed in percent inhibition: #### ###### #### ### ####### ###### # ### (mean + deviation) type) are summarized in Table II below
TABLE II

<tb><SEP> Final <SEP> Molarities <SEP> 0.1 <SEP> x <SEP> 10-6 <SEP> 0.2 <SEP> x <SEP> 10-6 <SEP> 0.4 <MS> x <SEP> 10-6 <SEP> 0.8 <SEP> x <SEP> 10-6 <SEP><SEP> RPMI Control
<tb> Porduits
<tb><SEP> Chimera <SEP>Fab'-IgG<SEP> * <SEP> 11872 # 4118 <SEP><SEP> 7971 # 1706 <SEP><SEP> 7184 # 849 <SEP><SEP> 7491 # 2245 <SEP><SEP> 19545 # 1822
<tb> 39 <SEP>% <SEP> 59 <SEP>% <SEP> 63 <SEP>% <SEP> 62 <SEP>%
<tb> Chimera <SEP>Fab'-IgG<SEP> ** <SEQ ID> 6763 # 1174 <SEP><SEP> 4957 # 666 <SEP><SEP> 4978 # 942 <SEP><SEP> 4267 # 962 <SEP><SEP> 18275 # 1408
<tb><SEP> 64 <SEP>% <SEP> 73 <SEP>% <SEP> 73 <SEP>% <SEP> 77 <SEP>%
<tb> Control <SEP>: <SEP> Ig2a <SEP> rat <SEP> 33B31) <SEP> 11032 # 8608 <SEQ> 10914 # 7216 <SEQ> 9109 # 4668 <SEQ> 7041 # 1683 <SEP><SEP> 17875 # 742
<tb><SEP> 38 <SEP>% <SEP> 39 <SEP>% <SEP> 49 <SEP>% <SEP> 60 <SEP>%
<tb> Control <SEP> Fab '<SEP> 33B31 <SEP> 7455 # 698 <SEP><SEP> 6613 # 997 <SEP><SEP> 7661 # 2341 <SEP><SEP> 7071 # 1473 <SEP><SEP> 17512 # 1209
<tb> 58 <SEP>% <SEP> 62 <SEP>% <SEP> 56 <SEP>% <SEP> 60 <SEP>%
<tb><SEP> Control <SEP>: <SEP> IgG <SEP> human <SEP> 14477 # 1144 <SEP><SEP> 16472 # 1589 <SEP><SEP> 17860 # 2934 <SEP><SEP> 16846 # 2028 <SEP> 17218 # 2480
<tb> 16 <SEP>% <SEP> 5 <SEP>% <SEP> 0 <SEP>% <SEP> 3 <SEP>%
<tb> * obtained in Example I (Lot 03) ** obtained in Example 1 (Lot 02)

Claims (8)

 1. Chimeric antibodies in which the variable part of an animal monoclonal antibody is grafted on divalent or monovalent human IgG or on human IgG Fc fragments or on the constant part of human IgG, characterized in that said variable portion recognizes the human interleukin-2 receptor, and that the Fab 'fragments of said animal antibody are capable of inhibiting the primary allogeneic response.  2. chimeric antibodies according to claim 1, characterized in that they are of the type IgG-Fab ', Fab / c-Fab', Fc-Fab 'or Fc- (Fab') 2.  3. Chimeric antibodies according to claim 1 or 2, characterized in that said IgG, Fab / c or Fc are polyclonal antibodies or are derived from polyclonal antibodies.  4. chimeric antibodies according to any one of claims 1 and 2, characterized in that said IgG or said Fc fragments are IgG1, 2, 3 or 4 or are derived from these isotypes.  5. chimeric antibodies according to any one of claims 1 to 4, characterized in that said Fab 'fragment is a rat monoclonal antibody fragment.  6. Process for the preparation of chimeric antibodies as defined in any one of the preceding claims, characterized in that on the one hand divalent or monovalent human IgGs or human FG IgG fragments are subjected to 11 a reducing agent in an amount calculated to cut a portion of the disulfide bridges connecting the two heavy channs of the IgG molecule or the Fc fragment, on the other hand is prepared Fab 'fragments, capable of inhibiting the allogeneic response from a monoclonal antibody capable of recognizing RIL2 and that said Fab 'fragments are grafted onto said Reduced IgG or reduced Fc fragments, via a bifunctional coupling agent.  7. Method according to claim 6, characterized in that said coupling agent is a diimide such as orthophenylenedimaleimide.  8. Pharmaceutical compositions, characterized in that they contain as active ingredient at least one chimeric antibody as defined in any one of claims 1 to 5.