Process for the potentiation of immunotoxins

The present invention relates to a process for potentiating the activity of a conjugate consisting of the A chain of ricin coupled with an antibody directed against human T cells, the said process consisting in adding an effective quality of ammonium chloride to the said conjugate.

In U.S. Pat. No. 4 340 535, there is described the preparation of 
anticancer products, called conjugates, obtained by coupling the A chain 
of ricin, by means of a covalent bond, with a protein structure, such as 
an antibody, an immunoglobulin or an immunoglobulin fragment, capable of 
selectively recognizing a given antigen on the surface of the target 
carrier cells, such as cancer cells. The principal property of these 
conjugates, which are also called immunotoxins, is that they are specific 
cytotoxic agents for the intended target cells. 
The use of antibodies directed against cancer cell differentiation antigens 
had already made it possible to obtain conjugates exhibiting a 
considerable specificity towards the target cells. 
The present invention relates to a process for potentiating the activity of 
these conjugates, which consists in adding an effective quantity of 
ammonium chloride to the said conjugates. 
Conjugates are understood to mean artificial mixed molecules in which the A 
chain of ricin is associated, by a covalent bond of the disulfide type, 
with an antibody or an antibody fragment directed against human T cells, 
capable of selectively recognizing an antigen associated with target 
cells. 
The preparation of the pure A chain of ricin has been described in our 
earlier patents. The preparation of monoclonal antibodies directed against 
human T leukemia cells has been mentioned in the scientific literature. 
(Reference may be made in particular to Journal of Immunology 125 (2), 
725-731, (1980).) 
To prepare the conjugates, the proteins to be coupled must each carry at 
least one sulfur atom which is naturally capable or has been artificially 
rendered capable of creating the desired disulfide bond. 
The A chain of ricin naturally contains a single sulfur atom which permits 
the desired coupling. It belongs to the thiol group of the cysteine 
residue included in the A chain, which linked this A chain to the B chain 
in the complete toxin. 
The whole antibody whose specificity is directed against human T cells 
contains neither a free thiol group nor other sulfur atoms capable of 
being used for coupling. If this whole antibody is used, one or more 
sulfur atoms capable of being subsequently incorporated into the disulfide 
bond to be formed with one or more molecules of A chain of ricin will 
therefore have to be introduced artificially into the immunoglobulin 
molecule. 
If, on the other hand, the fragment Fab' of this antibody is used, as 
conventionally obtained by restricted proteolysis in the presence of 
pepsin and then reduction of the disulfide bridge (or bridges) between 
high-molecular chains, this fragment then possesses at least one thiol 
group available for creating the disulfide bridge with the A chain of 
ricin. In this latter case, according to the invention, the thiol group 
available on the antibody fragment will generally be converted to 
activated mixed disulfide by known methods before being reacted with the 
thiol of the A chain to create the desired disulfide group. 
According to the invention, if the whole antibody is used, the conjugate is 
prepared by bringing the A chain of ricin, carrying its free SH group, 
into contact with the antibody into which the SH group has been 
artificially introduced in the activated form and especially in the form 
of a mixed disulfide with a suitable organic sulfur-containing radical. 
The preparation of the conjugate can thus be represented by the equation: 
EQU RA-SH+AC-R-S-S-X.fwdarw.RA-S-S-R-AC+XSH 
in which: 
RA denotes the A chain of ricin 
AC denotes the antibody 
X denotes the activating radical. 
The antibody substituted by an activated sulfur atom is obtained from the 
antibody itself by substitution with a reagent (itself carrying an 
activated sulfur atom) according to the equation: 
EQU A+Y-R-S-S-X.fwdarw.AC-R-S-S-X 
in which: 
AC denotes the antibody 
Y represents a group permitting the covalent attachment of the reagent to 
the protein 
R denotes a group capable of carrying the substituents Y and -S-S-X 
simultaneously 
X denotes the activating radical. 
The functional group Y is a group capable of bonding covalently with any 
one of the groups carried by the side chains of the constituent amino 
acids of the protein to be substituted. Of these groups, the terminal 
amino groups of the lysyl radicals contained in the protein are especially 
indicated. In this case, Y may represent in particular: 
a carboxyl group which may bond to the amino groups of the protein in the 
presence of a coupling agent such as a carbodiimide and especially a 
water-soluble derivative such as 
1-ethyl-3-(3-diethylamino-propyl)carbodiimide, 
a carboxylic acid chloride which is capable of reacting directly with the 
amino groups to acylate them, 
a so-called "activated" ester, such as an ortho- or para-nitrophenyl or 
-dinitrophenyl ester or an N-hydroxysuccinimide ester, which reacts 
directly with the amine groups to acylate them, 
an internal anhydride of a dicarboxylic acid, for example succinic 
anhydride, which reacts spontaneously with the amine groups to create 
amide bonds, or 
an imidoester group: 
##STR1## 
in which R.sub.1 is an alkyl group reacting with the amine groups of the 
protein according to the equation: 
##STR2## 
The radical -S-S-X denotes an activated mixed disulfide capable of reacting 
with a free thiol radical. In particular, in this mixed disulfide, X may 
denote a pyridin-2-yl or pyridin-4-yl group optionally substituted by one 
or more alkyl, halogen or carboxyl radicals. X can also denote a phenyl 
group preferably substituted by one or more nitro or carboxyl groups. X 
can also represent an alkoxycarbonyl group such as the methoxycarbonyl 
group. 
The radical R denotes any radical capable of carrying the substituents Y 
and S-S-X simultaneously. It must be chosen so as not to contain groups 
which are liable to interfere, during the subsequent reactions, with the 
reagents used and the products synthesized. In particular, the group R can 
be a group --(CH.sub.2).sub.n, where n is between 1 and 10, or a group: 
##STR3## 
in which R.sub.4 denotes hydrogen or an alkyl group having from 1 to 8 
carbon atoms and R.sub.3 denotes a substituent which is inert towards the 
reagents subsequently used, such as a carbamate group: 
##STR4## 
in which R.sub.5 denotes a linear or branched alkyl group having from 1 to 
5 carbon atoms, especially the tert.-butyl group. 
The reaction of the compound Y-R-S-S-X with the immunoglobulin is carried 
out in the homogeneous liquid phase, most frequently in water or a buffer 
solution. If the solubility of the reagents requires it, it is possible 
for up to 20% by volume of a water-miscible organic solvent, such as an 
alcohol, especially tertiary butanol, to be added to the reaction medium. 
The reaction is carried out at room temperature for a period varying from a 
few hours to 24 hours. After this, the low-molecular products and in 
particular the excess reagents can be removed by dialysis. This process 
makes it possible to introduce between 1 and 5 substituent groups per mol 
of protein if the protein is a class G immunoglobulin or between 1 and 15 
if the protein is a class M immunoglobulin. 
When using such compounds, the coupling with the A chain of ricin is 
carried out by bringing the two proteins into contact in aqueous solution 
at a temperature not exceeding 30.degree. C. and for a period varying from 
a few hours to one day. The solution obtained is dialyzed to remove the 
low-molecular products and the conjugate can then be purified by a variety 
of known methods. 
By themselves, the conjugates prepared by the above process have a strong 
specific cytotoxic activity towards human T cell lines. They can therefore 
be used in human therapy to treat T leukemia or any other cancerous or 
non-cancerous complaint calling for the selective destruction of cells 
which would be sensitive to the conjugate prepared according to the 
invention. This can be envisaged in transplantations or in certain 
autoimmune diseases for reducing the activity of the T lymphocytes or this 
type of subpopulation of T lymphocytes. 
Ammonium chloride has a very substantial potentiating effect on the 
activity of these conjugates towards the corresponding target cells. It 
has in fact been observed that the presence of NH.sub.4 Cl considerably 
accelerates the kinetics of expression of the cytotoxicity of these 
conjugates. 
This kind of accelerating effect is of the greatest importance for all 
therapeutic applications because the speed of action of the drug is always 
a favorable factor in the efficacy of the treatment. 
It is possible to utilize this new property to enhance the therapeutic 
efficacy of the conjugate in the treatment of the diseases to which it is 
applied. This improvement can be exploited according to two different 
therapeutic schemes: 
(a) Samples of human bone marrow, taken especially from leukemia patients, 
can be treated in vitro with the conjugate in the presence of ammonium 
chloride. As this association has a very high and very specific cytotoxic 
efficacy, any cell carrying the antigen recognized by the conjugate, in 
particular any tumoral cell, can be removed from the marrow treated in 
this way. Furthermore, the patient can be treated by any appropriate 
therapy, such as radiotherapy and/or chemotherapy, at a supralethal dose, 
so as to remove the tumoral cells from his organism. Finally, the marrow 
purified in the manner indicated is transplanted back into the patient's 
organism in the form of an autologous graft to allow the reconstitution of 
the populations of blood cells destroyed by the supralethal treatment. 
(b) The application of the potentiating property of ammonium chloride to 
the patient in vivo can also be exploited to obtain the maximum efficacy 
of the conjugate. In fact, it has been shown that mice into which tumoral 
cells have previously been transplanted, and to which the conjugate is 
administered at the same time as 3 injections of 7 mg of ammonium chloride 
at 15-minute intervals, exhibit a better percentage survival rate and a 
better inhibition of tumor growth than control mice which have only 
received the conjugate. 
These conjugates are packaged for administration by injection. They can be 
used by themselves, or associated with ammonium chloride, or associated 
with another treatment for the cancerous complaint in question and, in 
particular, associated with other immunodepressant drugs so as to delay 
and weaken the natural immune reaction of the patient towards the protein 
foreign to his organism, which the conjugate represents. 
With the aim of eliminating all the cancer cells, the treatment must be 
carried out with a sufficient dose of conjugate and the duration of the 
treatment must be determined in each case according to the subject and the 
nature of the complaint to be treated. 
The examples which follow provide a clearer understanding of the invention 
without limiting its scope. 
The A chain of ricin used in each of the examples below was prepared and 
purified by the process described in French Pat. Nos. 78 27838 and 79 
24655, to which U.S. Pat. No. 4 340 535 corresponds. 
Moreover, a study performed by cytofluorometry made it possible to show 
that the anti-human T cell antibody used, the corresponding activated 
antibody and the conjugate of this antibody with the A chain of ricin had 
superimposable fluorescence histograms, allowing the assertion that the 
antibody had not undergone any significant degradation during the 
activation and coupling reactions to which it had been subjected and, in 
particular, that it was still capable, even within the conjugate, of 
recognizing the human T antigen against which it was directed. 
Furthermore, each conjugate obtained was studied for its biological 
properties and, more especially, its anticancer action.

EXAMPLE 1: 
Conjugate obtained by reacting an antihuman T cell antibody (antibody 
directed against the antigen T65), substituted by an activated disulfide 
group, with the A chain of ricin 
(a) Anti-human T cell antibody (or antibody T101) 
This antibody was obtained by the method described in Journal of Immunology 
125 (2), 725-731, (1980). 
It undergoes a final purification by dialysis against PBS buffer (10 mM of 
phosphate, 140 mM of sodium chloride, pH 7.4). 
(b) Activated anti-human T cell antibody 0.1 ml of a solution containing 
42.7 mg/ml of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is added to 
0.5 ml of a solution containing 14.2 mg/ml of 
3-(pyridin-2-yldisulfanyl)propionic acid in tert.-butanol and the mixture 
is left at room temperature for 3 minutes. 
180 .mu.l of the resulting solution are added to 5.6 ml of an antibody 
solution containing 3.6 mg/ml in PBS buffer. Incubation is allowed to 
proceed for 20 hours at 30.degree. C. 
The solution is then dialyzed continuously for 3 days against 21 liters of 
PBS buffer at 4.degree. C. This gives 16 mg of activated antibody at a 
concentration of 2.6 mg/ml. 
By spectrophotometric analysis at 343 nm of the pyridine-2-thione released 
by exchange with reduced glutathion, it is found that the antibody 
obtained carries 3.1 activating groups per mol of antibody. 
(c) Conjugate 
0.87 ml of a solution of A chain of ricin in PBS buffer (concentration 6.6 
mg/ml) is added to 4.6 ml of a solution of activated antibody in the same 
buffer (concentration 2.6 mg/ml, i.e. 12 mg of activated antibody) and 
incubation is carried out for 20 hours at 25.degree. C. 
The reaction mixture is chromatographed on a column of Sephadex G100 gel. 
In each fraction, the antibody concentration is determined by 
spectrophotometry at 280 nm and the A chain concentration is determined by 
its power to inhibit protein synthesis measured on an acellular system. 
The identical fractions containing the conjugate are combined to give 
about 11 mg of the conjugate at a concentration of 0.8 mg/ml. 
The analytical determinations performed make it possible to show that the 
solution contains 140 .mu.g/ ml of biologically active A chain, i.e. about 
1.1 mol of A chain per mol of antibody. 
(1) INHIBITION OF PROTEIN SYNTHESIS 
The fundamental biological property of the A chain of ricin is to inhibit 
protein synthesis in cells by degradation of the ribosomal subunit 60S. 
A cellular model was used here. This test measures the effect of the 
substances studied on the incorporation of .sup.14 C-leucine into cancer 
cells in culture. 
The cells used belong to the CEM cell line derived from a human T leukemia 
which carries the antigen T65. The cells are incubated in the presence of 
the substance to be studied, and then, when incubation has ended, the 
degree of incorporation of .sup.14 C-leucine by the cells treated in this 
way is measured. 
This measurement is made by a technique adapted from the one described in 
Journal of Biological Chemistry 1974, 249 (11), 3557-62, using the tracer 
.sup.14 C-leucine to determine the degree of protein synthesis. The 
radioactivity incorporated is determined here on the whole cells isolated 
by filtration. 
On the basis of these determinations, it is possible to draw the 
dose/effect curves, plotting, on the abscissa, the molar concentration of 
A chain in the substances studied, and, on the ordinate, the incorporation 
of .sup.14 C-leucine expressed as a percentage of the incorporation by 
control cells in the absence of any substance affecting protein synthesis. 
It is thus possible to determine, for each substance studied, the 
concentration which causes a 50% inhibition of incorporation of .sup.14 
C-leucine, or "50% inhibitory concentration" (IC.sub.50). 
FIG. 1 shows the curves obtained in the same experiment with ricin, with 
its free A chain and with the conjugate RT2 (a compound prepared according 
to Example 1), in the presence and absence of 10 mM ammonium chloride in 
the incubation medium. Even in the absence of ammonium chloride and after 
incubation for 48 h, it can be seen on this figure that the conjugate 
studied, RT2, has a strong cytotoxic activity (IC.sub.50 
=7.times.10.sup.-12 M) which is about 7000 times greater than that of the 
A chain of ricin. 
(2) - POTENTIATION OF THE ACTIVITY OF THE CONJUGATE RT2 BY AMMONIUM 
CHLORIDE 
FIG. 1 also shows that the presence of 10 mM ammonium chloride in the 
medium for incubating the cells with the conjugate RT2 very greatly 
increases--by a factor of about 80--the cytotoxic activity of the 
conjugate on the target cells. This potentiating effect is not obtained 
with ricin, with the A chain or with a conjugate which is non-specific for 
the cells studied. Thus, in the presence of ammonium chloride as a 
potentiator, the cytotoxic activity of the conjugate (IC.sub.50 
=8.5.times.10.sup.-14 M) becomes about 600,000 times higher than that of 
the A chain by itself and even potentially exceeds the activity of ricin, 
which has never been described for any conjugate between the A chain of 
ricin and any type of antibody. 
EXAMPLE 2: 
Conjugate obtained by reacting an anti-human T cell antibody (antibody 
directed against an antigen of MW=40 KD), substituted by an activated 
disulfide group, with the A chain of ricin 
A - Anti-human T cell antibody (or antibody T3-A1) 
This antibody, deposited at the ATCC under the number HB2, is an IgG1; it 
is directed against CD7 (in: Leucocyte typing, A. Bernard, L. Boumssell, 
C. Milstein J. Dausset and S. F. Schlossman editors, Springer Verlag, 
Berlin, 1984, ref. 1). 
This antibody was obtained by the method described in Proc. Natl. Acad. 
Sci. U.S.A., 1979, 76, 5829. It undergoes a final purification by dialysis 
against phosphate buffer (125 mM, pH 7.0). 
B - Activated anti-human T cell antibody 
94 mg of antibody T3-A1 are modified with 7.6 mg of pyridyldithiopropionic 
acid, activated beforehand by reaction with 4.4 mg of 
ethyldimethylaminopropylcarbodiimide, in a total volume of 13 ml of 
phosphate buffer (125 mM, pH 7.0) for 15 min at room temperature. The IgGs 
modified in this way are purified by dialysis against the same phosphate 
buffer (125 mM, pH 7.0) to remove the excess reagents. 
C - Conjugate (immunotoxin) 
82.5 mg of modified antibodies are incubated for 20 h at 25.degree. C. with 
28.4 mg of A chain of ricin. The reaction medium is then purified y 
chromatography on Sephadex G100. The immunotoxin is obtained in the first 
chromatography peak. 
D - Biological properties 
(1) Inhibition of protein synthesis in cells and potentiation of this 
effect 
The cytotoxicity is evaluated by measuring the incorporation of .sup.14 
C-leucine by the cells after incubation for 18 h at 37.degree. C. in the 
presence of known quantities of the immunotoxin studied, or reference 
cytotoxic substances, in the presence or absence of ammonium chloride as a 
potentiator. 
The results of the experiments performed are presented in the form of 
dose/effect curves, plotting, on the ordinate, the cytotoxic effect 
evaluated as indicated above and calculated in % of the value obtained on 
control cells in the absence of any cytotoxic substance, and, on the 
abscissa, the concentrations of the cytotoxic substances studied, 
expressed as molar concentrations of the toxic subunits of these 
substances (molar concentration of A chain of ricin). 
Ammonium chloride was tested at a concentration of 10 mM. A previous check 
had been carried out to ensure that NH.sub.4 Cl is not spontaneously 
cytotoxic to the cells employed at the concentrations indicated. 
FIG. 2 shows the respective results obtained on human T lymphoblastoid 
cells of the CEM line. 
The experimental conditions used and the symbols employed to characterize 
the curves obtained are indicated in the table below: 
______________________________________ 
A chain of ricin A 
ricin R 
A chain + NH.sub.4 Cl 10 mM 
AN 
ricin + NH.sub.4 Cl 10 mM 
RN 
______________________________________ 
FIG. 2 shows the effects of ammonium chloride on the inherent cytotoxicity 
of ricin and isolated A chain, taken as reference substances. The values 
of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are indicated in Table I. 
TABLE I 
______________________________________ 
on CEM cells 
Substances tested 
With NH.sub.4 Cl 10 mM 
Without activator 
______________________________________ 
Ricin 3.8 .multidot. 10.sup.-13 
2 .multidot. 10.sup.-12 
A chain 3.8 .multidot. 10.sup.-8 
2.2 .multidot. 10.sup.-7 
______________________________________ 
FIG. 3 shows the respective results obtained on the same CEM cells for: 
______________________________________ 
A chain of ricin A 
immunotoxin anti-T T 
immunotoxin anti-T + NH.sub.4 Cl 10 mM 
TN 
______________________________________ 
The figure shows the comparative potentiating effect of the ion 
NH.sub.4.sup.+ (10 mM) on the cytotoxicity of the immunotoxin anti-T 
towards cells of the CEM line. 
The values of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are recorded in Table II. 
TABLE II 
______________________________________ 
on CEM cells 
Molar concentrations corresponding to 50% inhibition of 
incorporation of the tracer 
IC.sub.50 of IC.sub.50 of immunotoxin 
non-potentiated 
potentiated 
Example 
Antibody immunotoxin by NH.sub.4 Cl 10 mM 
______________________________________ 
2 T3-A1 5 .multidot. 10.sup.-11 
7 .multidot. 10.sup.-13 
3 LAU A1 6 .multidot. 10.sup.-10 
2 .multidot. 10.sup.-11 
4 RFT2 4 .multidot. 10.sup.-11 
5 .multidot. 10.sup.-12 
5 8H8.1 3 .multidot. 10.sup.-11 
4 .multidot. 10.sup.-12 
6 8A6 3 .multidot. 10.sup.-10 
2 .multidot. 10.sup.-11 
7 Fab 8A6 4 .multidot. 10.sup.-10 
7 .multidot. 10.sup.-12 
8 6D9 3 .multidot. 10.sup.-10 
2 .multidot. 10.sup.-12 
9 Fab T101 3.5 .multidot. 10.sup.-10 
2.6 .multidot. 10.sup.-12 
10 F(ab').sub.2 T101 
5 .multidot. 10.sup.-11 
6 .multidot. 10.sup.-13 
______________________________________ 
The results obtained show that, in the CEM cell model, the potentiating 
effect of the ammonium ion is of the order of 71. This factor is 
considerably higher than those observed on ricin or isolated A chain. The 
result of this is that, in the presence of NH.sub.4 Cl, the specific 
cytotoxicity of the immunotoxin anti-T (3A1) towards its target, in the 
presence of NH.sub.4 Cl, is equivalent to that of ricin itself. 
Moreover, the ammonium ion has the remarkable properties of not only 
potentiating the activity but also increasing the selectivity of the 
immunotoxin. In fact, if the ratio of the IC.sub.50 values of the free A 
chain and the immunotoxin is taken as the criterion for selectivity of 
action of the immunotoxin, this ratio is 4,400 in the absence of activator 
and 54,000 in the presence of NH.sub.4 Cl. 
(2) Acceleration of the cytotoxicity kinetics 
The effect of ammonium chloride is not restricted to considerably 
increasing the cytotoxic activity of the immunotoxins; it also makes it 
possible very substantially to accelerate the kinetics of the 
immunotoxins, as demonstrated by the following experiment. 
By way of example, this experiment measured, as previously, the 
incorporation of radioactive tracer into the cells incubated with the 
immunotoxin, in the absence or presence of NH.sub.4 Cl 10 mM as a 
potentiator. 
This experiment was carried out on the cellular model consisting of the CEM 
human T lymphoblastoid line with the immunotoxin anti-T at a concentration 
of 10.sup.-8 M. The results are presented in FIG. 4. This figure shows the 
results obtained by plotting the percentage incorporation of .sup.14 
C-leucine (% of the control values) on the ordinate and the time in hours 
on the abscissa. 
It is seen that, in the absence of potentiation, the expression of the 
cytotoxicity is very slow, as shown in curve (a). The value T10, which is 
the time required to obtain a 90% reduction in the incorporation of the 
tracer, is of the order of 30 h. On the other hand, in the presence of 
NH.sub.4 Cl 10 mM, a considerable acceleration of the kinetics of 
expression of the cytotoxicity is apparent--curve (b)--since the value T10 
is only 3 h here. 
(3) Inhibition of the proliferation of stimulated human T lymphocytes 
In physiological and pathological situations, as in numerous experimental 
models, the T lymphocytes isolated from peripheral blood or from bone 
marrow have the property of responding to a variety of stimulations by 
proliferating. It is this proliferative response which we studied. 
By way of example, lymphocytes from human peripheral blood, purified by 
Ficoll gradient centrifugation, are incubated in the presence of known 
concentrations of immunotoxin or reference cytotoxic substance and a final 
concentration of 10 mM of ammonium chloride for 24 h at 37.degree. C. The 
cells are then washed and brought into contact with a mitogenic agent 
specific for human T cells, which consists of a mixture of 
phytohemagglutinin A (PHA) (Wellcome Ltd., 1% final concentration) and "T 
cell growth factor" (or TCGF or interleukine 2 or IL2) at a final 
concentration of 0.5 unit/ml. The residual cells capable of proliferating 
are analyzed 72, 96 and 120 h after the cytotoxic treatment has ended by 
means of indirect immunofluorescence using a flux cytofluorometer (FACS IV 
Becton Dickinson). 
The results are presented in FIG. 8 (curve IT 3A1). 
EXAMPLE 3: 
Conjugate obtained by reacting an anti-human T cell antibody (antibody 
directed against an antigen of MW =40 KD), substituted by an activated 
disulfide group, with the A chain of ricin 
A - Anti-human T cell antibody (or antibody LAU A1) 
This antibody, which is directed against CD7 (ref. 1), is an IgG2. It was 
obtained by the method described in Molecular Immunology 21 (10), 831-840, 
1984. It undergoes a final purification by dialysis against phosphate 
buffer (125 mM, pH 7.0). 
B - Activated anti-human T cell antibody 
32.5 mg of antibody LAU A1 are modified with 2.1 mg of 
pyridyldithiopropionic acid, activated before-hand with 1.25 mg of 
ethyldimethylaminopropylcarbodiimide, in a total volume of 5 ml of 
phosphate buffer (0.125 M, pH 7.0) for 15 min at room temperature. The 
IgGs modified in this way are purified by dialysis against the same 
phosphate buffer (125 mM, pH 7.0) to remove the excess reagents. 
C - Conjugate (immunotoxin) 
27.6 mg of modified antibodies are incubated for 5 h at 25.degree. C. with 
11.0 mg of A chain of ricin. The reaction medium is then purified by 
chromatography on Sephadex G100. The immunotoxin is obtained in the first 
chromatography peak. 
Moreover, a study performed by cytofluorometry made it possible to show 
that the anti-human T cell antibody used, the corresponding activated 
antibody and the conjugate of this antibody with the A chain of ricin had 
superimposable fluorescence histograms, allowing the assertion that the 
antibody had not undergone any significant degradation during the 
activation and coupling reactions to which it had been subjected and, in 
particular, that it was still capable, even within the conjugate, of 
recognizing the human T antigen against which it was directed. 
The conjugate according to the invention, obtained above, was studied for 
its biological properties and, more especially, its anticancer action. 
D - Biological properties 
(1) Inhibition of protein synthesis in cells and potentiation of this 
effect 
The study is performed according to D(1) of Example 2 of the present patent 
application. 
The values of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are indicated in Table II. 
They show that the potentiating effect of the ammonium ion is 30 and that 
it is greater than that of the same ion on isolated A chain. 
(2) Acceleration of the cytotoxicity kinetics 
Following the procedure described in Example 2, it is seen that, in the 
absence of potentiation, the expression of the cytotoxicity is very slow. 
The value T10, which is the time required to obtain a 90% reduction in the 
incorporation of the tracer, is of the order of 20 h. On the other hand, 
in the presence of NH.sub.4 Cl 10 mM, a considerable acceleration of the 
kinetics of expression of the cytotoxicity is apparent since the value T10 
is only 2 h here. 
(3) Inhibition of the proliferation of stimulated human T lymphocytes 
The studies are performed according to D(3) of Example 2 of the present 
patent and the results are expressed in FIG. 8 (curve IT A1). 
EXAMPLE 4: 
Conjugate obtained by reacting an anti-human T cell antibody (antibody 
directed against an antigen of MW=40 KD), substituted by an activated 
disulfide group, with the A chain of ricin 
A - Anti-human T cell antibody (or antibody RFT2) 
This antibody, which is directed against CD7 (ref. 1), is an IgG2a. It was 
obtained by the method described in Leucocyte typing: A. Bernard, L. 
Boumssell, J. Dausset, C. Milstein, S. F. Schlossman editors, Springer 
Verlag, 1984, 469-475 Ref. 3. It undergoes a final purification by 
dialysis against phosphate buffer (125 mM, pH 7.0). 
B - Activated anti-human T cell antibody 
10 mg of antibody RFT2 are modified with 0.65 mg of pyridyldithiopropionic 
acid, activated beforehand with 0.38 mg of 
ethyldimethylaminopropylcarbodiimide, in a total volume of 3.5 ml of 
phosphate buffer (0.125 M, pH 7.0) for 15 min at room temperature. The 
IgGs modified in this way are purified by dialysis against the same 
phosphate buffer (125 mM, pH 7.0) to remove the excess reagents. 
C - Conjugate (immunotoxin) 
6.8 mg of modified antibodies are incubated for 17 h at 25.degree. C. with 
3.4 mg of A chain of ricin. The reaction medium is then purified by 
chromatography on Sephadex G100. The immunotoxin is obtained in the first 
chromatography peak. 
D - Biological properties 
(1) Inhibition of protein synthesis in cells and potentiation of this 
effect 
The study is performed according to D(1) of Example 2 of the present patent 
application. 
The values of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are indicated in Table II. These 
values show that the potentiating effect of the ammonium ion is 10. 
Moreover, it increases especially the selectivity of the immunotoxin. In 
fact, if the ratio of the IC.sub.50 values of the isolated A chain and the 
immunotoxin is taken as the criterion for selectivity of action of the 
immunotoxin, this ratio is 5,500 in the absence of activator and 7,600 in 
the presence of NH.sub.4 Cl. 
(2) Acceleration of the cytotoxicity kinetics 
Following the procedure described in Example 2, section D(2), the 
expression of the cytotoxicity is seen to be very slow in the absence of 
potentiation. The value T10, which is the time required to obtain a 90% 
reduction in the incorporation of the tracer, is more than 100 h. On the 
other hand, in the presence of NH.sub.4 Cl 10 mM, a considerable 
acceleration of the kinetics of expression of the cytotoxicity is apparent 
since the value T10 is only 4 h here. 
EXAMPLE 5: 
Conjugate obtained by reacting an anti-human T cell antibody (antibody 
directed against an antigen of MW=40 KD), substituted by an activated 
disulfide group, with the A chain of ricin 
A - Anti-human T cell antibody (or antibody 8H8.1) 
This antibody, which is directed against CD7 (ref. 1), is an IgG2a. It was 
obtained by the method described by C. Mawas, Marseille Luminy, France. It 
undergoes a final purification by dialysis against phosphate buffer (125 
mM, pH 7.0). 
B - Activated anti-human T cell antibody 
20 mg of antibody 8H8.1 are modified with 1.3 mg of pyridyldithiopropionic 
acid, activated beforehand by reaction with 0.77 mg of 
ethyldimethylaminopropylcarbodiimide, in a total volume of 8.7 ml of 
phosphate buffer (0.125 M, pH 7.0) for 15 min at room temperature. The 
IgGs modified in this way are purified by dialysis against the same 
phosphate buffer (125 mM, pH 7.0) to remove the excess reagents. 
C - Conjugate (immunotoxin) 
18.2 mg of modified antibodies are incubated for 5 h at 25.degree. C. with 
8.2 mg of A chain of ricin. The reaction medium is then purified by 
chromatography on Sephadex G100. The immunotoxin is obtained in the first 
chromatography peak. 
D - Biological properties 
(1) Inhibition of protein synthesis in cells and potentiation of this 
effect 
The study is performed according to D(1) of Example 2 of the present 
application. 
The values of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are indicated in Table II. 
(2) Acceleration of the cytotoxicity kinetics 
Following the procedure of Example 2, section D(2), of the application, it 
is seen that, in the absence of potentiation, the expression of the 
cytotoxicity is very slow. The value T10, which is the time required to 
obtain a 90% reduction in the incorporation of the tracer, is of the order 
of 16 h. On the other hand, in the presence of NH.sub.4 Cl 10 mM, a 
considerable acceleration of the kinetics of expression of the 
cytotoxicity is apparent since the value T150 is only 3 h 30 min here. 
EXAMPLE 6: 
Conjugate obtained by reacting an anti-human T cell antibody (antibody 
directed against an antigen of MW=40 KD), substituted by an activated 
disulfide group, with the A chain of ricin 
A - Anti-human T cell antibody (or antibody 8A6) 
This antibody, which is directed against CD7 (ref. 1), is an IgG1. It was 
obtained by D. Carriere, Centre de Recherche Clin-Midy/Sanofi, 
Montpellier, France, in the following manner: 4 weeks after the 
immunization of Balb/c mice with 10.sup.7 cells of the CEM human T 
lymphoblastoid line by intraperitoneal administration, a booster is 
administered intravenously with the same number of immunizing cells. Three 
days after the booster, the spleen cells of the immunized mice are fused 
with myeloma cells of the NS2 murine line in the presence of PEG 40%. The 
clone F938A6 was selected because of its specificity for human T cells. 
This purified antibody undergoes a final dialysis against phosphate buffer 
(125 mM, pH 7.0). 
B - Activated anti-human T cell antibody 
20 mg of antibody 8A6 are modified with 0.5 mg of pyridyldithiopropionic 
acid, activated beforehand with 0.3 mg of 
ethylmethylaminopropylcarbodiimide, in a total volume of 3.03 ml of 
phosphate buffer (0.125 M, pH 7.0) for 30 min at room temperature. The 
IgGs modified in this way are purified by dialysis against the same 
phosphate buffer (125 mM, pH 7.0) to remove the excess reagents. 
C - Conjugate (immunotoxin) 
6.47 mg of modified antibodies are incubated for 20 h at 25.degree. C. with 
5.52 mg of A chain of ricin. The reaction medium is then purified by 
chromatography on Sephadex G100. The immunotoxin is obtained in the first 
chromatography peak. 
D - Biological properties 
(1) Inhibition of protein synthesis in cells and potentiation of this 
effect 
The study is performed according to D(1) of Example 2 of the present 
application. 
The values of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are recorded in Table II. They 
show that the potentiating effect of the ammonium ion is 15 on the CEM 
cells. 
(2) Inhibition of the proliferation of stimulated human T lymphocytes 
The studies are performed according to D(3) of Example 2 of the present 
application. 
The results are presented in FIG. 8 (curve IT 8A6). 
EXAMPLE 7: 
Conjugate obtained by reacting an anti-human T cell antibody fragment 
(antibody fragment directed against an antigen of MW=40 KD). substituted 
by an activated disulfide group, with the A chain of ricin 
A - Anti-human T cell antibody fragment (or Fab 8A6) 
The antibody fragment 8A6, or Fab 8A6, was obtained from the antibody 8A6 
described in Example 6 above. 100 mg of antibody 8A6 are hydrolyzed with 
1 mg of papain for 3 h at 37.degree. C. in the presence of cysteine 10 mM 
and EDTA 1 mM. The reaction is stopped with iodoacetamide 20 mM for 1 h at 
37.degree. C. and the reaction medium is dialyzed against phosphate buffer 
(10 mM, pH 7.0). The reaction medium is chromatographed on DEAE trisacryl. 
The antibody fragments 8A6 are recovered in the filtrate. 
B - Activated anti-human T cell antibody fragment 
40 mg of Fab 8A6 are modified with 2.01 mg of SPDP (N-hydroxysuccinimide 
ester of pyridin-2-yldithiopropionic acid) in a total volume of 7.5 ml of 
phosphate buffer (0.125 M, pH 7.0) for 30 min at room temperature. The 
IgGs modified in this way are purified by dialysis against the same 
phosphate buffer (125 mM, pH 7.0) to remove the excess reagents. 
C - Conjugate (immunotoxin) 
29 mg of modified antibody fragments 8A6 are incubated for 17 h at 
25.degree. C. with 55 mg of A chain of ricin. The reaction medium is then 
purified by chromatography on Biogel P100. The immunotoxin is obtained in 
the first chromatography peak. 
D - Biological properties 
(1) Inhibition of protein synthesis in cells and potentiation of this 
effect 
The study is performed according to D(1) of Example 2 of the present 
application. 
The values of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are recorded in Table II. These 
values show that the potentiating effect of the ammonium ion is 57. 
Moreover, it increases especially the selectivity of the immunotoxin. In 
fact, if the ratio of the IC.sub.50 values of the isolated A chain and the 
immunotoxin is taken as the criterion for selectivity of action of the 
immunotoxin, this ratio is 550 in the absence of activation and 5,400 in 
the presence of NH.sub.4 Cl. 
EXAMPLE 8: 
Conjugate obtained by reacting an anti-human T cell antibody (antibody 
directed against an antigen of MW=65 KD), substituted by an activated 
disulfide group, with the A chain of ricin 
A - Anti-human T cell antibody (or antibody 6D9) 
This antibody, which is directed against CD5 (ref. 1), is an IgG2a. It was 
obtained by D. CarrieCentre de Recherche Clin-Midy/Sanofi, as for Example 
6 of the present patent application. 
The clone F936D9 was selected because of its specificity for human T cells. 
This purified antibody underwent a final dialysis against a phosphate 
buffer (125 mM, pH 7.0). 
B - Activated anti-human T cell antibody 
31 mg of antibody 6D9 are modified with 0.033 mg of SPDP in a total volume 
of 1.270 ml of buffer (pH 9.0) for 15 min at room temperature. The IgGs 
modified in this way are purified by dialysis against the same buffer (pH 
9.0) to remove the excess reagents. 
C - Conjugate (immunotoxin) 2.1 mg of modified antibodies are incubated for 
17 h at 25.degree. C. with 2 mg of A chain of ricin. The reaction medium 
is then purified by chromatography on Sephadex G100. The immunotoxin is 
obtained in the first chromatography peak. 
D - Biological properties 
(1) Inhibition of protein synthesis in cells and potentiation of this 
effect 
The study is performed according to D(1) of Example 2 of the present patent 
application. 
The values of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are indicated in Table II. These 
values show that the potentiating effect of the ammonium ion is 150. This 
factor is much higher than that observed for ricin or the isolated A 
chain. Moreover, the ammonium ion has the remarkable property of 
increasing the selectivity of the immunotoxin. In fact, if the ratio of 
the IC.sub.5O values of the A chain and the immunotoxin is taken as the 
criterion for selectivity of action of the immunotoxin, this ratio is 730 
in the absence of activator and 19,000 in the presence of NH.sub.4 Cl. 
EXAMPLE 9: 
Conjugate obtained by reacting an anti-human T cell antibody fragment 
(antibody fragment directed against an antigen of MW=65 KD), substituted 
by an activated disulfide group, with the A chain of ricin 
A - Anti-human T cell antibody fragment (or Fab T101) 
The antibody fragment T101, or Fab T101, was obtained from the antibody 
T101 described in French Patent Application 81 21 836. 
100 mg of antibody T101 are hydrolyzed with 1 mg of papain for 3 h at 
37.degree. C. in the presence of cysteine 10 mM and EDTA 1 mM. The 
reaction is stopped with iodoacetamide 20 mM for 1 h at 37.degree. C. and 
the reaction medium is dialyzed against a phosphate buffer (10 mM, pH 
7.0). The reaction medium is chromatographed on DEAE trisacryl and the 
antibody fragments T101 are recovered in the filtrate. 
B - Activated anti-human T cell antibody 
15.3 mg of Fab T101 are modified with 1.740 mg of SPDP in a total volume of 
3.06 ml of phosphate buffer (0.125 M, pH 7.0) for 30 min at room 
temperature. The antibody fragments modified in this way are purified by 
dialysis against the same phosphate buffer (125 mM, pH 7.0) to remove the 
excess reagents. 
C - Conjugate (immunotoxin) 
12.7 mg of modified antibody fragments T101 are incubated for 17 h at 
25.degree. C. with 17 mg of A chain of ricin. The reaction medium is then 
purified by chromatography on ACA 44. The immunotoxin is obtained in the 
first chromatography peak. 
D - Biological properties 
(1) Inhibition of protein synthesis in cells and potentiation of this 
effect 
The study is performed according to D(1) of Example 2 of the present patent 
application. 
The values of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are recorded in Table II. These 
values show that the potentiating effect of the ammonium ion is 134. This 
factor is much higher than that observed for ricin or the isolated A 
chain. Moreover, the ammonium ion has the remarkable property of 
increasing the selectivity of the immunotoxin. In fact, if the ratio of 
the IC.sub.50 values of the A chain and the immunotoxin is taken as the 
criterion for selectivity of action of the immunotoxin, this ratio is 628 
in the absence of activator and 14,600 in the presence of NH.sub.4 Cl. 
EXAMPLE 10: 
Conjugate obtained by reacting an anti-human T cell antibody fragment 
(antibody directed against an antigen of MW=65 KD), substituted by an 
activated disulfide group, with the A chain of ricin 
A - Anti-human T cell antibody fragments (or F(ab').sub.2 T101) 
The antibody fragments T101, or F(ab')2 T101, were obtained from the 
antibody T101 described in French Patent Application 81 21 836. 
61.4 mg of antibody T101 are dialyzed against a sodium formate buffer (100 
mM, pH 3.5) and then hydrolyzed with pepsin (5% by weight of antibody) for 
1 h 30 min at 37.degree. C. The reaction is stopped by bringing the pH of 
the solution to 7.0 with Tris buffer 1 M. The reaction medium is purified 
by filtration on a column of Sephadex G100. 12.5 mg of F(ab').sub.2 are 
collected in the filtrate. 
B - Activated anti-human T cell antibody fragment 
5 mg of F(ab').sub.2 T101 are modified with 0.135 mg of SPDP for 30 min at 
25.degree. C. The IgGs modified in this way are purified by dialysis 
against the same phosphate buffer (125 mM, pH 7.0) to remove the excess 
reagents. 
C - Conjugate (immunotoxin) 
3.96 mg of modified antibody fragments T101 are incubated for 18 h at 
30.degree. C. with 3.24 mg of A chain of ricin. The reaction medium is 
then purified by chromatography on Sephadex G100. The immunotoxin is 
obtained in the first chromatography peak. 
D - Biological properties 
(1) Inhibition of protein synthesis in cells and potentiation of this 
effect 
The study is performed according to D(1) of Example 2 of the present patent 
application. 
The values of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are recorded in Table II. These 
values show that the potentiating effect of the ammonium ion is 83. 
Moreover, the ammonium ion has the remarkable property of increasing the 
selectivity of the immunotoxin. In fact, if the ratio of the IC.sub.50 
values of the A chain and the immunotoxin is taken as the criterion for 
selectivity of action of the immunotoxin, this ratio is 4,400 in the 
absence of activator and 63,000 in the presence of NH.sub.4 Cl. 
EXAMPLE 11: 
Conjugate obtained by reacting an anti-human T cell antibody (antibody 
directed against an antigen of MW=50 KD), substituted by an activated 
disulfide group, with the A chain of ricin 
A - Anti-human T cell antibody (or antibody RFT11) 
This antibody, which is directed against CD2 (ref. 1), is an IgG1. It was 
obtained by the method described in ref.3. It undergoes a final 
purification by dialysis against phosphate buffer (125 mM, pH 7.0). 
B - Activated anti-human T cell antibody 
19 mg of antibody RFT11 are modified with 1.23 mg of pyridyldithiopropionic 
acid, activated beforehand by reaction with 0.73 mg of 
ethyldimethylaminopropylcarbodiimide, in a total volume of 3 ml of 
phosphate buffer (125 mM, pH 7.0) for 15 min at room temperature. The IgGs 
modified in this way are purified by dialysis against the same phosphate 
buffer (125 mM, pH 7.0) to remove the excess reagents. 
C - Conjugate (immunotoxin) 
15.9 mg of modified antibodies are incubated for 5 h at 25.degree. C. with 
6.4 mg of A chain of ricin. The reaction medium is then purified by 
chromatography on Sephadex G100. The immunotoxin is obtained in the first 
chromatography peak. 
Moreover, a study performed by cytofluorometry made it possible to show 
that the anti-human T cell antibody used, the corresponding activated 
antibody and the conjugate of this antibody with the A chain of ricin had 
superimposable fluorescence histograms, allowing the assertion that the 
antibody had not undergone any significant degradation during the 
activation and coupling reactions to which it had been subjected and, in 
particular, that it was still capable, even within the conjugate, of 
recognizing the human T antigen against which it was directed. 
The conjugate according to the invention, obtained above, was studied for 
its biological properties and, more especially, its anticancer action. 
D - Biological properties 
(1) Inhibition of protein synthesis in cells and potentiation of this 
effect 
The cytotoxicity is evaluated by measuring the incorporation of .sup.14 
C-leucine by the cells after incubation for 18 h at 37.degree. C. in the 
presence of known quantities of the immunotoxin studied, or reference 
cytotoxic substances, in the presence or absence of ammonium chloride as a 
potentiator. 
The results of the experiments performed are presented in the form of 
dose/effect curves, plotting, on the ordinate, the cytotoxic effect 
evaluated as indicated above and calculated in % of the value obtained on 
control cells in the absence of any cytotoxic substance, and, on the 
abscissa, the concentrations of the cytotoxic substances studied, 
expressed as molar concentrations of the toxic subunits of these 
substances. 
Ammonium chloride was tested at a concentration of 10 mM. A previous check 
had been carried out to ensure that NH.sub.4 Cl is not spontaneously 
cytotoxic to the cells employed at the concentrations indicated. 
FIG. 5 shows the respective results obtained on human T lymphoblastoid 
cells of the P12/ICHIKAWA line, carrying the target antigen. 
The experimental conditions used and the symbols employed to characterize 
the curves obtained are indicated in the table below: 
______________________________________ 
A chain of ricin A 
ricin R 
A chain + NH.sub.4 Cl 10 mM 
AN 
ricin + NH.sub.4 Cl 10 mM 
RN 
______________________________________ 
FIG. 5 shows the effects of ammonium chloride on the inherent cytotoxicity 
of ricin and isolated A chain, taken as reference substances. The values 
of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are indicated in Table III. 
TABLE III 
______________________________________ 
on P12/ICHIKAWA cells 
Substances tested 
With NH.sub.4 Cl 10 mM 
Without activator 
______________________________________ 
Ricin 3 .multidot. 10.sup.-13 
3.7 .multidot. 10.sup.-13 
A chain 7.5 .multidot. 10.sup.-8 
2.5 .multidot. 10.sup.-7 
______________________________________ 
FIG. 6 shows the respective results obtained on P12/ICHIKAWA cells for: 
______________________________________ 
A chain of ricin A 
immunotoxin anti-T T 
immunotoxin anti-T + NH.sub.4 Cl 10 mM 
TN 
______________________________________ 
The figure shows the potentiating effect of the ion NH.sub.4.sup.+ (10 mM) 
on the cytotoxicity of the immunotoxin anti-T towards cells of the 
P12/ICHIKAWA line. 
The values of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are recorded in Table IV. 
TABLE IV 
______________________________________ 
on P12/ICHIKAWA cells 
Molar concentrations corresponding to 50% inhibition of 
incorporation of the tracer 
IC.sub.50 of immunotoxin 
Exam- IC.sub.50 of non-potentiated 
potentiated by 
ple Antibody immunotoxin NH.sub.4 Cl 10 mM 
______________________________________ 
11 RFT11 4.5 .multidot. 10.sup.-11 
1.7 .multidot. 10.sup.-11 
12 IIB5 2.2 .multidot. 10.sup.-10 
5.5 .multidot. 10.sup.-11 
13 RL1T11 2.1 .multidot. 10.sup.-10 
1.5 .multidot. 10.sup.-11 
______________________________________ 
(2) Acceleration of the cytotoxicity kinetics 
The effect of potentiating substances is not restricted to increasing the 
cytotoxic activity of the immunotoxins; it also makes it possible very 
substantially to accelerate the kinetics of the immunotoxins, as 
demonstrated by the following experiment. 
By way of example, this experiment measured, as previously, the 
incorporation of radioactive tracer into the cells incubated with the 
immunotoxin, in the absence or presence of NH.sub.4 Cl 10 mM as a 
potentiator. 
This experiment was carried out on the cellular model consisting of the 
P12/ICHIKAWA human T lymphoblastoid line with the immunotoxin anti-T at a 
concentration of 10.sup.-8 M. The results are presented in FIG. 7. This 
figure shows the results obtained by plotting the percentage incorporation 
of .sup.14 C-leucine (% of the control values) on the ordinate and the 
time in hours on the abscissa. 
It is seen that, in the absence of potentiation, the expression of the 
cytotoxicity is very slow, as shown in curve (a). The value T10, which is 
the time required to obtain a 90% reduction in the incorporation of the 
tracer, is greater than 100 h. On the other hand, in the presence of 
NH.sub.4 Cl 10 mM, a considerable acceleration of the kinetics of 
expression of the cytotoxicity is apparent--curve (b) --since the value 
T10 is only 14 h 30 min here. 
(3) Inhibition of the proliferation of stimulated human T lymphocytes 
The studies are performed according to D(3) of Example 2 of the present 
patent and the results are shown in FIG. 8. 
EXAMPLE 12: 
Conjugate obtained by reacting an anti-human T cell antibody (antibody 
directed against an antigen of MW=50 KD), substituted by an activated 
disulfide group, with the A chain of ricin 
A - Anti-human T cell antibody (or antibody IIB5) 
This antibody, which is directed against CD2 (ref. 1) is an IgG2a. It was 
obtained by the method described by S. Carrel, Lausanne, Switzerland. It 
undergoes a final purification by dialysis against phosphate buffer (125 
mM, pH 7.0). 
B - Activated anti-human T cell antibody 
8 mg of antibody IIB5 are modified with 0.64 mg of pyridyldithiopropionic 
acid, activated beforehand by reaction with 0.38 mg of 
ethyldimethylaminopropylcarbodiimide, in a total volume of 4 ml of 
phosphate buffer (125 mM, pH 7.0) for 15 min at room temperature. The IgGs 
modified in this way are purified by dialysis against the same phosphate 
buffer (125 mM, pH 7.0) to remove the excess reagents. 
C - Conjugate (immunotoxin) 
8 mg of modified antibodies are incubated for 24 h at 25.degree. C. with 
3.4 mg of A chain of ricin. The reaction medium is then purified by 
chromatography on Sephadex G100. The immunotoxin is obtained in the first 
chromatography peak. 
D - Biological properties 
(1) Inhibition of protein synthesis in cells and potentiation of this 
effect 
This study is performed in a manner identical to D(1) of Example 11 of the 
present patent application. 
The values of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are indicated in Table IV. 
The ammonium ion has the remarkable property of increasing the selectivity 
of the immunotoxin. In fact, if the ratio of the IC.sub.50 values of the A 
chain and the immunotoxin is taken as the criterion for selectivity of 
action of the immunotoxin, this ratio is 1,000 in the absence of activator 
and 6,900 in the presence of NH.sub.4 Cl. 
(2) Inhibition of the proliferation of stimulated human T lymphocytes 
The studies are performed according to D(3) of Example 2 of the present 
patent and the results are shown in FIG. 8 (curve IT IIB5). 
EXAMPLE 13: 
Conjugate obtained by reacting an anti-human T cell antibody (antibody 
directed against an antigen of MW=50 KD), substituted by an activated 
disulfide group, with the A chain of ricin 
A - Anti-human T cell antibody (or antibody RL1T11) 
This antibody, which is directed against CD2, is an IgG2a. It was obtained 
by J. C. Laurent (Centre de Recherche Clin-Midy/Sanofi - Montpellier - 
France) in the following manner: 4 weeks after the immunization of Balb/c 
mice with 10.sup.7 cells of the P12/ICHIKAWA human T lymphoblastoid line 
by intraperitoneal administration, a booster is administered intravenously 
with the same number of immunizing cells. Three days after the booster, 
the spleen cells of the immunized mice are fused with myeloma cells of the 
X63 Ag 8.653 murine line in the presence of PEG 40%. The clone 3A11 was 
selected because of its specificity for human T cells. This purified 
antibody undergoes a final dialysis against phosphate buffer (125 mM, pH 
7.0). 
B - Activated anti-human T cell antibody 
3.25 mg of antibody RL1T11 are modified with 0.04 mg of SPDP for 30 min at 
room temperature in a total volume of 1.5 ml of buffer, pH 9.0. The IgGs 
modified in this way are purified by dialysis against the same buffer, pH 
9.0, to remove the excess reagents. 
C - Conjugate (immunotoxin) 
1.87 mg of modified antibodies are incubated for 17 h at 25.degree. C. with 
0.94 mg of A chain of ricin. The reaction medium is then purified by 
chromatography on Sephadex G100. The immunotoxin is obtained in the first 
chromatography peak. 
D - Biological properties 
(1) Inhibition of protein synthesis in cells and potentiation of this 
effect 
The study is performed according to D(1) of Example 11 of the present 
patent application. 
The values of the molar concentrations corresponding to a 50% inhibition of 
incorporation of the tracer (IC.sub.50) are recorded in Table IV. 
The ammonium ion increases the selectivity of the immunotoxin. In fact, if 
the ratio of the IC.sub.50 values of the isolated A chain and the 
immunotoxin is taken as the criterion for selectivity of action of the 
immunotoxin, this ratio is 1,000 in the absence of activator and 2,500 in 
the presence of NH.sub.4 Cl. 
EXAMPLE 14: 
Association of several immunotoxins for the allografting of bone marrow 
and/or immunosuppression in vivo for the treatment of certain autoimmune 
diseases and/or the treatment in vivo of malignant T proliferations and/or 
for the autographing of bone marrow in the case of patients suffering from 
malignant T proliferation 
(1) Anti-human T cell antibodies and immunotoxins 
Immunotoxin anti-CD5 or IT anti-T65 or IT T101 described in French Patent 
Application No. 81 21 836 
Immunotoxin anti-CD7 or IT 3A1 described in Example 2 
Immunotoxin anti-CD7 or IT A1 described in Example 3 
Immunotoxin anti-CD2 or IT RFT11 described in Example 11 
Immunotoxin anti-CD2 or IT IIB5 described in Example 12 
Immunotoxin anti-CD7 or IT 8A6 described in Example 6 
(2) Cytotoxic properties of the association of several immunotoxins for the 
depletion of stimulated human T cells 
The association of several immunotoxins directed against different T cell 
antigens must make it possible, according to these associations, to 
destroy all or part of the T lymphoid population. 
In physiological and pathological situations, as in numerous experimental 
models, the T lymphocytes isolated from peripheral blood or from bone 
marrow have the property of responding to a variety of stimulations by 
proliferating. It is this proliferative response which we studied. 
By way of example, lymphocytes from human peripheral blood, purified by 
Ficoll gradient centrifugation, are incubated in the presence of known 
concentrations of immunotoxin or reference cytotoxic substance and a final 
concentration of 10 mM of ammonium chloride for 24 h at 37.degree. C. The 
cells are then washed and brought into contact with a mitogenic agent 
specific for human T cells, which consists of a mixture of 
phytohemagglutinin A (PHA) (Wellcome Ltd., 1% final concentration) and "T 
cell growth factor" (or TCGF or interleukine 2 or IL2) at a final 
concentration of 0.5 unit/ml. The residual cells capable of proliferating 
are analyzed 72, 96 and 120 h after the cytotoxic treatment has ended by 
means of indirect immunofluorescence using a flux cytofluorometer (FACS IV 
Becton Dickinson). 
FIG. 8 shows the respective results obtained on human T lymphocytes from 
peripheral blood, stimulated with: 
A chain of ricin 10.sup.-8 M+NH.sub.4 Cl 10.sup.-2 M : A 
immunotoxin 3A1 10.sup.-8 M+NH.sub.4 Cl 10.sup.-2 M : IT 3A1 
immunotoxin A1 10.sup.-8 M+NH.sub.4 Cl 10.sup.-2 M : IT A1 
immunotoxin T101 10.sup.-8 M+NH.sub.4 Cl 10.sup.-2 M : IT T101 
immunotoxin RTF11 10.sup.-8 M+NH.sub.4 Cl 10.sup.-2 M: IT RFT11 
immunotoxin IIB5 10.sup.-8 M+NH.sub.4 Cl 10.sup.-2 M : IT IIB5 
immunotoxin 8A6 10.sup.-8 M+NH.sub.4 Cl 10.sup.-2 M : IT 8A6 
The association IT 3A1+IT T101 10.sup.-8 M+NH.sub.4 Cl 10.sup.-2 M=curve 1. 
The association IT 3A1+IT T101+IT RFT11 10.sup.-8 M+NH.sub.4 Cl 10.sup.-2 
M=curve 2. 
The association IT 3A1+IT T101+IT IIB5 10.sup.-8 M +NH.sub.4 Cl 10.sup.-2 
M=curve 3. 
The association IT A1+IT T101+IT RFT11 10 .sup.-8 M +NH.sub.4 Cl 10.sup.-2 
M=curve 4. 
The association IT T101+IT RFT11 10.sup.-8 M +NH.sub.4 Cl 10.sup.-2 M=curve 
5. 
The association IT T101+IT IIB5 10.sup.-8 M+NH.sub.4 Cl 10.sup.-2 M=curve 
6. 
The association IT T101+IT 8A6 10.sup.-8 M+NH.sub.4 Cl 10.sup.-2 M=curve 7.