Use of anti-CD45 leukocyte antigen antibodies for immunomodulation

A method for the prevention or reversal of transplant rejection, or for therapy for autoimmune diseases, is provided comprising administering compounds such as monoclonal antibodies, that bind specifically to one or more preselected CD45R leukocyte antigens, including the CD45RB epitope.

BACKGROUND OF THE INVENTION 
Organ, cell and tissue transplant rejection and the various autoimmune 
diseases are thought to be primarily the result of a T-cell mediated 
immune response. This T-cell mediated immune response is initially 
triggered by helper T-cells which are capable of recognizing specific 
antigens. These helper T-cells may be memory cells left over from a 
previous immune response or naive cells which are released by the thymus 
and may express any of an extremely wide variety of antigen receptors. 
When one of these helper T-cells recognizes an antigen present on the 
surface of an antigen presenting cell (APC) or a macrophage in the form of 
an antigen-MHC complex, the helper T-cell is stimulated to produce IL-2 by 
signals emanating from the antigen-specific T-cell receptor, co-receptors, 
and IL-1 secreted by the APC or macrophage. The helper T-cells then 
proliferate. Proliferation results in a large population of T-cells which 
are clonally selected to recognize a particular antigen. T-cell activation 
may also stimulate B-cell activation and nonspecific macrophage responses. 
Some of these proliferating cells differentiate into cytotoxic T-cells 
which destroy cells having the selected antigen. After the antigen is no 
longer present, the mature clonally selected cells will remain as memory 
helper and memory cytotoxic T-cells, which will circulate in the body and 
recognize the antigen should it show up again. If the antigen triggering 
this response is not a foreign antigen, but a self antigen, the result is 
autoimmune disease; if the antigen is an antigen from a transplanted 
organ, the result is graft rejection. Consequently, it is desirable to be 
able to regulate this T-cell mediated immune response. 
CD45 antigen (CD45) is expressed on most leukocytes. Indeed, it was 
previously thought that a common CD45 antigen was present on all 
leukocytes, for which reason the receptor was originally known as the 
Leukocyte Common Antigen (LCA). Monoclonal antibodies (mAbs) to CD45 were 
proposed as a means of effectively eliminating all leukocytes where 
desirable, for example, purging an organ to be transplanted of passenger 
leukocytes prior to transplantation using nonspecific CD45 monoclonal 
antibody. See, e.g., WO 91/05568. 
It has recently been shown that different isoforms of CD45 are generated by 
alternate splicing of a single primary transcript of the CD45 gene. These 
CD45 isoforms include CD45RA, CD45RB, CD45RC, and CD45RO. CD45RA contains 
the expression product of exon 4 (sometimes referred to as R.sup.A) of the 
CD45 gene; CD45C contains the expression product of exon 6; CD45RB 
contains the expression product of exon 5; CD45RO does not contain the 
expression products of any of the three exons 4, 5, or 6. See Hall et al, 
"Complete Exon-Intron Organization of the Human Leukocyte Common Antigen 
(CD45) Gene," J. Immunol., 141, 2781(1988), herein incorporated by 
reference and Streuli et al, "Characterization of CD45 and CD45R 
Monoclonal Antibodies Using Transfected Mouse Cell Lines that Express 
Individual Human Leukocyte Common Antigens," J. Immunol., 141, 3910 
(1988). The significance of this variable expression, however, has been 
unclear. 
Increased success in clinical organ transplantation has paralleled 
improvements in techniques for immunosuppression. However, increasingly 
potent immunosuppressant drugs often produce complications due to their 
lack of specificity. For example, recipients can become very susceptible 
to infection. Highly specific immunosuppression is therefore desired. 
The ideal specific immunosuppression method would be a treatment which 
suppresses the action of the lymphocytes responsible for rejection of the 
particular graft the patient receives without otherwise affecting the 
immune system. 
Therefore, a need exists to durably and selectively suppress or otherwise 
modulate the immune response in humans, particularly transplant recipients 
or those afflicted with autoimmune diseases. 
SUMMARY OF THE INVENTION 
The present invention provides a method for in vivo immunosuppression in 
humans and mammals. The methods include pretreatment in vivo therapies to 
prevent rejection of transplanted cells, tissues and organs and 
post-transplant in vivo therapies to reverse a pathological immune 
response. Preferably, the present method can impart durable tolerance, 
rather than just delayed rejection to the recipient. The methods also 
include in vivo treatment of autoimmune diseases. 
Specifically, the method of the present invention comprises administering 
to a patient in need of such treatment, an effective immunosuppressive 
amount of at least one compound which binds specifically to a CD45 
leukocyte antigen present on T-cells. For example, the method of the 
present invention can be used to treat a patient undergoing transplant 
rejection, including graft-versus host disease or afflicted with an 
autoimmune disease. Preferably, the compound binds to the CD45RB receptor. 
The present invention additionally provides pharmaceutical compositions 
comprising an effective immunosuppressive amount of at least one compound 
which specifically binds to a CD45 antigen in combination with a 
pharmaceutically acceptable carrier. The term "compound" is meant to 
indicate, for example, antibodies as defined herein, and molecules having 
antibody-like function such as synthetic analogues of antibodies, e.g., 
single-chain antigen binding molecules, small binding peptides, or 
mixtures thereof. 
Preferably, the compound of the present method is an antibody. More 
preferably, the antibody administered will be capable of binding to the 
CD45RB leukocyte antigen, the CD45RO leukocyte antigen, the CD45RA 
leukocyte antigen or the CD45RC leukocyte antigen. Most preferably, the 
antibody is capable of binding to the CD45RB or CD45RO leukocyte antigen. 
As mentioned hereinabove, the method of the present invention is useful in 
the treatment of transplant rejection. More specifically, the method may 
be employed for the treatment of a patient that has undergone cell tissue 
or organ transplantation that is either allogeneic or xenogeneic. 
Furthermore, the method of the present invention may be utilized prior to, 
following or concurrently with the transplant procedure, or any 
combination thereof. 
The method of the present invention is contemplated to be beneficial in a 
variety of transplant situations, even those situations where a recipient 
may receive sequential transplants of the same or different cells, 
tissues, or organs. For example, the method of the present invention may 
be utilized during a heart, liver, bone marrow or kidney transplant, or 
during the transplantation of pancreatic islets or vascular tissue, e.g., 
a coronary bypass procedure. 
The method of the present invention may also be useful in the treatment or 
prevention of autoimmune disease, inflammatory conditions and arthritic or 
rheumatoid diseases. For example, the method of the present invention may 
be employed for the treatment of autoimmune hematological disorders, 
systemic lupus erythematosus, inflammatory bowel disease, ulcerative 
colitis, Crohn's disease, multiple sclerosis, diabetes mellitus type 1, 
and the like. 
In a further embodiment of the method of the present invention, an 
anti-inflammatory or immunosuppressive drug may be administered prior to, 
following, or concurrently with the compound described hereinabove. For 
example, suitable drugs for this purpose include, but are not limited to, 
cyclosporin, FK-506, rapamycin, corticosteroids, cyclophosphamide, 
mycophenolate, mofetil, leflunomide, anti-lymphocytc globuline, 
deoxyspergualin OKT-3 and the like. 
In yet another embodiment, the method of the present invention may further 
comprise administering an amount of the patient's lymphocytes to the 
patient, i.e., in combination with the CD45 leukocyte antigen binding 
compound(s). 
This invention is based on the discovery that leukocytes such as different 
types of T lymphocytes, or "T-cells" may predominantly express one or 
another CD45 isoform. Naive helper T-cells and memory T-cells express 
predominately CD45RA and CD45RO respectively. CD 45RB expression is also 
variable; it is highly expressed (bright) on naive helper T-cells (Th0) 
and on T-cells that produce predominantly interleukin 2 and can induce 
inflammatory and cytotoxic responses (Th1). T cells that predominantly 
produce interleukin 4 and induce humoral immune responses (Th2) have low 
CD45RB expression (dim). 
It has now been shown that some antibodies which react with CD45RB (MB23G2 
in mouse, 6G3 in primates) are capable of selectively inhibiting the 
inflammatory and cytotoxic T-cell mediated immune response without 
destroying the pool of memory T-cells. Consequently, CD45RB suppressors 
have a great advantage over current immunosuppressants in that (i) they 
act on a particular T-cell population rather than having an overall 
immunosuppressive effect, thereby avoiding the risk of side effects 
associated with over-suppression of the immune system; and (ii) they are 
capable of conferring long term tolerance to a particular antigen when 
they are administered contemporaneously with exposure to antigen, e.g., 
just before and after an organ transplant or during an acute phase of an 
autoimmune disease. 
As used herein, the term "immune tolerance" or simply "tolerance" is 
intended to refer to the durable active state of unresponsiveness by 
lymphoid cells to a preselected or specific antigen or set of antigens. 
The immune response to other immunogens is thus unaffected, while the 
requirement for sustained exogenous immunotherapy is either reduced or is 
eliminated. Additionally, tolerance enables subsequent transplantation of 
material comprising the same antigen or set of antigens without increasing 
the need for exogenous immunotherapy. 
Generally, it is believed that the present methods may lead to T-cells 
having a receptor for the antigen becoming anergized, so that the T-cell 
clones are functionally, if not actually, deleted. For a fully functional 
activation of T-cells two signals are necessary. The first signal requires 
recognition of an antigen via the T-cell receptor. The second signal 
requires interaction between co-stimulatory molecules, such as B7, on 
antigen presenting cells and receptors, such as CD28, on the T-cells. It 
is generally accepted that lack of this second signal through CD28 leads 
to anergy. However, CD45 is required for the activation through the T-cell 
receptor and interference with this process through CD45RB interrupts the 
first signal and can also lead to anergy. This is in fact a more 
fundamental approach than blocking of the B7-CD28 interaction, since there 
are a number of different co-stimulatory pathways, whereas there is only 
the one T-cell receptor complex. Since CD45 is differentially expressed it 
is also possible to selectively affect specific subsets of T-cells. Thus, 
the observed effects are not due solely to depletion of a subset, or of 
the general T-cell population, but rather involve a combination of 
transitory depletion and a durable effect that leads to tolerization of 
the recipients. For example, in a mouse kidney transplant model, allograft 
tolerance following initial treatment with anti-CD45RB monoclonal antibody 
persists indefinitely, with survival well in excess of 100 days. In mice 
surviving over 100 days following a kidney allograft, the present method 
permits skin grafts syngeneic with the donor kidney to be tolerated, while 
skin grafts allogeneic with both the recipient and the donor kidney were 
rejected. 
The term "antibody", includes human and animal mAbs, and preparations of 
polyclonal antibodies, as well as antibody fragments, synthetic 
antibodies, including recombinant antibodies, chimeric antibodies, 
including humanized antibodies, anti-idiotopic antibodies and derivatives 
thereof. 
As used herein, the term "treating", with respect to an autoimmune disease 
or condition includes preventing the onset or flare-up of the disease or 
condition, as well as reducing or eliminating one or more symptoms of the 
disease or condition.

DETAILED DESCRIPTION OF THE INVENTION 
The compounds, antibodies and compositions of the invention are preferably 
produced as described in the following examples, or by equivalent means as 
would be apparent to one skilled in the art. 
It will be understood by those skilled in the art that the hybridomas 
herein referred to may be subject to genetic mutation or other changes 
while still retaining the ability to produce monoclonal antibody of the 
same desired specificity. The present invention therefore encompasses 
mutants, other derivatives and descendants of the hybridomas. 
It will be further understood by those skilled in the art that a monoclonal 
antibody can be subjected to the techniques of recombinant DNA technology 
to produce other derivative antibodies, humanized or chimeric molecules or 
antibody fragments which retain the specificity of the original monoclonal 
antibody. Such techniques may involve combining DNA encoding the 
immunoglobulin variable region, or the complementarity determining regions 
(CDRs), of the monoclonal antibody with DNA coding the constant regions, 
or constant regions plus framework regions, of a different immunoglobulin, 
for example, to convert a mouse-derived monoclonal antibody into one 
having largely human immunoglobulin characteristics (see EP 184187A, GB 
2188638A, both herein incorporated by reference). 
Antibodies Against CD45R Isoforms 
S. Poppema, et al., J. Iminunol., 147, 218 (1991), previously described the 
monoclonal antibody MT3. This publication, however, does not disclose a 
detailed method for making this antibody, nor does it disclose any 
pharmnaceutical use for this antibody and, therefore, necessarily fails to 
disclose the T-cell population recognized by this antibody. A. Lazarovits, 
et al., Transplantation, 54, 724 (1992), characterized the in vitro effect 
of this antibody. Lazarovits et al., for the first time, showed that MT3 
mAb inhibits proliferation of and generation of T-cells by interfering 
with CD45RB. In addition to MT3 mAb, Lazarovits et al. reported that 
monoclonal antibodies to CD45RB, such as an antibody produced by the cell 
line HB220 which is publicly available from the ATCC in Rockville, Md. 
(now designated anti-CD45RB mAb (MB23G2)), bind to CD45RB and are 
effective agents in inhibiting immune function in vitro and in vivo (see 
U.S. Ser. No. 08/071,009 filed Jun. 2, 1993 herein incorporated by 
reference). 
The inventors have now found that 6G3 monoclonal antibody binds to CD45RB. 
This is a murine IgG1 directed against human CD45RB. It cross-reacts with 
monkey CD45RB. The inventors have also now found that the MB23G2, 6G3, and 
MT3 monoclonal antibodies bind to neuraminidase sensitive epitopes on 
leukocytes including T-cells and that at least MB23G2 and 6G3 increase the 
tyrosine phosphorylation of phospholipase C-.gamma.1. It is of interest to 
note that HB223 (now designated MB4B4), an analogous anti-CD45RB antibody 
to those of the invention, is found not to bind to neuraminidase sensitive 
epitopes. It is also observed that MB4B4 mAB binds to a neuraminidase 
insensitive epitope and does not alter the tyrosine phosphorylation of 
phospholipase C-.gamma.1. MB4B4 was also ineffective at preventing renal 
allograft rejection in mice. The specific anti-CD45R mAbs that can be used 
in the summarized present invention are in Table I, hereinbelow; from 
Streuli et al., J. Immunol., 141, 3910 (1988) which is incorporated herein 
by reference. 
TABLE I 
______________________________________ 
Classification of Anti-CD45R mAb.sup.a 
______________________________________ 
CD45RC DNL-1.9.sup.c, OX22 
CD45RA Anti-2H4.sup.b, 3AC5, F8-11-13, E7, HI 100.sup.c, CMRF.11 
73.5.17, G1-15, 10G3, 111-1C5, Leu18, 2A10, 5A9 
MMT-1, 5E7, 4F4, HB-11 
CD45RO UCHL1.sup.c 
CD45RB PD-7/26/16, 6B6.sup.c, 6G3, MT3, MEM93.sup.d, MB2362, 
______________________________________ 
MB4B4 
.sup.a AntiCD45R antibodies were classified into three groups: CD45RA, 
CD45RO, and CD45RB, based on their binding to the 30019(LCA) cell lines. 
.sup.b Available from Coulter, Nialeah, FL. 
.sup.c Available from Pharmingen, San Diego, CA 
.sup.d See Bazil et al., Immunogenetics, 29, 202 (1989). 
Chimeric and Reshaped Antibodies 
EP-A-0 120 694 (Boss et al/Celltech, herein incorporated by reference) 
describes the cloning and expression of chimeric antibodies. In these 
derivatives, the variable domains from one immunoglobulin are fused to 
constant domains from another immunoglobulin. Usually, the variable 
domains are derived from an immunoglobulin gene from one species, for 
example a mouse or a rat, and the constant domains are derived from an 
immunoglobulin gene from a different species, perhaps a human. This 
technology is now very well known in the art. A later European Patent 
Application, EP-A-0 125 023 (Cabilly/Genetech, herein incorporated by 
reference), also U.S. Pat. No. 4,816,567, describes much the same subject 
as the Boss patent application, but describes production of other 
variations of immunoglobulin-type molecules using recombinant DNA 
technology. 
Another possibility is to attach just the variable region of the monoclonal 
antibody to another non-immunoglobulin molecule, to produce a derivative 
chimeric molecule (see WO 86/01533, Neuberger and Rabbits/Celltech, herein 
incorporated by reference). A further possibility would be to produce a 
chimeric immunoglobulin having different specificities in its different 
variable regions, e.g., the monoclonal antibodies of the present invention 
(see EP 68763A). 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. 
The Winter patent application EP-A-0 239 400 (herein incorporated by 
reference) describes how it is possible to make an altered, derivative, 
antibody by replacing the complementarity determining regions (CDRs) of a 
variable region of an immunoglobulin with the CDRs from an immunoglobulin 
of different specificity, using recombinant DNA techniques--so called 
"CDR-grafting". This enables altering the antigen-binding specificity of 
an antibody. (In the present case it might be the CDRs of MT3, 6G3, 
MB23G2, an antibody with the same binding specificity as these anti-CD45RB 
antibodies, or an antibody which is cross-reactive with MT3, 6G3, or 
MB23G2 which are transferred to another antibody.) Thus, CDR grafting 
enables "humanization" of antibodies, in combination with alteration of 
the framework regions. 
Human antibodies can also be directly provided by reconstituting the human 
immune system in mice lacking their native immune system, then producing 
human antibodies in these "humanized mice." 
A "humanized" antibody containing the CDRs of a rodent antibody specific 
for an antigen of interest might well be less likely to be recognized as 
foreign by the immune system of a human. It follows that a "humanized" 
antibody with the same binding specificity as, e.g., MT3 or 6G3, or an 
antibody that cross-reacts with either (see later), might well be of 
particular use in human therapy and/or diagnostic methods. 
As discussed, the state of the art is such that the person skilled in the 
art well knows how to manipulate and alter any given antibody or gene(s) 
encoding for the same to generate a derivative to suit his or her 
particular needs. 
Anti-idiotopic Antibodies 
The provision of an antibody such as MT3 or 6G3 allows persons skilled in 
the art to obtain binding partners, e.g., antigens/epitopes or 
antibody/paratopes which bind to it. Therefore, the present invention also 
provides binding partners, e.g., antigens and/or antibodies which bind 
with an antibody or derivatives thereof as hereby provided, such as MT3 
and 6G3. 
The binding partners obtained by use of the MT3 mAb and 6G3 mAb may also be 
used to produce additional ligands, e.g., antibodies other than MT3 or 6G3 
(or molecules having antibody-like binding function, e.g., fragments, 
derivatives and synthetic analogues of antibodies such as single-chain 
antigen-binding molecules). Therefore, also provided are ligands, e.g., 
mAbs which are able to bind with a binding partner which is able to bind 
with the MT3 mAb and 6G3 mAb. Such ligands ("cross-reactive ligands"), 
e.g., mAbs may recognize the same epitope as recognized by MT3 mAb and 6G3 
mAb on said binding partner. 
The present invention also provides derivatives, functional equivalents 
(e.g., a molecule having an antibody-like binding specificity) and 
fragments of said cross-reactive ligands, perhaps produced using one or 
more of the techniques of recombinant DNA technology referred to and 
discussed above. Also included are single domain ligands (mAbs) as 
described in WO 90/05144 (herein incorporated by reference). 
Antigen Isolation 
Using standard techniques, it is possible to use a ligand, e.g., antibodies 
of the present invention and derivatives thereof, in immunopurification of 
a binding partner antigen. Techniques for immunoaffinity column 
purification are well known, see for instance "Current Protocols in 
Immunology," ed. J. E. Coligan et al, John Wyley and Sons, Unit 8.2 
(herein incorporated by reference). For instance, it is now known that the 
epitope identified by the CD45RB mAb MB23G2 is encoded by the B exon of 
the leukocyte common antigen gene. Isolation of the epitope and compounds 
binding to the epitope are contemplated by this invention. 
In fact, it should be possible to use an immunoaffinity column to isolate 
cross-reactive ligands as discussed above, without needing to isolate the 
antigens themselves. A first round of immunoaffinity purification uses a 
ligand, e.g., MT3, 6G3, etc. mAb to remove from a sample the 
antigen-containing binding partner, which may then be used in the column 
to select, from a heterogeneous population of ligands, those ligands which 
are cross-reactive with the MT3 mAb, 6G3 mAb, etc. and recognize the same 
binding partners. 
A binding partner, such as a peptide or small binding molecule, isolated 
using the ligand, e.g., the MT3, 6G3, etc. mAb may be used to select 
cross-reactive ligands from a repertoire or heterogenous population of 
antibodies generated by a whole variety of means. One way is to select 
monoclonal antibodies and cell lines producing them by the standard 
hybridoma techniques. Also provided by the present invention are 
immortalized cells, e.g., hybridomas producing said cross-reactive 
ligands. 
Another way of selecting ligands which are cross-reactive with a ligand 
such as the MT3 mAb or 6G3 mAb is to use the methods for producing members 
of specific binding pairs disclosed in WO 92/01047 (Cambridge Antibody 
Technology Limited and MRC/McCafferty et al, herein incorporated by 
reference). This publication discloses expression of polypeptide chain 
components of a genetically diverse population of specific binding pair 
members, such as antibodies, fused to a component of a secreted replicable 
genetic display package (RGDP), such as a bacteriophage, which thereby 
displays the polypeptide on the surface. Very large repertoires of 
displayed antibodies may be generated, and screened by means of antigen 
binding to obtain one or more antibodies of interest, along with their 
encoding DNA. DNA encoding for a polypeptide displayed on the surface of 
an RGDP is contained within the RGDP and may therefore be easily isolated 
and cloned for expression. The antibody repertoire screen may of course be 
derived from a human source. 
Recombinant Antibodies 
Obviously, once one has an immortalized cell line, e.g., a hybridoma, or an 
RGDP containing DNA encoding at least a polypeptide component of a binding 
ligand, one skilled in the art is in a position to obtain (according to 
techniques well known in the art, see EPA 449,769) the entire nucleotide 
sequence encoding the ligand, e.g., the mAb secreted by the cell. 
Therefore, the present invention also encompasses primary nucleotide 
sequences which encode the ligands, e.g., mAbs as defined above, together 
with fragments of these primary sequences and secondary nucleotide 
sequences comprising derivatives, mutations and hybridizing partners of 
said primary nucleotide sequences. 
These nucleotide sequences may be used in a recombinant system to produce 
an expression product according to standard techniques. Therefore, the 
present invention includes vectors (cloning and expression vectors) 
incorporating said nucleotide sequences, transformed cells incorporating 
said vectors and expression products produced by use of a recombinant 
system utilizing any such vectors or transformed cells. 
The production of fusion proteins is also contemplated. See, for instance, 
Stamenkovic et al, "The B Lymphocyte Adhesion Molecule CD22 Interacts with 
Leukocyte Common Antigen CD45RO on T Cells and .alpha.2-6 
Sialytransferase, CD75, on B Cells," CELL, Vol. 66, pp. 1133-1144 (1991), 
herein incorporated by reference. 
The present invention also includes methods for expressing a ligand, e.g., 
a mAb, derivative, functional equivalent or fragment thereof, which 
comprises using a nucleotide sequence, vector or transformed cell as 
defined above. 
More specifically, MT3 and 6G3 which are mAbs directed against the human 
CD45RB antigen will bind to an epitope on CD45RB in human cells expressing 
CD45RB. This epitope may then be purified, for instance utilizing an 
immunoaffinity column (as discussed), and partially or wholly sequenced, 
for instance using repeated rounds of Edman degradation. 
Immunosuppression and Inducing Immune Tolerance 
The antibodies and pharmaceutical compositions of the invention are useful 
in immunomodulation, especially immunosuppression, e.g., in the following 
indications: 
a) Treatment and prevention of organ, cells or tissue allo-or 
xeno-transplant rejection, e.g., for the treatment of human recipients of, 
e.g., heart, lung, islets, bone marrow, chromaffin or dopamine producing 
cells, combined heart-lung, liver, kidney, pancreatic, skin, small bowel, 
vascular tissue grafts or corneal transplants. They are also indicated for 
the prevention of graft-versus-host disease (GvH), such as sometimes 
occurs following bone marrow transplantation. The methods and compositions 
of the invention also reverse and prevent rejection of organ transplants 
in mammals such as rodents and primates. For example, the antibodies 
prevent mice from rejecting kidney transplants and induce long term 
survival. 
b) Treatment and prevention of autoimmune disease and of inflammatory 
conditions, in particular inflammatory conditions with an etiology 
including an autoimmune component such as arthritis (for example 
rheumatoid arthritis, arthritis chronica progrediente and arthritis 
deformans) and rheumatic diseases. Specific autoimmune diseases for which 
the methods of the invention may be employed include, autoimmune 
hematological disorders (including, e.g., hemolytic anemia, aplastic 
anemia, pure red cell anemia and idiopathic thrombocytopenia), systemic 
lupus erythematosus, polychondritis, scleredema, Wegener granulomatosis, 
dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, 
Steven-Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel 
disease (including, e.g., ulcerative colitis and Crohn's disease) 
endocrine ophthalmopathy, Graves disease, sarcoidosis, multiple sclerosis, 
primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type 1), 
uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal 
keratoconjunctivitis, interstitial lung fibrosis, psoriatic arthritis, 
glomerulonephritis (with and without nephrotic syndrome, e.g., including 
idiopathic nephrotic syndrome or minimal change nephropathy) and juvenile 
dermatomyositis. 
c) Treatment of leukemias characterized by over-proliferation of 
T-lymphocytes, including virally induced leukemias, e.g., HTLV-1-induced 
leukemia and acute lymphocytic leukemia. 
Dosages and Dosage Forms 
The invention provides a kit containing one or more compounds capable of 
binding to CD45 for administration to patients who have received 
xenografts. The kit can include said compounds in an appropriate 
pharmaceutical formulation such as a unit dosage form, along with one or 
more drugs used to suppress rejection induced by preexisting antibodies. 
Such drugs could include cyclophosphonamide, Deoxyspergualin and the like. 
Appropriate dosages of said compounds will of course vary, e.g., depending 
on the condition to be treated (for example the disease type or the nature 
of resistance), the effect desired, and the mode of administration. 
Dosages effective in humans can be derived from dosages effective in mice 
and other mammals by methods known to the art, i.e., U.S. Pat. No. 
5,035,878. 
In general however satisfactory results are obtained on administration 
parenterally, e.g., intravenously, for example by iv drip or infusion, at 
dosages on the order of from 0.01 to 2.5 up to 5 mg/kg, e.g., on the order 
of from 0.05 or 0.1 up to 1.0 mg/kg. Suitable dosages for human patients 
are thus on the order of from 0.5 to 125 up to 250 mg iv, e.g., on the 
order of from 2.5 to 50 mg i.v. The compounds may be administered daily or 
every other day or less frequently at diminishing dosages to maintain a 
minimum level of compound in the blood during the antigen challenge, e.g., 
following organ transplant or during the acute phase of an autoimmune 
disease. 
The pharmaceutical compositions of the present invention may be 
manufactured in conventional manner. A composition according to the 
invention is preferably provided in lyophilized form. For immediate 
administration it is dissolved in a suitable aqueous carrier, for example 
sterile water for injection or sterile buffered physiological saline. If 
it is considered desirable to make up a solution of larger volume for 
administration by infusion rather as a bolus injection, it is advantageous 
to incorporate human serum albumin or the patient's own heparinized blood 
into the saline at the time of formulation. The presence of an excess of 
such physiologically inert protein prevents loss of antibody by adsorption 
onto the walls of the container and tubing used with the infusion 
solution. If albumin is used, a suitable concentration is from 0.5 to 
4.50% by weight of the saline solution. 
In clinical tests, for example, patients about to undergo kidney, liver or 
heart transplantation are selected for prophylactic therapy. On the day of 
transplantation, 2 hours prior to surgery, a first intravenous infusion of 
the compound, antibody or mixture thereof, is administered at a dose of 
0.2 mg of each compound or antibody per kg of body weight. Two days after 
surgery an identical infusion of the compound and/or antibody at 0.4 mg/kg 
of body weight is administered and then repeated at weekly intervals for 
one month. The intravenous infusions are prepared as follows: the 
lyophilized compounds and/or antibodies are mixed together and dispersed 
into 100 ml sterile buffered saline containing 4.51% by weight of human 
albumin. This saline dispersion is administered to the patients over a 30 
minute period. 
The compounds of the invention are also useful as diagnostic aids, as 
diagnostic reagents or as components of a diagnostic kit to identify 
particular sub-populations of leukocytes. The compounds may be labeled, 
e.g., fluorolabeled or radiolabeled, using conventional techniques. For 
example, 25 micrograms of monoclonal antibody in 0.25 ml of 0.12 M sodium 
phosphate, pH 6.8 is iodinated using 2 mCi .sup.125 I and 10 micrograms 
chloramine T. After 5 minutes at 23.degree. C., the reaction is stopped by 
the addition of 20 micrograms of sodium metabisulfite, 3 mg of KI and 1 mg 
of BSA. lodinated protein is separated chromatographically. The labeled 
compounds are exposed to a frozen tissue section, e.g., from a patient 
exhibiting symptoms of graft rejection or acute autoimmune disease, 
exhibiting infiltration of leukocytes. Excess compound is washed away, and 
bound compound is assayed. Substantial binding of the compounds to 
leukocytes present in the tissue section suggests that the majority of 
leukocytes involved are naive rather than memory leukocytes, thereby 
indicating that therapy with the compounds and/or with immunosuppressants 
acting primarily on the T-cell mediated immune response, e.g., Cyclosporin 
or FK-506, is appropriate. 
Finally, the compounds are useful in a screening assay to identify drugs 
capable of modulating the biological activity of CD45RB. 
Adjuvant Agents 
It is also contemplated that an anti-CD45 CD4RB compound may be given alone 
or with standard immunosuppressant or anti-inflammatory agents. These 
would include cyclosporin, FK-506, Leflunomide, Rapamycin, 
cyclophosphamide, mycophenolate mofetil, Deoxyspergualin, corticosteroids, 
anti-lymphocyte globulin, OKT-3 and the like, and others. Use of the 
compounds and/or antibodies of the invention is expected to reduce the 
dosage requirements for such drugs and thereby to reduce undesired side 
effects. The compounds may also be used in combination with other 
monoclonal antibodies or other compounds specifically recognizing 
particular lymphocyte sub-populations, e.g., CD25 mAbs, CTLA4-Ig fusion 
peptide, etc. 
Ex Vivo, Conditioning of Recipient's Lymphocytes 
In some cases, immune suppression and/or tolerization may be enhanced by 
administering an amount of lymphocytes derived from the recipient that 
have been conditioned in vivo or ex vivo with the anti-CD45R antibodies 
useful in the present invention. The conditioned or anergized lymphocytes 
can be given before, simultaneously with, or following transplantation 
and/or administration of the anti-CD45R antibodies, in an amount effective 
to induce or assist in inducing immune tolerance in the recipient. The 
lymphocytes preferably are obtained from the recipient prior to 
transplantation or other treatment, preconditioned by exposure to the 
antibodies employed in the present method, and exposed to the antigens on 
the donor material, prior to re-introduction into the recipient. 
EXAMPLE 1 
Murine Monoclonal Antibody to CD45RB 
Murine monoclonal antibody to human CD45RB is produced by using 
conventional techniques, essentially as described by Kohler and Milstein 
in Nature 256: 49. Female BALB/C mice (20-25 g) each receive 100 .mu.g of 
antigen containing human CD45RB, e.g., Hodgkin cell line DEV (publicly 
available), by i.p. injection. (Alternatively, the antigen may comprise 
murine cells which have been transformed to express human CD45RB). After 2 
weeks a second booster injection comprising 50 .mu.g of the antigen is 
administered, again by i.p. injection. The presence of antibodies reactive 
to the antigen in the animals' blood serum is confirmed by 
immunohistologic screening. Mice displaying maximum blood serum levels of 
CD45RB antibody receive another booster injection comprising 20 .mu.g of 
antigen. Four days later, they are sacrificed and their spleen cells are 
isolated and fused with a suitable myeloma line, e.g., myeloma X63 
(publicly available). The resulting hybridoma are cultured and selected 
for expression of antibody having a high affinity to CD45RB. 
A hybridoma line producing murine monoclonal antibody to human CD45RB is 
the MT3 hybridoma line, which was deposited on Mar. 29, 1993 under the 
Budapest Treaty with the American Type Culture Collection (ATCC), 12301 
Parklawn Drive, Rockville, Md. 20852/U.S.A. 
A second hybridoma cell line, which produces rat monoclonal antibody to 
murine CD45RB, is HB220 (now designated MB23G2). This cell line has been 
deposited with the ATCC and is available by purchase from the ATCC. 
A third hybridoma cell line (deposited with the ATCC as HB-11873), produces 
antibodies of the invention (6G3 mAb). This hybridoma cell line was 
produced by the fusion of myeloma cell line SP2/O and spleen cells from a 
mouse immunized with human large cell B cell non-Hodgkin lymphoma cell 
line VER. The resulting clones were screened by an immunoperoxidase 
procedure on frozen tissue sections of human tonsil and rhesus monkey 
spleen. Clone 6G3 was selected because of the high reactivity of 6G3 mAb 
with subsets of T and B lymphocytes in both tissues. The antibody 
reactivity of 6G3 was characterized as anti-CD45RB by its selective 
reactivity with human CD45RB expressing transfectants and by the 
characterization of the molecular weight of the antigen immunoprecipitated 
by 6G3 as three bands with molecular weights of 220, 204 and 190 kD. The 
reactivity of the antibody could be abolished by pretreatment of tissues, 
cells or blots with neuraminidase, indicating the sialic acid dependence 
of the antigen. 
A fourth hybridoma cell line, HB223, produces analogous monoclonal 
antibodies to MB23G2; it is also deposited and available through the ATCC. 
EXAMPLE 2 
Chimeric Monoclonal Antibody to CD45RB 
a) Cloning of the Gene Encoding the Variable Domain of the Heavy Chain 
The genomic DNA of the desired hybridoma, in this example the MT3 or 6G3 
hybridomas of Example 1, and of the parental myeloma cell lines of the 
hybridomas (myeloma X63 or SP2/O) is isolated and digested with EcoRI. 
Each digested DNA is then fractionated on the same agarose gel. After 
migration, the agarose gel is analyzed by Southern blot using as probe a 
.sup.32 P labeled 0.7 kb XbaI-EcoRI DNA fragment which encodes the murine 
heavy chain enhancer E.mu. (Heinrich et al, J. OF IMMUNOL. (1989) 143: 
3589) to identify the desired variable heavy chain fragment, i.e., the 
desired fragment is present in the MT3 and 6G3 hybridomas but not in the 
X63 or SP2/O myelomas. Further purification of this fragment is then 
carried out by preparative agarose gel electrophoresis. 
DNA fragments of the same size as the desired fragment are cloned in the 
EcoRI restriction site of bacteriophage ZAP (Stratagene). Using the probe 
described above, the recombinant phages are screened and clones selected 
which hybridize to the probe. The DNA inserts of the selected clones are 
amplified on phage plate lysate by polymerase chain reaction (PCR) using 
as primers, a first oligonucleotide encoding the murine J.sub.Z gene and a 
second oligonucleotide encoding the beginning of the MT3 or the 6G3 heavy 
chain. The DNA fragments obtained from each of the selected clones are 
analyzed by Southern blot using as probe an oligonucleotide encoding a 
portion of the E.mu. probe described above. 
b) Construction of a Chimeric Heavy Chain Gene 
The EcoRI fragment (comprising the gene of the MT3 or 6G3 heavy chain 
variable domain (including the promoter and the enhancer)) is obtained by 
digestion of the DNA of one of the phage clones selected in step a) is 
then cloned into the EcoRI restriction site of the eukaryotic expression 
vector pSV2 neo-human .gamma..sub.1, constant part (Heinrich et al, 
supra). Following propagation of the resulting plasmid, the nucleotide 
sequence of the gene encoding the MT3 or 6G3 heavy chain variable domain 
is redetermined to exclude the possibility that a mutation in this gene 
has occurred. 
c) Cloning of the Gene Encoding the Variable Domain of the Light Chain 
The genomic DNA of the MT3 or 6G3 hybridomas and of the parental cell lines 
X63 or SP2/O is isolated and digested with EcoRI. Each digested DNA is 
then fractionated on the same agarose gel. After migration, the agarose 
gel is analyzed by Southern blot using as probe a .sup.32 P-labeled DNA 
fragment comprising the five mouse J.sub..kappa. genes and the mouse 
C.sub..kappa. gene. Size fractionated EcoRI fragments corresponding in 
size to the desired MT3 or 6G3 light chain variable domain are cloned in 
phage EMBL4 (Stratagene). 
A clone containing the DNA fragment encoding the MT3 or 6G3 light chain is 
identified by screening the recombinant phage clones with the probe 
described immediately above. The desired DNA fragment is then subcloned 
into the EcoRI-XbaI site of pGEM4 (Promega) and its sequence determined. 
d) Construction of a Chimeric Light Chain Gene 
An XbaI--XbaI fragment containing the sequence encoding the murine heavy 
chain enhancer (Heinrich et al; supra) and a HindIII-SphI DNA fragment 
containing the sequence for the human .kappa. constant part (huC.kappa.) 
are cloned together into phage mpl8 (Stratagene). Site-directed 
mutagenesis is performed on the resulting recombinant phage to disrupt the 
HindIII site in the desired coding region and followed by digestion with 
EcoRI and HindIII to generate a DNA fragment containing the sequences for 
both (E.mu.) and (huC.kappa.). After filling in the ends of this fragment, 
the fragment is subcloned into the blunt-ended EcoRI-BamHI site of 
pSV2-DHFR to generate pSV2-DHFR-E.mu.-huC.sub..kappa.. The plasmid 
pSV2-DHFR is obtained by replacing the BamHI-HindIII fragment of pSV2-neo 
with a BamHI-HindIII fragment encoding the dihydrofolate reductase gene. 
Lastly, an EcoRI-XbaI DNA fragment containing the MT3 or 6G3 light chain 
sequence is isolated from the recombinant pGEM4 plasmid of step 3 and 
subcloned into pSV2-DHFR-E.mu.-huC.sub..kappa. to generate 
pSV2-DHFR-E.mu.-huC.sub..kappa. -MT3.sub.L or 
pSV2-DHFR-E.mu.-huC.sub..kappa. -6G3.sub.L. 
e) Expression of Chimeric Antibody 
The plasmids obtained in steps b) and d) are co-transferred into the mouse 
myeloma cell line SP2/0 (ATCC CRL 1581) by electroporation using a gene 
pulser apparatus from Biorad. This technique is known to create stable 
transfectants at a high frequency. The SP2/0 cell line fails to produce 
endogenous heavy and light chains and is sensitive to Geneticin (G 418) at 
a concentration of 0.8 mg/l. 
SP2/0 cells are grown in the usual growth medium (RPMI+10% FCS 
5.times.10.sub.-5 .beta.-mercaptoethanol) harvested in the log phase of 
growth and washed with the electroporation buffer (Bio-Rad). Cell 
concentration is adjusted to 2.times.10.sup.7 cells/ml. To 0.8 ml of the 
cell suspension is added 15-20 .mu.g of each plasmid. The mixture is 
placed on ice and left to stand for 10 min. Then the cells are subjected 
to an electrical pulse (280 Volt; 25.degree. F.) and again left to stand 
for 15 min. Cells are transferred to the usual growth medium and incubated 
at 37.degree. C. in a C0.sub.2 incubator. 
After a 3-day incubation, selection for G 418 resistance is started. Cells 
are re-suspended in fresh medium containing 1.4 mg/ml G 418. The cultures 
yield growing cells after 10-14 days incubation in the presence of G 418. 
After the 2-week incubation, supernatants of the confluent cultures are 
tested for human IgG expression in a sandwich-type ELISA (anti-human 
.kappa.-light chain/supernatant/anti-human IgG-alkaline phosphatase 
conjugate). 
This test indicates that complete antibody molecules are secreted in all 
cultures at varying concentrations in the range of 50-500 ng/ml. 
To select cells in which the DHFR gene is amplified and which therefore 
secrete high amounts of the desired antibody, two selection procedures for 
methotrexate (MTX) resistance are carried out as described below. For this 
purpose, the G 418 resistant cell pools are each divided and amplification 
is preformed either according to procedure A (MTX increase by a factor of 
2 or 2.5) or procedure B (MTX increase by a factor of 5) (Table II). 
TABLE II 
______________________________________ 
Procedure A Procedure B 
______________________________________ 
100 nM MTX 200 nM MTX 
250 nM MTX 1 .mu.M MTX 
500 nM MTX 5 .mu.M MTX 
1 .mu.M MTX 25 .mu.M MTX 
2.5 .mu.M MTX 100 .mu.M MTX 
5 .mu.M MTX 
10 .mu.M MTX 
25 .mu.M MTX 
100 .mu.M MTX 
______________________________________ 
Each amplification step comprises inoculating the cells at a density of 
2.times.10.sub.5 cells/ml in the usual growth medium supplemented with G 
418 at 1.4 mg/ml and with MTX at the concentration of choice. After 72 
hour incubation, cells and the supernatant are separated. Antibody 
secretion is monitored either by ELISA or by HPLC using a protein A 
column. Most of the pools reach a maximum of specific antibody production 
at a certain MTX concentration. The best producing pools are cloned by 
limiting dilution. Out of several hundred analyzed clones, 15 best 
producing clones are selected. Productivity of the clones ranges from 30 
to 50 mg mAb/10.sup.9 cells in 72 hours. 
The antibody is purified from a culture supernatant by elution on a protein 
A affinity column. 
EXAMPLE 3 
In Vivo Prevention of Rejection of Kidney Transplants in Mice 
In this experiment a right nephrectomy was performed on 18 mice at the same 
time an allograft (kidney transplant from a different strain of mouse) was 
performed. A contralateral nephrectomy was followed on the seventh 
postoperative day (POD 7), so that from that point on, the animals relied 
only on the allografted kidney. Nine of the mice were treated with 50 
.mu.g of a mixture of rat anti-mouse CD45RB monoclonal antibodies produced 
from cell lines HB220 and HB223 iv for the first two days (POD 0 and POD 
1) followed by 100 .mu.g of each antibody intraperitoneally (i.p.) for 9 
days (POD 2 to POD 10). Of the nine control animals which did not receive 
the anti-CD45RB antibodies, seven were dead three days after the second 
kidney was removed, and the remaining two exhibited severe rejection one 
week later. 
Of the nine animals treated with the anti-CD45RB antibodies, there were 
three deaths due to surgical complications unrelated to any immune 
response, but remarkably, the remaining six animals survived long term 
(e.g., over 100 days) without any further treatment and without any 
evidence of allograft rejection. In a third group of 10 untreated isograft 
recipients, the incidence of death due to surgical complication was the 
same. There was no significant difference between the serum creatinine 
levels of the allograft group receiving monoclonal antibody and the 
isograft group, indicating that the kidneys in both groups were 
functioning normally. 
EXAMPLE 4 
Reversal of the Rejection of Kidney Transplants in Mice 
In this experiment a right nephrectomy was performed on 10 mice at the same 
time an allograft (kidney transplant from a different strain of mouse) was 
performed. 
All ten of the animals were observed for five days without 
immunosuppression therapy. These animals were known to be experiencing 
severe rejection at this stage because sacrificed control animals, also 
subjected to a nephrectomy and an allograft kidney transplantation, 
exhibited severe rejection on day 5. 
On POD 5, four of the animals were given three daily 25 .mu.g doses, 
intraperitoneally, of anti-CD45RB antibody (a mixture of monoclonal 
antibodies from cell lines HB220 (anti-CD45RB MB23G2 mAb) and HB223 
(anti-CD45RB MB4B4 mAb)) for the next three days. All four of the animals 
experienced rapid reversal of their rejection symptoms, including a return 
to normal levels of creatinine, and lived greater than 100 days. The 
untreated animals died by day nine due to organ rejection. Table III 
summarizes the results of this experiment: 
TABLE III 
______________________________________ 
REVERSAL OF THE REJECTION OF KIDNEY 
TRANSPLANTS IN MICE 
SURVIVAL 
MOUSE THERAPY DAYS CAUSE OF DEATH 
______________________________________ 
1 NONE 8 REJECTION/UREMIA 
2 NONE 9 REJECTION/UREMIA 
3 NONE 8 REJECTION/UREMIA 
4 NONE 9 REJECTION/UREMIA 
5 NONE 9 REJECTION/UREMIA 
6 NONE 9 REJECTION/UREMIA 
7 CD45RB &gt;100 -- 
8 CD45RB &gt;100 -- 
9 CD45RB &gt;100 -- 
10 CD45RB &gt;100 -- 
______________________________________ 
This data with respect to reversal is significant in confirming that the 
antibody therapy is highly effective in suppressing an immune response. 
Treatment and cures are accomplished with antibody therapy. 
EXAMPLE 5 
Confiming Results Using MB23G2 and MB4B4 Separately for Kidney Transplants 
in Mice 
Recipient Balb/c (h-2d) mice had the right kidney removed before receiving 
a transplanted kidney from donor C57B1 (h-2b) mice. A left native 
nephrectomy was subsequently performed on day 7. There were four groups of 
animals. Thirteen received isografts, 17 received allografts with no 
immunosuppression (vehicle control), 44 received allografts and were given 
two doses of purified rat anti-mouse CD45RB mAb MB23G2 1 mg/kg (30 .mu.g) 
intravenously on days 0 and 1, and 16 received allografts but were treated 
with two doses of purified rat anti-mouse CD45RB mAb MB4B4 1 mg/kg (30 
.mu.g) intravenously on days 0 and 1. No further antibodies were given. 
As expected, the MB23G2-treated animals exhibited prolonged survival 
compared to the untreated group (p&lt;0.002) (see FIG. 1) and was comparable 
to the isograft group. Remarkably, the anti-CD45RB MB4B4 mAb was no better 
than the vehicle alone at preventing rejection. Both MB23G2 and MB4B4 are 
IgG2a but a difference exists between them. Both mAbs bind to Balb/C 
leukocytes as assayed by FACS. However, MB23G2 binding is inhibited by 
neuraminidase, while binding of MB4B4 is not affected by such treatment. 
FIG. 2 shows the serum creatinine levels in animals from each group at the 
time of sacrifice, or beyond day 100 for the long-term survivors. There 
were no differences between the isograft and MB23G2-treated groups, while 
the untreated and MB4B4-treated animals died from uremia. Therefore, the 
glycosylated epitope for MB23G2 is either involved in, or is near to, 
sites involved in the biochemical activity of CD45RB. The non-glycosylated 
epitope for MB4B4 appears to be non-critical for CD45RB activity. 
Immunoperoxidase microscopic studies were performed on renal allografts at 
7 days in three groups of mice: untreated, MB4B4-treated, and 
MB23G2-treated. Sections were stained with rat anti-mouse mAb reactive 
with mouse CD3, CD4, CD8, CD45RB and Ia. Slides were evaluated in a masked 
manner with respect to aggregates and diffuse infiltrates as described in 
Ibrahim et al., TRANSPLANTATION Vol. 59, pp. 724-728, herein incorporated 
by reference. The numbers of cells in the diffuse infiltrates were counted 
in ten (.times.400) high power fields (HPF) in each section of 5 mice per 
group and the data are shown in Table IV below. 
TABLE IV 
______________________________________ 
DIFFUSE CELLULAR INFILTRATES 
IN KIDNEY ALLOGRAFTS AT 7 DAYS 
(MEDIANS/HPF) 
MB4B4- MB23G2- 
Phenotype 
mAb Untreated Treated 
Treated 
______________________________________ 
CD3 KT3 33 38 22* 
CD4 GK1.5 10 11 9 
CD8 3.155 27 26 13* 
CD45RB MB23G2 12 10 9 
______________________________________ 
*P &lt;0.05. Statistically significant differences between MB23G2treated and 
nontreated groups and between MB23G2treated and MB4B4treated groups. 
Interestingly, differences between the three groups of mice became evident 
after separately counting cells in aggregates and diffuse infiltrates. 
Staining for Ia was clearly less in the MB23G2-treated allografts than in 
the other groups, but due to positive staining of other interstitial cell 
types the numbers of lymphocytes could not reliably be quantified. The 
aggregates contained high numbers of CD4+ and CD8+ cells in all three 
groups. While the numbers of CD4+ and CD45RB+ cells in the diffuse 
infiltrates were approximately equivalent in all three groups (Table III, 
second and fourth rows) the numbers of CD3+ cells and CD8+ cells in the 
diffuse infiltrates were statistically different between MB23G2-treated 
and the other groups (Table III, first and third rows). Thus, the 
MB23G2-treated animals demonstrated an elevated CD4:CD8 ratio compared to 
MB4B4-treated and untreated animals. Remarkably, few of the infiltrating 
cells were CD45RB positive, a particularly notable finding considering 
that the CD45RB mAb MB23G2 could reverse acute rejection. 
EXAMPLE 6 
Immunotolerance 
To assess the possibility of antigen specific tolerance, skin transplants 
were performed on 13 animals from Example 5 which had maintained a kidney 
transplant beyond 100 days after receiving 2 doses of MB23G2 mAb at the 
time of renal allografting. Each animal received full thickness skin 
allografts from a C57B1/6 mouse (isogeneic with the donor of the renal 
allograft) and a control skin transplant: 9 received a Balb/C isograft and 
4 received a CBA allograft (third party donor). No further 
immunosuppression was given. Of the 13 animals with kidney specific 
tolerance, there was a subset of 4 which demonstrated donor alloantigen 
specific tolerance since they did not reject the C57B1/6 skin. All 4 
animals rejected the third party CBA skin, while all 9 Balb/C isografts 
survived indefinitely. No renal allograft rejection was stimulated by the 
skin transplants. 
EXAMPLE 7 
Reversal of Allograft Rejection 
To determine whether MB23G2 mAb could reverse acute rejection, seven 
allografts were performed as described in Example 5, but no immunotherapy 
was administered until day 4. Untreated allografted kidneys demonstrated 
rejection at this time. The treated animals received MG23G2, 1.5 mg/kg (50 
.mu.g) i.v. daily on days 4, 5 and 6 and no further therapy thereafter. 
Three animals died of ureteric complications in the MG23G2 treated 
group--the graft histology did not show rejection at the time of death on 
days 8, 9 and 25. All the animals had their rejection reversed and the 
remaining 4 survived &gt;60 days with a normal serum creatinine. 
EXAMPLE 8 
Effect of MB23G2 on Blood Composition 
The pharmacologic effects of MB23G2 on the peripheral blood in mice were 
assessed using multiparameter FACS analysis. Mice were treated with 30 
.mu.g of MB23G2 mAb intravenously on two consecutive days. As shown in 
FIG. 3A, MB23G2 induced a significant depletion of circulating lymphocytes 
which returned to normal one week after stopping the mAb. MB23G2 bound to 
almost all the remaining circulating T and B lymphocytes (FIG. 3B and 3C). 
FACS analysis revealed no excess MB23G2 antibody in the plasma by day 8. 
FACS analysis of the spleen demonstrated that the administered therapy 
penetrated the lymphoid tissue 24 hours after the second dose of MB23G2. A 
FACS inhibition assay did not reveal sensitization of any of the mice to 
the MB23G2 mAb up to 2 weeks after therapy. 
Since CD45 is a protein tyrosine phosphatase, tests were designed to show 
that induction of allograft tolerance by MB23G2 mAb is related to an 
alteration in tyrosine phosphorylation of T cell substrates necessary for 
signal transduction to occur. 
EXAMPLE 9 
Mechanism of Tolerance Induction by CD45RB Monoclonal Antibody: Increased 
Tyrosine Phosphorylation of Phospholipase C-.gamma.1 and Decreased 
Expression of inflammatory Cytokines 
Murine T cell hybridoma A1.1 cells were stimulated with the CD3 mAb 2C11 in 
the presence or absence of MB23G2 or MB4B4 mAb. The cells were lysed in 
Brij 96 and the phosphotyrosine-containing proteins were 
immunoprecipitated with anti-phosphotyrosine mAb PY72. The 
immunoprecipitated proteins were extracted and separated on 10% 
SDS-polyacrylamide gels, transferred electrophoretically to PVDF membranes 
and submitted to an immunoblotting procedure (See Lazarovits et al., J. 
Immunol., 153, 3956 (1994)) using the anti-phosphotyrosine mAb 4G10. 
Augmentation of tyrosine phosphorylation of a 145 kDa substrate in the 
presence of the CD45RB MB23G2 mAb was found. The MB4B4 mAb did not alter 
the tyrosine phosphorylation of this substrate. The identity of this 145 
kDa band as PLC-.gamma.1 was confirmed by stripping the 4G10 mAb from the 
blots and reprobing with monoclonal antibody to phospholipase C-.gamma.1 
(PLC-.gamma.1) (Upstate Biotech, Lake Placid, N.Y.). Thus, in the presence 
of MB23G23, substantially more tyrosine phosphorylated PLC-.gamma.1 could 
be identified than in its absence, while MB4B4 did not alter the amount of 
(PLC-.gamma.1) which could be immunoprecipitated. The increased tyrosine 
phosphorylation of (PLC-.gamma.1) has been noted by Gajewski et al (PROC. 
NATL. ACAD. SCI. USA, Vol. 91, pp. 38-42 (1994)) to be a property of 
anergic T cells. 
Since gene activation is a consequence of signal transduction in T cells, 
experiments were performed to investigate whether the MB23G2 mAb could 
alter the expression of cytokine genes in vivo known to be increased in 
rejecting allografts. It is generally believed that a so-called TH1 
cytokine profile (IL-2, .gamma.-interferon) is associated with rejection, 
while a TH2 phenotype (IL-4, IL-5, IL-6, IL-10) may be associated with 
non-responsiveness (see T. R. Mosmann, et al., J. Immunol., 136, 2348 
(1986)). 
To examine gene expression in mouse renal allografts, steady state levels 
of specific mRNA transcripts were assessed by Northern Blot analysis using 
.sup.32 P-labeled cDNA probes. Gene expression in four groups of animals 
was examined: isografts on postoperative day 7, allografts from untreated 
animals on postoperative day 7, and allografts from MB23G2-treated animals 
on postoperative day 7 and day 28. No specific pattern of IL-1, IL-2, 
IL-4, IL-5, IL-6 or IL-10 was detected. However, there was a selective 
decrease on day 28 in mRNA transcripts for .gamma.-interferon and tumor 
necrosis factor .alpha. compared to untreated allografts. There was no 
difference noted on day 7. Interestingly, intercellular adhesion 
molecule-1 (ICAM) mRNA was also decreased in the MB23G2-treated animals on 
day 28, with no difference observed on day 7. Thus, the MB23G2 therapy may 
induce tolerance in part by inhibiting the expression of inflammatory 
cytokines because of interference with the signal transduction cascade. 
In yet other experiments, use of anti-CD45RB antibodies were found to be 
effective when administered to primates. 
EXAMPLE 10 
Prevention of Organ Rejection in Primates and Reversal of Organ Rejection 
Renal allografts were performed on two Cynomolgous monkeys using a CD45RB 
monoclonal antibody (which binds to a neuraminidase sensitive epitope), as 
an immune suppressor and the details are set forth below: 
Detailed Experimental Procedures: 
1. Animal Care: 
The animals were housed in the University of Western Ontario primate 
facility. They were provided with squeeze cages which allow for drug 
injections and sample collection without having to anesthetize the animal, 
thereby reducing stress. They were maintained on standard monkey feed, and 
other foods for diversity. They were allowed regular exercise in the 
exercise cage. The animal care followed the standard operating procedures 
for non-human primates provided by veterinary services. 
Animals were typed for blood groups. On arrival, the animals were rested 
for at least 2 weeks. The animals were anesthetized with atropine and 
ketamine for physical examination, including inspection for oral B virus, 
TB testing, and de-wormed with Ivernectin 2828. Animals fasted the night 
prior to any anesthetics. 
2. Kidney Transplantation: 
1. Donor procedure: 
Two donor animals were injected with ketamine, taken to the OR, intubated 
and put on insofluorane/nitrous oxide. A three stage surgical prep is 
used. After a midline incision of the two, the left renal artery, vein and 
ureter were carefully isolated and divided. Grafts were ex-vivo perfused 
and stored in 4.degree. C. University of Wisconsin solution. The wounds 
were closed and the animals returned to the cage to recover from the 
anesthetic. 200-300 ml of saline were given by continuous i.v. during the 
surgery. During the surgery, the animals were kept warm using a heating 
lamp, heated saline and heating pad, etc. 
Postoperative care followed Standard Operating Procedures. Briefly, the 
animals remained on a warm water blanket and under a heating lamp for 24 
hours. Buprenorphine was given q6h after surgery for 24 hours. The animals 
were monitored daily. The well recovered donor animals are used as the 
recipient in future transplantations. The interval between the two 
surgeries is at least two weeks. 
2. Recipient procedure: 
The recipient was anesthetized and prepared preoperatively as described for 
the donor. After a midline incision, abdominal aorta and inferior vena 
cava were exposed. End-to-side anastomoses were performed between the 
donor renal artery and the recipient aorta, as well as between the donor 
renal vein and the recipient inferior vena cava. The donor ureter was 
sutured to the recipient's bladder. The right kidney was removed and the 
wound was closed. 3. Post-operative care: 
The post-operative care is the same as described for the donor. Animals 
were monitored continually post-operatively for at least 24 hours, more if 
necessary. They were monitored closely (i.e., several times per day) until 
feeding and grooming normally. Thereafter, they were monitored at least 
daily when they received their monoclonal antibody. 
Animals were given 4 mg of anti-CD45RB 6G3 mAb 4 i.v. daily for 7 days. The 
outcome of kidney grafts was measured by percutaneous biopsy weekly and 
blood creatinine levels twice per week. For these procedures animals were 
anesthetized with ketamine. Criteria for early euthanasia would include 
lethargy, lack of grooming or feeding, significant weight loss (&gt;20%) and 
renal failure (elevated creatinine levels). 
As discussed above, the recipient animals received 4 mg (1 mg/kg) of 
anti-CD45RB 6G3 mAb post operatively for 7 days. There were no side 
effects associated with such infusions. Both animals survived normally 
until day 16 when each experienced an acute rejection crises. The first 
animal was euthanized 2 days later on day 18. The second animal was 
re-treated with 4 mg/kg (16 mg) of anti-CD45RB 6G3 mAb. This therapy was 
given daily i.v. for four days. Remarkably the rejection crises completely 
reversed as the animal was observed to resume normal activities and 
creatine levels were observed to decrease from a "crises" level of 738 
.mu.mol/L to 366 .mu.mol/L. The animal remained well until day 36 when 
another rejection crises developed. The animal was than euthanized. 
Histology of the allograft revealed that there was profound endothelitis 
on post-operative day 15 just before additional therapy was administered 
leading to reversal. A biopsy of the allograft of this animal was 
performed on post-operative day 23 revealing the endothellitis had 
cleared. 
It is known that control animals will die by day 10 if therapy is not 
administered in this type of model. (Lazarovits et al., Kidney Inter., 25, 
344 (1984)). 
The data suggests two conclusions: 
[1] Because both monkeys lived past the known date of controls, it is shown 
the therapy of the invention exhibits significant graft-survival in a 
primate. 
[2] Even more dramatic is the observation that one can reverse acute 
rejection with anti-CD45RB monoclonal antibodies. 
EXAMPLE 11 
Additional Monkey Experiments 
Two additional Cynomolgus monkeys (#3 and #4) have received renal 
allografts and have been treated with the CD45RB monoclonal antibody 6G3 
as the sole form of immunosuppression. Blood grouping was performed to 
control for ABO compatibility and major histocompatibility complex 
profiles were obtained using PCR based DNA typing to confirm that 
allogeneic renal transplants were being performed. 
On day zero, a nephrectomy was performed in the recipient animal and the 
renal allograft was performed. On day seven the second native kidney was 
removed and from that point on the animal relied on its transplanted 
kidney. Animals which do not receive immunosuppression or which receive 
ineffective immunosuppression will reject at a mean of ten days +/-two 
days (Lazarovits et al., Kidney Intern., 25, 344 (1984)). 
Monkey #3 
This animal was treated with 6G3 antibody 2 mg/kg/day (8 mg).times.7 days 
and then 6 more doses given on Monday, Wednesday and Friday for each of 
the next two weeks was to be given. Thus, 8 mg of 6G3 antibody was planned 
to be given over three weeks. The animal developed rejection on day 14 and 
was euthanized. 
Monkey #4 
This animal received the same therapy as monkey #3. That is 8 mg of 6G3 
antibody was given intravenously for 13 doses over three weeks. This 
animal has done remarkably well and continues to be alive beyond 70 days. 
No rejection has been diagnosed. 
Thus, animals #3 and #4 treated with 6G3 have had significantly prolonged 
allograft survival which is illustrated in FIG. 4. Monkey #2 of Example 8 
is of particular interest because the antibody successfully reversed acute 
rejection which was predicted by the mouse kidney transplant experiments. 
Additional experiments are underway to try to determine the cause for 
relatively early graft failure in monkeys 1 and 3, although both of these 
animals also had significantly prolonged allograft survival. 
EXAMPLE 12 
In Vivo Prevention of the Rejection of Heart Transplants in Mice 
Heterotopic heart transplants from C57B1/6 mice into BALB/C donors were 
performed essentially as described in R. L. Kirkman et al (1985) 
Transplantation 40: 719-722. Seven of the mice received 30 .mu.g iv of rat 
anti-mouse CD45RB mAb MB23G2 on days 0 and 1 following heart 
transplantation. Four other mice received 30 .mu.g i.v. on days 0 and 1 
and 100 .mu.g ip of rat anti-mouse CD45RB mAb MB23G2 daily on days 2 to 11 
following heart transplantation. Fourteen control mice received no 
antibody. Survival of the heterotopic graft was determined by whether the 
heart was beating and rejection was confirmed by histological analysis. 
All of the control mice had rejected their hearts by day 14 post-operation, 
with a mean survival time of 9 days. Mean survival time of the hearts in 
the group receiving antibody for two days only was 20 days and mean 
survival time of the hearts in the group receiving antibody for 11 days 
was 34 days. Table V summarizes the results from this experiment. 
TABLE V 
______________________________________ 
MOUSE CARDIAC ALLOGRAFTS 
Groups Number Survival (days) Mean 
______________________________________ 
Untreated 14 8, 8, 9, 9, 9, 9, 9, 9, 9, 10, 
9 
11, 11, 11, 14 
CD45RB mAb 7 16, 16, 17, 22, 24, 23, 24 
20 
30 .mu.g D0, D1 
CD45RB mAb 4 15, 30, 38, 38 34 
30 .mu.g IV D0, D1 
AND 100 .mu.g ip .times. 
9 days 
______________________________________ 
Additional evidence of the utility of anti-CD45RB was demonstrated in the 
following studies. 
EXAMPLE 13 
In Vivo Pevention of the Rejection of Pancreatic Islet Allograft 
Transplants in Mice 
Pancreatic islet allografts were transplanted under the kidney capsule from 
CBA/J donors into streptozotocin-treated BALB/C recipients essentially as 
described in M. C. Fabian et al., Transplantation, 56, 1137 (1993). Five 
control mice received no antibody while eleven mice received 30 .mu.g i.v. 
of rat anti-mouse CD45RB mAb MB23G2 on days 0 to 1 post-operation. 
Rejection was defined as onset of glycosuria. All islet allografts from 
control mice had been rejected by day 24 with a mean rejection time of 17 
days. Antibody-treated mice showed a mean rejection time of 34 days with 
two mice having no signs of rejection at day 50 when the experiment was 
stopped. 
Table VI summarizes the result from this experiment. 
TABLE VI 
______________________________________ 
MOUSE PANCREATIC ISLET ALLOGRAFTS 
Groups Number Survival (days) Mean 
______________________________________ 
Untreated 5 12, 12, 15, 24, 20 
17 
CD45RB mAb 11 23, 32, 20, 30, 30 
34 
0 .mu.g iv &gt;50, &gt;50, 21, 23, 47, 50 
D0, D1 
______________________________________ 
EXAMPLE 14 
Induction of Xenograft Tolerance in Rat to Mouse Transplant Models 
To assess whether anti-CD45RB monoclonal antibody could prevent xenogeneic 
renal graft rejection, orthotopic kidney xenografts were performed in 
BALB/c mice with Lewis rats as donors. Five groups of recipients were 
studied: no treatment (Controls), cyclosporin (CsA) treatment (5 mg/kg 
S.C. daily), splenectomy (Spl), cyclophosphamide (CyP) treatment (20 mg/kg 
on POD 0, 2, 4 & 7), MB23G2 mAb treatment (100 .mu.g daily.times.11 days, 
I.P.), and combined treatment with MB23G2 mAb (100 .mu.g.times.11 days) 
and CyP (20 mg/kg I.V. on POD 0, 2, 4 & 7). As shown below in Table VII, 
animals treated with MB23G2 mAb and CyP had a significantly longer median 
survival time than animals treated with mAb alone or CyP alone, 
demonstrating that CD45RB mAb and CyP have a synergistic effect on 
prolonging renal xenografts in the mouse. 
TABLE VII 
______________________________________ 
RAT-TO-MOUSE KIDNEY XENOGRAFTS 
Survival Median 
Group n Treatment (days) Survival (Days) 
______________________________________ 
Control 6 None 6(4), 7, 16 
6 
CsA 3 Cyclosporin 4, 6, 8 6 
Spl 5 Splenectomy 4, 5, 6, 7, 11 
6 
CyP 8 Cyclophosphamide 
10, 18, 23, 24, 
26 
28, 49, 58, 
&gt;100 
mAb 6 MB23G2 4, 6, 8, 8, 11, 
8 
13 
mAb + CyP 
9 CyP + MB23G2 9, 11, 23, 24, 
70* 
70, 76, 
&gt;100(3) 
______________________________________ 
*P &lt;0.01, the mAb group vs the control group. 
The ultimate goal for clinical xenotransplantation is to induce xenograft 
tolerance, thereby eliminating the continuous use of toxic, high-dose 
immunosuppression. It was recently demonstrated in a hamster heart to 
mouse xenograft model that the combination of pulse therapy with CyP and 
continuous CsA treatment induced prolonged graft survival. See Hasan et 
al., Transplantation, 54, 408 (1992). However, when the cyclosporin 
therapy was stopped, the xenografts were rejected within a short period of 
time, i.e., the median survival time was less than three weeks. Thus, 
therapy with CyP and CsA was unable to induce xenograft tolerance. 
In contrast, as shown in Table VII above, xenografts in animals treated 
with CD45RB mAb and CyP continued to survive several months after 
cessation of immunosuppressive therapy. Moreover, the long term surviving 
kidney xenografts treated with mAb and CyP had a normal renal function and 
a normal pathology at sacrifice on POD 100. These results demonstrate that 
treatment with CD45RB mAb and CyP can induce functional xenograft 
tolerance in a rat-to-mouse model. This therapy may be applicable in the 
prevention of transplant rejection in man. 
EXAMPLE 15 
In Vivo Treatment of NOD Mice to Inhibit the Onset of Diabetes 
Five female NOD mice were treated with 30 .mu.g iv of rat anti-mouse CD45RB 
mAb MB23G2 on days 28, 29 and 30 after birth. Five control mice received 
no antibody treatment. By week 27 all control mice were dead as a result 
of diabetes. Of the antibody-treated mice 2 died of diabetes while the 
other 3 remained alive and well until week 35 at which point they were 
killed and their pancreas examined histologically. There was no sign of 
insulitis in any of the three surviving animals. 
Table VIII summarizes the results from this experiment. 
TABLE VIII 
______________________________________ 
ONSET OF DIABETES IN NOD MICE 
Survival Without 
Groups Number Diabetes 
______________________________________ 
Untreated 5 0 (all dead by 27 weeks) 
CD45RB mAb 30 .mu.g iv 
5 3 &gt; 35 weeks 
days 28, 29, 30 
______________________________________ 
Additional experiments have been conducted since this preliminary 
experiment was conducted. The therapy in these experiments comprised 
administering 100 ug of MG23G2 two times per week from 2-35 weeks, at 
which time all remaining mice were euthanized. The results are shown in 
Table IX below: 
TABLE IX 
______________________________________ 
NOD MOUSE SURVIVAL (WEEKS) 
INSULITIS SCORE* 
THERAPY SURVIVAL WEEKS 
(MEAN) 
______________________________________ 
MB23G2 (N = 9) 
35(6), 20(2), 13 
0.99 
CONTROL (N = 12) 
34 (2), 30 (2), 28, 23, 
1.81 
18, 16, 15, 14, 13, 12 
______________________________________ 
*Insulitis score obtained from 6 additional animals in each group at 15 
weeks. 
As seen from the data a significantly greater number of animals survived 
indefinitely, i.e., until the end of the 35 week experiment compared to 
the controls. The improvement induced by MB23G2 is evident not only in 
animal survival but also in blood sugar where all of the animals who 
survived to 35 weeks in the treated group had no evidence of 
hyperglycemia. The beneficial effect of MB23G2 was also confirmed by the 
insulitis score which is a careful histologic assessment of the pancreas. 
The difference between 1.81 and 0.99 is both biologically and 
statistically meaningful. 
EXAMPLE 16 
Prevention of Skin Graft Rejection 
Four groups of A-strain mice (white) will receive skin grafts from 
C57BL/10-strain mice (black): groups I and II will receive prior treatment 
with rat anti-mouse CD45RB mAb and groups III and IV will receive no prior 
treatment with rat anti-mouse CD45RB mAb. Following the skin graft 
operation, groups I and III are treated with anti-mouse CD45RB mAb on 
varying days, while groups II and IV receive no further antibody 
treatment. The effectiveness of the antibody treatment in preventing 
rejection of the skin graft is determined by comparing the length of time 
the black skin graft survives on the white recipient mice in the four 
groups. 
EXAMPLE 17 
Localized Graft-Versus-Host (GvH) Reaction 
In vivo efficacy of the compounds will be demonstrated in a suitable animal 
model, as described, e.g., in Ford et al., Transplantation, 10, 258 
(1970). Spleen cells (1.times.10.sup.7) from 6 week old female mice are 
injected subcutaneously on day 0 into the left hind-paw of mice of a 
different strain weighing about 100 g. Animals are treated for 4 
consecutive days and the popliteal lymph nodes are removed and weighed on 
day 7. The difference in weight between the two lymph nodes is taken as 
the parameter for evaluating the reaction. 
EXAMPLE 18 
Freund's Adjuvant Arthritis 
Efficacy against experimentally induced arthritis can be demonstrated using 
the procedure described, e.g., in Winter & Nuss, ARTHRITIS & RHEUMATISM 9 
(1966) 394; Billingham Davies, HANDBOOK OF EXPERIMENTAL PHARMACOL. (Vane & 
Ferreira Eds, Springer-Verlag, Berlin) 50/II (1979) 108-144. Mice (male or 
female, 150 g body weight) are injected, i.e., at the base of the tail or 
in the hind paw, with 0.1 ml of mineral oil containing 0.6 mg of 
lyophilized heat-killed Mycobacterium smegmatis. In the developing 
arthritis model, treatment is started immediately after the injection of 
the adjuvant (days 1-18); in the established arthritis model treatment is 
started on day 14, when the secondary inflammation is well developed (days 
14-20). At the end of the experiment, the swelling of the joints is 
measured by means of a micro-caliper. ED.sub.50 is the oral dose in mg/kg 
which reduces the swelling (primary or secondary) to half of that of the 
controls. 
EXAMPLE 19 
In Vivo Treatment of NZB Mice to Inhibit Onset of Lupus-Like Autoimmune 
Disease 
Mice of the New Zealand black-strain (NZB) die with widespread and diverse 
symptoms of hemolytic anemia, glomerulonephritis, and vasculitis, all very 
reminiscent of human systemic lupus erythematosus (SLE). The effectiveness 
of the present invention in treating SLE is evaluated in this mouse model 
by treating newborn NZB mice with rat anti-mouse CD45RB mAb MB23G2 at 
varying times after birth and then analyzing treated and untreated mice 
for the onset of autoimmune disease, particularly for glomerulonephritis, 
which is also a prominent feature of human SLE. 
EXAMPLE 20 
In-Vitro Evaluation of the Imunomodulatory Activity of CD45 Reagents 
The ability of anti-human CD45 antibodies to suppress human T-cell 
activation was evaluated in vitro by the following method. 
Mononuclear cells were isolated from the peripheral blood of volunteers by 
Ficoll-Hypaque density gradient centrifugation. The cells were stimulated 
in RPMI1640 with 10% FCS with OKT3 or a non-reactive control antibody for 
periods of 1-4 days. The percentages of CD3, CD4 and CD8 cells positive 
for CD69 as a marker of early and CD25 as a marker of late activation were 
determined by FACScan analysis. Poppema et al., Leukemia and Lymphoma, 20, 
217 (1996). The effect of CD45 and CD45R antibodies was measured by adding 
these reagents as singles or as cocktails. Western blots of the cell 
lysates were stained with anti-phosphorylated tyrosine antibody 4G10 to 
investigate the potential role of dephosphorylation as a result of the 
tyrosine phosphatase activity of CD45. June et al., J. Immunol., 144, 
1591(1990). 
As shown in Table XIII below, OKT3 caused CD25 expression on approximately 
70% of the T cells on day 4, whereas exposure to a non-reactive control 
antibody resulted in approximately 10% CD25 expression. None of the 
CD45(R) antibodies alone led to T-cell activation. The results of 
co-incubation of the CD45(R) antibodies with OKT3 stimulated peripheral 
blood mononuclear cells are summarized in FIG. 5. The four CD45RA reagents 
tested had no effect or resulted in slight stimulation. Of four CD45RB 
reagents tested 2 (6B6, 6G3) gave significant inhibition whereas the other 
two (MT3, PD7) only had minimal effects. Of three CD45RO reagents tested 
one gave significant inhibition (UCHL1) whereas the other two (A6, OPD4) 
showed much less inhibition. Of four CD45 reagents tested one (4C9) had no 
effect, two (4D 11, 2G1) gave inhibition and one (4F9) resulted in 
stimulation clearly above the effect of OKT3 alone. 
TABLE X 
______________________________________ 
CORRELATION OF ACTIVATION AND TYROSINE 
PHOSPHORYLATION 
Tyrosine Phosphorylation 
Antibodies % CD25 110 kDa Band 
______________________________________ 
OKT3 plus control ab 
70% yes 
OKT3 plus 6B6 (RB) 
45% no 
OKT3 plus 6G3 (RB) 
45% no 
OKT3 plus MT3 (RB) 
65% yes (slightly weaker) 
OKT3 plus PD7 (RB) 
70% yes 
______________________________________ 
The expression of CD25 and CD69 was also analyzed after gating for CD4+ or 
CD8+ T cells. The results indicate that anti-CD45RB reagent 6G3 has a much 
more pronounced inhibitory effect on CD8 cells than on CD4 cells. See FIG. 
6. Combinations of reagents were also tested and as is shown in FIG. 7, 
clear synergy of a CD45, CD45RB and CD45RO mixture was found, which 
reduced the level of CD25 expression to that not significantly different 
from the non-stimulated controls. 
The results of this study demonstrate that CD45(R) specific antibodies can 
modulate CD3 mediated T-cell activation in vitro. The results of 
combinations of antibodies indicate that different mechanisms may 
contribute to the measured effect. 
The finding that some, but not other, anti-CD45RB antibodies inhibit T-cell 
activation is in accordance with the finding that one CD45RB specific 
antibody does enhance renal allograft survival in a mouse model while 
another does not. See Example 5. The predominant inhibitory effect of the 
CD45RB antibodies on CD8 cells is in agreement with the finding that the 
major immunophenotypic finding in the successfully treated mice is a 
reduction of the number of CD8 cells in the allograft. 
In conclusion, analysis of CD25 expression of in vitro CD3 stimulated 
peripheral blood T cells demonstrates the immunomodulatory activity of 
anti-CD45(R) antibodies. A mixture of anti-human CD45(R) antibodies is a 
powerful inhibitor of T-cell activation in vitro and may well be suitable 
for the prevention and reversion of allograft rejection. 
All patents, patent applications and publications are incorporated herein 
by reference, as though fully set forth. Thus, it is apparent that there 
has been provided, in accordance with the present invention, methods and 
products which will substantially benefit those with autoimmune diseases 
and those receiving organ transplants. While the invention has been 
described in conjunction with specific embodiments thereof, it is evident 
that many modifications and variations will be apparent to those skilled 
in the art. Accordingly, it is intended to include all such alternatives, 
modifications and variations set forth within the spirit and scope of the 
appended claims.