FR2748483A1 - New gp120 receptors or binding sites - Google Patents

Abstract

The following are claimed: (1) new gp120 receptors or binding sites, characterised by the fact that they are found on CD4+ cells, but differs from the CD4 binding receptor which is blocked when reacting CD4+ cell with a monoclonal antibody directed against the gp120 binding site of CD4, and that they give interactions with gp120, such as those obtd. when reacting gp120 with CD4+ cells in the absence of serum; (2) new gp120 receptors or binding sites, such as involved in a process comprising incubating gp120 with CD4+ cells under duration and temp. conditions allowing binding of gp120 to CD4+ cells with blocked CD4 receptor; (3) polyclonal or monoclonal antibodies directed against the above receptors or binding sites; (4) the part of gp120 such as bound to membrane sites on living cells, distinct from the CDR2 binding gp120 site of CD4, when incubating CD4+ cells with monoclonal antibodies specific for the gp120 binding domain of CD4; (5) the binding site on the gp120 mol. which is localised by inhibition of anti B3 monoclonal antibodies near the V3 and the CD4 binding region; (6) polyclonal or monoclonal antibodies directed against the binding site situated on the gp120 mol. and their diagnostic on therapeutic applications; and (7) serum factors capable of at least partially inhibiting gp120 binding to the above gp120 receptors or interaction domains.

Description

MEANS FOR THE FIGHT OR DIAGNOSIS
FROM HIV INFECTION
The subject of the invention is means for combating or diagnosing an infection with the human immunodeficiency virus, HIV, the causative agent of AIDS.

 As early as 1984, several authors have described the binding of HIV to the CD4 receptor of CD4 + T lymphocytes and of macrophages (see Dalgleish et al., 1984, Nature, 312, 763-767 and Klatzman et al., 1984, Nature, 312, 767 -768).

 This binding of the virus to the CD4 receptor involves the envelope protein gpl20 which has a high affinity for the CD4 molecule (Lasky et al.

1987, Cell, 50, 975-985).

Other gap120 receptors, different from
CD4, have been described, such as galactose-ceramide on nerve cells (Harouse et al, 1991, Science, 253, 320-323), or the CR complement receptor (Montefiori et al, 1993, J. Virol. 67 , 2699-2706).

Different CD4 secondary receptors and those already reported have been demonstrated by the inventors in ELISA and FACS experiments carried out on living cells
By blocking the CD4 receptor with anti-CD4 monoclonal antibodies, the inventors found that the binding of gap120 to peripheral blood mononuclear cells (PBMC) and to CD4 + CEM cells, living or adherent to microplates, was not not limited to the CD4 receptor, but that there was also interaction with another receptor.

 This interaction could be demonstrated in the absence of serum and for certain gpl20 / cell ratios.

 On the contrary, in the presence of serum, an inhibition of the interaction of gp120 with the secondary receptor of whole cells could be observed, indicating the involvement of serum factors.

 Thus, in ELISA, it turned out that the binding of gpl20 to target cells could be practically completely prevented by prior blocking of the CD4 receptor with an anti-CD4 monoclonal antibody, when a pre-incubation with gp120 had been performed, and the presence of serum (10% fetal calf serum) maintained during the experiment.

 On the other hand, the binding of gpl20 to PBMC or CEM cells was only partially reduced (by 50 to 80%) by blocking of CD4 by the anti-CD4 antibody, when the gp120 was pre-incubated with BSA 3% and that contact with serum has been avoided.

 These results show that the "residual" gap120 must be linked to binding sites present on whole cells, differing from the CDR2 region of CD4. These sites are designated by the inventors "secondary binding sites" or even "serum inhibited receptors" (RIS for short).

 The phenomenon mentioned above appeared even more pronounced with dried CEM cells, adherent to microplates, having accessible intracellular binding sites.

 The binding of gap120 to secondary binding sites also increases when cells are permeabilized with paraformaldehyde. FACS analysis confirms the existence of secondary binding sites on the outer cell membrane of living CEM cells.

 The simultaneous presence of CD4 and secondary receptors, but in distinct regions, has been demonstrated by double labeling of CEM cells for CD4 and gap120, under conditions where the CD4 receptor was completely blocked.

 The presence of secondary binding sites was revealed in more than 95% of the cells.

 Likewise, the inventors identified two different binding sites on gp120.

 By using iodine, it was thus found that the binding activity of gap120 to CD4 could be completely suppressed while the binding activity to receptors inhibited by serum was only reduced by 30%.

 The binding of gp120 to cells was partially inhibited by an anti-CD4 antibody or by serum and these effects were found to be cumulative the combined use of antibodies and serum led to greater inhibition, but however incomplete, allowing the formation of a bridge between CD4 and RIS by the gpl20 molecules.

 The continued work of the inventors in this area has enabled them to characterize the RIS region involved in the link with gap120, thus providing specific means for inhibiting HIV infection or for detecting the presence of HIV when the infection has already produced or to characterize the immune response against HIV.

 The inventors have indeed demonstrated a strong interaction between the gap120 and the proteins of families of proteins with multiple transmembrane passages such as those present on the surface of human red blood cells or of lymphocyte cells, such as CEM.

 The proteins of the family of proteins of the band 3 type are taken as an example to illustrate the invention. They are known to be ubiquitous, which can be found in the plasma membrane, the Golgi or the mitochondria in various cells. The proteins of this family, hereinafter generally called the band 3 protein family, have a PM of the order of 102, 112 and 137 Kd in SDS-PAGE according to the SWISS-PROT bank on the INTERNET.

 The protein band 3 is a transmembrane protein which crosses the membrane 12 or more times and serves as anion exchange being very strongly positively charged. In fact, it carries on its extracellular surface 3 times more positive than negative charges, which is interpreted as an aid for the transport of anions (Jay et al. 1986 Amm. Rev. Biochem. 55: SM-538).

 The receptors or binding sites of the HIV gap120 are characterized in that they comprise at least part of the proteins of the family of the type of the band 3 protein, as present on the surface of human red blood cells.

 These receptors or binding sites are also characterized in that they are capable of interacting with the gap120, in the absence of human serum, at 37 ° C.

These receptors are erythrocyte in nature or are as present on cells infectable by
HIV like CEM CD4 +.

 It will be noted that the interaction of gpl20 with the band 3 protein is inhibited by serum.

 The invention relates to transmembrane proteins with several passages from the inside to the outside of the cell, in purified form, and the fragments thereof, in particular the regions outside relative to the membrane, especially the charged regions. positively, since these fragments and regions exhibit specific binding activity with respect to the gap 120 present on HIV.

 The term "protein", as used in the description and the claims, denotes a protein of the family of transmembrane proteins, in purified form, or in vesicle, or in reconstituted liposomes, as its fragments or regions as identified. above. It also covers the recombinant forms of this protein, for example fusion proteins, or the forms devoid of their intramembrane sequence.

 The invention also relates to polyclonal or monoclonal antibodies directed against this protein.

The fragments of these antibodies, in particular the fragments corresponding to the invariable Fab part, also come within the scope of the invention.

 The invention also relates to anti-antibody or anti-idiotype antibodies, characterized by their capacity to react, according to a reaction of the antigen-antibody type, with the antibodies defined above. Likewise, the invention covers the fragments of these antibodies.

 The polyclonal antibodies are obtained according to conventional methods by immunization of animals using the band 3 protein, fragments or regions thereof, recovery of the antibodies produced from the spleen cells, and purification.

 For the production of monoclonal antibodies, cultures of hybridomas obtained in a conventional manner are established, by fusion of spleen cells secreting antibodies with myeloma cells, the clones of secretory hybridomas are selected and injected into mice for produce tumor ascites from which the sought-after monoclonal antibodies are recovered and purified.

 The proteins and antibodies defined above are used, in accordance with the invention, for the development of detection means, therapeutic compositions or prophylactic compositions.

 The invention thus relates to compositions, methods and kits for detection or diagnosis characterized in that they comprise these proteins or these antibodies in an amount sufficient to characterize an HIV infection and, if necessary, to quantify the level of HIV in a sample to be analyzed or the immune response against HIV.

 In the detection or diagnostic compositions, the proteins or the antibodies advantageously comprise a marker allowing the revelation of an immunological reaction.

According to the invention, the method for detecting the presence of HIV or for detecting the immune response is characterized by
- bringing a sample to be analyzed into contact with the secondary receptor as defined above, capable of binding to the product of the HIV reaction
RIS that the sample may contain, or at the site
RIS or alternatively, with the protein defined above capable of binding to HIV, where the inhibition of the binding of viral proteins to the RIS will indicate the presence of antibodies capable of preventing the binding of the virus to its second receptor ,
- the revelation of the antigen-antibody reaction possibly produced.

 The contacting step is carried out under sufficient conditions, in particular as regards the duration, the temperature, the buffer, to obtain the fixation of HIV when it is present. The sample to be analyzed consists in particular of a bodily fluid such as blood, plasma, urine, saliva, cerebrospinal fluid, seminal fluid. The antibody or protein is in solution or fixed on a support.

 For revealing the reaction, labeling means are used, for example labeling with fluorescent agents such as fluorescein, enzymes such as peroxidase, or coloring agents.

 To detect the presence of HIV or anti-HIV antibodies for the purposes of studies or diagnosis, use will advantageously be made of a kit comprising the protein or the antibody, if necessary fixed on a microplate, in a sufficient quantity. for carrying out a test, the reagents and buffers to carry out the detection, as well as a user manual.

 According to another aspect, the invention relates to compositions which can be used in therapy comprising, in association with pharmaceutically acceptable vehicles, an effective amount of at least one antibody, or a protein, as defined above.

 Thus, the antibodies are used as inhibitory agents vis-à-vis the interaction with the HIV virus.

 The band 3 protein, or its fragments, is used as a decoy protein so as to compete with the transmembrane protein constituting the cells of the RIS site.

 Advantageously, the pharmaceutical compositions of the invention contain agents capable of protecting the antibodies or proteins administered against the action of cellular proteases.

 The compositions of the invention can also be used in combination with antiviral agents.

 For these therapeutic applications, the antibodies or the proteins are administered in galenical forms usable by intravenous or parenteral route.

 They can also be administered in the form of liposomes.

 In the examples which follow, other characteristics and advantages of the invention are reported.

The following products are used:
gal20: this is the glycosylated recombinant gap120 of HIV-1 / IIIB produced in baculovirus (Intracell, marketed by Neosystem, Strasbourg,
France).

Anti-gpl20 antibody: These antibodies are obtained after a first immunization with gap120 in the complete Freund's adjuvant, followed by hyperimmunizations every month, without adjuvant, at doses of 1 pg / kg. Anti-rabbit IgG antibodies coupled to peroxidase are sold by Amersham, Grande
Brittany.

Example 1: Fixation of the gap120 on human red cells
A) Dose response curve for the fixation of the gap120.

AB red cells from the Transfusion Center
Regional Sanguine were washed in PBS (3x) and incubated in Maxisorb R microtiter plates (NUNC) at 4 x 10 per well to make them adhere. The wells were then saturated with 3% BSA and after washing incubated with different doses of gp120 / HIV1 in PBS-BSA 3% for 1 h at 371. After 3 washes in 0.3% PBS-BSA, the gap 120 was detected by an anti-gpl20 rabbit antiserum followed by an anti-rabbit Ig labeled with alkaline phosphatase and revealed by pNPP. The wells were evaluated in a 414 nm plate reader. The results obtained are illustrated in FIG. 1A which gives the variation of the OD as a function of increasing doses of gpl2O. A dose-dependent fixation of gpl20 is observed on human red blood cells. This fixation is visible in ELISA from a concentration of 0.25 pg / ml of gap120. It is linear up to a concentration of 4, ug / ml and reaches a saturation plateau for higher concentrations.

B) Inhibition by human serum
Red cells were adsorbed on plates and saturated as described above. To a single dose of 1 μg / ml of gp120 were added different concentrations of human serum or fetal calf serum for 1 h at 37 ° C. This mixture was then incubated with human red cells for 1 hour at 37 C. The washings and the revelation of gp120 were identical to the method described above for the alkaline phosphatase method. FIG. 1B gives the variation of the OD as a function of the concentration in human serum (curve and in fetal calf serum (curve-n,). There is an inhibition of the fixation of the gap 120, which reaches practically 100% in the presence of human serum.

EXAMPLE 2 Fixation of gp120 on purified transmembrane proteins of red blood cells
To demonstrate the nature of a erythrocyte receptor, two major transmembrane proteins of the membrane which are accessible on the surface of red blood cells, were studied with regard to their binding capacity for gp120:
a) glycophorin which carries 60% of the sugars in the red blood cells which are responsible for its negative charge and for the blood groups,
b) the band 3 protein, which alone represents more than 25% of the transmembrane proteins and serves as an anion exchanger.

 Purified glycophorin, obtained commercially, was fixed in increasing doses on MaxisorbE microtiter wells using a commercial anti-glycophorin mouse monoclonal antibody (DAKO) adsorbed beforehand on microtiter plates. It was incubated with recombinant gpl20 and revealed by an anti-rabbit gpl 20 antibody, then an anti-rabbit Ig coupled to peroxidase, followed by OPD. Fixation of gp120 is observed on the insolubilized glycophorin, but only with a low OD after 30 minutes of incubation with OPD.

This weak reaction cannot therefore explain the extent of the binding of gpl20 to red cells.

The system was also reversed by first insolubilizing gpl20 on an anti gap120 antibody. An anti-gpl20 antibody (Aalto Bioreagents,
Dublin, Ireland), directed against a peptide of its part
C-terminal, was previously adsorbed on Maxisorb plates, then incubated with the gap120. After a second incubation with glycophorin at different concentrations, its presence was revealed by the anti-glycophorin monoclonal antibody, then by a sheep anti-Ig coupled with peroxidase. Under these conditions, binding of the gap120 to glycophorin was observed, but it also remained very weak. These two approaches have therefore not shown an interaction of the gap120 with purified glycophorin which can sufficiently explain the fixation of the gap120 on red cells.

 In order to test the interaction of gpl20 with the second most abundant transmembrane protein in the red blood cell, the band 3 protein, this protein was purified from red blood cell ghosts according to WOLOSIN et al, 1977 J Biol. Chem.

252.2419-2427. This method has the particularity of maintaining this transmembrane protein in the erythrocyte membrane by removing most of the other transmembrane proteins or those associated with the membrane.

Starting from red blood cell ghosts, a large part of the proteins associated with the membrane was eliminated by an alkaline pH with low ionic strength. Then the glycophorin was removed using a mild detergent, octyl-glycopyranoside (3 x CMC). Other proteins which still remained associated with the inner membrane were removed by a short passage at pH 12 at low ionic strength. After these treatments, there remained vesicles, much smaller than the starting red cells which were harvested after centrifugation at 50,000 g and which were highly enriched in protein band 3 at approximately 3 mg / ml. There remained, however, contamination of a few other proteins with a high molecular weight, therefore rather localized inside the red blood cell vesicle.

 In order to verify, if these vesicles rich in band 3 protein could fix the gap 120, they were adsorbed on wells of MaxisorbE plates and, after saturation with BSA 3%, incubated with gpl20 which was revealed as described above. After insolubilization of the vesicles in decreasing concentrations, it was found that the gap120 fixed strongly on the vesicles at 3 mg / ml of protein band 3. The fixation decreases for lower concentrations and disappears at concentrations below 0.3 mg / ml of protein band 3.

EXAMPLE 3 Study of the fixation of the gap120 on CEM cells with CD4
A) Attaching gpl20 to EMF
In order to verify, if the gap120 restored a different CD4 receptor on CEM cells, these cells were adsorbed on microtitration plates using a mixture of monoclonal anticopros directed against CD5 and CD100. Gpl20 in increasing doses was then incubated with these living cells, adsorbed on microtitration plates using monoclonal antibodies. Its presence has been demonstrated as described above with a rabbit antibody, an anti-rabbit Ig-POD, followed by
OPD. Strong fixation of the gap120 on these cells with an accessible CD4 receptor was thus observed.

 B) Inhibition by human serum.

CEM cells (10 / well) are made to adhere to anti-CD5 and CD100 antibodies previously fixed on microtiter plates and saturated with
BSA 3%.

The CD4 was then blocked by incubation with the antibody F101.69 (10ug / ml), specially directed against the binding site of the gap120. Gpl20 was pre-incubated with different concentrations of sera (1h at 37C) and then the mixture incubated with the cells
EMF members (1h to 37C). The gpl20 was detected by a rabbit anti-gpl20 antiserum, followed by a rabbit anti-Ig labeled with peroxidase, revealed by the OPD and read at 492 nm. The results obtained are illustrated in FIG. 2A which gives the variation of the OD as a function of increasing concentrations in fetal calf serum (curve) and in human serum (curve). These experiments demonstrate the inhibitory effect of the serum. The inhibition is of the order of 90% with human serum.

 C) Inhibition of the binding of gpl2O / HIV on target cells by the protein band 3, enriched in red blood cell vesicles.

 Adherent and saturated CEM cells were blocked at the CD4 level as described above. The gap120 was previously incubated with the vesicles containing the band 3 protein (1 h at 37 ° C.) and then incubated with the blocked CEM cells. The prior contact of the gap120 with the band 3 protein at doses of approximately 1 mg / ml inhibited the binding of gpl20 to the RIS by approximately 75-80% and was dose dependent.

 Binding of the gap120 was detected as described above by ELISA with antibodies labeled with peroxidase. In FIG. 1B, the variation of the D.O. is reported as a function of increasing concentrations of protein band 3.

 A plateau is observed up to a concentration of 12 μg / ml of protein band 3, then there is a decrease in the OD being interpreted as the inhibition of gpl20 by the protein band 3.

The same experiments as those described above can be carried out in order to fix
- virus concentrated on a transmembrane protein such as that of the band 3 family,
- virus concentrated on red blood cells previously digested with proteolytic enzymes,
- virus concentrated on dried membranes of red blood cells.

 In each case, strong binding of gp120 is observed, this being able to be verified with functional HIV.

Claims (7)

 1. HIV gp120 receptors or binding sites characterized in that they comprise at least a portion of proteins from families of proteins with several transmembrane passages, such as proteins of the band 3 type as present on the surface of human red blood cells or lymphocyte cells, such as EMF, for example.  2. Receptors or binding sites according to claim 1, characterized in that they are capable of interacting with the gap120, at 37il.  3. Receptors or binding sites according to claim 1 or 2, characterized in that they are erythrocytic in nature or as present on the surface of cells infectable by HIV, such as CEM cells CD4 +.  4. Polyclonal antibodies and monoclonal antibodies directed against the receptors or binding sites according to any one of claims 1 to 3.  5. Applications of receptors or binding sites according to any one of claims 1 to 3 and antibodies according to claim 4 to the development of detection means.  6.- Applications of receptors or binding sites according to any one of claims 1 to 3 and antibodies according to claim 4 to the development of therapeutic compositions.  7. Applications of receptors or binding sites according to any one of claims 1 to 3 and antibodies according to claim 4 to the preparation of prophylactic compositions.