Non-depleting CD4-specific monoclonal antibodies for the treatment of insulin-dependent diabetes mellitus (IDDM)

Non-depleting CD4 monoclonal antibodies may be used in the treatment of insulin-dependent diabetes mellitus.

The present invention relates to the use of certain antibodies in the 
prevention and treatment of insulin-dependant diabetes mellitus. 
Insulin-dependant diabetes mellitus (hereafter IDDM) is the juvenile-onset 
form of diabetes. At present there is no available cure for the disease 
and treatment consists of maintenance of insulin levels by oral or 
intramuscular administration and palliation of the inevitable side-effects 
both of the disease itself and of the treatments. 
There has been a continuing need to find a means to arrest the loss of 
insulin-producing .beta.-cells in the pancreas which is the immediate 
result of the underlying abnormalities, but little progress has been made. 
The present invention is founded upon the surprising observation that 
administration of a non-depleting CD4 monoclonal antibody (hereafter nd 
CD4 mAb) can arrest the loss of insulin-producing cells in an animal model 
of IDDM. It is now believed that the use of nd CD4 mAbs will be effective 
in arresting IDDM in humans and that this treatment will also permit 
regeneration of the .beta.-cells such that the course of the disease may 
even be reversed. 
WO-A-90/15152 describes the use of nd CD4 mAbs in conjunction with 
non-depleting CD8 monoclonal antibodies in inducing tolerance to an 
antigen and suggests that this may be useful in surgery and therapy, for 
instance in preventing transplant rejection, treating autoimmune diseases 
and in avoiding undesirable immune reactions to peptide and hormone 
therapeutic agents. However this treatment is intended to block CD4.sup.+ 
and CD8.sup.+ cells and there is no indication that nd CD4 mAbs alone 
would be useful in any treatment. 
Accordingly the present invention provides a method for treating 
insulin-dependant diabetes mellitus comprising administering an effective, 
non-toxic amount of at least one non-depleting CD4 monoclonal antibody to 
a human or non-human patient in need thereof. 
As used herein the term "antibody" is intended to include any binding 
member having a binding domain which reacts with an epitope of the CD4 
cell surface antigen. Thus the invention also covers derivatives and 
homologues of CD4 antibodies, fragments of antibodies containing at least 
one antigen binding site such as Fab and F(ab')2 fragments and "single 
domain antibodies" also known as dabs. Monoclonal antibodies according to 
the invention are antibodies or fragments thereof produced by a clone of 
cells all derived from a single antibody-producing cell, which may have 
been obtained from a mammal immunised against the antigen recognised by 
the antibody or by transformation of a cell with expressible DNA encoding 
the antibody or fragment thereof, such DNA having been removed from a cell 
obtained from a mammal immunised against the antigen recognised by the 
antibody or constructed by recombinant techniques. Techniques of 
recombinant DNA technology may be used to produce antibodies or chimeric 
molecules with appropriate specificity for CD4. Such techniques may 
involve introducing DNA encoding an immunoglobulin variable region, or one 
or more complementarity determining regions capable of binding CD4, to the 
constant regions, or constant regions plus framework regions, of a 
different immunoglobin, for example to convert a mouse-derived monoclonal 
antibody into one having largely human immunoglobulin characteristics (see 
EP 184187A, GB 2188638A). Another possibility is to attach just the 
variable region of an antibody to another non-immunoglobulin molecule, to 
produce a chimeric molecule (see WO 86/01533). Yet another possibility 
would be to produce a mutation in the DNA encoding the monoclonal 
antibody, so as to alter certain of its characteristics without changing 
its essential specificity. This can be done by site-directed mutagenesis 
or other techniques known in the art. Monoclonal antibodies and 
derivatives and homologues thereof and fragments thereof are produced by 
conventional techniques. 
As used herein the term "CD4 monoclonal antibody" refers to a monoclonal 
antibody, or fragment thereof containing at least one antigen-binding 
site, capable of specifically binding an epitope of a CD4 cell surface 
antigen. The term "CD4 cell surface antigen" includes the human CD4 cell 
surface antigen and corresponding cell surface antigens of other mammals 
such as the L3T4 antigen of mice. 
As used herein the term "non-depleting CD4 monoclonal antibody" refers to 
CD4 monoclonal antibodies which deplete fewer than 50% of target cells in 
vitro. Preferred nd CD4 mAbs deplete fewer than 25% and most probably less 
than 10% of target cells in vitro. 
A simple test to ascertain wherein a CD4 mAb should be regarded as 
non-depleting is to take a sample of peripheral blood, count the target 
cells, ie CD4.sup.+ cells, in an aliquot as a control, treat a further 
aliquot with the CD4 mAb and count the target cells after treatment; if 
there are 50% or greater target cells in the treated aliquot compared with 
the control aliquot, the CD4 mAb is non-depleting in accordance with the 
invention. 
For use in the present invention nd CD4 mAbs may be obtained by 
conventional techniques for raising mAbs against CD4 and screening and 
selecting clones with secrete non-depleting antibodies. Typically such 
antibodies will be of the IgG.sub.2 class such as rat IgG.sub.2a, mouse 
IgG.sub.2b or human IgG.sub.2 but human IgG.sub.4 are also useful. A 
preferred nd CD4 mAb for use in accordance with the present invention is 
YTS177.9 produced by a hybridoma deposited with the European Collection of 
Animal Cell Cultures Vaccine Research and Production Laboratory, Public 
Health Laboratory Service, Centre for Applied Microbiology and Research, 
Porton Down, Salsibury, Wiltshire SP4 OJG, United Kingdom under accession 
number ECACC 90053005 on 30th May 1990 in connection with International 
patent Application No PCT/GB90/00840. 
Other preferred nd CD4 mAbs for use in the present invention include nd CD4 
mAb fragments of YTS 177.9 (ECACC 90053005) and nd CD4 mAbs and fragments 
thereof which have similar or higher affinity for the same epitope as YTS 
177.9. 
The treatment of diabetes patients in accordance with the present invention 
may commence in individuals identified as being at risk (for instance 
because of a family history of IDDM) prior to emergence of the disease but 
this is generally less preferred as it is believed that this will lead to 
a generalised tolerising of the patient to irrelevant antigens leading to 
a state of immunosuppression. More preferably the treatment is commenced 
once the disease has become patent since at this stage loss of 
.beta.-cells has already commenced and the treatment will then primarily 
interfere with the cause of the disease. Ideally treatment will be 
commenced soon after the disease has become patent so that the patient 
will retain the majority of .beta.-cells and, even if there is no 
regeneration of .beta.-cells, insulin levels will be susceptible of 
management by conventional techniques but avoiding administration of 
insulin. Even when the disease has progressed to a late stage, the 
treatment will be beneficial in protecting the patient's remaining 
.beta.-cells. 
Treatment according to the invention comprises administration of at least 
one dose of nd CD4 mAb and preferably comprises a course of several doses. 
The exact amount to be administered in any single dose and the number and 
timing of any subsequent doses will be determined by many factors 
including the age, sex, weight and size of the patient, the patient's 
general health and level of nutrition and the stage to which the patient's 
disease has progressed. As a general guide, it is believed that saturating 
amounts of the nd CD4 mAb will be most effective and that such amounts may 
be determined by routine pharmacokinetic studies; administration of 
greater than such amounts is uneconomic but unlikely to be harmful whereas 
administration of less than saturating amounts may also be effective but 
is likely to be less effective such that additional doses may be required. 
In a model mouse system, doses of from 1 .mu.g to 2 mg, preferably from 400 
.mu.g to 1 mg of nd CD4 mAb are administered. In humans, doses of from 1 
to 400 mg, such as from 3 to 30 mg, for example from 5 to 20 mg of nd CD4 
mAb may be contemplated in an otherwise normal healthy adult of about 75 
kg. 
Typical treatment regimes involve repeat doses being administered several 
times per week, for instance from 1 to 7 times per week, preferably from 1 
to 4 times per week, for example 3 times per week and such regimes may 
continue for several weeks or even months, for instance for at least 2 
weeks and up to 6 months, more preferably up to two months. It is 
presently envisaged that a single course of treatment will cure the 
disease for the lifetime of the patient but relapses and recurrences of 
the disease may be treated with further courses of treatment. 
Preferably the nd CD4 mAbs are administered in accordance with the 
invention by conventional routes such as orally or parenterally, for 
instance by subcutaneous or intravascular, preferably intravenous, 
injection. Where large doses are to be administered or the chosen route of 
administration limits the amount that may be delivered to a patient in a 
single injection, several sub-doses may be administered to achieve the 
desired total dose or the dose may be infused for instance intravenously. 
Conveniently the nd CD4 mAbs are administered in the form of a composition 
comprising a pharmaceutically acceptable diluent or carrier. Preferred 
compositions are pharmaceutical formulations for oral administration or 
for injection. Such formulations will generally comprise, in addition to a 
conventional diluent or carrier, suitable accessory ingredients. For oral 
administration the formulations may be presented in the form of tablets, 
capsules and other discrete dosage units or in multi-dose form such as 
bulk powders and liquids. For parental administration the formulations may 
be presented as solutions for injection, concentrated solutions to be 
diluted with a solvent for instance with pyrogen-free demineralised water 
or water for injection) prior to injection or as dry powders for 
dissolution in a solvent (for instance water for injection) prior to 
injection. The accessory ingredients, which will be selected according to 
the type of presentation, may be fillers, flavours, tabletting aids and 
coatings, preservatives, antioxidants, stabilisers, buffers, antimicrobial 
agents, surfactants, salts for adjusting tonicity and other conventional 
ingredients well known in the art of pharmacy. 
As previously mentioned, it is sufficient for the present treatment to 
administer a single nd CD4 mAb species. Use of a cocktail of nd CD4 mAbs 
may also be contemplated but the use of CD8 mAbs and depleting mAbs is not 
required and preferably is avoided. In a particular aspect the invention 
therefore provides a method as hereinbefore described consisting 
essentially of administering a single nd CD4 mAb species or a composition 
thereof. Preferably the method consists of the administration of a 
composition of a single nd CD4 mAb species unaccompanied by administration 
of any CD8 mAb and unaccompanied by administration of any depleting mAb. 
In a further aspect the present invention provides the use of and CD4 mAb 
in the preparation of a medicament for use in the treatment of IDDM in a 
human or non-human. Preferably the medicament is a composition or 
pharmaceutical formulation as hereinbefore described. The treatment will 
be as hereinbefore described, preferably using nd CD4 mAb YTS 177.9

The invention will now be illustrated by the following Example which is not 
intended to limit the scope of protection in any way. 
EXAMPLE 1 
The NOD mouse is considered by many to be a good model for IDDM since the 
spontaneous incidence among females is 60 to 80% by 30 weeks in most 
colonies. Among males, the incidence is much lower (for instance less than 
10%) and this makes them an ideal recipient, when immunocompromised by 
irradiation, in which to induce disease by the transfer of spleen cells 
from diabetic donors. 
In this study it is shown that YTS177 strongly protects NOD mice from IDDM 
transferred by diabetic donor spleen cells. YTS177 is a non-depleting 
IgG.sub.2a anti-CD4 rat monoclonal antibody which although recognising the 
same epitope as the depleting IgG.sub.2b monoclonal anti-CD4 YTS191.1 
(ECACC 87072282) has a different mode of action since CD4.sup.+ T cells 
are not eliminated but appear to be permanently anergised. 
Groups of 4 or 5 mice were irradiated using a cobalt source (650 rads per 
mouse) and spleen cells (2.times.10.sup.7 per mouse) from diabetic donors 
were transferred intravenously the following day. A control group (Group 
1) received no antibody treatment. Two groups of mice were treated with 
YTS177, given i.v. (2 mg per mouse) 3 days before transfer of spleen 
cells, and i.p. (2 mg per mouse on each occasion) on the next 2 days and 
then the same dose i.p. 3 times weekly for a total of 10 days following 
transfer of spleen cells (Group 3) or until sacrifice at week 4 (Group 2). 
The experiment was repeated for groups 1 and 2 only. 
As can be seen from Table 1 (Experiments 1 and 2) below, none of the mice 
in Groups 1 and 2 were hyperglycaemic by week 4 compared with 7/9 control 
animals. Three months later all mice in Group 3 were still normoglycaemic. 
In addition to repeating Groups 1 and 2, Experiment 2 included a group of 
mice in which YTS177 treatment was delayed until 12 days after transfer of 
spleen cells when it was given i.v., i.p. and i.p. on 3 consecutive days 
and then 3 times weekly as before (Group 4). In Experiments 3 and 4 Groups 
1 and 4 were repeated and administration of the depleting CD8 mAb 
(YTS169.4; ECACC 87072284) to a further group of mice (400 .mu.g i.v., 
i.p. and i.p. on 3 consecutive days) starting 12 days after transfer of 
spleen cells was included for comparison (Group 5). Table 1 shows that no 
animal in Experiments 2,3 or 4 given YTS177 became diabetic whereas 9/13 
untreated animals and 1/10 animals treated with depleting CD8 mAb were 
hyperglycaemic by week 5. In Experiment 4, a further group of mice (Group 
6) were given the depleting CD4 mAb (YTS191.1 ECACC 87072282) starting 12 
days after transfer and any protection conferred on these animals was 
barely significant. 
The doses of the depleting antibodies administered in Experiments 3 and 4, 
although much less than those of the non-depleting YTS177, were found 
previously to deplete animals of virtually all CD4.sup.+ or CD8.sup.+ T 
cells. 
To ascertain the protective effect of YTS177 on islet morphology, cryostat 
sections were prepared from the pancreata of Experiment 3 mice and stained 
for infiltrating T cells. Examination of pancreata from untreated mice 
sacrificed at day 12 after transfer revealed that peri-islet infiltration 
was already extensive with some intra-islet lymphocytes also present 
although the number of residual islets was still relatively low (23%). 
Pancreata from animals given YTS177 from day 12 onwards and killed at 4 
weeks after transfer were indistinguishable from the previous group (19% 
residual islets) suggesting that the antibody had arrested the 
infiltration and prevented any further .beta. cell destruction. 
In contrast, the animals given no antibody treatment and killed at week 4 
had extensive intra-islet infiltration and 70% of the islets were 
residual. Group 5 (treated with depleting CD8 mAb from day 12 onwards) 
although not yet overtly diabetic, nevertheless displayed evidence of 
considerably more B cell destruction (51% residual islets) than the mice 
treated with YTS177. In Experiment 4, where animals similarly treated were 
allowed to go on beyond 4 weeks, it was seen that by 8 weeks, 3/5 of the 
Group 5 animals were diabetic whereas none of the YTS177 treated mice 
showed any signs of overt disease. In the same experiment it was seen that 
the depleting CD4 mAb afforded no significant or lasting protection. 
It could be argued that YTS177 is effective simply as a result of general 
immunosuppression, but when SRBC were administered i.p. to Group 4 mice 
two weeks after cessation of YTS177 treatment, or to a group of NOD mice 
irradiated and reconstituted with 2.times.10.sup.7 normal spleen cells, 
the level of agglutinating anti-SRBC antibody found in the sera 5 days 
later, were comparable in both groups. 
The protection afforded by antibody YTS177 was unexpectedly powerful when 
compared with the effect of monoclonal antibodies depleting either of the 
two major T cell subsets. In the case of the latter, it may be expected 
that newly emerging T cells will eventually be able to mount a response 
against .beta. cells and any protection would thus be of limited duration. 
The nd CD4 mAb, by switching off .beta. cell specific effectors and, 
perhaps, by initiating a protective idiotypic network, seems to offer 
permanent protection. 
TABLE 1 
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Number of mice diabetic at week 4 or 5 after transfer 
Group 2 
Group 3 
Group 4 
Group 5 
Group 6 
(nd CD4 
(nd CD4 
(nd CD4 
(d CD8 
(d CD4 
Group 1 
mAb mAb mAb mAb mAb 
Exp No 
(Control) 
d 0-30) 
d 0-10) 
d 12+) 
d 12+) 
d 12+) 
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1 4/5 0/4 0/4 N.D. N.D. N.D. 
2 3/4 0/4 N.D. 0/5 N.D. N.D. 
3 4/4 N.D. N.D. 0/5 0/5 N.D. 
4 2/5 N.D. N.D. 0/4 1/5 2/4 
Total 
13/18 
0/8 0/4 0/14 1/10 2/4 
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