Monoclonal antibodies to cyclosporins

Novel monoclonal antibodies capable of distinguishing between cyclosporins, e.g. Cyclosporine, and metabolites, e.g. Cyclosporins 17 and 18, are produced, e.g. starting from novel cyclosporins having an activated coupling group, e.g. activated carboxy group, e.g. (i) [(O-succinimidooxysuccinyl)-Thr].sup.2 -Cyclosporine and (ii) [(N-.epsilon.-succinimidooxysuccinyl)-(D)Lys].sup.8 -Cyclosporine. Cyclosporin starting materials required for the production of cyclosporins of type (ii), e.g. [(D)Lys].sup.8 -Cyclosporine are also new and additionally have utility in the preparation of novel labelled cyclosporin derivatives, as well as antibodies and antisera generally. Also claimed are novel antigenic conjugates and hybridoma cell lines used in the production of antibodies and antisera as aforesaid as well as assay kits comprising novel antisera, antibodies and/or labelled cyclosporins as aforesaid.

The present invention relates to monoclonal antibodies to cyclosporins, in 
particular to monoclonal antibodies capable of distinguishing between 
cyclosporins and metabolites thereof and suitable for use in 
diagnostic/assay kits, as well as to novel hybridoma cell lines used in 
the production of said monoclonal antibodies and diagnostic/assay kits 
comprising said monoclonal antibodies. In addition the invention relates 
to novel cyclosporins and immunogenic conjugates comprising them, used for 
the generation of monoclonal antibodies as aforesaid and also useful for 
the generation of regular polyclonal antisera suitable for 
diagnostic/assay kit use, as well as to the product antisera and 
antibodies and diagnostic/assay kits comprising them. The invention also 
relates to labelled derivatives of said novel cyclosporins, themselves 
suitable for diagnostic/assay kit use, as well as to diagnostic/assay kits 
comprising them. 
The cyclosporins comprise a class of structurally distinctive, cyclic, 
poly-N-methylated undecapeptides commonly possessing pharmacological, in 
particular immunosuppressive, anti-inflammatory and anti-parasitic 
activity. The first of the cyclosporins to be isolated was the naturally 
occurring fungal metabolite Cyclosporine, also known as cyclosporin A, of 
formula A 
##STR1## 
wherein -MeBmt- represents the 
N-methyl-(4R)-4-but-2E-en-1-yl-4-methyl-(L)threonyl residue of formula B 
##STR2## 
in which --x--y-- is --CH.dbd.CH-- (trans). 
Since the original discovery of Cyclosporine, a wide variety of naturally 
occurring cyclosporins have been isolated and identified and many further 
non-natural cyclosporins have been prepared by total- or semi-synthetic 
means or by the application of modified culture techniques. The class 
comprised by the cyclosporins is thus now substantial and includes for 
example the naturally occurring cyclosporins A through Z [c.f. Kobel et 
al. European Journal of applied Microbiology and Biotechnology 14, 237-240 
(1982) and poster presented by Traber et al., 24th. Interscience 
Conference on Antimicrobial Agents and Chemotherapy, Washington, Oct. 
8-10, (1984)]; as well as various non-natural or artificial cyclosporins, 
including dihydro-cyclosporins (in which the group --x--y-- of the -MeBmt- 
residue (see formula B above) is saturated, e.g. as disclosed in the U.S. 
Pat. Nos. 4,108,985; 4,210,581 and 4,220,641, cyclosporins in which the 
-MeBmt- residue is present in isomeric or N-desmethyl form [c.f. European 
patent no. 0 034 567 and "Cyclosporin A", Proc. Internat. Conference on 
Cyclosporin A, Cambridge (U.K.) Sep. 1981, Ed. D. J. G. White, Elsevier 
Press (1982)13 both describing the total-synthetic method for the 
production of cyclosporins developed by R. Wenger] and cyclosporins in 
which incorporation of variant amino acids at specific positions within 
the peptide sequence is effected. Examples of such cyclosporins as 
disclosed in the above art references include e.g. [Thr].sup.2 -, [Val] 
.sup.2 -, [Nva].sup.2 - and [Nva].sup.2 -[Nva].sup.5 -Cyclosporine (also 
known as cyclosporins C, D, G and M respectively) and dihydro-[Val].sup.2 
-Cyclosporine (also known as dihydrocyclosporin D). 
[In accordance with now conventional nomenclature for the cyclosporins, 
these are defined throughout the present specification and claims by 
reference to the structure of Cyclosporine (i.e. cyclosporin A). This is 
done by first indicating those residues in the molecule which differ from 
those present in Cyclosporine and then applying the term "Cyclosporine" to 
characterise the remaining residues which are identical to those present 
in Cyclosporine. At the same time the prefix "dihydro" is employed to 
designate cyclosporins wherein the -MeBmt- residue is hydrogenated 
(-dihydro-MeBmt-), i.e. wherein --x--y-- in formula B is --CH.sub.2 
--CH.sub.2 --. Thus [Thr].sup.2 -Cyclosporine is the cyclosporin having 
the sequence shown in formula A, but in which -.alpha.Abu- at the 
2-position is replaced by -Thr-, and dihydro-[Val].sup.2 -Cyclosporine is 
the cyclosporin having the sequence shown in formula A but in which 
-MeBmt- at position 1 is hydrogenated and -.alpha.Abu- at the 2 position 
is replaced by -Val-. 
In addition, amino acid residues referred to by abbreviation, e.g. -Ala-, 
-MeVal- etc . . . are, in accordance with conventional practice, to be 
understood as having the (L)-configuration unless otherwise indicated. 
Residue abbreviations preceded by "Me", as in the case of -MeLeu- 
represent N-methylated residues. The individual residues of the 
cyclosporin molecule are numbered, as in the art, clockwise and starting 
with the residue -MeBmt- (or -dihydro-MeBmt-) in position 1. The same 
numerical sequence is employed throughout the present specification and 
claims.] 
Because of their unique immunosuppressive activity, the cyclosporins have 
attracted very considerable attention not only in medical and academic 
circles, but also in the lay press. Cyclosporine itself is now 
commercially available and commonly employed to prevent rejection 
following allogenic organ, e.g. heart, heart-lung, kidney and bone-marrow 
transplant, as well as more recently in the treatment of various 
auto-immune and related diseases and conditions. Both dihydro-[Val].sup.2 
-Cyclosporin and [Nva].sup.2 -Cyclosporin are under extensive clinical 
investigation as potential successors to Cyclosporine. 
Dosaging of cyclosporins, e.g. Cyclosporine, however presents particular 
difficulties. Since metabolic conversion rates tend to be patient specific 
and the therapeutic range narrow, effective dosaging is highly subject 
specific and requires the establishment of appropriate individual serum 
levels. Regular monitoring of cyclosporin plasma concentrations is thus an 
essential prerequisit for effective treatment. To this end a number of 
high pressure liquid chromatography (HPLC), radioimmunoasssay (RIA) and 
fluoroimmunoassay (FIA) systems have been developed. However, HPLC 
methods, whilst highly specific are difficult and cumbersome to use in 
practice and the current commercially available RIA system based on sheep 
polyclonal antiserum has met with criticism because of its lack of 
specificity. Development of cyclosporin, e.g. Cyclosporine, specific 
monoclonal antibodies capable of distinguishing between therapeutically 
administered cyclosporins and their metabolites in man has accordingly for 
a long time been an urgent practical as well as purely scientific goal, 
since these would have the advantage of offering the same potential 
specificity as HPLC methodology, whilst retaining the advantage of ease of 
application provided by conventional immunoassay systems. In addition the 
provision of such cyclosporin-specific monoclonal antibodies would provide 
a vital new research tool permitting e.g. the comparative investigation of 
cyclosporin conformation and definition of cyclosporin receptor 
requirements etc . . . . 
Since the original discovery of Cyclosporine, numerous attempts have been 
made to produce monoclonal antibodies reactive to cyclosporins. Since 
cyclosporins, e.g. Cyclosporine, themselves have little immunogenic 
activity, a common approach has been to proceed employing an immunogenic, 
e.g. hapten-protein, conjugate, e.g. derived by coupling of 
immunoglobulins via the hydroxy group available at -Thr.sup.2 - in 
[Thr].sup.2 -Cyclosporine employing conventional coupling techniques, e.g. 
with EDCI [N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide.2HCl] or MCDI 
[N-cyclohexyl-N'-[.beta.-(N-methylmorpholino)ethyl]-carbodiimide.p.toluene 
sulfonate] as coupling agent. Attempts in this manner have however failed 
and where monoclonal antibodies have been obtained, these have been found 
to have relatively low specificity for Cyclosporine, or to be specific 
with respect to the carrier protein or the coupling reagent employed 
rather than Cyclosporine, or to be highly cross-reactive with the coupling 
agent. In no instance has it proved possible to produce monoclonal 
antibodies identifiable as distinguishing between e.g. Cyclosporine and 
metabolites thereof, e.g. the metabolites Cyclosporine 17 and Cyclosporine 
18 hereinafter specifically described In addition such attempts have led 
to the production of monoclonal antibodies to Cyclosporine of the type IgM 
only and hence in any event essentially inappropriate for use in any form 
of regular, e.g. clinical, assay kit. The production of monoclonal 
antibodies having specific reactivity with cyclosporins and capable of 
distinguishing between individual cyclosporins and their metabolites, e.g. 
between Cyclosporine and its metabolites in man, and suitable for use in 
an assay system has thus remained a major goal. 
In accordance with the present invention it has now surprisingly been found 
that monoclonal antibodies reactive to cyclosporins and meeting the 
various objectives discussed above, in particular capable of 
distinguishing between cyclosporins and metabolites thereof, can be 
produced via essentially conventional immunisation/fusion/cloning 
techniques, employing immunogenic conjugates comprising a cyclosporin as 
hapten at the initial immunisation step, if the conjugate is prepared by 
coupling of the carrier to the cyclosporin by the agency of an activated 
coupling group, e.g. if conjugate synthesis is effected employing a 
cyclosporin having an activated coupling group as starting material. In 
particular using such immunogenic conjugates it is possible to obtain 
monoclonal antibodies capable of fine discrimination between cyclosporins 
and metabolites thereof bearing even single variant groupings on 
individual residues, e.g. in the case of Cyclosporine, being reactive with 
Cyclosporine while exhibiting low cross-reactivity with, for example, its 
metabolites Cyclosporine 17 and/or Cyclosporine 18. 
In addition to at last providing the means for development of convenient 
monoclonal assay systems, e.g. for use in clinic, the present invention 
also provides a means for the further purification of cyclosporin 
metabolites and, since it may be anticipated that monoclonal antibodies 
will be obtainable by application of the general methods of the invention, 
which may mimic receptor sites, the characterisation of potential 
endogenous cyclosporin-like molecules. The significance of the present 
invention from both a practical and a purely scientific stand point will 
be thus readily apparent. 
As indicated above, the immunogenic conjugates required in the practice of 
the invention are prepared by direct coupling of a carrier, e.g. protein 
molecule, with a cyclosporin by the agency of an activated coupling group. 
This may be effected, either by reaction of a carrier bearing an activated 
coupling group with a cyclosporin bearing an appropriate co-reactive 
substituent, e.g. hydroxy or amino group, e.g. as in the case of 
[Thr].sup.2 -Cyclosporine or [(D)Lys].sup.8 -Cyclosporine hereinafter 
described, or by reaction of a carrier with a cyclosporin having an 
activated coupling group, e.g. cyclosporin in which one of the amino acid 
residues present in the cyclosporin molecule has a side chain at the 
.alpha.-carbon atom comprising or bearing an activated coupling group. The 
said conjugates thus comprise a cyclosporin, hapten moiety, directly 
linked to a carrier moiety, rather than via an intervening coupling agent 
residue, as in the case of immunogenic conjugates comprising a cyclosporin 
as hapten previously employed in the art, e.g. for raising regular 
polyclonal antisera. 
By the term "activated coupling group" as used herein and throughout the 
accompanying claims is to be understood any group capable of direct 
reaction with an appropriate, co-reactive grouping, e.g. amino, hydroxy, 
thio group or the like, so as to provide a co-valent linkage, without 
requirement for use of a coupling agent to enable, effect or promote 
reaction. Thus in the case of cyclosporins bearing an "activated coupling 
group" this will be any group capable of direct reaction with a carrier 
molecule, e.g. protein molecule, to provide a co-valently linked conjugate 
with said carrier molecule, without requirement for use of a coupling 
reagent to enable, effect or promote coupling or reaction with said 
carrier molecule. 
Groups suitable as activated coupling groups are well known in the art and 
include for example i) activated ester or activated carboxy groups, i.e. 
of formula --CO--OZ wherein Z is a carboxy activating group such as o- or 
p-nitrophenyl, 1-benztriazole, pentafluorophenyl or N-succinimido; ii) 
activated dithio groups, i.e. of formula --S--S--X wherein X is a dithio 
activating group such as 2-pyridyl; and iii) epoxy groups. 
Suitable immunogenic conjugate carrier molecules, bearing an activated 
coupling group, e.g. epoxy group, as aforesaid, may be prepared in 
accordance with techniques known in the art, e.g. as described by Laumen 
et al., Tetrahedron Letters, 26 (4), 407-410(1985). In accordance with the 
general methods of the present invention it is however preferred that the 
activated coupling group be provided on the cyclosporin which is to be 
coupled with the carrier, rather than vice versa. 
In principle the activated coupling group may be present at any position 
around the cyclosporin molecule. In so far as transformations at the 
1-position are of particular significance in cyclosporin metabolism, or in 
so far as major cyclosporin metabolites, e.g. in the case of Cyclosporine, 
Cyclosporine 17 and Cyclosporine 18, exhibit structural variation at the 
1-position as described below, it is preferred that the activated coupling 
group be present at one or other of positions 2 to 11 inclusive, thus 
leaving the residue at the 1-position intact, preferably "unmasked" by the 
carrier, in the immunogenic conjugate subsequently obtained, and hence 
free to elicit specific antibody response. Generally it is appropriate if 
the activated coupling group is at the 2-position or at any of the 
positions 3, 5 to 8 or 10, especially 5 to 8 inclusive, whereby the 2- and 
8-positions are particularly favoured. 
In the case of Cyclosporine major metabolic conversions occurring in man 
are: 
I Terminal hydroxylation of -MeLeu.sup.9 - to give the residue of formula E 
##STR3## 
II Terminal hydroxylation of -MeBmt.sup.1 - to give the residue of formula 
F 
##STR4## 
III Des-N-methylation of -MeLeu.sup.4 - to give -Leu-; IV Terminal 
hydroxylation of -MeLeu.sup.4 - to give the residue of formula E above; 
V Terminal hydroxylation of -MeLeu.sup.6 - to give the residue of formula E 
above; 
VI Terminal hydroxylation and ring-closure in -MeBmt.sup.1 - to give the 
residue of formula G 
##STR5## 
Thus known metabolites of Cyclosporine (identified as Cyclosporine 1, 
Cyclosporine 8 etc . . . ) exhibit the following metabolic variations. 
Cyclosporine 1:I. Cyclosporine 8:I+II. Cyclosporine 9:I+III+V. Cyclosporine 
10:I+IV. Cyclosporine 16:I+V. Cyclosporine 17:II. Cyclosporine 18:VI. 
Cyclosporine 21:III. 
[See G. Maurer et al, "Drug Metab. Disposit" 12, 120-126 (1984)]. 
Accordingly, for the preparation of monoclonal antibodies capable of 
distinguishing between Cyclosporine and metabolites thereof in man, it 
will be appropriate that the activated coupling group in the cyclosporin 
employed for immunogenic conjugate formation, be situated in a position 
other than the 1-, 4-, 6- or 9-position, and, in so far as Cyclosporine 17 
and 18 represent major metabolites, at least in a position other than the 
1-position. Thus again in the particular case of Cyclosporine, the 2- and 
8-position are especially favoured. 
Cyclosporins having an activated coupling group as described above may be 
prepared e.g. either: 
i) by activation of an appropriate pre-existing precursor group (i.e. 
coupling group in non-activated form), e.g. conversion of the carboxy 
group of a cyclosporin having a carboxy-substituted .alpha.-amino acid 
residue (i.e. .alpha.-amino acid residue having a side chain at the 
.alpha.-carbon atom comprising or bearing a carboxy group), e.g. at the 2- 
or 8-position, into an activated carboxy group, by reaction with a carboxy 
activating agent; or 
ii) by acylation or etherification of a cyclosporin having an amino- or 
hydroxy-substituted .alpha.-amino acid residue (i.e. .alpha.-amino acid 
residue having a side chain at the .alpha.-carbon atom comprising or 
bearing a hydroxy or amino group), e.g. hydroxy-substituted .alpha.-amino 
acid residue at the 2-position or amino- or hydroxy-substituted 
.alpha.-amino acid residue at the 8-position, with an acylating or 
alkylating agent bearing an activated coupling group. 
Process step i) above may be carried out in accordance with standard 
techniques known in the art, e.g. for the activation of carboxy groups by 
reaction with a regular carboxy activating agent such as o- or 
p-nitrophenol, 1-hydroxy-benztriazole, pentafluorophenol or 
N-hydroxy-succinimide. Reaction is suitably carried out in the presence of 
a condensing agent such as EDCI. 
Process step ii) may also be carried out in accordance with essentially 
conventional techniques. Thus amino or hydroxy groups may be suitably 
acylated by reaction with a derivative of a carboxylic acid in which the 
carboxy group is activated and which additionally bears an activated 
coupling group which is non-reactive with amino or hydroxy as the case may 
be, for example N-[(2-pyridyl)dithio-propion-1-yl]-succinimide, [the 
(2-pyridyl)dithio moiety providing the activated coupling group (non 
reactive, in this instance, with both amino and hydroxy groups) and the 
-COO-succinimido moiety the activated carboxy group for effecting 
acylation]. Reaction is suitably performed in an inert solvent or diluent 
such as dichloromethane at e.g. ambient temperature. Alternatively hydroxy 
groups may be etherified, e.g. to introduce an epoxy bearing moiety of 
formula 
##STR6## 
[the epoxy moiety providing the activated coupling group] employing any 
of the various agents known in the art for such purpose, such as 
epichlorhydrin or epibromhydrin, e.g. in accordance with the general 
procedures described by Laumen et al. Loc. cit.. 
Cyclosporin starting materials for process step i) above may be prepared 
analogously to process step ii), e.g. for the production of a cyclosporin 
having a carboxy-substituted .alpha.-amino acid residue, e.g. at the 2- or 
8-position: 
iii) by reaction of a cyclosporin having an amino- or hydroxy-substituted 
.alpha.-amino acid residue, e.g. hydroxy-substituted .alpha.-amino acid 
residue at the 2-position or amino- or hydroxy-substituted .alpha.-amino 
acid residue at the 8-position, either a) with a dicarboxylic acid in 
which one of the carboxy groups present is in protected form, or b) with a 
dicarboxylic acid anhydride e.g. succinic anhydride, reaction in case a) 
being followed by deprotection of the carboxy group in the product 
cyclosporin. 
Reaction step iii) may also be carried out employing essentially 
conventional procedures, e.g. in the presence of an acid binding agent 
such as 4-dimethylaminopyridine, in an inert organic solvent or diluent, 
at ambient or slightly elevated temperature. Where carboxy protecting 
groups are employed as in variant a) these may be entirely conventional 
and removed by entirely conventional technique. 
Cyclosporin starting materials for process steps ii) and iii) a 
hydroxy-substituted .alpha.-amino acid residue include the known 
cyclosporins: [Thr].sup.2 -Cyclosporine and [(D)Ser].sup.8 -Cyclosporine, 
the latter being disclosed and claimed e.g. in European Patent No. 0 056 
782, together with processes for its production in accordance with the 
general techniques of the total-synthetic method for the production of 
cyclosporins referred to above, or by fermentation technique. Other 
cyclosporins having a hydroxy-substituted .alpha.-amino acid residue, e.g. 
in the 8-position, may be prepared or obtained analogously and various 
further such Cyclosporins including [(D)Thr].sup.8 -Cyclosporine, 
[Nva].sup.2 -[(D)Ser].sup.8 -Cyclosporine, and [Thr].sup.2 -[(D)Ser].sup.8 
-Cyclosporine have been described and claimed in U.S. patent application 
Ser. No. 713 259 (filed 19 Mar., 1985)=W. German Appn. No. P 3 509 809.0 
(filed 19 Mar., 1985)=French Appn. No. 8 404 172 (filed 19 Mar., 
1985).ident.Australian Appn. No. 40 272/85 (filed 22 Mar., 1985).ident.UK 
Appn. No. 8 507 270 (filed 20 Mar., 1985).ident.New Zealand Appn. No. 211 
526 (filed 21 Mar., 1985).ident.South African Appn. No. 85/2195 (filed 22 
Mar., 1985). 
Preferred cyclosporins having an amino-substituted .alpha.-amino acid 
residue are those wherein the said amino acid residue is at the 
8-position, cyclosporins wherein the residue at the 8-position is -(D)Lys- 
being especially preferred. Such cyclosporins may also be prepared in 
accordance with the general techniques of the total-synthetic method for 
the production of cyclosporins developed by R. Wenger, e.g. 
iv) by deprotection of a cyclosporin having an amino-substituted 
.alpha.-amino acid residue at the 8-position said cyclosporin being in 
protected form, e.g. by deprotection of a cyclosporin wherein the residue 
at the 8-position is -(D)Lys- in N-.epsilon.-protected form; or 
v) cyclising a straight chain undecapeptide having the sequence of the 
product cyclosporin, said undecapeptide being in free or protected form, 
e.g. undecapeptide comprising a -(D)Lys-residue in free or 
N-.epsilon.-protected form at the position corresponding to the 8-position 
of the product cyclosporin, and when required carrying out precess step 
iv). 
Process steps iv) and v) may in particular be carried out in accordance 
with the general procedure hereinafter illustrated in example 1. 
As will be appreciated from the description of process steps i), ii) and 
iii) above, the products of steps i) or ii) will generally comprise 
cyclosporins having an acylamino-, acyloxy- or alkoxy-substituted 
.alpha.-amino acid residue (i.e. .alpha.-amino acid residue having a side 
chain at the .alpha.-carbon atom comprising or bearing an acylamino-, 
acyloxy- or alkoxy- group), e.g. cyclosporin having an acyloxy- or 
alkoxy-substituted .alpha.-amino acid residue at the 2-position or 
acylamino-, acyloxy- or alkoxy-substituted .alpha.-amino acid residue at 
the 8-position, in which the activated coupling group is present on the 
acyl/alkyl moiety. 
This may be more readily appreciated by reference to the following reaction 
schemes, illustrating the production of particular groups of cyclosporins 
in accordance with the general methods of process steps i) to v) above: 
##STR7## 
It may at this point be noted that the hydroxy group at the 3'-position in 
-MeBmt- and -dihydro-MeBmt- is of relatively low reactivity. Thus where 
processes described above involve reaction of cyclosporins having a 
hydroxy substituted .alpha.-amino acid residue at any one of positions 2 
to 11, e.g., -Thr- in the 2-position, reaction with the hydroxy group of 
said residue will be in preference to reaction with the hydroxy group in 
-MeBmt- or -dihydro-MeBmt-, unwanted side reaction with the latter thus 
being readily avoidable. 
In the formulae Ib, IIb, Id, IId, Ie and III above, A.sup.2 and B.sup.2 
preferably represent -MeBmt- and -.alpha.Abu- respectively. 
Whenever throughout the whole of the foregoing description, cyclosporins 
are referred to as having a specified residue at the 8-position, but the 
configuration of said residue is not recited, the (D)-configuration is 
preferred. 
Cyclosporins having an activated coupling group described above as well as 
cyclosporins having an amino-substituted .alpha.-amino acid residue at the 
8-position in which the amino substituent is in free or protected form, or 
is otherwise derivatised, e.g. acylated are novel and comprise a part of 
the present invention. The present invention accordingly provides: 
1.1 A cyclosporin having an .alpha.-amino acid residue bearing an activated 
coupling group. 
1.2 A cyclosporin according to 1.1 wherein the said .alpha.-amino acid 
residue is present at one of positions 2 through 11 inclusive. 
1.3 A cyclosporin according to 1.2 wherein the said .alpha.-amino acid 
residue comprises an acylamino-, acyloxy- or alkoxy-substituted 
.alpha.-amino acid residue in which, the activated coupling group is 
present on the acylamino-, acyloxy- or alkoxy substituent. 
1.4 A cyclosporin according to any one of 1.1 to 1.3 wherein the activated 
coupling group is an activated ester, activated dithio, or epoxy group. 
1.5 A cyclosporin according to 1.3 wherein the said .alpha.-amino acid 
residue comprises: an acylamino substituted .alpha.-amino acid residue, 
wherein the acylamino substituent is substituted in the acyl moiety 
thereof by an activated carboxy or activated dithio group; an acyloxy 
substituted .alpha.-amino acid residue, wherein the acyloxy substituent is 
substituted in the acyl moiety thereof by an activated carboxy group; or 
an alkoxy substituted .alpha.-amino acid residue, wherein the alkoxy 
substituent is substituted by an epoxy group. 
1.6 A cyclosporin according to any one of 1.3 to 1.5 wherein the said 
.alpha.-amino acid residue is an (0-acyl)-threonyl residue at the 
2-position. 
1.7 A cyclosporin according to 1.6 wherein the acyl moiety has the formula 
ZO--CO--CH.sub.2 --CH.sub.2 --CO-- wherein Z is a carboxy activating 
group. 
1.8 A cyclosporin according to 1.2 wherein the said .alpha.-amino acid 
residue is present at the 5-, 6-, 7- or 8-position. 
1.9 A cyclosporin according to 1.8 wherein the said .alpha.-amino acid 
residue is a (D).alpha.-amino acid residue in the 8-position. 
1.10 A cyclosporin according to 1.9 wherein the said .alpha.-amino acid 
residue is an acylamino substituted (D).alpha.-amino acid residue in which 
the activated coupling group is present on the acylamino substituent. 
1.11 A cyclosporin according to 1.10 wherein the activated coupling group 
is an activated carboxy or activated dithio group. 
1.12 A cyclosporin having an amino substituted (D).alpha.-amino acid 
residue at the 8-position the amino substituent being in free or protected 
form. 
1.13 A cyclosporin having an acylamino substituted (D).alpha.-amino acid 
residue at the 8-position wherein the acylamino substituent is substituted 
in the acyl moiety thereof by a free carboxy group. 
1.14 A cyclosporin according to any one of 1.10 to 1.13 of formula 
##STR8## 
wherein X is hydrogen, an amino protecting group or an acyl group 
substituted by a free carboxy group or an activated coupling group, for 
example an activated carboxy or dithio group, e.g. an acyl group of 
formula 
EQU Y--CH.sub.2 --CH.sub.2 --CO-- 
wherein Y is carboxy, an activated carboxy group or an activated dithio 
group. 
1.15 A cyclosporin of formula IIa, Ib, IIb, IIc, Id, IId or Ie as 
hereinbefore defined. 
As hereinbefore discussed it has, in accordance with the present invention, 
now surprisingly been found that immunogenic conjugates comprising a 
carrier and a cyclosporin coupled by the agency of an activated coupling 
group, in particular, obtained employing cyclosporins having an activated 
coupling group as described above, e.g. as defined under 1.1 to 1.11, 1.14 
or 1.15, enable, for the first time, the production of monoclonal 
antibodies capable of distinguishing between cyclosporins and metabolites 
thereof. Thus immunogenic conjugates comprising the reaction products of 
such cyclosporins as aforesaid as hapten component are capable of 
eliciting an antibody response in animals challenged, e.g. inoculated 
therewith, such that antibody producing cells, e.g. spleen or lymph-node 
cells, subsequently recoverable therefrom may be used for the preparation 
of hybridoma lines providing monoclonal antibodies capable of 
distinguishing between therapeutically administered cyclosporins, e.g. 
Cyclosporine, and metabolites thereof, in particular metabolites thereof 
in man, e.g. Cyclosporine 17 and Cyclosporine 18. Such antigenic 
conjugates being hitherto unknown, the present invention further provides: 
2.1 An immunogenic conjugate comprising a carrier coupled to a cyclosporin 
by the agency of an activated coupling group, for example comprising a 
carrier coupled to a cyclosporin having an .alpha.-amino acid residue 
bearing an activated coupling group, e.g. as hereinbefore described, in 
particular a cyclosporin as hereinbefore defined under any one of 1.1 to 
1.11, 1.14 or 1.15 (formulae IIa, IIb, IIc or IId) above. 
2.2 An immunogenic conjugate obtained or obtainable by coupling of a 
cyclosporin having an .alpha.-amino acid residue bearing an activated 
coupling group, e.g. as hereinbefore described, in particular a 
cyclosporin as hereinbefore defined under any one of 1.1 to 1.11, 1.14 or 
1.15 (formulae IIa, IIb, IIc or IId) above. 
2.3 An immunogenic conjugate, e.g. as defined under 2.1 or 2.2, capable of 
use in the production of a monoclonal antibody capable of distinguishing 
between a cyclosporin and a metabolite thereof, e.g. a monoclonal antibody 
as hereinafter described and, in particular, as hereinafter defined under 
any one of 3.1 to 3.10 below. 
Suitable carriers for the immunogenic conjugates of the invention include 
any of those known and commonly employed in the art in particular high 
molecular weight polypeptides, especially proteins such as serum albumins, 
e.g. bovine serum albumin and chicken ovalbumin, immunoglobulins, in 
particular of the class IgG such as chicken or guinea pig IgG and 
synthetic polymers such as polyglutamic acid. 
In addition the present invention provides a process for the production of 
an immunogenic conjugate as defined above, which process comprises: 
vi) Coupling a carrier, e.g. as hereinabove described, bearing an activated 
coupling group with a cyclosporin having an .alpha.-amino acid residue 
bearing an appropriate co-reactive, e.g. hydroxy or amino, group, e.g. 
with [Thr].sup.2 -Cyclosporine or [(D)Lys].sup.8 -Cyclosporine, or 
coupling a carrier, e.g. as hereinabove described, with a cyclosporin 
having an .alpha.-amino acid residue bearing an activated coupling group, 
e.g. as hereinbefore described, in particular a cyclosporin as 
hereinbefore defined under any one of 1.1 to 1.11, 1.14 or 1.15 (formulae 
IIa, IIb, IIc or IId) above. 
The above process step is carried out by direct reaction of the cyclosporin 
component, i.e. without use of a coupling agent. Reaction is suitably 
effected by addition of the cyclosporin component dissolved in an 
appropriate inert diluent or carrier such as dimethyl formamide to a 
buffered preparation of the carrier, e.g. carrier protein, e.g. solution 
or supension in bicarbonate buffer, at ambient temperature. The obtained 
immunogenic conjugate is suitably purified by dialysis, e.g. against 
phosphate buffered saline. 
The above described immunogenic conjugates, e.g. as defined under 2.1 to 
2.3, may be employed to produce monoclonal antibodies by essentially 
standard techniques, e.g. via a stepwise procedure comprising: a) 
administration of an immunogenic conjugate, e.g. as defined under any one 
of 2.1 to 2.3 above, to an appropriate animal species; b) recovery of 
antibody producing, e.g. spleen or lymph-node, cells sensibilised to the 
immunogenic conjugate; c) immortalization of recovered cells, e.g. by 
fusion with an appropriate myeloma cell line to produce hybridoma cell 
lines; and d) selection of an immortalized cell, e.g. hybridoma line , 
producing monoclonal antibodies as required. 
Step a) is suitably carried out using rats or mice, e.g. .female. Balb/c 
mice as recipient, and administration of the immunogenic conjugate by s.c. 
or i.p. injection in an amount of from ca. 50 to 200, e.g. ca. 100 .mu.g 
followed by booster injections, i.p., s.c. or i.m., 14 to 21 days later. 
Mice showing high-titred antisera of appropriate isotype distribution, 
e.g. as determined by regular RIA and/or ELISA technique, are given 
further booster injections e.g. in accordance with the specific procedures 
hereinafter described in example 9, and antibody producing, e.g. spleen 
cells, collected [step b)]. Step c) may be performed in accordance with 
any of the techniques practiced in the art, e.g. using the method 
described by S. Fazekas et al., "J. Immunol. Methods" 35, 1-32 (1980), a 
preferred myeloma line being a mouse (Balb/C) line. In step d), growing 
myeloma lines are screened for antibody production against a cyclosporin, 
e.g. in a regular RIA system using a radiolabelled derivative thereof or 
in a regular ELISA system, e.g. as hereinafter described in example 9. 
By application of the above procedures using the particular immunogenic 
conjugates of the present invention, it is possible to obtain monoclonal 
antibodies which exhibit a degree of specificity such that they are 
capable of distinguishing between individual cyclosporins differing from 
one another in only minor structural elements, e.g. presence of a single 
hydroxy group in place of a hydrogen atom. More importantly the present 
invention makes it possible, for the first time, to obtain monoclonal 
antibodies capable of distinguishing between cyclosporins, e.g. 
Cyclosporine, and metabolites thereof, in particular metabolites thereof 
in man. Thus monoclonal antibodies obtainable in accordance with the 
methods of the invention are found to be reactive with cyclosporins, e.g. 
Cyclosporine, while exhibiting relatively low cross-reactivity with 
metabolites thereof. Moreover employing immunogenic conjugates in 
accordance with the invention, e.g. as defined under 2.1 to 2.3 above, in 
which the cyclosporin hapten component corresponds to a selected "target" 
cyclosporin, the present invention enables the obtention of monoclonal 
antibodies capable of distinguishing between the "target" cyclosporin and 
structurally closely related metabolites, e.g. human metabolites, thereof. 
Thus starting from immunogenic conjugates obtained by coupling of a 
carrier with a cyclosporin having an activated coupling group at the 
2-position, e.g. as defined under 1.6 above, and in which the 
.alpha.-amino acid residues at the remaining positions 1 and 3 to 11 are 
the same as those in Cyclosporine, it is possible to prepare monoclonal 
antibodies reactive with Cyclosporine as "target" cyclosporin in 
preference to metabolites thereof in man, e.g. Cyclosporines 1, 8, 9, 10, 
16, 17, 18 and/or 21, in particular 17 and/or 18 and especially 17. 
Similarly, starting from immunogenic conjugates obtained by coupling of a 
carrier with a cyclosporin having an activated coupling group at the 5-, 
6-, 7- or 8-position, e.g. as defined under 1.8 above, in particular the 
8-position, e.g. as defined under any one of 1.9, 1.11, 1.14 or 1.15 
(formulae IIb or IId) above, and in which the residues at the remaining 
positions, e.g. 1 to 7 and 9 to 11, correspond to those in Cyclosporine, 
dihydro-[Val].sup.2 -Cyclosporine or [Nva].sup.2 -Cyclosporine, monoclonal 
antibodies may be prepared reactive with Cyclosporine, dihydro-[Val].sup.2 
-Cyclosporine or [Nva].sup.2 -Cyclosporine as "target" cyclosporin, in 
preference to metabolites thereof, e.g. metabolites thereof in man, such 
as, in the case of Cyclosporine, those recited immediately above. 
Monoclonal antibodies obtainable in accordance with the methods of the 
invention are, in particular, capable of distinguishing between "target" 
cyclosporins and metabolites thereof exhibiting structural transformation 
of the .alpha.-amino acid residue at the 1-position, e.g. metabolites 
which differ from the non-metabolised cyclosporin from which they are 
derived by substitutional or other chemical modification of the -MeBmt- or 
-dihydro-MeBmt- residue at the 1-position, in particular exhibiting 
structural transformation at a terminal position in the residue at the 
1-position, e.g. comprising hydroxylation at of the terminal (C.sup.9) 
-MeBmt- methyl group, as in the case of monoclonal antibodies described in 
the accompanying example 9, which are reactive with Cyclosporine while 
having relatively low cross-reactivity with its metabolite Cyclosporine 
17. In so far as such metabolic transformation of cyclosporin is of 
especial significance, e.g. as characteristic of major metabolites in man, 
as in the case of Cyclosporins 17 and 18, ability of monoclonal antibodies 
obtainable in accordance with the methods of the invention to distinguish 
between cyclosporins and metabolites thereof exhibiting such 
transformation is in particular to be noted. 
Cross-reactivity with metabolites, e.g. as described above, is preferably 
ca. 5% or less, more preferably ca. 3% or less, more preferably ca. 2% or 
less, of reactivity with the non-metabolised cyclosporin, e.g. as measure 
by RIA or ELISA, e.g. competitive ELISA, technique, suitably employing a 
buffer e.g. of ca. pH 6 to 8, in particular 7 to 8, and appropriately also 
containing a minor amount, e.g. 0.01 to 0.1% e.g. 0.01 to 0.05%, of a 
non-ionic surfactant or tenside such as Tween, for example phosphate 
buffered saline at pH 7.5 and containing 0.03% surfactant, e.g. Tween 20. 
Thus monoclonal antibodies obtainable in accordance with the methods of 
the invention and reactive with Cyclosporine exhibit a distinction in 
IC.sub.50 ratio for Cyclosporine 17 as compared with Cyclosporine, 
measured by competitive ELISA technique under conditions as set forth 
above of the order of 35 fold or greater. 
Monoclonal antibodies obtainable in accordance with the methods of the 
invention are also characterised by high affinity for the "target" 
cyclosporin, e.g. Cyclosporine. Thus preferred monoclonal antibodies in 
accordance with the invention will exhibit an affinity constant 
[equilibrium dissociation constant] in respect of the "target" 
cyclosporin, e.g. Cyclosporine, of the order of 10.sup.-9 mol/L or less, 
preferably 10.sup.-10 mol/L or less,e.g. at normal RIA temperatures (ca. 
4.degree. to 37.degree. C.) as determined by standard methods, e.g. in 
accordance with the method described by Muller et al., Methods in 
Enzymology, 92, 589-601 (1983). 
The present invention further permits the ready obtention of monoclonal 
antibodies of the class IgG, e.g. of the sub-class IgG.sub.1. In so far as 
such antibodies are especially suited to use in diagnostic/assay kits, 
e.g. as described below, these are preferred. 
Monoclonal antibodies as described above, as well as hybridoma lines 
producing them are entirely novel and, as will be appreciated from the 
foregoing description of their general and specific properties, well 
adapted for use in diagnostic/assay kit systems, e.g. for monitoring of 
cyclosporin drug plasma-blood levels in patients receiving cyclosporin 
therapy. The present invention accordingly also provides: 
3.1 A monoclonal antibody capable of distinguishing between a cyclosporin, 
e.g. a predetermined cyclosporin, and a metabolite thereof, in particular 
at least one metabolite thereof in man, especially at least one major 
metabolite thereof in man. 
3.2 A monoclonal antibody according to 3.1 reactive with a cyclosporin, 
e.g. a predetermined cyclosporin, and exhibiting relatively low 
cross-reactivity with a metabolite thereof, in particular at least one 
metabolite thereof in man, especially at least one major metabolite 
thereof in man. 
3.3 A monoclonal antibody according to 3.1 or 3.2 wherein the cyclosporin 
is Cyclosporine, dihydro-[Val].sup.2 -Cyclosporine or [Nva].sup.2 
-Cyclosporine, especially Cyclosporine. 
3.4 A monoclonal antibody according to any one of 3.1 to 3.3 wherein the 
metabolite is a metabolite exhibiting structural transformation of the 
.alpha.-amino acid residue at the 1-position, in particular at a terminal 
position on the residue at the 1-position, e.g. exhibiting terminal 
hydroxylation of the .alpha.-amino acid residue -MeBmt- at the 1-position. 
3.5 A monoclonal antibody according to 3.4 wherein the cyclosporin is 
Cyclosporine and the metabolite is Cyclosporin 1, 8, 9, 10, 16, 17, 18 or 
21, especially Cyclosporine 17 or 18, most especially Cyclosporine 17. 
3.6 A monoclonal antibody according to any one of 3.2 to 3.5 wherein cross 
reactivity with the metabolite is of the order of ca. 5% or less, 
preferably 3% or less, more preferably 2% or less, e.g. as measured by RIA 
or ELISA technique, for example under conditions as hereinbefore set 
forth. 
3.7 A monoclonal antibody according to any one of 3.1 to 3.6 wherein the 
affinity constant with respect to the (predetermined) cyclosporin, e.g. 
Cyclosporine, is of the order of 10.sup.-9 mol/liter or less, preferably 
10.sup.-10 mol/liter or less, e.g. as measured under conditions as 
hereinbefore set forth. 
3.8 A monoclonal antibody according to any one of 3.1 to 3.7 of the class 
IgG. 
3.9 A monoclonal antibody, e.g. according to any one of 3.1 to 3.8, 
obtained or obtainable by: 
a) coupling of a cyclosporin having an .alpha.-amino acid residue bearing 
an activated coupling group, e.g. as hereinbefore described, in particular 
as hereinbefore defined under any one of 1.1 to 1.11, 1.14 or 1.15 
(formulae IIa, IIb, IIc or IId) above, to a carrier to obtain an 
immunogenic conjugate; 
b) administration of said immunogenic conjugate to an appropriate animal 
species to effect immunogenic challenge, and recovery of antibody 
producing cells sensitised to said conjugate; 
c) immortalisation of said antibody producing cells, e.g. by fusion with an 
appropriate myeloma cell line; and 
d) recovery of monoclonal antibody from a selected immortalised cell line, 
e.g. hybridoma cell line, thus established. 
3.10 A monoclonal antibody, e.g. according to any one of 3.1 to 3.8 
obtained or obtainable by: 
a) recovery of antibody producing cells sensitised to an immunogenic 
conjugate according to any one of 2.1 to 2.3 above; 
b) immortalisation of said antibody producing cells, e.g. by fusion with an 
appropriate myeloma cell line, and 
c) recovery of the required monoclonal antibody from a selected 
immortalised cell line, e.g. hybridoma cell line, thus established. 
4.1 A hybridoma cell line producing a monoclonal antibody according to any 
one of 3.1 to 3.8 above. 
4.2 A hybridoma cell line obtained or obtainable in accordance with steps 
a) to c) of 3.9 above or steps a) and b) of 3.10 above. 
As will be appreciated, monoclonal antibodies in accordance with the 
invention may distinguish between any given cyclosporin, e.g. 
Cyclosporine, and a plurality of its metabolites, e.g. exhibit low-cross 
reactivity with respect to more than one of its metabolites. 
In addition to the foregoing the present invention also provides: 
vii) A method for the production of a monoclonal antibody as defined under 
any one of 3.1 to 3.8 above, which method comprises culturing a hybridoma 
cell line producing such antibody and recovering the antibody thus 
produced; and 
viii) A method for the production of a hybridoma cell line producing a 
monoclonal antibody as defined under any one of 3.1 to 3.8 above, which 
method comprises immortalizing an antibody producing cell, e.g. spleen or 
lymph-node cell, producing such antibody, e.g. by fusion with an 
appropriate myeloma cell line. 
The above process steps may be performed in accordance with now standard 
techniques, e.g. as hereinabove described, or as described in the 
accompanying examples, preferred myeloma cell lines for use in process 
viii) being a mouse (Balb/C) myeloma cell line. 
In accordance with a yet further aspect of the present invention it has 
also surprisingly been found that cyclosporins having a -(D)Lys- residue 
at the 8-position, i.e. as defined under 1.14 or 1.15 (formula Id) above, 
as well as derivatives in which the N-.epsilon.-atom thereof is further 
derivatised, exhibit e.g. cell binding characteristics which parallel 
those of the corresponding "parent" cyclosporin (e.g. the corresponding 
cyclosporin having -(D)Ala- at the 8-position) to a surprising and 
remarkable degree. This finding is of especial significance since the 
N-.epsilon.-nitrogen atom of -(D)Lys- provides an ideal position at which 
labelling may be effected, e.g. at which label or tracer groups may be 
introduced. Such labelled cyclosporins provide a further key tool for 
study of the mechanism of action of "parent" cyclosporins (e g. in the 
case of [(D)Lys].sup.8 -Cyclosporine of Cyclosporine) and/or for 
identifying binding sites of the "parent" cyclosporin, e.g. in in vitro 
tissue culture preparations. Thus radioactively labelled derivatives, e.g. 
.sup.125 I labelled derivatives, are useful for rapid autoradiography of 
tissues, e.g. as in kidney micro-autoradiography. 
In addition labelled, e.g. radioactively or fluorescently labelled, 
derivatives obtainable from cyclosporins having a -(D)Lys- residue at the 
8-position provide ideal components for use e.g. in RIA and FIA diagnostic 
kits. [(D)Lys].sup.8 -cyclosporins thus provide a means for the ready 
obtention of labelled analogues of the "parent" cyclosporin having 
equivalent binding properties, e.g. in relation to monoclonal antibodies 
to the parent cyclosporin, e.g. as obtained in accordance with the present 
invention or as hereinabove defined, and hence emminently useful as 
diagnostic/assay kit component or co-component. 
Accordingly the present invention also provides: 
5.1 A labelled derivative of a cyclosporin wherein the residue at the 
8-position is -(D)Lys-; in particular 
5.2 A labelled derivative of a cyclosporin of formula Id as defined above. 
By the term "labelled derivative" as used herein is meant a derivative 
bearing a tracer or marker atom or group, e.g. enabling or facilitating 
quantitative assay or location of said derivative. Such derivatives 
include derivatives, e.g. wherein one or more atoms of the -(D)Lys- 
residue functions as a tracer or marker atom, e.g. radioactive atom, as 
well as derivatives wherein a tracer or marker group is attached to the 
N-.epsilon.-atom of the -(D)Lys- residue either by direct linkage of the 
tracer or marker group to said N-.epsilon.-atom or by linkage of the 
tracer or marker group to said N-.epsilon.-atom via an intervening linking 
moiety. Examples of labelled derivatives include radioactively labelled 
derivatives, fluorescent and chemiluminescent derivatives and derivatives 
suitable for photoaffinity labelling, i.e. provided with a substituent 
which will react with a protein to which the cyclosporin is bound on 
illumination. Radioactively labelled derivatives as aforesaid include 
derivatives wherein the N-.epsilon.-atom of the -(D)Lys- residue at the 
8-position attaches to e.g. an .sup.125 I labelled p-OH-phenyl-propionyl 
residue. Fluorescent and chemiluminescent derivatives as aforesaid include 
derivatives wherein the N-.epsilon.-atom of the -(D)Lys- residue at the 
8-position attaches to a fluorescent group, such as a dansyl or rhodamine 
group, or chemiluminescent group such as an acridinium ester group, e.g. 
as described in Clin. Chem. 29, 8, pp 1474-1479 (1983). A particular group 
of cyclosporins as defined under 5.1 and 5.2 above are accordingly 
5.3 those wherein the residue at the 8-position is a residue of formula 
##STR9## 
wherein Q is or comprises a tracer or marker group, in particular 
radioactively labelled, fluorescent or chemiluminescent group, e.g. as 
specifically described above. 
Labelled derivatives as aforesaid may be prepared analogously to process 
steps iv) and v) above, e.g. employing starting materials in which the 
-(D)Lys- residue at the 8-position is pre-labelled. Alternatively they may 
be prepared by introduction of an appropriate labelling substituent, e.g. 
at the N-.epsilon.-atom of the -(D)Lys- residue at the 8-position. Thus 
fluorescently labelled derivatives may be prepared by coupling of a 
fluorescent moiety to the N-.epsilon.-atom e.g. by 
N-.epsilon.-dansylation. Similarly radioactively labelled derivatives may 
be prepared by coupling of a radioactively labelled substituent, e.g. 
.sup.125 I labelled p-OH-phenyl-propionyl, to the N-.epsilon.-atom. In the 
latter case the substituent may either be in labelled form prior to 
introduction or may be labelled subsequent to introduction. For example 
the N-.epsilon.-atom of the lysine residue in the 8-position may either be 
reacted directly with .sup.125 I-labelled p-OH-phenyl-propionic acid or 
with unlabelled p-OH-phenyl-propionic acid and the obtained 
N-.epsilon.-amide subsequently labelled in the p-OH-phenyl moiety with 
.sup.125 I. Coupling may be effected in accordance with standard 
techniques known in the art for example by reaction with 
p-OH-phenyl-propionic acid (labelled or unlabelled) in the form of its 
N-hydroxy-succinimide ester. 
.sup.125 I labelled-p-OH-phenyl-propionic acid may itself be prepared by 
the chloramine T-method [Hunter and Greenwood, Nature, 194, 495 (1962)]. 
Where labelling is effected subsequent to coupling this may be carried out 
using the chloramin T-method or the iodogen-method [Good, 
J.Clin.-Chem.Clin.Biochem., 19, 1051 (1981)]. Derivatives of the 
cyclosporins of the invention which are susceptible to labelling, e.g. as 
described above, for example derivatives wherein the N-.epsilon.-atom of 
-(D)Lys- at the 8-position is substituted by a group, such as 
p-OH-phenyl-propionyl, susceptible to 125iodination, are immediate 
precursors of the labelled derivatives of the invention and are also to be 
understood as being within the purview of the present invention. In 
accordance with the foregoing the present invention also provides: 
ix) A process for the production of a labelled derivative as defined under 
any one of 5.1 to 5.3 above, in free or protected form, which process 
comprises labelling the corresponding unlabelled free or protected 
cyclosporin, e.g. introducing a labelling substituent, for example as 
hereinbefore described, at the N-.epsilon.-atom of the -(D)Lys- residue at 
the 8-position thereof, and when required carrying out process step iv) 
above. 
It may at this point additionally be noted, that the cyclosporins having a 
free amino group as defined under 1.12 above, for example the compound 
L(D)Lys].sup.8 -Cyclosporine, also possess pharmaceutical, in particular 
immunosuppressive, anti-inflammatory and anti-parasitic (e.g. 
anti-malarial and anti-coccidiomycotic), activity, as can be demonstrated 
in standard in vivo and in vitro tests, for example in the various test 
methods described in European Patent No. 0 056 782. 
In accordance with a yet further aspect of the present invention it has 
been found that immunogenic conjugates in which the carrier molecule is 
coupled to a cyclosporin as hapten via the residue at the 5-, 6-, 7- or 
8-position, in particular the 8-position, including such conjugates as 
defined under 2.1 to 2.3 above, as well as conjugates obtained by coupling 
of a cyclosporin bearing an amino acid having a reactive group at any one 
of these positions other than an activated coupling group to a carrier by 
means of a coupling agent, are capable of generating regular polyclonal 
antisera of higher specificity than hitherto obtainable e.g. employing 
conjugates obtained by coupling of a carrier to -(Thr).sup.2 - in 
(Thr).sup.2 -Cyclosporine. 
(By the term "reactive group" as used above is to be understood any group 
which permits or enables coupling with a carrier, e.g. polypeptide or 
other appropriate macromolecule. Generically the term thus embraces 
activated coupling groups as hereinbefore defined as well as other groups 
capable of reaction, e.g. free amino, carboxy or hydroxy groups). 
Immunogenic conjugates comprising a cyclosporin as hapten, linked to a 
carrier via the .alpha.-amino acid residue at the 5-, 6-, 7- or 8-position 
are novel as such. Preferred cyclosporins providing the hapten moiety of 
such conjugates are in particular those having an activated coupling group 
as defined under 1.8 to 1.11, 1.14 and 1.15 (formulae IIb and IId) above. 
The present invention accordingly also provides: 
2.4 An immunogenic conjugate comprising a carrier coupled to a cyclosporin 
via the .alpha.-amino acid residue at position 5-, 6-, 7- or 8- of said 
cylosporin. 
2.5 An immunogenic conjugate comprising a carrier coupled with a 
cyclosporin having an activated coupling group as defined under any one of 
1.8 to 1.11, 1.14 or 1.15 (formulae IIb and IId) above. 
2.6 An immunogenic conjugate comprising a carrier coupled with a 
cyclosporin having a free amino or carboxy group as defined under any one 
of 1.12 to 1.14 and 1.15 (formulae Ib and Id) above, in particular a 
cyclosporin as defined under 1.14 above or of formula Id as defined above. 
3.11 A monoclonal antibody according to 3.9 above, characterised in that 
the cyclosporin employed at step a) is a cyclosporin as defined under 2.5 
above. 
6.1 Antibody reactive with a cyclosporin (including polyclonal antiserum 
containing antibodies reactive with a cyclosporin) generated in response 
to an immunogenic conjugate as defined under any one of 2.4 to 2.6 above. 
6.2 Antibody according to 6.1 reactive with Cyclosporine, 
dihydro-[Val].sup.2 -Cyclosporine or [Nva].sup.2 -Cyclosporine, especially 
Cyclosporine. 
Immunogenic conjugates as defined under 2.5 may be prepared in accordance 
with the methods of process step vi) above. Immunogenic conjugates as 
defined e.g. under 2.6 may be prepared by methods as hereinbefore 
described or by a process comprising: 
x) Coupling a carrier to a cyclosporin having an .alpha.-amino acid residue 
bearing a reactive group (i.e. .alpha.-amino acid residue having a side 
chain at the .alpha.-carbon atom comprising or bearing a reactive group), 
e.g. a cyclosporin bearing a free amino or carboxy group as defined under 
any one of 1.12 to 1.14 and 1.15 (formulae Ib and Id) above. 
The above process step may be effected in accordance with techniques known 
in the art, by linkage of the carrier to the cyclosporin via the 
intermediary of a coupling reagent. Thus in the case of cyclosporins 
having a -(D)Lys- residue at the 8-position, conjugates may be obtained by 
linkage of the carrier, e.g. polypeptide, for example immunoglobulin, to 
the -(D)Lys-N-.epsilon.-atom, employing the carbodiimide procedure [Kellie 
et al., "Steroid Immunology", ed. Cameron et al., Alpha. Omega, Cardiff, 
1975] or by Mannich reaction employing formaldehyde as the coupling 
reagent. 
Suitable carriers include those of the type already referred to in relation 
to the preparation of conjugates for the production of monoclonal 
antibodies, in particular high molecular weight polypeptides, especially 
proteins such a serum albumins, immungobulins and synthetic polymers such 
as polyglutamic acid. 
Regular polyclonal antisera as defined under 6.1, while lacking the fine 
specificity of monoclonal antibodies as hereinbefore described and 
defined, are also useful, e.g. as diagnostic/assay kit components, in 
particular having regard to their improved specificity with respect to 
cyclosporins as compared with such antisera known from the art. They may 
be prepared employing essentially conventional techniques, e.g. by a 
process comprising: 
xi) Elliciting an immune response in an appropriate animal species by 
administration of an immunogenic conjugate as defined under any one of 2.4 
to 2.6 above and recovering antisera thus generated. 
The above defined process step xi) may be performed e.g. by administration 
of the immunogenic conjugate to e.g. a mouse, sheep or chicken, e.g. by 
injection. After an appropriate incubation period the antiserum is 
recovered and is suitably lyophylised, e.g. for later use in kits, for 
example as hereinafter described. The period of incubation is suitably 
chosen to give an antiserum titre of greater than 1:2,000, e.g. in the 
range of from about 1:7,000 to about 1:10,000, in e.g. regular RIA. 
As previously indicated the monoclonal antibodies and polyclonal antisera, 
as well as labelled cyclosporins of the invention are all of particular 
utility as components of diagnostic/assay kit systems, e.g. immuno assay 
kits. 
Accordingly, in a yet further aspect the present invention provides: 
7. An immuno assay kit or system, e.g. RIA or FIA kit or system, for 
cyclosporine assay, for example for the assay of a cyclosporin, e.g. 
Cyclosporine, in subjects receiving cyclosporin, e.g. Cyclosporine, 
therapy, said kit or system comprising: 
A) Antibody or antiserum as defined under any one of 3.1 to 3.11 or 6.1 to 
6.2 above, in particular a monoclonal antibody as defined under any one of 
3.1 to 3.11 above, and/or 
B) a labelled derivative of a cyclosporin as defined under any one of 5.1 
to 5.3 above, 
as component of said kit or system. 
Kits as defined under 7 are useful for diagnostic purposes, e.g. for 
determining quantities of a cyclosporin present in blood, blood plasma or 
urine, e.g. as a means of establishing an appropriate dosaging regimen for 
patients receiving cyclosporin therapy. Such kits provide an assay means 
for cyclosporins, e.g. Cyclosporine, of hitherto unmatched sensitivity. 
Kits, e.g. RIA and FIA, kits in accordance with the invention may be of 
entirely conventional type for use in accordance with conventional RIA and 
FIA assay techniques. Thus RIA kits will suitably comprise in addition to 
antiserum or antibody, e.g. A) above, an appropriate labelled cyclosporin 
derivative, e.g. B) above, and C) cyclosporin standard The labelled 
cyclosporin derivative will be complementary to the cyclosporin to be 
assayed. Suitably it will be a labelled derivative as defined under B) 
above, e.g. where Cyclosporine is to be assayed it will suitably be a 
labelled derivative of [(D)Lys].sup.8 -Cyclosporine. However it may also 
be any other labelled complementary cyclosporin, for example where 
Cyclosporine is to be assayed, tritiated Cyclosporine. The cyclosporin 
standard C) will generally be a solution or the like comprising a known 
quantity of the cyclosporin to be assayed. 
In use, e.g. lyophilised, antiserum/antibody is dissolved and incubated 
together with e.g. component B) and with either the sample to be assayed 
or component C). Incubation is preferably effected with cooling e.g. at 
4.degree. C. The pH of the incubating mixture is preferably kept in the 
range of from about 5 to 8, e.g. at about pH 7 or 8, preferably with the 
aid of a buffering agent such as a citrate or tris buffer. 
Incubation conveniently lasts for at least 2 hours, e.g. from about 6 to 
about 12 hours. After incubation the fraction of e.g. component B) bound 
to the antibody is separated from the unbound fraction, e.g. by the use of 
charcoal such as dextran-coated charcoal. The unbound fraction adsorbs 
onto the charcoal and may then be separated by filtration or by 
centrifugation. The amount of radioactivity in one fraction is then 
measured by standard techniques, e.g. by liquid scintillation counting 
after the addition of a secondary solute. The proportion of component B) 
bound to the antibody is inverseley proportional to the amount of 
cyclosporin in the unknown plasma sample. For quantitive analysis, it is 
usual to prepare a standard calibration curve by analysing solutions of 
the cyclosporin of known concentration. 
FIA kits in accordance with the invention may be e.g. of the kind wherein 
antibodies are bound to a light scavenger and which depend upon 
competition between a fluorescent cyclosporin (e.g. a fluorescently 
labelled derivative in accordance with the invention) and the antibody. 
Alternatively assay kits/systems as defined under 7 above may be based on 
any of the conventional ELISA systems known in the art.

The following examples are illustrative of the present invention: 
EXAMPLE 1 
Preparation of [(D)Lys.sup.8 ]-Cyclosporine 
a) A solution of 6.4 g H-MeLeu-MeLeu-MeVal-OBzl maleinate in CH.sub.2 
Cl.sub.2 (200 ml) and H.sub.2 O (100 ml) is adjusted to pH 8 using solid 
K.sub.2 CO.sub.3. After extracting 2.times., each time with CH.sub.2 
Cl.sub.2 (200 ml), the organic phase is dried over Na.sub.2 SO.sub.4 
filtered and evaporated to dryness to yield free H-MeLeu-MeLeu-MeVal-OBzl 
as a crystalline residue. 
b) (N-.epsilon.-BOC)-FMOC-(D)Lys (6.25 g) is dissolved in CHCl.sub.3 (100 
ml) and N-methylmorpholine (2.95 g) is added with stirring. After cooling 
the solution to -20.degree., pivaloyl chloride (1.75 g) is added dropwise, 
and the reaction mixture is stirred for 6 hours at -20.degree.. A solution 
of H-MeLeu-MeLeu-MeVal-OBzl (6.34 g) in CHCl.sub.3 (20 ml) is added to the 
anhydride solution dropwise and stirred for 17 hours at -20.degree. to 
complete reaction. After diluting the CHCl.sub.3 solution with further 
CHCl.sub.3 (200 ml), the mixture is shaken with saturated NaHCO.sub.3 
solution (100 ml). The organic phase is dried over Na.sub.2 SO.sub.4, 
filtered and the solvent evaporated to dryness. 
The obtained oily residue is purified chromatographically, using 25.times. 
the amount of silica gel (particle size 0.063-0.20 mm) and methylene 
chloride with additional 3% methanol as eluant:[.alpha.].sub.D.sup.20 
=-114.2.degree. (c=1.0 in CHCl.sub.3). 
c) (N-.epsilon.-BOC)-FMOC-(D)Lys-MeLeu-MeLeu-MeVal-OBzl (8.8 g) dissolved 
in absolute ethanol (400 ml) is hydrogenated with 10% palladium/C catalyst 
(0.6 g) in a Gastar hydrogenator until the theoretical quantity of H.sub.2 
is taken up (214 ml). After evaporating off the solvent, the residue is 
purified chromatographically using 50.times. the quantity of silica gel 
(0.063-0.20 mm) and methylene chloride plus 7% methanol as eluant: 
[.alpha.].sub.D.sup.20 =129.1.degree. (c=1.0 in CHCl.sub.3). 
d) (N-.epsilon.-BOC)-FMOC-(D)Lys-MeLeu-MeLeu-MeVal-OH (7.1 g) and 
H-MeBmt-.alpha.Abu-Sar-MeLeu-Val-MeLeu-Ala-OBzl (7.4 g) are dissolved in 
methylene chloride (100 ml), and N-methylmorpholine (1.72 g) and Castro 
reagent (Bt-Op(NMe.sub.2).sub.3.sup.+ PF.sub.6.sup.31 ) (5.6 g) are added 
at room temperature (25.degree.). reaction mixture is stirred for 3 days 
at room temperature, then the solution is diluted with methylene chloride 
(200 ml) and shaken with saturated NaHCO.sub.3 solution (100 ml). The 
organic phase is dried over Na.sub.2 SO.sub.4, filtered and evaporated to 
dryness. The obtained oily residue is purified chromatographically on 
silica gel (500 g) (0.06-0.20) using methylene chloride plus 3% methanol 
as eluant: [.alpha.].sub.D.sup.20 =-143.8.degree. (c=1.0 in CHCl.sub.3). 
e) 
(N-.epsilon.-BOC)-FMOC-(D)Lys-MeLeu-MeLeu-MeVal-MeBmt-.alpha.Abu-Sar-MeLeu 
-Val-MeLeu-Ala-OBzl (5.54 g) are stirred for a total of 4 hours at room 
temperature in a solution of methylene chloride (50 ml) and piperidine (10 
ml). The solvent is evaporated and the obtained oil chromatographed on 
Sephadex LH20 (300 g) using methylene chloride plus 3% methanol as eluant: 
[.alpha.].sub.D.sup.20 =-165.2.degree. (c=1.0 in CHCl.sub.3). 
f) 0.2N NaOH (24 ml) is added to 
(N-.epsilon.-BOC)-H-(D)Lys-MeLeu-MeLeu-MeVal-MeBmt-.alpha.Abu-Sar-MeLeu-Va 
l-MeLeu-Ala-OBzl (6.48 g) dissolved in ethanol (75 ml). After 7 hours the 
solution is adjusted to pH 4 by dropwise addition of 2N HCl with cooling. 
After evaporation of the solvent, the obtained residue is shaken in 
CH.sub.2 Cl.sub.2 (200 ml) and saturated NaHCO.sub.3 (200 ml) solution. 
After extraction of the aqueous phase 2.times., each time using methylene 
chloride (200 ml), the organic phase is dried over Na.sub.2 SO.sub.4, 
filtered off and evaporated. The product is purified chromatographically 
on silica gel (300 g) (0.06-0.20 mm) using methylene chloride plus 20% 
methanol as eluant: [.alpha.].sub.D.sup.20 =-169.9.degree. (c=1.0 in 
CHCl.sub.3). 
g) [Process step v)] 
Dimethylaminopyridine (147 mg) is added with stirring to 
(N-.epsilon.-BOC)-H-(D)Lys-MeLeu-MeLeu-MeVal-MeBmt-.alpha.Abu-Sar-MeLeu-Va 
l-MeLeu-Ala-OH (413 mg) dissolved in methylene chloride (2000 ml). Propane 
phosphonic acid anhydride [(0.19 g) 50% solution in CH.sub.2 Cl.sub.2 ] is 
added and the reaction mixture is stirred for 24 hours at 25.degree.. The 
obtained solution is washed with saturated NaHCO.sub.3 solution (200 ml), 
the organic phase is dried over NaSO.sub.4, filtered off and evaporated, 
and purified chromatographically on silica gel (300 g) (0.062-0.20) using 
methylene chloride plus 5% methanol as eluant: [.alpha.].sub.D.sup.20 
=-198.3 (c=1.0 in CHCl.sub.3). 
h) [Process step iv)] 
[(N-.epsilon.-BOC)-(D)Lys].sup.8 -Cyclosporine (842 mg) is cooled to 
-20.degree. with trifluoroacetic acid (25 ml) and stirred together for 4 
hours at -20.degree.. The reaction solution is mixed with ice, sat. 
K.sub.2 CO.sub.3 (10 ml) and extracted 3.times. with methylene chloride 
(200 ml). The organic phase is dried over Na.sub.2 SO.sub.4, filtered and 
the solvent is evaporated. The obtained crude product is chromatographed 
on Sephadex LH20 (200 g) using methylene chloride plus 1% methanol as 
eluant to yield the title compound [(D)Lys].sup.8 -Cyclosporine: 
[.alpha.].sub.D.sup.20 =-204.3.degree. (c=1.0 in CHCl.sub.3). 
The product compound may also be converted into salt form in accordance 
with standard techniques. Typical salts include [(D)Lys].sup.8 
-Cyclosporine hydrochloride: [.alpha.].sub.D.sup.20 =-203.degree. (c=1.0 
in CHCl.sub.3), and [(D(Lys].sup.8 -Cyclosporine trifluoroacetate: 
[.alpha.].sub.D.sup.20 =-203.degree. (c=1.0 in CHCl.sub.3). 
EXAMPLE 2 
Preparation of [(N-.epsilon.-hydroxysuccinyl)-(D)Lys].sup.8 -Cyclosporine: 
[Process step iii)] 
255 mg of [(D)Lys].sup.8 -Cyclosporine produced in accordance with example 
1 are dissolved in 20 ml pyridine and 36 mg succinic anhydride are added. 
The obtained solution is stirred for ca. 14 hrs. at room temperature and 
the pyridine fully evaporated under vacuum at max. 40.degree. C. The 
obtained oily residue is chromatographed on 55 g Sephadex LH 20 employing 
methylene chloride+2% methanol and collected in 10 ml fractions. The pure 
title compound is obtained from fractions 15 through 23. 
NMR spectroscopy shows succinyl protons at 2.50 and 2.70 ppm (broad 
signals) and a signal for --CH.sub.2 --NH--COCH.sub.2 CH.sub.2 COOH at 
3.25 ppm. 
EXAMPLE 3 
Preparation of [(O-hydroxysuccinyl)-Thr].sup.2 -Cyclosporine: [Process step 
iii)] 
6.05 g [Thr].sup.2 -Cyclosporine is dissolved in 20 ml pyridine and 3.66 g 
4-dimethylamino-pyridine and 1.5 g succinic anhydride are added at 
75.degree. C. The reaction mixture is stirred for 4 hours at 75.degree. C. 
and then diluted with 500 ml CH.sub.2 Cl.sub.2, washed 5.times., each time 
with 50 ml 2N HCl, and 1.times. with 150 ml H.sub.2 O. The organic phase 
is extracted, dried over Na.sub.2 SO.sub.4 and evaporated and purified 
chromatographically using 250 g silica gel (0.040-0.062 mm) with acetic 
acid as eluant. 
EXAMPLE 4 
Preparation of [(N-.epsilon.-succinimidooxysuccinyl)-(D)Lys].sup.8 
-Cyclosporine: [Process step i)] 
50 mg of [(N-.epsilon.-hydroxysuccinyl)-(D)Lys].sup.8 -Cyclosporine 
produced in accordance with example 2, 14 mg 
N-ethyl-N'-(dimethylaminopropyl)-carbodiimide HCl, 23.8 mg 
N-hydroxysuccinimide and 24.6 mg triethylamine are stirred for 2 hrs. at 
room temperature in 2 ml methylene chloride to yield a clear colourless 
solution. The obtained solution is diluted with 50 ml methylene chloride 
and 10 ml H.sub.2 O and 1N HCl is added dropwise until pH 6. A two phase 
mixture develops and this is shaken thoroughly between each addition of 
HCl. The organic phase is finally shaken with 10 ml dilute NaHCO.sub.3 
solution and dried over Na.sub.2 SO.sub.4, filtered and the solvent 
evaporated off to yield the title compound. 
The NMR spectrum exhibits succinyl protons at 2.50 and 2.75 ppm (J=5 cps) 
and N-succinimido protons at 2.18 and 2.19 ppm (2S). The residue at the 
8-position has the following structure: 
##STR10## 
EXAMPLE 5 
Preparation of [(O-succinimidooxysuccinyl)-Thr].sup.2 -Cyclosporine: 
[Process step i)] 
54 mg triethylamine, 98 mg N-hydroxysuccinimide and 102 mg 
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide are added to a solution of 
200 mg of [(O-hydroxysuccinyl)-Thr].sup.2 -Cyclosporine produced in 
accordance with example 3 in 10 ml methylene chloride, addition being 
effected at 20 .degree. C. with rigorous exclusion of moisture. The 
reaction mixture is stirred for 6 hours at room temperature, diluted with 
200 ml methylene chloride and shaken with 50 ml H.sub.2 O. The aqueous 
phase in adjusted to pH 5-6 by drop-wise addition of 1N HCl and shaken. 
The aqueous phase is extracted with 100 ml methylene chloride and the 
organic phases washed with 0.1N NaHCO.sub.3, dried over Na.sub.2 SO.sub.4, 
filtered and evaporated. The residue is purified chromatographically using 
110 g silica gel with ethylacetate as eluant to yield the title compound: 
[.alpha.].sub.D.sup.20 =-178.degree. (c=1.0 in CHCl.sub.3). .sup.1 H-NMR 
in CDCl.sub.3 shows succi protons at 2.80 as a singlet and succinyl 
protons at 2.60 ppm as a multiplet. The residue at the 2-position has the 
following structure: 
##STR11## 
EXAMPLE 6 
Preparation of [(N-(3-(2-pyridyl)dithio)propion-1-yl)-(D)Lys].sup.8 
-Cyclosporine: [Process step ii)] 
35 mg succinyl-3-[(2-pyridyl)dithio]propionate are added to a solution of 
126 mg [(D)Lys].sup.8 -Cyclosporine in 10 ml methylene chloride at 
20.degree. C. and with rigorous exclusion of moisture. The reaction 
mixture is stirred for 6 hours at room temperature, diluted with 200 ml 
methylene chloride and shaken with 50 ml saturated NaHCO.sub.3. The 
aqueous phase is extracted with 150 ml methylene chloride, the organic 
phases washed with H.sub.2 O, dried over Na.sub.2 SO.sub.4, filtered and 
evaporated. The amorphous residue is purified chromatographically using 
100 g silica gel with methylene chloride/methanol (95:5) as eluant to 
yield the pure title compound: [.alpha.].sub.D.sup.20 =-165.degree. (c=1.0 
in CHCl.sub.3). 
The residue at the 8-position has the formula 
##STR12## 
EXAMPLE 7 
Preparation of immunogenic cyclosporin-carrier conjugates of the type 
defined under 2.1 above: [Process step vi)] 
7.1 Conjugate with Chicken Y-Globulin 
10 mg of [(N-.epsilon.-succinimidooxysuccinyl)-(D)Lys].sup.8 -Cyclosporine 
produced in accordance with the method of example 4 in 0.2 ml 
dimethylformamide are added to 100 mg chicken .gamma.-globulin in 4 ml 
NaHCO.sub.3 (1.5% w/v, pH 8.1). The reaction mixture is stirred for ca. 2 
hours at room temperature and the obtained conjugate purified by dialysis 
against phosphate buffered saline. 
7.2 Conjugate with Chicken Ovalbumin 
10.7 mg [(O-succinimidooxysuccinyl)-Thr].sup.2 -Cyclosporine produced in 
accordance with example 5 and additionally containing 10% ditritrated 
material (1 - 2 .mu.Ci/mg - obtained analogously to example 5, but using 
tritrated [Thr].sup.2 -Cyclosporine as starting material) in 100 .mu.l 
dimethylformamide are added with vigorous stirring to 30.45 mg chicken 
ovalbumin in 2 ml, 1.5% NaHCO.sub.3 buffer (molar excess 
cylosporin/ovalbumin=10.68). The reaction mixture is stirred for 2 hours 
at ambient temperature and the obtained conjugate purified by dialysis 
3.times. against phosphate buffered saline for 18 hours at 4.degree. C. 
For the conjugate product 55.7% of input radioactivity is found bound to 
ovalbumin, indicating a binding ratio of 5.95 cyclosporin/ovalbumin. 
Covalent binding of cyclosporin to ovalbumin was evaluated by acetone 
precipitation of 3 conjugate aliquots . 39.5% of radioactivity 
corresponding to non-covalently bound cyclosporin is determined in the 
acetonic supernatant, giving a final covalent coupling ratio of 3.6 
cyclosporin/ovalbumin. The obtained conjugate was aliquoted and kept at 
-20.degree. C. 
Similar conjugates may be prepared analogously to examples 7.1 and 7.2 
above, but employing the product of example 6 as the cyclosporin starting 
material. 
EXAMPLE 8 
Preparation of immunogenic cyclosporin-carrier conjugates of the type 
defined under 2.4/2.6 above: [Process step (x)] 
8.1 Conjugate with Guinea pig IgG 
Guinea pig IgG (30 mg) are dissolved in 0.1M bicarbonate buffer (pH 9, 0.5 
ml) and added to a solution of [(D)Lys].sup.8 -Cyclosporine (1 mg) 
prepared in accordance with the method of example 1, in the same buffer 
(0.5 ml) and a few drops of 1% acetic acid are added. Coupling is effected 
by subsequent addition of 2 portions of 
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (each 
portion=100 mg). The reaction product is prepared for injection 24 hours 
later by multiple dialysis against water followed by lyophilisation. 
8.2 Conjugate with Rabbit IgG 
[(D)Lysl].sup.8 -Cyclosporine (10 mg), prepared in accordance with the 
method of example 1, is dissolved in 0.1 ml of an ethanolic solution of 
6.9 .mu.g (=0.1 mCi) tritiated [(D)Lys].sup.8 -Cyclosporine. 0.1 ml 
pyridine and 0.1 ml 35% formaldehyde are added and the whole is held for 
30 mins. at room temperature. 20 mg rabbit IgG in 0.02M phosphate-buffered 
saline (1 ml) and 0.3 ml pyridine are then added and the reaction allowed 
to stand for ca. 14 hrs. at room temperature. The obtained conjugate is 
dialysed against 30% pyridine, 10% pyridine and, finally, 0.005M 
phosphate-buffered saline. The coupling rate of the conjugate is 1:11.4. 
EXAMPLE 9 
Production of hybridoma cell-lines, producing monoclonal antibodies 
reactive with Cyclosporine: [Process step viii)] 
9.1 Employing the conjugate of example 7.1 
a) Immunisation 
Female Balb/c mice (20-25 g) each receive 100 .mu.G of the immunogenic 
conjugate product of example 7.1 in 0.2 ml complete Freund adjuvant, 
administered by i.p. injection. After 2 weeks a second booster injection 
comprising 50 .mu.g of the product of example 7.1 emulsified in 0.2 ml 
complete Freund adjuvant is administered, again by i.p. injection. The 
presence of antibodies reactive to Cyclosporine in the serum of treated 
mice is confirmed by regular RIA assay employing tritium labelled 
Cyclosporine as tracer. 
b) Hybridoma Generation 
Mice obtained in step a) exhibiting maximum Cyclosporine reactive antibody 
titres receive a booster injection comprising 20 .mu.g of the product of 
example 3.2 in saline (0.85% w/v) administered i.v. The mice are 
sacrificed on the 4th. day, and spleen cells, isolated and fused with 
mouse (Balb/C) myeloma cells in accordance with the methods described by 
S. Fazekas et al., J. Immunol. Methods, 35, 1-21 (1980). 
Growing hybridomas are screened for production of antibody reactive to 
Cyclosporine by regular RIA assay technique employing tritium labelled 
Cyclosporine as tracer, and exhibiting low cross-reactivity with 
Cyclosporine 17, again using regular RIA assay technique with tritum 
labelled Cyclosporine as tracer and Cyclosporine 17 as competitive ligand. 
One selected hybridoma line, J19.2, is found to produce a monoclonal 
antibody reactive with Cyclosporine and having low cross-reactivity with 
Cyclosporine 17. The antibody is characterised as belonging to the class 
IgG, subclass IgG.sub.1. The obtained IC.sub.50 value for reactivity with 
Cyclosporine in RIA is 6.7 ng/ml, compared with 280 ng/ml for Cyclosporine 
17. Cross-reactivity with Cyclosporine 17 is thus of the order of 2% only. 
Determined affinity constant with respect to Cyclosporine is of the order 
of 10.sup.-9 mol/liter. 
The said hybridoma line has been deposited with the National Collection of 
Animal Cell Cultures, (now known as the European Collection of Animal Cell 
Cultures) PHLS Centre for Applied Microbiology and Research, Porton Down, 
Salisbury, SP4 OJG. U.K. under the accession no. 85 06 14 01 (date deposit 
13. Jun. 1985). 
It will be appreciated that by application of the techniques of the present 
invention as generally taught herein, in particular the employment of 
cyclosporins having an activated coupling group, e.g. as hereinbefore 
defined under any one of 1.1 to 1.11, 1.14 or 1.15 (formulae IIa, IIb, IIc 
or IId) for the preparation of immunogenic conjugates, and proceeding e.g. 
analogously to the general methods of this example, hybridoma 
lines/monoclonal antibodies may. readily be prepared which, though not 
identical with the specific product hybridoma line/monoclonal antibody of 
this example, will meet the same essential criteria, e.g. exhibit 
equivalent or even improved characteristics to those described above. This 
will be apparent from results evidenced in the following example. 
9.2 Employing the conjugate of example 7.2 
a) Immunisation 
Mice (Balb/c) each receive 100 .mu.g of the immunogenic conjugate product 
of example 7.2 in 200 .mu.l phosphate buffered saline/Freund adjuvent 
(1:1). The first administration (complete Freund adjuvent) is effected 
s.c. in the hind foot pad, near the tail and near the neck. After three 
weeks, second and third administrations follow (incomplete Freund 
adjuvent) effected s.c. on the back and i.m. in the hind legs 
respectively. Blood samples are collected 1 week after both the 2nd and 
3rd administrations. 
Mice are selected for further use on the basis of the following measured 
antisera criteria: 
1. Titre in liquid phase RIA and in ELISA; 
2. Apparent isotype distribution (IgG.sub.1 only or IgG.sub.1+2a+2b in 
ELISA); 
3. Relative avidity in ELISA; 
4. Capacity to discriminate between Cyclosporine and Cyclosporine 17 and 
Cyclosporine 18 in competitive ELISA. 
Selected mice are given booster injections on days -3, -2 and -1 prior to 
fusion using 100 .mu.g of the immunogenic conjugate product of example 7.2 
in 200 .mu.l, 9% NaCl, by i.p. (50%) and i.v. (50%) injection on day -3, 
and i.p. (100%) on days -2 and -1. 
b) Hybridoma generation 
2.5.times.10.sup.-7 or 5.times.10.sup.-7 spleen cells from each mouse are 
fused with 5.times.10.sup.-7 mouse (Balb/c) myeloma cells using PEG 4000 
and distributed into 24.times.24 wells. 
Culture supernatants are screened in ELISA for the presence of antibodies 
recognising [Thr].sup.2 -Cyclosporine coupled to bovine serum albumin 
(prepared analogously to example 7.2) and/or [(D)Lys].sup.8 -Cyclosporine 
coupled to bovine serum albumine (prepared analogously to example 7.1) in 
preference to free bovine serum albumin as negative control. Selected IgG 
producing hybridoma lines are cloned to guarantee monoclonality. 
Ability of monoclonal antibodies produced by hybridoma lines obtained, to 
distinguish/discriminate between (a) Cyclosporine and (b) Cyclosporine 17 
and Cyclosporine 18 is tested in a competition format of indirect ELISA as 
described by Quesniaux et al., Immunology Letters, 9, 99-104, (1985), in a 
variety of buffer systems including: phosphate buffered saline at pH 7.5, 
with and without 0.03% Tween 20; and Tris at pH 7.5, with 0.03% Tween and 
without NaCl. Optimal conditions for discrimination are generally observed 
in phosphate buffered saline at pH 7.5, with 140 mM NaCl and 0.03% Tween 
20. Of 9 clone lines examined 7 produce monoclonal antibodies 
discriminating between (a) Cyclosporine and (b) Cyclosporines 17 and 18. 
For 6 the IC.sub.50 ratio of Cyclosporine 17 compared to Cyclosporine is 
ca. 35.times. or greater. 
EXAMPLE 10 
Production of regular polyclonal antisera reactive with Cyclosporine 
[Process step xi)] 
Sheep are immunised by hind limb, intramuscular injections 10.times. at 
intervals of approx. 14 days. The injections comprise a lyophylisate of 
the immunogenic protein conjugate product of example 8.1 (3 mg), Alugel S 
(0.2 ml) and Freund adjuvant (0.6 ml). The final titre obtained against 
tritiated Cyclosporine is 1/100,000 as measured by RIA. 
Polyclonal antisera recovered are found to exhibit improved discrimination 
between Cyclosporine and its metabolite Cyclosporine 17 as compared with 
polyclonal antisera obtained using [Thr].sup.2 -Cyclosporine-IgG 
conjugates known in the art, e.g. as employed in current Cyclosporine RIA 
assay kits. Thus for antisera obtained in accordance with the present 
example, cross-reactivity with Cyclosporine 17 is of the order of ca. 18% 
as compared with ca. 42.0% for known polyclonal antisera. 
EXAMPLE 11 
Preparation of labelled derivatives of cyclosporins as defined under 5.1 
above: [Process step ix)] 
11.1: 
Preparation of [N- -TRITC-(D)Lys].sup.8 -Cyclosporine 
[(D)Lys].sup.8 -Cyclosporine (15 mg) produced in accordance with example 1 
are dissolved in methylene chloride (2 ml). Rhodamine isothiocyanate 
(TRITC) (5.3 mg) is added and the reaction mixture allowed to stand at 
-7.degree. C. for 17 hours. The intensively-red-coloured solution is 
directly chromatographed on Sephadex LH20 (20 g) with methylene chloride 
and 0.5% methanol. Fractions are collected in 10 ml portions. 
The title compound is recovered as an oil from fractions 5 to 7 and 10 to 
14: UV absorption: 300 nm/fluorescence emission: 540 nm. The residue at 
the 8-position has the structure: 
##STR13## 
11.2: 
Preparation of [N-.epsilon.-Dansyl-(D)Lys].sup.8 -Cyclosporine 
[(D)Lys].sup.8 -Cyclosporine (232 mg) produced in accordance with example 1 
is dissolved in chloroform (15 ml). Ethyl diisopropylamine (7.3 mg) and 
dansyl chloride (99.5 mg) are added and the reaction mixture is stirred 
for 2 hours. The product is chromatoraphed directly on Sephadex LH20 (100 
g) with methylene chloride and 0.5% methanol. Fractions are collected in 
10 ml portions. 
The collected fractions are evaporated and the resulting product 
re-chromatographed using silica gel (0.06-0.20 mm) (100 g) with methylene 
chloride and 5% methanol. Fractions are collected in 15 ml portions. 
Fractions 28-44 yield pure product: [.alpha.].sub.D.sup.20 =-183.8.degree., 
c=1.08 in CHCl.sub.3. 
11.3: 
Preparation of .sup.125 Iodinated Derivative of [(D)Lys].sup.8 
-Cyclosporine 
Title compound is prepared analogously to the methods described by Bolton 
and Hunter [Biochem. J. 133, 529 (1973)] by attachment of a 
p-OH-phenylpropionyl residue to the N-.epsilon.-atom of the residue at the 
8-position of [(D)Lys].sup.8 -Cyclosporine prepared in accordance with 
example 1. The .sup.125 I label is carried in the phenyl ring of the 
p-OH-phenylpropionyl residue 
##STR14## 
Purification is effected by HPLC on a 4.times.250 column of RP18 with a 
linear gradient and using 10-30% n-propanol/0.2% trifluoroacetic acid in 
5% acetic acid/0.2% trifluoroacetic acid as liquid phase.