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A METHOD OF PREPARING AN UNDIFFERENTIATED CELL
' The present invention relates to a method of preparing an undifferentiated
cell.
In particular, the present invention relates to a method of preparing an
undifferentiated cell from a more committed cell.
In addition the present invention relates to the use of the undifferentiated
cell of the
present invention for the preparation of a new more committed cell - i.e. a
recommitted cell.
The present invention also relates to the use of the undifferentiated cell of
the present
invention or the recommitted cell of the present invention to have an effect
(directly
or indirectly via the use of products obtained therefrom) on the immune
system. such
as the alleviation of symptoms associated with, or the partial or
complete.cure from,
an immunological condition or disease.
By way of introduction, differentiation is a process whereby structures and
functions
of cells are progressively committed to give rise to mare specialised cells.
such as the
formation of T cells or B cells. Therefore, as the cells become more
committed. the~~
become more specialised.
In contrast. retro-differentiation is a .process whereby structures and
functions of cells
are progressively changed to give rise to less specialised cells.
Undifferentiated cells are capable of multilineage differentiation - i.e. they
are capable
of differentiating into two or more t<pes of specialised cells. A typical
example of
an undifferentiated cell is a stem cell.
In contrast, differentiated cells are incapable-of multilineage
differentiation. A typical
example of a differentiated cell is a T cell.
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There are many undifferentiated cells and differentiated cells found in vivo
and the
general art is replete with general teachings on them.
By way of example, reference may be made to inter alia Levitt and Mertelsman
1995
(Haematopoietic Stem Cells, published by Marcel Dekker Inc - especially pages
45-
59) and Roitt et al (Immunology, 4th Edition, Eds. Roitt, Brostoff and Male
1996,
Publ. Mosby - especially Chapter 10).
In short, however, examples of undifferentiated cells include
lymphohaematopoietic
progenitor cells (LPCs). LPCs include pluripotent stem cells (PSCs), lymphoid
stem
cells (LSCs) and myeloid stem cells (MSCs). LSCs and MSCs are each formed by
the differentiation of PSCs. Hence.. LSCs and MSCs are more committed than
PSCs.
Examples of differentiated cells include T cells, B cells, eosinophils,
basophils,
15. neutrophils, megakaryocytes, monocytes, erythrocytes, granulocytes, mast
cells, and
lymphocytes.
T cells and B cells are formed by the differentiation of LSCs. Hence. T cells
and B
cells are more committed than LSCs.
Eosinophils, basophils, neutrophils, megakaryocytes, monocytes, en~throcytes.
granulocvtes. mast cells, IVI~s, and lymphocytes are formed by the
differentiation of
MSCs. Hence, each of these cells are more committed than MSCs.
Antigens are associated with undifferentiated and differentiated cells. The
term
"associated" here means the cells expressing or capable of expressing, or
presenting
or capable of being induced to present. or comprising, the respective
antigen(s).
Most undifferentiated cells and differentiated cells comprise Major
Histocompatability
Complex (MHC) Class I antigens and/or Class II antigens. If these antigens are
associated with those cells then they are called Class I' and/or Class Il*
cells.
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3
Each specific antigen associated with an undifferentiated cell or a
differentiated cell
can act as a marker. Hence, different types' of cells can be distinguished
from each
other on the basis of their associated particular antigens) or on the basis of
a
particular combination of associated antigens.
. 5
Examples of these marker antigens include the antigens CD34, CD 19 and CD3. If
these anigens are present then these particular cells are called CD34+, CD19*
and
CD3* cells respectively. If these antigens are not present then these cells
are called
CD34-, CD 19' and CD3' cells respectively.
In more detail, PSCs are CD34* cells. LSCs are DR+, CD34* and TdT* cells.
MSCs are CD34*, DR*, CD13+, CD33+, CD7* and TdT* cells. B cells are
CD19*, CD21*, CD22+ and DR* cells. T cells are CD2*, CD3*, and either CD4*
or CD8* cells. Immature lymphocytes are CD4* and CD8* cells. Activated T cells
are DR* cells. Natural killer cells (NKs) are CD56* and CD16* cells. T
Lymphocytes are CD7+ cells. Leukocytes'are CD45 * cells: Granulocytes are CDI3
*
and CD33* cells. Monocyte macrophage cells are CD14+ and DR* cells
Hence, by looking for the presence of the above-listed antigen markers it is
possible
to identify certain cell types (e.g. whether or not a cell is an
undifferentiated cell or
a differentiated cell) and the specialisation of that cell type (e.g. whether
that cell is
a T cell or a B cell).
The general concept of retrodiffererniation is not new. In fact, in 1976 Jose
Uriel
(Cancer Research 36, 4269-4275. November 1976) presented a review on this
topic,
in which he said:
"retrodiffereraiation appears as a common adaptive process for the
maintenance of cell integrity against deleterious agents of varied
etiology (physical, chemical, and viral). While preserving the entire
information encoded on its genome, cells undergoing
retrodifferenticuion lose morphological and functional complexity by
WO 96/23870 CA 02395452 2002-08-30 PCTIGB96100208 -
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virtue of a process of self deletion of cytoplasmic structures and the
transition to a more juvenile pattern of gene expression. This results
in a progressive uniformization of originally distinct cell phenotypes
and ro a decrease of responsiveness to regulatory signals operational
in adult cells. Retrodifferentiation is normally counterbalanced by a
process of reontogeny that tends to restore the terminal phenotypes
where the reversion started. This explains why retrodifferentiation
remains invariably associated to cell regeneration and tissue repair. "
Uriel (ibid) then went on to discuss cases of reported retrodifferentiation -
such as the
work of Gurdon relating to nuclei from gut. epithelial cells of Xenopus
tadpoles
(Advances in Morphogenesis [1966] vol 4, pp 1-43. New York Academic Press, Eds
Abercrombie and Bracher), and the work of Bresnick relating to regeneration of
liver
(Methods in Cancer Research [1971) vol 6, pp 347-391).
Uriel (ibid) also reported on work relating to isolated liver parenchyma)
cells for in
vitro cultures. According to Uriel:
"Contrary to the results with fetal or neonatal hepatocvtes, with
hepatocvtes from regenerating liver, or from established hepatomas, it
has been difficult to obtain permanent class lines from resting adult
hepatocytes. "
Uriel (ibid) also reported on apparent retrodifferentiation in cancer. wherein
he stated:
"the biochemical phenotypes of many tumours show analogous changes
of reversion toward immaturity ... during the preneoplastic phase of
liver carcinogenesis, cells also retrodifferentiate. "
More recent findings on .retrodifferentiation include the-work of Minoru
Fukunda
(Cancer Research (1981] vol 41, pp 4621-4628). Fukunda induced specific
changes
in the cell surface glycoprotein profile of K562 human leukaemic cells by use
of the
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tumour-promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA).
According to Fukunda TPA appeared to induce the K562 human leukaemic cells
into
a retrodifferentiated stage.
. 5 Also, Hass et al (Cell Growth & Differentiation [1991] vol 2, pp 541-548)
reported
that long term culture of TPA-differentiated U-937 leukaemia cells in the
absence of
phorbol ester for 32-36 days resulted in a process of retrodifferentiation and
that the
retrodifferentiated cells detached from the substrate and reinitiated
proliferation.
Another case of retrodifferentiation is the work of Curtin and Snell (Br. J.
.Cancer
[1983] vol 48, pp 495-505]. These workers compared enzymatic changes occurring
during diethylnitrosamine-induced hepatocarcinogenesis and liver regeneration
after
partial hepatectomy to normal liver differentiation. Theses workers found
changes
in enzyme activities during carcinogenesis that were similar to a step-wise
reversal
of differentiation. According to these workers, their results suggest that an
underlying retrodifferentiation process is common to both the process of
hepatocarcinogenesis and Liver regeneration.
More recently, Chastre et al (FEBS Letters [1985] vol 188, number 2, pp 2810-
?811]
reported on the retrodifferentiation of the human colonic cancerous subclone
HT29
18.
Even more recently, Kobayashi et al (Leukaemia Research [1994] vol 18, no. 12,
pp
929-933) have reported on the establishment of a retrodifferentiated cell line
(R.D-1)
from a single rat myelomonocyticleukemia cell which differentiated into a
macrophage-like cell by treatment with lipopolysaccharide (LPS).
According to the current understanding, as borne out by the teachings found on
page
911 of Molecular Biology of the Cell (pub. Garland Publishers Inc. 1983) and
more
recently Levitt and Mertelsman (ibid), a stem cell, such as a PSC, has the
following
four characteristics:
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i. it is an undifferentiated cell - i.e. it is not terminally differentiated;
ii. it has the ability to divide without limit;
iii. it has the ability to give rise to differentiated progeny, such as the
differentiated cells mentioned earlier; and
iv. when it divides each daughter has a choice: it can either remain as
PSC like its parent or it can embark on a course leading irreversibly
to terminal differentiation.
Note should be made of the last qualification, namely that according to the
general
teachings in the art once an undifferentiated cell has differentiated to a
more
committed cell it can not then retrodifferentiate. This understanding was even
supported by the teachings of Uriel (ibic~, Fukunda (ibia~, Hass er al (ibic~,
Curtin
and Snell (ibic~, Chastre et al (ibid), and Kobayashi et al (ibic~ as these
workers
retrodifferentiated certain types of differentiated cells but wherein those
cells
remained committed to the same lineage and they did not retrodifferentiate
into
undifferentiated cells.
Therefore, according to the state of the art before the present invention, it
was
believed that it was not possible to form undifferentiated cells, such as
.stem cells,
from more committed cells. However, the present invention shows that this
belief
is inaccurate and that it is possible to form undifferentiated cells from more
committed cells.
Thus, according to a first aspect of the present invention there is provided a
method
of preparing an undifferentiated cell, the method comprising contacting a more
committed cell with an agent that causes the more committed cell to
retrodifferentiate
into an undifferentiated cell.
According to a second aspect of the present invention there is provided a
provided a
method comprising contacting a more committed cell with an agent that causes
the
more committed cell to retrodifferentiate into an undifferentiated cell; and
then
committing the undifferentiated cell to a recommitted cell.
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The term "recommitted cell" means a cell derived from the undifferentiated
cell - i.e.
a new more committed cell.
According to a third aspect of the present invention there is provided an
S undifferentiated cell produced according to the method of the present
invention.
According to a fourth aspect of the present invention there is provided an
undifferentiated cell produced according to the method of the present
invention as or
in the preparation of a medicament.
According to a fifth aspect of the present invention there is provided the use
of an
undifferentiated cell produced according to the method of of the present
invention in
the manufacture of a medicament for the treatment of an immunological disorder
or
disease.
According to a sixth aspect of the present invention there is provided a
recommitted
cell produced according to the method of the present invention.
According to a seventh aspect of the present invention there is provided a
recommitted cell produced according to the method of the present invention as
or in
the preparation of a medicament.
According to an eighth aspect of the present invention there is provided the
use of a
recommitted cell produced according to the method of of the present invention
in the
manufacture of a medicament for the treatment of an immunological disorder or
disease.
According to a ninth aspect of the present invention there is provided a more
committed cell having attached thereto an agent that can cause the more
committed
cell to retrodifferentiate into an undifferentiated cell.
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According to a tenth aspect of the present invention there is provided a CD 19
T and
CD3+ cell.
Thus, in its broadest sense, the present invention is based on the highly
surprising
finding that it is possible to form an undifferentiated cell from a more
committed cell. -
The present invention is highly advantageous as it is now possible to prepare
undifferentiated cells from more committed cells and then use those
undifferentiated
cells as, or to prepare, medicaments either in vitro or in vivo or
combinations thereof
for the treatments of disorders.
The present invention is also advantageous as it is possible to commit the
undifferentiated cell prepared by retrodifferentiation to a recommitted cell,
such as
a new differentiated cell, with a view to correcting or removing the original
more
committed cell or for correcting or removing a product thereof.
Preferably, the more committed cell is capable of retrodifferentiating into an
MHC
Class I'" andlor an MHC Class II+ undifferentiated cell.
Preferably, the more committed cell is capable of retrodifferentiating into an
undifferentiated cell comprising a stem cell antigen.
Preferably. the more committed cell is capable of retrodifferentiating into a
CD34T
undifferentiated cell.
2~
Preferably, the more committed cell is capable of retrodifferentiating into a
Iymphohaematopoietic progenitor cell.
Preferably, the more committed cell is capable of retrodifferentiating into a
pluripotent stem cell.
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The undifferentiated cell may comprise any components that are concerned with
antigen presentation, capture or recognition. Preferably, the undifferentiated
cell is
an MHC Class I'" and/or an MHC Class II+ cell.
. 5 Preferably, the undifferentiated cell comprises a stem cell antigen.
Preferably. the undifferentiated cell is a CD34+ undifferentiated cell.
Preferably, the undifferentiated cell is a lymphohaematopoietic progenitor
cell.
Preferably. the undifferentiated cell is a pluripotent stem cell.
The more committed cell may comprise any components that are concerned with
antigen presentation, capture or recognition. Preferably, the more committed
cell is
an MHC Class I+ andlor an MHC Class II+ cell.
Preferably, the agent acts extracelluarly of the more committed cell.
Preferably, the more committed cell comprises a receptor that is operably
engageable
by the agent and wherein the agent operably engages the receptor.
Preferably. the receptor is a cell surface receptor.
Preferably, the receptor comprises an a- component and/or a (3- component.
Preferably, the receptor comprises a l3-chain having homologous regions.
Preferably, the receptor comprises at least the homologous regions of the Q-
chain of
HLA-DR.
Preferably. the receptor comprises an a-chain having homologous regions.
WO 96123870 ~ 02395452 2002-08-30 PCTlGB96100208
Preferably, the receptor comprises at least the homologous regions of the a-
chain of
HLA-DR.
5
15
Preferably, the agent is an antibody to the receptor.
Preferably, the agent is a monoclonal antibody to the receptor..
Preferably, the agent is an antibody, preferably a monoclonal antibody, to the
homologous regions of the a-chain of HLA-DR.
Preferably, the agent is an antibody, preferably a monoclonal antibody, to the
homologous regions of the a-chain of HLA-DR.
Preferably, the agent is used in conjunction with a biological response
modifier.
Preferably, the biological response modifier is an alkylating agent.
Preferably, the alkylating agent is or comprises cyclophosphoamide.
In one preferred embodiment, the more committed cell is a differentiated cell.
Preferably, the more committed cell is any one of a B cell or a T cell.
In an alternative preferred embodiment, the more committed cell is a more
mature
undifferentiated cell.
In one preferred embodiment, when the undifferentiated cell is committed to a
recommitted cell the recommitted cell is of the same lineage as the more
committed
cell prior to retrodifferentiation.
In another preferred embodiment, when the undifferentiated cell is committed
to a
recommitted cell the recommitted cell is of a different lineage as the more
committed
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cell prior to retrodifferentiation.
Preferably, the recommitted cell is any one of a B cell, a T cell or a
granulocyte.
Preferably, the method is an in vitro method.
Preferably, the agent modulates MHC gene expression, preferably wherein the
agent
modulates MHC Class I+ andlor MHC Class II'" expression.
/O The agent operably engages the more committed cell in order to
retrodifferenciate that
cell into an undifferentiated cell. In this regard, the agent for the
retrodifferentiation
of the more committed cell into the undifferentiated cell may act in direct
engagement
or in indirect engagement with the more cotramitted cell.
An example of direct engagement is when the more committed cell has at least
one
cell surface receptor on its cell surface; such as a f3-chain having
homologous regions
(regions that are commonly found having the same or a similar sequence) such
as
those that may be found on B cells, and wherein the agent directly engages the
cell
surface receptor. Another example, is when the more committed cell has a cell
surface receptor on its cell surface such as an a-chain having homolpgous
regions
such as those that may be found on T cells, and wherein the agent directly
engages
the cell surface receptor.
An example of indirect engagement is when the more committed cell has at least
two
cell surface receptors on its cell surface and engagement of the agent with
one of the
receptors affects the other receptor which then induces retrodifferentiation
of the more
committed cell.
The agent for the retrodifferentiation of the more committed cell into an
undifferentiated cell may be a cherilical compound or composition. Preferably,
however, the agent is capable of engaging a cell surface receptor on the
surface of
the more committed cell. For example, preferred agents include any one or more
of
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cyclic adenosine monophosphate (cAMP), a CD4 molecule, a CD8 molecule, a pan
or all of a T-cell receptor, a ligand (fixed or free), a peptide, a T-cell
receptor
(TCR), an antibody, a cross-reactive antibody, a monoclonal antibody, or a
polyclonal
antibody.
If the anent is an antibody, a cross-reactive antibody, a monoclonal antibody,
or a
polyclonal antibody, then preferably the agent is any one or more of an
antibody, a
cross-reactive antibody, a monoclonal antibody, or a polyclonal antibody to
any one
or more of: the p chain of a MHC class II antigen, the (3 chain of a MHC HLA-
DR
antigen, the a chain of a MHC class I or class II antigen, the a chain of HLA-
DR
antigen, the a and the Q chain of MHC class a antigen or of a MHC class I
antigen.
An example of a suitable antibody is CR3/43 (supplied by Dako).
The more committed cell is any cell derived or derivable from an
undifferentiated
IS cell.
Thus, in one preferred emdodiment, the more committed cell is also an
undifferentiated cell. By way of example therefore the undifferentiated cell
can be
a lymphoid stem cell or a myeloid stem cell, and the undifferentiated cell is
a
piuripotent stem cell.
In another preferred embodiment, the more committed cell is a differentiated
cell,
such as a CFC-T cell, a CFC-B cell, a CFC-Eosin cell, a CFC-Bas cell, a CFC-
Bas
cell, a CFC-GM cell, a CFC-MEG cell, a BFC-E cell, a CFC-E cell, a T cell, a B
cell, an eosinophil, a basophil, a neutrophil, a monocyte, a megakaryocyte or
an
erythrocyte: and the undifferentiated cell is a myeloid stem cell, a lymphoid
stem cell
or a pluripotent stem cell.
If the more committed cell is a differentiated cell then preferably the
differentiated
cell is a B lymphocyte (activated or non-activated), a T Lymphocyte (activated
or non-
activated), a cell from the macrophage monocyte lineage, a nucleated cell
capable of
expressing class I or class II antigens, a cell that can be induced to express
class I or
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class II antigens or an enucleated cell (i.e. a cell that does not contain a
nucleus -
such as a red blood cell).
In alternative preferred embodiments, the differentiated cell is selected from
any one
S of a group of cells comprising large granular lymphocytes, null lymphocytes
and
natural killer cells, each expressing the CD56 and/or CD16 cell surface
receptors.
.The differentiated cell may even be formed by the nucleation of an enucleated
cell.
The agent may act intracellularly within the more committed cell. However,
preferably, the agent acts extracelluarly of the more committed cell.
In a preferred embodiment, agent operably engages a receptor present on the
surface
of the more committed cell - which receptor may be expressed by the more
committed cell, such as a receptor that is capable fo being expressed by the
more
committed cell.
Preferably, the receptor is a Class I or a Class II antigen of the major
histocompatibility complex (MHC). In preferred embodiments the cell surface
receptor is any one of: an HLA-DR receptor, a DM receptor, a DP receptor, a DQ
receptor, an HLA-A receptor, an HLA-B receptor, an HLA-C receptor, an HLA-E
receptor, an HLA-F receptor, or an HLA-G receptor.
In more preferred embodiments the cell surface receptor is an HLA-DR receptor.
2~
Preferably the contacting step comprises the agent engaging with any one or
more of
the following: homologous regions of the a-chain of class I antigens,
homologous
regions of the a-chain of class II antigens, a CD4 cell surface receptor, a
CD8 cell
surface receptor, homologous regions of the I3-chain of class II antigens in
the
presence of lymphocytes, homologous regions of the a-chain of class I antigens
in the
presence of lymphocytes, or homologous regions of the a-chain of class II
antigens
in the presence of lymphocytes.
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Preferably the contacting step occurs in the presence of the biological
response
modifier.
Preferably the biological response modifier is any one or more of a modulator,
such
S as an immunomodulator, a growth factor, a cytokine, a cell surface receptor,
a
hormone, a nucleic acid, a nucleotide sequence, an antigen or a peptide.
In a preferred embodiment of the present invention the undifferentiated cell
is then
committed into a recommitted cell, such as a differentiated cell.
The recommitted.cell may be of the same lineage to the more committed cell
from
which the undifferentiated cell was derived.
Alternatively, the recommitted cell may be of a different lineage to the more
committed cell from which the undifferentiated cell was derived.
In addition, the present invention also encompasses the method of the present
invention for preparing an undifferentiated cell, wherein the method includes
committing the undifferentiated cell into a recommitted cell and then fusing
the
recommitted cell to a myeloma. This allows the expression in vitro of large
amounts
of the desired product, such as an antibody or an antigen or a hormone etc.
Other aspects of the present invention include:
The use of any one of the agents .of the present invention for preparing an
undifferentiated cell from a more committed cell.
The use of an undifferentiated cell produced according to the method of the
present invention for producing any one of a monoclonal or a polyclonal or a
specific
antibody from a B-lymphocyte or a T-lymphocyte; a cell from the macrophage
monocyte lineage; a nucleated cell capable of expressing class I or class II
antigens;
a cell capable of being induced to express class I or class II antigens; an
enucleated
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cell; a fragmented cell; or an apoptic cell.
The use of an undifferentiated cell produced according to the method of the
present invention for producing effector T-lymphocytes from B-lymphocytes
and/or
vice versa.
The use of an undifferentiated cell produced according to the method of the
present invention for producing any one or more of: a medicament, such as a
medicament comprising or made from a B-lymphocyte, a T-lymphocyte, a cell from
the macrophage monocyte lineage, a nucleated cell capable of expressing a
class I or
a class II antigen, a cell capable of being induced to express a class I or a
class II
antigen, or an enucleated cell.
The present invention also encompasses processes utilising the afore-mentioned
uses
1 S and products or compositions prepared from such _ processes.
The present invention also encompasses a medicament comprising an
undifferentiated
cell according to the present invention or a product obtained therefrom
admixed with
a suitable diluent, carrier or excipient.
In one preferred embodiment the medicament comprises an antibody or antigen
obtained from an undifferentiated cell according to the present invention
admixed with
a suitable diluent, carrier or excipient.
Preferably the medicament is for the treatment of any one of: cancer.
autoimmune
diseases, blood disorders, cellular or tissue regeneration, organ
regeneration, the
treatment of organ or tissue transplants, or congenital metabolic disorders.
In a preferred embodiment the present invention relates to a process of
introducing
a gene into the genome of an undifferentiated cell, wherein the process
comprises
introducing the gene into a more committed cell, and then preparing an
undifferentiated cell by the method according to the present invention,
whereby the
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- 16 -
gene is present in the undifferentiated cell.
In a more preferred embodiment the present invention relates to a process of
introducing a gene into the genome of an undifferentiated cell, wherein the
process
comprises inserting the gene into the genome of a more committed cell; and
then
preparing an undifferentiated cell by the method according to the present
invention,
whereby the gene is present in the undifferentiated cell.
In an even more preferred embodiment the present invention relates to a
process of
introducing the genome of a gene into an undifferentiated cell, wherein the
.process
comprises inserting the gene into the genome of a more committed cell, and
then
preparing an undifferentiated cell by the method according to the present
invention,
whereby the gene is present in the genome of the undifferentiated cell.
IS The present invention encompasses an undifferentiated cell prepared by any
one of
these processes of the present invention.
As already mentioned, the present invention also encompasses a medicament
comprising an undifferentiated cell prepared by any one of these processes
admixed
with a suitable diluent, carrier or excipient. With such a medicament the
undifferentiated cell could be used to produce a beneficial more committed
cell, such
as one having a correct genomic structure, in order to alleviate any symptoms
or
conditions brought on by or associated with a more committed cell having an
incorrect genomic structure.
2S
Thus, the present invention also provides a process of removing an acquired
mutation
from a more committed cell wherein the method comprises forming an
undifferentiated cell by the method according to the present invention,
committing the
undifferentiated cell into a recommitted cell, whereby arrangement or
rearrangement
of the genome and/or nucleus of the cell causes the mutation to be removed.
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Preferably the gene is ,inserted into the immunoglobulin region or TCR region
of the
genome.
Alternatively, the undifferentiated cell could be used to produce a more
committed
cell that produces an entity that cures any symptoms or conditions brought on
by or
associated with a more committed cell having an incorrect genomic structure.
For example, the present invention may be used to prepare antibodies or T cell
receptors to an antigen that is expressed by the more committed cell which has
retrodifferentiated into the undifferentiated cell. In this regard, the
antigen rnay be
a fetospecific antigen or a cross-reactive fetospecific antigen.
The present invention also includes a process of controlling the levels of
undifferentiated cells and more committed cells. For example, the present
invention
1,5 includes a method comprising forming an undifferentiated cell by the
method
according to the present invention and then activating an apoptosis gene to
affect the
undifferentiated cell, such as bring about the death thereof.
In one preferred embodiment of the present invention, the more committed cell
is not
a cancer cell. In another preferred embodiment of the present invention, the
agent
is neither carcinogenic nor capable of promoting cancer growth.
The present invention also covers a method of treating a patient suffering
from a
disease or a disorder resulting from a defective cell or an unwanted cell, the
method
comprising preparing an undifferentiated cell by contacting a more committed
cell
with an agent that causes the more committed cell to retrodifferentiate into
the
undifferentiated cell, and then optionally committing the undifferentiated
cell into a
recommitted cell; wherein the undifferentiated cell, or the recommitted cell.
affects
the defective cell or the unwanted cell to alleviate the symptoms of the
disease or
disorder or to cure the patient of the disease or condition.
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In summation, the present invention relates to the preparation of an
undifferentiated
cell From a more committed cell.
The present invention will now be described by way of example, in which
reference
shaft be made to the following Figures:
Figure 1 which is a microscope picture of cells before the method of the
present invention;
Figure 2 which is a microscope picture of cells prepared by the method of the
present
invention;
Figure 3 which is a microscope picture of cells prepared by the method of the
present
invention but at a lower magnification;
Figure 4 which is a microscope picture of cells, before the method of the
present
invention;
Figure 5 which is a microscope picture of cells prepared by the method of the
present
invention; and
Figure 6 which is a microscope picture of cells prepared by the method of the
present
invention.
A. MATERIALS AND METHODS
PATIENTS
Blood samples were obtained in lavender top tubes containing. EDTA from
patients
with B-cell chronic lymphocytic leukaemia's, patients with antibody deficiency
(including IgA deficiency and X-linked infantile hypogammaglobulinaemias),
patients
with HIV infections and AIDS syndrome, a patient with CMV infection, a patient
with Hodgkin's lymphomas, a patient with acute T-cell leukaemia, a 6-days old
baby
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19
with Hodgkin's lymphomas, a patient with acute T-cell Leukaemia, a 6-days old
baby
with blastcytosis, various patients with various infections and clinical
conditions, cord
. blood, bone marrow's, and enriched B-lymphocyte preparations of healthy
blood
donors.
CLINICAL AND EXPERIMENTAL CONDITIONS
The clinical and experimental treatment conditions of patients, including
various types
of treatment applied to their blood samples, are described in Table 1.
Differential
white blood cell (WBC) counts were obtained using a Coulter Counter and these
are
included in the same Table.
TREATMENT OF BLOOD
Blood samples, once obtained, were treated with pure monoclonal antibody to
the
1~ homologous region of the ~3-chain of the HLA-DR antigen (DAKO) and left to
mix
on a head to head roller at room temperature for a maximum of 24 hours. Some
samples were mixed first on a head to head roller for 15 minutes after which
they
were left to incubate in an incubator at 22°C. The concentration of
monoclonal
antibody added to blood samples varied from 10-50~,1/mI of blood.
In addition, other treatments treatments were applied at the same
concentrations and
these included addition of a monoclonal antibody to the homologous of the a-
chain
of the HLA-DR antigen, a monoclonal antibody to the homologous region of class
I
antigens, a monoclonal antibody to CD4, a monoclonal antibody to CDB, and a PE
conjugated monoclonal antibody to the homologous region of the ,Q-chain of the
HLA-
DR antigen.
Other treatments included the simultaneous addition of monoclonal antibodies
to the
homologous regions of the a and ~3-chains of the HLA-DR antigen to blood
samples.
Furthermore, alkylating agents such as cyclophosphoamide were added to blood
samples in combination with pure monoclonal antibody to the homologous region
of
WO 96123870 CA 02395452 2002-08-30 PCTIGB96100208
the /3-chain of the HLA-DR antigen.
Following these treatments blood samples were stained with panels of labelled
monoclonal antibodies as instructed by the manufacturer's instructions and
then
analyzed using flow cytometry.
Incubation periods with monoclonal antibodies ranged from 2 hour, 4 hour, 6
hour.
12 hour to ?4 hour intervals.
10 LABELLED ANTIBODIES
The following monoclonal antibodies were used to detect the following markers
on
cells by flow cytometry: CD19 and CD3, CD4 and CDB, DR and CD3, CD56 &
16 and CD3. CD45 and CD14, CD8 and CD3, CD8 and CD28, simultest control
15 (IgGI FITC + IgG2a PE), CD34 and CD2, CD7 and CD13 & 33, CDIO and CD2~,
CDS and CD10, CDS and CD21, CD7 and CDS, CD13 and CD20, CD23 and CD57
and CD2~ and CD45 RA (Becton & dickenson and DAKO).
Each patient's blood sample, both treated and untreated, was analyzed using
the
20 majority of the above panel in order to account for the immunophenotypic
changes
that accompanied different types of treatments and these were carried out
separately
on different aliquots of the same blood sample. Untreated samples and other
control
treatments were stained and analyzed simultaneously.
FLOW CYTOMETRY
Whole blood sample was stained and lysed according to the manufacturer
instructions.
Flow cytomery analysis was performed on a FACScan(c~ with either simultest or
PAINT A GATE software (BDIS) which included negative controls back tracking.
10,000 to 20.000 events were acquired and stored in list mode files.
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21
MORPHOLOGY
Morgholojy was analyzed using microscopy and Wright's stain.
B. RESULTS
CD19 AND CD3 PANEL
Treatment of blood samples with monoclonal antibody to the homologous region
of
the ;Q-chain of the HLA-DR antigen always decreased the relative number of .CD
I9T
cells. This marker is a pan B-cell antigen (see Table). This antigen is
present on all
human B lymphocytes at all stages of maturation but is lost on terminally
differentiated plasma cells. Hence, this is an indication that B cells were
retrodifferentiating into undifferentiated cells.
1~
The same treatment caused the relative number of CD3 * cells to increase
drarriatically
especially in blood of patients with B-CLL, which was always accompanied by an
increase in the relative number in CD3'CDI9' cells. CD3 is present on all
mature
T-lymphocytes and on 65 %-85 % of thymocytes. This marker is always found in
association with a-/~3- or gamma/delta T-cell receptors (TCR) and together
these
complexes are important in transducing signals to the cell interior. Hence,
this is an
indication that B cells were retrodifferentiating into undifferentiated cells
and then
being committed to new differentiated cells, namely T cells.
A novel clone of cells appeared in treated blood of B-CLL patients co-
expressing the
CD19 and CD3 markers - i.e. CD19'' and CD3* cells (see Charts 1, patient 2, 3
&
4 at 2hr. 6hr & 24hr of starting treatment). Other patients with different
conditions
' showed an increase in the relative number of these clones of cells. These
cells were
exceptionally large and heavily granulated and extremely high levels of CD 19
were
expressed on their cell membrane. The CD3 marker seems to be expressed on
these
cells at similar levels to those expressed on normal mature lytriphocytes.
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22
In Table 2, patient numbers 2, 3 and 4 are actually numbers representing the
same
patient and their delineation was merely to show the effect of treatment on
blood
with time (See Table 1 for experimental and clinical condition of this
patient).
The CD19~CD3+ clones in treated samples seem to decrease with time, reaching
original levels to those determined in untreated sample at 2hrs, 6hrs and
24hrs time.
Another type of cell of the same size and granuliry was detected in treated
samples
and these cells had high levels of CD 19 expressed on their surface but were
negative
IO for the CD3 marker and rich in FC receptors. However, the relative number
of these
cells appeared to decrease in time. Of interest, at 24 hours treatment of
blood sample
(?. 3 and 4) there was a decrease in the relative number of CD19'CD3' cells in
a
group of cells that were initially observed to increase after ? and 6 hr's
treatment of
blood samples. However, Coulter counts of WBC populations were reduced on
15 treatment of blood with monoclonal antibody to the homologous region of the
~i-chain
of the HLA-DR antigen. This finding suggests that this type of treatment gives
rise
to atypical cells that cannot be detected by Coulter (Table 1) but can be
accounted for
when measured by flow cytometry which counts cells on the basis of surface
markers,
size and granulity. Furthermore, these atypical cells were accounted for by
analysing
20 morphology using Wright's stain under a microscope. Flow cytometric charts
of
these phenomena are represented in Charts (1, 2, 3 & 4) and the
immunophenotypic
changes obtained on treatment of blood samples seems to suggest that CD19+ and
CD3+ lymphocytes are an interconnected group of cells but remain distinct on
the
basis of CD19 and CD3 relative expression compared to stem cells.
as
In Table 2, patient numbers 5 and 6 represent the same patient but analysis of
created
and untreated blood samples were monitored with time and at the same time (see
Table 1).
30 Patients blood with no B-cell malignancy showed similar trends of
immunophenotypic
changes when compared to blood of B-CLL patients but the changes were not to
the
same extent. However, the relative and absolute number of B-lymphocytes and
MHC
CA 02395452 2002-08-30
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23
class II positive cells in the blood of these patients are extremely low
compared to
those found in the blood of B-CLL patients.
Two brothers both with X-linked infantile hypogammaglobulinemia who were B
cell
deficient showed different immunophenotypic changes in the relative number of
CD3+
cells on treatment of their blood. The younger brother who was 2 months old
and
not ill, on treatment of his blood, showed a slight increase in the relative
number of
CD3'' cells which was accompanied by a decrease in the relative number of CD3'
CD19' cells. On the other hand, the other brother who was Z years old and was
extremely sick and with a relatively high number of activated T cells
expressing the
DR antigens showed a decrease in the number of CD3+ cells on treatment of his
blood. No other markers were used to measure other immunophentypic changes
that
might have occurred because the blood samples obtained from these two patients
were
extremely small (Table 2, ID 43/BD and 04/BD).
Patient 91 in Table 2 shows a decrease in the relative number of CD3~+ cells
following treatment of blood which was accompanied by an increase in the
relative
number of CD3'CD19' cells. However, on analysis of other surface markers such
as
CD4 and CD8 (see Table 3) the patient was observed to have a high relative
number
of CD4+CD8+ cells in his blood and this was noted prior to treatment of blood
samples with monoclonal antibody to the ~3-chain of the DR antigen and these
double
positive cells decreased appreciably following treatment of blood.
Furthermore, when
further markers were analyzed the relative number of CD3'' cells were seen to
have
elevated (See Table 4).
An enriched preparation of B-lymphocytes obtained from healthy blood donors
when
treated with monoclonal antibody to the ~i-chain of DR antigens showed a
dramatic
increase in the relative number of CD3" cells which were always accompanied by
a
decrease in the relative number of CD19+ cells and by an increase in the
relative
number of CD19'CD3' cells. Further analysis using markers such as CD4 and CD8
show a concomitant increase in the relative number of these markers. However,
an
enriched preparation of T lymphocytes of the same blood donors when treated
with
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24
the same monoclonal antibody did not show the same changes.
CD4 AND CD8 PANEL
The CD4 antigen is the receptor for the human immunodificiency virus. The CD4
molecule binds MHC class II antigen in the B2 domain, a region which is
similar to
the CD8 binding sites on class I antigens, Binding of CD4 to class n antigen
enhances T cell responsiveness to antigens and so does the binding of CD8 to
class
I antigens. The CD8 antigens are present on the human supressor/cytotoxic T-
lymphocytes subset as well as on a subset of natural killer (NK) lymphocytes
and a
majority of normal thymocytes. The CD4 and CD8 antigens are coexpressed on
thymocytes and these cells lose either markers as they mature into T-
lymphocytes.
On analysis of the CD4 and CD8 markers - see below - and from a majority of
blood
1 ~ samples presented in Table 2, a pattern of staining emerges which supports
the
presence of a retrodifferentiation process of B=lymphocytes into
undifferentiated cells
and the subsequent differentiation into T-lymphocytes.
CD4+CD8' cells, which are double positive cells, always appeared following
treatment of blood samples with monoclonal antibody to the homologous region
of the
(3-chain and these types of cells were markedly increased in the blood of
treated
samples of patients with B-CLL and which were absent altogether in untreated
samples (See Table 3 and Charts 1, 2 3 & 4). In the same specimens the
relative
number of single positive cells such as CD8'' and CD4* cells was also noted to
increase simultaneously. Furthermore, a decrease in the relative number of
CD4'
CD8' cells which, at least in the case of B-CLL correspond to B cells was
noted to
fall dramatically in treated samples when compared to untreated specimens
which
remained at the same level when measured with time. However, measurement of
the
relative number of CD4+CD8+ cells with time in treated samples showed that
there
was a concomitant increase in the number of single positive cells with a
decrease in
the relative number of double positive cells. This type of immunophenotypic
change
is characteristic of thymic development of progenitor cells of the T-
lymphocyte
CA 02395452 2002-08-30
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lineage in the thymus (Patient number 2,3 and 4). The CD4 antigen is present
on the
helperlinducer T- lymphocyte subsets (CD4+CD3'") and a majority of normal
. thymocytes. However, this antijen is present in low density on the cell
surface of
monocytes and in the cytoplasm of monocytes and macrophages (CD3'CD4+).
S
IO
The relative number of CD4* low cells was affected differently in different
blood
samples following treatment. The relative number of this type of cells seems
unaffected in blood samples of patients with B-CLL following treatment when
compared to untreated samples. Such low levels of CD4 expression is found on
monocytes and very early thymocytes. .
Patient HIV+25 on treatment showed a substantial increase in the number of
double
positive cells expressing CD4 and CD8 simultaneously. On the other hand,
patient
91 on treatment showed a decrease in this subtype of cells and the observation
of such
15 . phenomenon is time dependent. The relative number of CD8+ cells was.
observed to
increase in untreated blood samples of patients with B-CLL when measured with
time
whereas the relative number of CD4+ and CD4+ low cells was observed to
decrease
at the same times (Table 3 patient 2,3 and 4).
20 DR AND CD3 PANEL
The DR markers are present on monocytes, dendritic cells, B-cells and
activated T-
lymphocytes.
25 Treated and untreated samples analysed with this panel showed similar
immunophenotypic changes to those obtained when blood samples were analysed
with
the CD19 and CD3 markers (see Table 2) and these antigens as mentioned earlier
are
pan B and T-cell markers respectively.
Treatment of blood with monoclonal antibodies seems to affect the relative
number
of DR' B-lymphocytes so that the level of DR+ cells decrease. In contrast, the
relative number of CD3+ (T-cells) cells increase significantly (see Table 4
and Chart).
CA 02395452 2002-08-30 pCTIGB96100208
26 '
Furthermore, the relative number of activated T cells increased in the
majority of
treated blood samples of patients with B-CLL and these types of cells were
affected
variably in treated samples of patients with other conditions. Furthermore,
the
relative number of DR high positive cells appeared in significant numbers in
treated
samples of patients with B-CLL and a 6 day old baby with increased DR* CD34*
blasts in his blood. However, it should be noted that the blasts which were
present
in this patient's blood were negative for T and B-cell markers before and
after
treatment but became more positive for myeloid lineage antigens following
treatment.
The relative number of CD3'DR' cells increased in the majority of treated
blood
samples and was proportional to increases in the relative number of CD3 ~
cells (T-
cells) and was inversely proportional to decreases in the relative number of
DR+
cells (B-cells).
CD56&16 AND CD3 PANEL
The CD56&CD16 markers are found on a heterogeneous group of cells, a subset of
lymphocytes known generally .as large granular lymphocytes and natural killer
(NK)
lymphocytes. The CD16 antigen is expressed on virtually all resting NK
lymphocytes
and is weakly expressed on some CD3' T lymphocytes from certain individuals.
This antigen is found on granulocytes in lower amount and is associated with
lymphocytes containing large azurophilic granules. The CD16 antigen is the IgG
FC
receptor III.
A variable number of CD16' lymphocytes coexpress either the CD57 antigen or
low-
density CD8 antigen or both. In most individuals, there is virtually no
overlap with
other T-lymphocyte antigens such as the CDS, CD4, or CD3 antigens. The CD56
antigen is present on essentially all resting and activated CDlb'' NK
lymphocytes and
these subsets of cells carry out non-major histocompatibiliry complex,
restricted
cytotoxiciry.
Immunophenotyping of treated and untreated blood samples of B-CLL and some
other
patients with other conditions showed an increase in the relative number of
cells
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_. 27
coexpressing the CD56&CD 16 antigens which were heavily granulated and of
medium size (see Table 5 and Charts 1, 2, 3 & 4). These observations were also
accompanied by a marked increase in the relative number of cells expressing
the CD3
antigen only (without the expression of CD56 and CD16 markers) and cells
coexpressing the CD56&CD16 and CD3 markers together.
In Table 5, patient numbers 2, 3, and 4 represent the same blood sample but
being
analysed at 2 hours, 6 hours and 24 hours respectively (before and after
treatment}.
This sample shows that treatment of blood with monocionaI antibody to the
homologous region of the Q-chain of DR antigen seems to cause spontaneous
production of CD66y and CD16' cells, CD3+ cells and CD56* and CD16* CD3*
cells and these observations were always accompanied by the disappearance of B-
cell
markers (CD19, DR, CD56, CD16'CD3').
. Onward analysis of this blood sample before and after treatment showed the
levels of
CD56j and CD16+ cells fo decrease v~iith time and the Level of CD3* cells to
increase
with time.
Blood samples of patient 7 with B-CLL, did not show any changes in the number
of
cells expressing the CD56, CD16 and CD3 antigens when compared to
immunophenotypic changes observed in treated and untreated samples and this is
because the amount of monoclonal antibody added was extremely low relative to
the
number of B lymphocytes. However, treatment of this patient's blood sample on
a
separate occasion with an appropriate amount of monoclonal antibody showed
2~ significant increases in the relative number of CD3+, CD56' & CD16* and
CD56+
and CD16+ CD3+ cells.
Blood samples of other patients with other conditions showed variable changes
in the
IeveI of these cells and 'this seems to be dependent on the number of B-
lymphocytes
present in blood before treatment, duration of treatment and probably the
clinical
condition of patients.
WO 96123870 CA 02395452 2002-08-30 PCTIGB96100208 "
28
CD45 AND CD14 PANEL
The CD4~ antigen is present on all human leukocytes, including lymphocytes,.
monocytes, polymorphonuclear cells, eosinophils, and basophils in peripheral
blood,
thymus, spleen, and tonsil, and leukocyte progenitors in bone marrow.
The CD 14 is present on 70 % to 93 % of normal peripheral blood monocytes, 77
% to
90% of pleural or peritoneal fluid phagocytes. This antigen is weakly
expressed on
granulocytes and does not exist on unstimulated lymphocytes, micogen-activated
T
lymphocytes, erythrocytes, or platelets.
The CD4~ antigen represents a family of protein tyrosine phosphatases and this
molecule interacts with external stimuli (antigens) and effects signal
transduction via
the Scr-family members leading to the regulation of cell growth and
differentiation.
Engagement of the ~3-chain of the DR antigens in treated blood samples
especially
those obtained from patients with B-CLL suggests that such a treatment affects
the
level of CD45 antigens on B-lymphocytes. The overall immunophenotypic changes
that took place on stimulation of the ~i-chain of the DR antigen seem to give
rise to
different types of cells that can be segregated on the basis of the level of
CD45 and
CD14 expression as well as morphology as determined by forward scatter and
side
scatter (size and granulity respectively) and these results are presented in
Table 6 and
Charts(1,?,3,4&5).
On treatment the relative number of CD45 low cells (when compared to untreated
samples) increased significantly and so did the relative number of cells co-
expressing
the CD4~ and CD14 antigens. This type of immunophenotypic chances coincided
with a decrease in the relative number of CD45 high cells (compared to
untreated
samples). However. this latter population~of cells can be further divided on
the basis
of morphology and the degree of CD45 expression. One .type was extremely large
and had extremely high levels of CD45 antigen when compared to the rest of
cells
present in the charts (see charts 1, 2, 3 and 4). On analysis of this panel
following
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WO 96/23870 PCTlGB96/00208
29
treatment with time (see Table patient 2,3 and 4 and charts) the relative
number of
CD45* cells initially fell drastically with time to give rise to CD4~ low
cells.
However, analysis of blood 24 hours later showed the opposite situation.
Samples 5 and 7 reveal apposite immunophenotypic changes to those obtained
with
other samples obtained from other B-CLL patients and this is because the
samples
were analysed at a much earlier incubation time with the monoclonal antibody.
In
fact the sequential analysis of blood samples after treatment seems to suggest
that the
immunophenotypic changes undertaken by B lymphocytes is time dependent because
it represents a stage of development and the immunophenorypic changes measured
at time X is not going to be the same at time X plus (its not fixed once
induced).
However, these types of changes must be occurring in a more stringent manner
in
the body otherwise immunopathology would ensue. The effect of treatment of
blood
samples from other patients with no B-cetl malignancy show variable changes in
immunophenotypes of cells and this because B-lymphocytes are present in lower
amount. However, treatment of enriched fractions of B-lymphocytes obtained
from
healthy blood donors show similar immunophenorypic changes to those obtained
with
B-CLL with high B lymphocyte counts.
CD8 AND CD3 PANEL
The CD8 antigenic determinant interacts with class I MHC molecules, resulting
in
increased adhesion between the CD8+ T lymphocytes and the target cells. This
type
of interaction enhances the activation of resting lymphocytes. The CD8 antigen
is
coupled to a protein tyrosine kinase (p56ick) and in turn the CD8/p56ick
complex
may play a role in T-lymphocyte activation.
Treatment of blood samples obtained from patients with B-CLL with monoclonal
antibody to the B chain causes a significant increase iti the relative number
of
CD3CD8 and CD3 (highly likely to be CD4CD3) positive cells thus indicating
more
clearly that double positive cells generated initially are undergoing
development into
mature T-lymphocytes. This is a process that can be measured directly by CD19
and
WO 96/23870 ~ 02395452 2002-08-30 pCT/GB96100208
by DR and indirectly by CD8'CD3' antigens. Serial assessment of treated blood
samples of the same patient with time seems to agree with a process which is
identical
to thymocyte development (Table 7, patient 2, 3 and 4 and Chart 1).
The relative number of CD8+ cells increased with time in treated and untreated
samples but to a higher extent in untreated samples. On the other hand, the
relative
number of CD8+CD3 ~ cells decreased with time in untreated samples: However,
the
relative number of CD3+ cells increased in treated blood samples when measured
with time and these types of cells highly correspond to CD4+CD3+ single
positive
10 cells; a maturer form of thymocytes. In addition, since these samples were
also
immunophenoryped with other panels (mentioned above in Tables 3, 4, 5 and 6)
the
overall changes extremely incriminate B cells in the generation of T
lymphocyte
progenitors and progenies.
15 Blood samples from a patient with B-CLL (number 2. 3 and 4 Tables 1. 2, 3,
4, 5,
6, 7) in separate aliquots were treated with nothing, PE conjugated monoclonal
antibody to the homologous region of the ~i-chain of DR antigen and
unconjugated
form of the same monoclonal antibody. On comparison of PE conjugated treatment
clearly indicates no change in the relative number of CD3 positive cells and
20 associated markers such as CD4 which have been observed in significant
levels when
the same blood sample was treated with unconjugated form of the antibody.
However, an increase in the number of CD45 positive cells with no DR antigen
being
expressed on their surface was noted when measured with time (see Table 8). A
finding that was similar to that noted in untreated samples when
immunophenoryped
25 with time (Table 6). Furthermore, the relative number of cells expressing
CD45 low
decreased in time, a phenomenon which was also noted in the untreated samples
(when measured with time) of the same patient (see chart 1A).
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C. COMPARISON OF THE EFFECT OF OTHER MONOCLONAL
ANTIBODIES WITH DIFFERENT SPECIFICITY ON T-
LYMPHOPHOIESIS
CD19 AND CD3 PANEL
Treatment of blood samples with monoclonal antibody to the homologous region
of
the a-chain of the DR antigen and the homologous region of MHC Class I
antigens
decreased the number of CD3+ cells and increased the number of CDl9y cells.
Treatment of the same blood with monoclonal antibody to the homologous region
of
the ~3-chain of the DR antigen decreased the number of CD19+ cells and
increased the
number of CD3' cells. Treatment with the latter monoclonal antibody with
cyclophosphoamide revealed the same effect (Table 14 patient ~/6 with B-CLL at
2hr
treatment).
Onward analysis of CD19+ and CD3+ cells in the same samples revealed further
increases in the relative number of CD3+ cells only in blood treated with
monoclonal
antibody to the homologous region of the (3-chain of DR antigen (Table 14
patient 5I6
at 24 hours following treatment). However, onward analysis (24 hours later
patient
5/6 Table 14) of blood samples treated with cyclophosphamide plus monoclonal
antibody to the ~i-chain of DR antigen show reversal in the relative number of
CD 19"
and CD3' cells when compared to that observed at 2 hour incubation time under
exactly the same condition.
In general, treatment of blood samples of the same patient with monoclonal
antibody
to the homologous region of the a chain of the DR antigen or monoclonal
antibody
to the homologous of the a-chain of the class I antigen shows an increase in
the
relative number of CD19' cells (pan B marker) when compared to untreated
sample.
The relative number of CD 19-CD3' cells decreased slightly in blood samples
treated
with monoclonal antibody to the a-chain of DR antigen or treated with
monoclonal
antibody to class I antigens (see Table 14 & Charts 2, 3 & 4}. Treatment of
blood
samples of patient 09 with monoclonal antibody to class I antigens increased
the
relative number of CD3' cells and decreased slightly the relative number of CD
19+
WO 96/23870 CA 02395452 2002-08-30 PCT/GB96/00208 '
32 -
and CD19'CD3' cells. However, treatment of an enriched preparation of B-
lymphocytes obtained from healthy blood donors with monoclonal antibody to the
(3-
chain or a-chain of DR antigen showed similar immunophenotypic changes to
those
obtained with patient with B-CLL.
Treatment of HIV'' and IgA deficient patients with monoclonal antibody to the
(3-
chain of the DR antigen increased the relative number of CD3' cells and
decreased
the relative number of CD19' cells. However, treatment of the same blood
sample
with monoclonal antibody to the homologous region of class I antigen did not
produce
the same effect. Treatment of blood samples obtained from patients (34/BD and
04/BD) with B-cell deficiency showed variable immunophenotypic changes when
treated with monoclonal antibodies to the /3-chain of the DR antigen, class I
antigens
and CD4 antigen.
CD4 AND CD8 PANEL
Blood samples analysed using the CD19 and CD3 panel (Table 14) were also
immunophenotyped with the CD4 and CD8 panel (Table 15). Both panels seem to
agree and confirm each other. Incubation for 2 hours of blood samples of
patients
~yith B-CLL (Table 15, patients 5/6 and 10, Charts 2, 3 & 4) with monoclonal
antibody to the homologous region of the /3-chain of the DR antigen or with
this
monoclonal antibody plus cyclophosphoamide increased the relative number of
CD8
and CD4' cells and cells coexpressing both markers. On the other hand,
treatment
of the same samples with monoclonal antibodies to the homologous region of the
a-
chain of the DR antigen or the homologous region of the a-chain of class I
antigen
did not produce the same effects.
Comparison of immunophenotypic trends obtained at 2 hours and 24 hours
incubation
periods with monoclonal antibody to the ~i-chain of the DR antigen plus
cyclophosphoamide revealed reverse changes in the relative number of CD4 and
CD8
positive cells (Table 1~, patient 5/6 with B-CLL at 2 hours and 24 hours) and
such
changes were in accordance with those obtained when the same blood sample was
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33
analysed with the CD19 and CD3 panel (Table 14 the same patient). The later
findings indicate that the subsequent differentiation is reversible as the
undifferentiated
cells can differentiate into T-lymphocytes or B-lymphocytes.
DR AND CD3 PANEL
The immunophenotypic changes obtained with DR and CD3 (Table 16) panel confirm
the findings obtained with CD19 and CD3 panel and CD4 and CD8 panel (Tables 14
& 1~ & Charts 2, 3 & 4) which followed treatment of the same blood samples
with
monoclonal antibodies to the homologous region of the beta- or alpha- side of
the DR
antigen or monoclonal antibody to class I antigens or monoclonal antibody to
the ~i-
chain of the DR antigen plus cyclophosphoamide at 2 hour analysis.
From the results, it would appear that the monoclonal antibody to the
homologous
region of the (3-chain of the DR antigen is extremely capable of driving the
production
of CD3 positive cells from-DR+ cells.
Furthermore, treatments such as those involving engagement of the a-chain of
DR
antigens or engagement of the (3-side of the molecule in conjunction with
cyclophosphoamide (prolonged incubation time) promoted increases in the
relative
number of CD19+ cells or DR+ cells.
CD~6&16 AND CD3 PANEL
Treatment of blood samples, especially of those of patients with B-CLL with
high B-
lymphocyte counts with monoclonal antibody to the homologous region of the (3-
chain
of the DR antigen increased the relative number of CD56&16 positive cells.
In these patients the relative number of CD3+ and CD56+ and CD16+CD3' cells
also
increased following treatment of blood samples with monoclonal antibody to the
~i-
chain, confirming earlier observations noted with the same treatment when the
same
blood samples were analysed with CD3 and CD19 and DR and CD3 panels.
WO 96123870 CA 02395452 2002-08-30 PCT/GB9G/00208
34
CD45 AND CD14 PANEL
Blood samples treated with monoclonal antibodies to the Vii- or alpha- chains
of the
DR antigen or to the (3-chain plus cyclophosphoamide or class I antigens were
also
analysed with the CD45 and CD14 panel (Table 18). The delineation of CD45 low,
CD45 high and CD45 medium is arbitrary. Treatment of blood sample ~/6 (at 2
hours) with monoclonal antibodies to the (3-chain of the DR antigen or with
this
monoclonal antibody plus cyclophosphoamide generated CD45' low cells and
increased the relative number of CD45+ medium cells. However, the former
treatment increased the relative number of CD45 * high cells and the latter
treatment
decreased the relative number of CD45'' medium cells and these changes
appeared
to be time dependent.
Blood samples of patient 5/6 and 10 (B-CLL) on treatment with monoclonal
antibody
to class I antigens showed a decrease in the relative number of CD45'' medium
cells
and similar observations were noted in blood samples 09 and HIV+ following the
same treatment when compared to untreated samples. Treatment of blood samples
of HIV + and IgA/D patients with monoclonal antibody to class I antigen
increased
the relative number of CD45+ low cells when compared to untreated samples or
samples treated with monoclonal antibody to the ~i-chain of the DR antigen.
However, blood samples of these patients showed a decrease in the relative
number
of CD45'" medium cells on treatment with monoclonal antibody to the homologous
regions of the ~3-chain of the DR antigen. Medium CD45j cells increased in
blood
samples of IgAID patient following monoclonal antibody to class I antigen
treatment.
2~ Cells that were extremely large, heavily granular and expressing intense
levels of
CD45 antigen were noted in treated blood samples with monoclonal antibody to
the
homologous region of the (3-chain of DR antigen of MHC class II antigens (see
Charts
1, 2, 3, 4 & 5).
CA 02395452 2002-08-30
' WO 96/23870 PCT /GB96100208
CD8 AND CD28 PANEL
The CD28 antigen is present on approximately 60 % to 80 % of peripheral blood
T
(CD3+) lymphocytes, 50% of CD8+ T lymphocytes and 5% of immature CD3-
. ~ thymocytes. During thymocyte maturation, CD28 antigen expression increases
from
low density on most CD4tCD8'" immature thymocytes to a higher density on
virtually
all mature CD3+, CD4'' or CD8T thymocytes. Cell activation further augments
CD28 antigen density. Expression of the CD28 also divides the CD8+ lymphocytes
into two functional groups. CD8+CD28+ lymphocytes mediate aIloantigen-specific
10 cytotoxicity, that is major histocompatibility complex (MHC) class I-
restricted.
Suppression of cell proliferation is mediated by the CD8+CD28' subset. The
CD28
antigen is a cell adhesion molecule and functions as a ligand for the B7lBB-1
antigen
which is present on activated B lymphocytes.
15 Treatment of blood samples of patients (Table 19, patients 5/6 and 8) with
B=C-LL
with monoclonal antibody to the homologous region of (3-chain of the DR
antigen
increased the relative number of CD8+, CD28+ and CD8+CD28+ cells and all other
types of treatments did not.
20 CD34 AND CD2 PANEL
The CD34 antigen is present on immature haematopoietic precursor cells and all
haematopoietic colony-forming cells in bone marrow, including unipotent (CFU-
GM,
BFU-E) and pluripotent progenitors (CFU-GEMM, CFU-Mix and CFU-blast). The
2~ CD34 is also expressed on stromal cell precursors. Terminal
deoxynucleotidyl
transferase (TdT)' B- and T-lymphoid precursors in normal bone are CD34-, The
CD34 antigen is present on early myeloid cells that express the CD33 antigen
but lack
the CD 14 and CD 15 antigens and on early erythroid cells that express the
CD71
antigen and dimly express the CD45 antigen. The CD34 antigen is also found on
30 capillary endothelial cells and approximately 1 % of human thymocytes.
Normal
peripheral blood lymphocytes, monocytes, granulocytes and platelets do not
express
the CD34 antigen. CD34 antigen density is highest on early haematopoietic
WO 96/23870 CA 02395452 2002-08-30 PCTlGB96/00208
36
progenitor cells and decreases as the cells mature. The antigen is absent on
fully
differentiated haematopoietic cells.
Uncommitted CD34+ progenitor cells are CD38', DR' and lack lineage-specific
antigens, such as CD71, CD33, CD10, and CDS, while CD34+ cells that are
lineage-committed express the CD38 antigen in high density.
Most CD34' cells reciprocally express either the CD45R0 or CD45RA antigens.
Approximately 60% of acute B-lymphoid leukaemia's and acute myeloid leukaemia
express the CD34 antigen. The antigen is not expressed on chronic lymphoid
leukaemia !B or T lineage) or lymphomas. The CD2 antigen is present on T
lymphocytes and a subset of natural killer lymphocytes (NK).
The results are shown in Charts 2, 3 and 5.
Analysis of blood samples of a patient with B-CLL (Table 20, patient 5/6 at
2hours)
after treatment with monoclonal antibodies to the (3-chain of the DR antigen
or the a-
chain of the same antigen revealed marked increases in the relative number of
CD34+
and CD34-CDZ' cells after treatment with the former antibody. Since the same
blood samples were immunophenotyped with the above mentioned panels (see
Tables
14 to 19 ) for other markers the increase in the relative number of CD34T and
CD34'CD?- cells observed here seems to coincide with increases in the relative
number of CD4+CD8T, CD8+CD3+ and CD4+CD3'" single positive (SP) cells.
Furthermore. these findings which seem exclusive to engagement of the /3-chain
of
the HLA-DR antigen, are in direct support that the process is giving rise to T-
lymphopoiesis via B lymphocyte regression.
On analvsina the same treatment 24 hours later the CD34+ cells seemed to
decrease
in levels to give rise to further increase in the relative number of T
lymphocytes.
The process of retrodifferentiation that initially gave rise to T-
lymphopoiesis can be
reversed to give rise to B-lymphopoiesis. The former phenomenon was observed
at
2 hours incubation time with monoclonal antibody to the a-chain of the HLA-DR
CA 02395452 2002-08-30
WO 96/23870 PCT/GB96/00208
37
antigen plus cylophosphoamide, whereas the latter process was noted at 24
hours
incubation time with the same treatment in the same sample (Chart 2).
Treatment of blood samples of HIV'' patient (Table 20 patient HIV+) with
S monoclonal antibody to the ~i-chain of the HLA-DR antigen markedly increased
the
relative number of CD34y and CD2+CD34'' cells and so did treatment of the same
blood sample with monoclonal antibody to the ~3-chain of the HLA-DR antigen
and
monoclonal antibody to the a-chain of the same antigen when added together.
However, treatment of this blood sample with monoclonal antibody to the a-
chain of
the HLA-DR antigen did not affect the level of CD34+ cells. Treatment of blood
samples obtained from a 6-day old baby (BB/ST Table 20) who was investigated
at
that time for leukaemia and who had very high number of atypical cells
(blasts) in his
blood with monoclonal antibody to the (3-chain of the HLA-DR antigen, or
monoclonal antibody to the a-chain of the same antigen or both monoclonal
antibodies
added together resulted in the following imrnunophenotypic changes.
On analysis of untreated blood samples the relative number of CD34+ and DR''
cells
were markedly increased and on treatment with monoclonal antibody to the (3-
chain
the relative number of CD34' cells further increased but were noted to
decrease on
' treatment with monoclonal antibody to the a-chain of the HLA-DR antigen or
treatment with monoclonal antibodies to the a and ~i-chains of the molecule
when
added together. However, the latter treatment increased the relative number of
CD34yCD?+ cells and the opposite occurred when the same blood sample was
treated with monoclonal antibody to the a-chain of the HLA-DR antigen alone.
On
analysis of treated and untreated blood aliquots of the same patient 24 hours
later the
relative number of CD34+ decreased with all above mentioned treatments except
it
was maintained at a much higher level with monoclonal antibody to the ~i-chain
of the
HLA-DR antigen treatment. The latter treatment continued to decrease the
relative
number of CD34+CD2+ cells 24 hours later.
These results indicate that engagement of the HLA-DR antigen via the (3-chain
promotes the production of more CD34+ cells from CD2+CD34' pool or from more
WO 96/23870 CA 02395452 2002-08-30 PCT/GB96/00208
3$ _
mature types of cells such as B-lymphocytes of patients with B-CLL and these
results
indicate that this type of treatment promotes retrodifferentiation. However,
immunophenotyping of blood samples 24 hours later suggests that these types of
cells
seem to exist in another lineage altogether and in this case cells seem to
exist or
rather commit themselves to the myeloid lineage which was observed on analysis
of
treated blood sample with the CD7 and CD 13&33 panel.
Morphology changes immunophenotypic characteristics of B-Lymphocytes of B-CLL
and enriched fractions of healthy individuals (using CD 19 beads) on treatment
with
monoclonal antibodies to homologous regions of the ~i-chain of MHC class II
antigens. These were accompanied by a change in the morphology of B-
lymphocytes.
B-lymphocytes were observed colonising glass slides in untreated blood smears
were
substituted by granulocytes, monocytes, Large numbers of primitive looking
cells and
nucleated red blood cells. No mitotic figures or significant cell death were
observed
1~ in treated or untreated blood smears.
The results of Table 20 also demonstrate a further important finding in that
according
to the method of the present invention it is possible to prepare an
undifferentiated cell
by the retrodifferentiation of a more mature undifferentiated cell.
D. MICROSCOPE PICTUES
In addition to the antigen testing as mentioned above, the method of the
present
invention was followed visually using a microscope.
In this regard, Figure 1 is a microscope picture of differentiated B cells
before the
method of the present invention. Figure 2 is a microscope picture of
undifferentiated
cells formed by the retrodifferentiation of the B cells in accordance with the
present
invention wherein the agent was a monoclonal antibody to the homologous
regions
of the (3-chain of HLA-DR antigen. The undifferentiated cells are the dark
stained
clumps of cells. Figure 3 is a microscope picture of the same undifferentiated
cells
but at a lower magnification.
CA 02395452 2002-08-30
~ WO 96/23870 PCT/GB96100208
' 39
Figures 1 to 3 therefore visually demonstrate the retrodifferentiation of B
cells to
undifferentiated stem cells by the method of the present invention.
Figure 4 is a microscope picture of differentiated B cells before the method
of the
~ 5 present invention. Figure 5 is a microscope picture of undifferentiated
cells formed
by the retrodifferentiation of the B cells in accordance with the present
invention
wherein the agent used was a monoclonal antibody to the homologous regions of
the
j3-chain of HLA-DR antigen. Again, the undifferentiated cells are the dark
stained
clumps of cells. Figure 6 is a microscope picture of the formation of
differentiated
granulocyte cells from the same undifferentiated cells of Figure 5.
Figures 4 to 6 therefore visually demonstrate the retrodifferentiation of B
cells to
undifferentiated stem cells by the method of the present invention followed by
commitment of the undifferentiated cells to new differentiated cells being of
a
different lineage as the original differentiated cells.
The retrodifferentiation of T cells to undifferentiated stem cells by the
method of the
present invention followed by commitment of the undifferentiated cells to new
differentiated cells being of a different lineage as the original
differentiated cells was
also followed by microscopy.
E. SUMMARY
In short, the examples describe in vitro experiments that reveal extremely
interesting
findings regarding the ontogeny and development of T and B lymphocytes which
can
be utilised in the generation of stem cells to affect lymphohaematopoiesis in
peripheral blood samples in a matter of hours.
Treatment of peripheral blood samples obtained from patients with B-cell
chronic
Iymphocytic leukaemia's (B-CLL) with high B lymphocyte counts. with monoclonal
antibody to the homologous region of the /3-chain of class-II antigens gave
rise to a
marked increase in the relative number of single positive (SP) T-lymphocytes
and
WO 96/23870 CA 02395452 2002-08-30 PCT/GB96/00208
, 40 '
their progenitors which were double positive for the thymocyte markers CD4 and
CD8 antigens and these were coexpressed simultaneously. However, these
phenomena were always accompanied by a significant decrease in the relative
number
of B-lymphocytes. These observations were not noted when the same blood
samples
were treated with monoclonal antibodies to the homologous region of the a-
chain of
class-II antigens or to the homologous region of class-I antigens.
Treatment of whole blood obtained from patients with B-cell chronic
lymphocytic
leukaemia (CLL) with monoclonal antibody to the homologous region of the B
chain
' 10 of the HLA-DR antigen appeared to give rise to T-lymphopoiesis. This
event was
marked by the appearance of double positive cells coexpressing the CD4 and CD8
markers, the appearance of cells expressing CD34 and the concomitant increase
in the
number of single positive CD4" CD3+ and CD8* CD3* lymphocytes. Furthermore,
the immunophenotypic changes that took place in the generation of such cells
were
identical to those cited for thymocyte development, especially when measured
with
time.
The percentages of double positive cells (DP) generated at 2 hour incubation
time of
whole blood with monoclonal antibody to the homologous region of the a-chain
of the
DR antigen, decreased with time and these events were accompanied by increase
in
the percentages of single positive CD4+ CD3'' and CD8* CD3 cells
simultaneously
and at later times too. TCR a and Q chains were also expressed on these types
of
cells.
B-lymphocytes were constantly observed to lose markers such as CD19, CD31,
CD23, IaM and DR and this coincided with the appearance of CD34* and CD34T
CD2' cells, increases in CD7' cells, increases in CD8* CD28' and CD28' cells.
increases in CD25* cells, the appearance of CD10' and CD34'' cells and CD34-
and
' CD19* cells increases in CDSt cells, and cells expressing low levels of CD45
antigen. These changes were due to treatment of blood with monoclonal antibody
to
the homologous region of the ~B-chain of HLA-DR antigen.
CA 02395452 2002-08-30
' WO 96/23870 PCT/GB96100208
41
The immunophenotypic changes associated with such treatment is consistent with
retrodifferentiation and subsequent commitment (i.e. recommitment) of B
lymphocytes, because the majority of white blood cells in blood of patients
with B-
CLL before treatment were B lymphocytes. Furthermore, B-lymphocytes of
patients
with B-CLL which were induced to become T-lymphocytes following treatment with
cyclophosphamide and monoclonal antibody to the p-chain of HLA-DR antigen,
were
able to revert back to B lymphocytes following prolonged incubation with this
treatment.
On analysis of treated samples with monoclonal antibody to the ,Q-chain of HLA-
DR
antigen, with CD16&~6 and CD3 and CD8 and CD3 panels, the relative number of
cells expressing these markers steadily increases in increments consistent
with those
determined with panels such as CD19 and CD3 and DR and CD3. Investigation of
the supernatant of treated and untreated samples of patients with HIV
infection using
1~ nephlometry and immunoelectrophoresis reveals increased levels of IgG
indicating
that the B-cells must have passed through the plasma cell stage. The increase
in the
relative number of all above-mentioned cells was also accompanied by the
appearance
of medium size heavily granulated cells expressing the CD56&16 antigens in
extremely high amounts. Other cells which were extremely large and heavily
granulated were observed transiently and these were positive for CD34 and
double
positive for CD4 CDS markers. Other transient cells were also observed and
these
were large and granular and positive for the CD3 and CDl9 receptors. CD?~
which
was present on the majority of B-lymphocytes was lost and became expressed by
newly formed T-lymphocytes which were always observed to increase in number.
CD28''CD8' and CD28' cells appeared after treatment of whole blood of patients
with B-CLL with monoclonal antibody to the homologous region of the B chain of
the DR antigen. These findings were due to treatment of blood with monoclonal
antibody to the homologous region of the ~i-chain of HLA-DR antigen.
T-lymphopoiesis generated in this manner was also observed in peripheral blood
of
healthy blood donors, cord blood, bone marrow, patients with various
infections
WO 96/23870 ~ 02395452 2002-08-30 PGT/GB96/00208
4~
including HIV' individuals and AIDS patients, enriched fractions for B
lymphocytes
obtained from blood samples of healthy blood donors, IgA deficient patients
and other
patients with various other conditions. Furthermore, analysis of myeloid
markers in
treated samples of two patients with B-CLL with monoclonal antibody to the
homologous region of the /3-chain of the HLA-DR antigen showed a signif cant
increase in the relative number of cells expressing the myeloid markers such
as CD13
and CD33. These markers were coexpressed with the CD56 & 16 or the CD7
antigens. However, the relative number of CD7i cells with T-lymphocyte markers
and without myeloid antigens was observed on a separate population of cells.
These
particular observations were not seen in untreated samples or in samples
treated with
monoclonal antibodies to class I antigens or the homologous region of the a-
chain of
HLA-DR antigen (see Charts 2 & 3). These final results suggest that B-
lymphocytes
once triggered via the /3-chain of the HLA-DR antigen are not only able to
regress
into T-lymphocyte progenitor cells but are also capable of existing into the
myeloid
and erythroid lineages.
It should be noted that the stem cells that are produced by the method of the
present
invention may be stem cells of any tissue and are not necessarily limited to
lymphohaematopoietic progenitor cells.
Other modif cations of the present invention will be apparent to those skilled
in the
an.
CA 02395452 2002-08-30
' WO 96123870 PGTIGB96~0208
43
TABLE 1
CLINICAL DIAGNOSIS OF PATIENTS AND EXPERIMENTAL CONDITIONS OF BLOOD SAMPLES
INCLUDING COULTER COUNTS (WBC) FOLLOWING AND PRIOR TREATMENT OF BLOOD
SPECIMENS
WITH VARIOUS MONOCLONAL ANTIBODIES AND OTHER AGENTS
PATIENT DIAGNOSIS EXPT WBC/L XLYMPH #LYMPH/L AGENT
ID COND X10-9 lOX-9 MLImL
B A B A 8 A
1 B-CLL 12HR 100 ND 86.1 86.1 ND ANTI-B
ND
AT 50
22C
2 B-CLL 2HR 39.1 74.4 29.9 ANTI-B
9.6 63.3
AT 6.1 50
22C
2HR 39.1 74.4 ANTI-B
AT 37.7 75.1
22C Z9.9 28.3PE
50
3 B-CLL 6HR 39.5 71.9 28.3 ANTI-d
9.3 67.2
AT 6.2 50
22C
6 HR 39.5 71.972_5 ANTI-B
37.7
AT 28.3 27.4PE
22C
50
4 B-CLL 24HR 73 60.5 28.4 ANTI-B
AT 39 9.3 6.2 50
22C
24HR 73 70.4 ANTI-g
AT 39 36.2 28.4 25.5PE
22C
50
~. ~ g-CLL 2HR
AT ANTI-B
22C
50
ANTI-A
50
ANTI-T
50
ANTI-B
TOXIC
AGENT
25+25
WO 96/23870 CA 02395452 2002-08-30 PCT/GB96/00208
44
PATIENT DIAGNOSIS EXPT WBC/L XLYMPH #L.YMPH/L AGENT
ID C0N0 X10-9 B A lOX~9 ML/ml
'
B A B A
6 B-CLL 24HR ANTI-8
AT 50
22C
7 B-Cl.L 24HR 170 128 95.4 16.9 11.6 ANTI-8
91.1
AT 10
22C
I78 94.2 I6.8 ANTI-I
10
130 90.4 11.9 aNTi-8
& TOx
IC
AGENT
10+20
8 8-CLL 24HR 16 7 81.9 14 3.0 ANTI-8
51.2
AT 20
22C
9 B-CLL 12HR +++ 89.587 85.i +-~ 76.2 ANTI-8
AT 30
22C
85.4 ANTI-I
+++ +++ 30
89.4 ANTI-4
30
+++ g4.g +++ A N T
I -
I+II+4
95.4 10+10+10
B=CLL 2HR 19.3 86 ND 16.7 NO ANTI-B
ND
AT 30
22C
ANTI
-I
3Q
92 OUT 2HR 5.4 NO 74.5 ND ANTI-8
ND
PATIENT AT 20
22C
87 OUT 2HR 4.8 ND 59.3 ND ANTI-B
ND
PATIENT AT 20
22C
91 OUT 2HR 4.2 NO 54.0 NO ANTI-B
ND
PATIENT AT 20
22C
21 OUT 2HR 3.9 NO 47.4 ND ANTI-B
N0
PATIENT AT 20
22C
CA 02395452 2002-08-30
WO 96!23870 PCTIGB96/00208
PATIENT DIAGNQSISEXPT WBC/L XLYMPH ~(-YMPH/LAGENT
ID COND X10-9 B A !0X-9 MLlml
B A B A
34 OUT 2HR 7.2 20.0 ND ND ANTI-8
ND
PATIENT AT 22C 20
3E CMV 4HR I3.4 7.3 ND ND ANTI-B
ND
INFANT AT 22C 20
93 HIV+ 4HR 5.6 43.4 ND ND ANTI-B
ND
INFANT AT 22C 20
BB/ST 40% BLAST2HR 20.2 NO 12.2 NG ANTI-8
AT
IN BL000 22C 60.5 50
ND
6 DA'(S 24HR ANTI-A
OLD
AT 22C 50
ANTI-AB
25*25
HIV25 AILS 2HR 7.5 34.8 ND 2.6 ND ANTI-B
NO
AT 22C 50
ANTI-A
SO
ANTI
-A.B
25r25
43/BD B CELL 4HR ANTI-B
DEFICIENTAT 22C 20
. ANTI-I
ANTI-4
20
OB!BD B CELL 4HR ANTI-B
DEFICIENTAT 22C 20
ANTI-i
20
ANTI.a
2C
WO 96123870 CA 02395452 2002-08-30 PC"TIGB96100208
46
PATIENT DIAGNOSIS EXPT WBC/L XLYMPH #LYMPH/L AGENT
ID COND X7.0-9 B A 10X-9 ML/mL
B A B A
HIV+ AIDS 6HR
AT ANTI-B
22C
20
ANTI-I
IgA-O IgA 6HR ANTI-B
DEFICIENT AT 20
22C
ANTI-I
20
EXPT COND : EXPERIMENTAL CONDITIONS
B : BEFORE
A : AFTER
ANTI-B : monoclonal antibody to the homologous region of the p-chain of HLA-DR
anigen
ANTI-A : monoclonal antibody to the homologous regiow of the cx-chain of HLA-
OR antigen
ANTI-I : monoclonal antibody to the homologous region of Class i antigens
ANTI-AB : both ANTI-8 and ANTI-A added togather
ANTI-4 : monoclonal antibody to the CD4 antigen
ANTI-I+II+4 : ANTI-I and ANTI-B and ANTI-4 added togather
Cytoxic agent : Cyclophophamide .
MLlml : micro litre per ml
L : litre
CA 02395452 2002-08-30
WO 96123870 PCTIGB96/00208
47
TABLE 2
IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONS
BEFORE AND AFTER
TREATMENT OF BLOOD SAMPLES WITH MON~LONAL ANTIBODY
TO THE HOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DR
WITH CD19 AND CD3 MONOCLONAL ANTIBODIES.
PATIENT X CD19+ X CD3+ XCD19+CD3+ X X
C 0019+HGCD
0 3~ FC+
3
-
CD19-
B A B A B A B A B A
1 88 40 5 19 1 2 6 26 0 12
2 73 15 10 33 2 7 15 41 0 5
3 73 11 11 33 2 2 14 52 0 2
4 71 13 11 37 2 2 16 47 0 2
85 40 5 16 1 1 6 26 3 18
6 85 43 5 18 1 1 6 27 3 10
7 90 72 2 4 0 2 7 8 0 14
8 62 25 7 13 0 1 29 55 2 6
9 90 85 2 3 0 0 2 I 1 4
78 50 7 ' 14 0 0 14 26.0 ~8
92 12 ~ 10 38 49 0 1 49 40 0 0
91 7 3 35 29 0 1 59 67 0 0
87 5 3 32 38 1 I 63 58 0 0
21 1 1 27 29 1 0 71 70 0 0
34 1 1 I3 13 0 2 86 84 0 0
39 10 6 23 25 0 0 67 69 0 0
93 6 3 26 27 1 1 68 70 0 0
BB/ST 1 1 12 13 0 0 87 86 0 0
HIV25 7 2 26 27 0 0 68 67 0 0
43/BD 0 0 40 42 0 1 58 54 0 0
04/BD 0 0 49 41 0 3 43 41 0 0
HIV+ 1 1 10 14 0 0 89 87 0 0
IgA/D 10 1 21 25 2 3 67 71 0 0
8: before treatment . ~: after treatment
WO 96/23870 CA 02395452 2002-08-30 PC'T/GB96I00208
48
T-ABIE 3
IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL
AND OTHER CONDITIONS BEFORE AND AFTER TREATMENT OF BLOOD SAMPLES
WITH MONOCLONAL ANTIBODY TO THE B CHAIN OF THE HOMOLOGOUS REGION
OF THE HLA-DR WITH MONOCLONAL ANTIBODIES TO CD4 AND CD8.
PATIENT XCDB+ XCD4+ XC04+CD8+XCD4-C08-C04+LOW
8 A B A B A 8 A B A
1 I 2.8 16 2.9 11.4 0 3.2 93.1 67.50 0
2 6.2 13.2 9.1 24.3 0 9.4 78.7 46 5.8
6.3
3 7.2 13.1 7.4 23.9 0 8.2 78.8 48.16.3
6.6
4 1C.1 24.27.6 24.9 0.3 2.8 77.5 42 4.0
5
2.9 16.2 1.8 7.6 0 2 95 62.3 0 0
6 ND 12 NC 8.i ND 1.7 ND 75.7 NO 0
7 1.y 2.6 1.9 2.8 0 0 95.8 94.30 0
8 3.2 7 3.9 6.9 0.1 2 87.3 79.84.3
6
9 2.8 2.9 3 3 0 0 94 94.1 0 0
5.7 9.4 ~4.7 9.I 0.6 0.8 88:7 79.20 p
92 2i 19 21.6 21 0.8 1.9 50.5 52.55.3
4.8
91 i5.4 18.113.6 17.96.2 2.6 57 57.3 7.3
3.5
87 16.8 21.813.4 20.42.9 2.6 59.5 48.97 5.6
2I 15 24.1 9.1 15.2 1 2.6 69.6 53.23.7
4.2
34 9 n Il.g 5.7 4.9 2 3.3 67.6 65.314.4
14.5
39 1?.i 12.G13.I 1n.60.4 I.3 62.3 66.711.9
4.3
93 18.9 20.39.7 10.3 1.8 1.4 65.5 65.93.4
1.8
BB/ST 6.3 l3 5.7 7.3 2.2 1.1 34.7 70.350.3
7.6
HIJ25 24.1 24.9T0.8 1.1~1.3 5 70.2 69.32.9
3.8
CA 02395452 2002-08-30
WO 96IZ3870 PCT/GB96I00208
49
TABLE 4
IMMUNOPHENOTYPING OF PATIENTS WITH B-~ D OTHER CONDITIONS BEFORE AND AFTER
TREATMENT OF SAMPLES WITH MONOCLONAL ANTIBODY TO THE B CHAIN OF THE HLA-DR
WITH
MONOCLONAL ANTIBODIES TO CD3 AND DR
PATIENT DR+ CD+ CD+DR+ DR-C03- DR+HC03-
B A B A B A B A B A
-
1 8? 45.5 3.5 20.8 2.5 4.2 6.9 21.6 0 7.6
2 76.2 19.4 9.6 29.2 3.9 8.7 10.3 36.8 0 5.5
3 77.7 18.3 8.4 29.4 4.I 8.8 9.6 38.1 0 4.7
76.8 19.2 7.6 29.5 6.2 10.5 9.i 37.2 0 3.3
ND 47.1 ND I1.5 ND 9.9 ND 22.4 ND ,'.3
6 ND
7 91.4 85.8 2.4 2.5 0.7 0.7 5.1 4.2 0 6
.
3
3 61.8 28.9 6.5 11.2 2 3.3 28.6 54.6 0 1
~ 5
I
9 ND
i0 82.6 44.7 4.3 9.8 3.3 5 9.8 22.2 0 17.9
92 23.8 14.1 39.3 41.9 4.5 3.5 32.4 40.5 0 0
91 13.3 7.9 29.6 32.5 3.4 2.9 53.4 56.5 0 0
8% 14.8 12.2 28.4 34.1 5.5 6.6 5i.1 45.5 0 0
ND
3~ 11.9 12.9 1D.4 13.7 C.8 0.6' 76.7 72.8 G 0
3 25.6 13.7 24.6 25.2 3 2.8 46.5 25.2 C 0
?3 13.3 8.9 18.4 18.9 9.9 10.1 58.2 61.7 0 0
3BIST 44.2 32.5 11.7 12.2 0.8 0.8 43 49.4 0 ~ 4.6
~ ~ ~ ~ ~ J ~ ~ ~
WO 96/23870 CA 02395452 2002-08-30 PCTIGB96100208
TABLE 5
IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONS BEFORE AND AFTER
TREATMENT OF BL000 SAMPLES WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION
OF THE
B CHAIN OF THE HLA-DR WITH MONOCLONAL ANTIBODIES TO CD16+56 AND CD3.
PATIENTS CD56+&16 CD3+ CD56+&16+CD3+ CD56+&16-CD3-
B A B A B A B A
I 2 4.3 5.7 19.7 0.7 1.7 91.3 73
2 11.5 38.9 12.:132.6 1 6,6 74.5 21
3 12 36.2 12.134.5 0.7 6 75.5 23
4 12.2 32.6 1Z.439.6 0.5 5 74.7 22.2
5 ND 13.I ND 9.4 ND 2.6 ND 73,5
6 ND
'7 . 0.8 0.8 2.8 2.4 0.3 0.2 96.2 96.4
. I
8 24.8 52 5.4 I2.4 0.9 4.1 68.3 31.1
I
9 NO
I
10 1 1.3 6.1 13.7 2.1 2.5 90.5 8 2
.1 .
4
92 23.8 34.5 44.344:8 2 1.5 29.2 18.6
91 a.6 3.9 28.829.4 3 3.2 63.3 63.3
87 47.9 46.4 28.836.5 5.8 3.7 16.9 13
21 9.4 9.a 19.723.6 4.2 6.7 66 59.5
CA 02395452 2002-08-30
WO 96!23870 PCTIGB96100208
51
PATIENTS CD56+&16 C03+ C056+&16+C03+ C056+&16-CD3-
34 21.5 IZ,B, 1.8 0,6 64,6 72.8
11.4 I3.7
39 7 2.7 23.4 26.1 1.i 0.1 68.2 71
93 55.8 54.926.2 26.3 1.7 2 16.i 16.8
BB/ST 28 . 8 ~ 12 -14 I - 1 ~9. 53
29 . , 3 4 . 4 .
9 . 8 6
8
WO 96/23870 CA 02395452 2002-08-30 PCTIGB96I00208
52
TABLE 6
IMMUNOPHENTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONS BEFORE AND AFTER
OF
TREATMENT OF BLOOD WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B
CHAIN
OF THE HLA-DR WITH MONOCLONAL ANTIBODIES TD CD45 AND CD14.
PATIENTS CD45+H CD45+L CD45+CD14+
~ ~
3 A B a B a
1 90.570.1 7.5 21.9 0.8 3.3
2 25.852.2 8.8 38.3 5.3 9.5
3 91.352.2 9.9 33.8 5.1 13.2
4 i.5 79.2 2.1 7 5.7 10.8
63.184.6 34.99.4 0.5 3.6
6 ~D
,' ~?.885.2 45.613.9 C.5 0.6
8 ''1.155 71..34.5 5.3 8.7
9 ~cE
79.747.3 16.348 2.1 1.9
92 ~I.754.7 27.426.0 5.9 3.5
~
91 19.449,2 40,444.3 6.5 3.2
87 ~ 52.461.5 36.128.7 7 6.5
21 15.843.3 44.347.6 6.2 3.3
34 2~..424.6 54.859.6 13.39.7
39 ~3.746.3 30.542.1 14.58.8
93 SEE
HIU+ 22.626.9 66.863.5 5.8 6 7
I4A/D =?.459.8 41.933.3 5.9 4.i
CA 02395452 2002-08-30
' WO 96/23870 PCTIGB96I00208
53
TABLE 7
IMMUNOPHENOTYPING OF PATIENT WITH B-CLL AND OTHER CONDITIONS BEFORE AND AFtER
TREATMENT OF BLOOD WITH MONOCLONAL ANTIBODIES TO THE HOMOLOGOUS REGION OF THE
B
CHAIN OF THE HLA-OR WITH MONOCLONAL ANTIBODIES TO CD8 AND C03.
PATIENTS C08+ CD3+ CD8+C03+ CD8-C03-
B A B A 8 A. 8 A
2 0.61.3 7.5 19.3 4.2 19.3 87.7 63.8
3 1.11.4 8.3 20.3 5.6 18.4 84.8 59.8
4 3.52.9 8.3 27 3.9 16.6 84.2 53.1
92 3.51.9 27.6 25.2 I8.4 19 50.3 52.8
91 4 3.I 18.2 19 14.1 I2.6 63.0 65.3
87 S.73.9 ~ 19.9 23.6 15.4 i7.4 58.8 55
I
21 4.87.4 16.3 17.3 13.7 13 65.2 62
~, 34 ~ 3 3. 6 5.2 6. 7 7 7 .5 _ -82.3-
~ . ~ 84.1
6
TABLE 8
IMtIUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTER TREATMENT OF BLOOD
WITH
PE CONJUGATED MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B-CHAIN OF
THE
HLA-OR MEASURE WITH MONOCLONAL ANTIBODIES TO CD45 AND C014.
TIME OR+C045+CDI4+r f CD45+L CD45+H
2HR 81.7 8.2 8.2
6HR 90.7 8.1 10.6
24HR 79 1.1 18.4
TABLE 9
IMMUNOPHENOTYPING OF A PATINENT WITH B-CLL WITH TIME AFTER TREATMENT OF BLOOD
WITH
PE CONJUGATED MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE 8-CHAIN OF
THE
HLA-OR MEASURED WITH MONOCLONAL ANTIBODIES TO CD19 AND CD3.
TIME CD19+DR+r C03+ CD3+DR+ CDI9-C03-DR-
2HR 87.4 10.. 1.8 10.7
6HR 75.5 10.a 3.1 10.7
24HR 74 11.? 2.9 11
V1~0 96/23870 CA 02395452 2002-08-30 PCTIGB96100208 '
54
TABLE 10
IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTER TREATMENT OF BLOOD
WITH
PE CONJUGATED MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B-CHAIN OF
THE
HLA-DR MEASURED WITH MONOCLONAL ANTIBODIES TO CD4 AND.CDB.
TIME C08+& OR+r C04+ C04+&C08+bOR+rC04+DR+ CD4-C08-OR-
2HR 77.6 6.8 5.4 1.3 8.8
6HR 75.8 6.7 6.4 1.8 9.3
24HR 77 6.4 4.8 1,9 11
TABLE 11
IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTER TREATMENT OF BLOOD
WITH
PE CONJUGATED MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B-CHAIN OF
THE
HLA-DR MEASURED WITH MONOCLONAL ANTIBODIES TO C03 AND DR.
TIME OR+ CD3+ CD3+DR+ CD3+DR-
2HR 75 9.5 4.2 10.9
6HR 74.8 8.8 4.8 10.9
24HR ND NO NO NO
TABLE 12
IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTER TREATMENT OF BLO00
WITH
PE CONJUGATED MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B-CHAIN OF
THE
HtA-OR MEASUR~0 WITH MONOCLONAL ANTI80DIES TO C016&56 AND CD3.
TIME CD56+&16+DR+r CD3+ CD56+C016+&CD3+DR+CD56-CD16-&CD16-
r DR-
2HR 82.5 9.5 4.1 3.5
6HR 84,3 7.5 4.1 3.3
24HR ND ND ND ND
TABLE 13
IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTER TREATMENT OF BL000
WITH
PE CONJUGATED MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B-CHAIN OF
THE
HLA-DR MEASURED WITH MONOCLONAL ANTIBODIES TO C08 AND CD3.
TIME CD8+OR+ CD3+ CD8+CD+3&DR+r CD8-CD3-DR-
2HR 76.2 6.6 6.7 I0.6 '
6HR 76.5 6.2 6.2 10.3 .
CA 02395452 2002-08-30
WO 96/23870 PCT/GB96I00208
SS
TABLE 14
IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL BEFORE AND AFTER TREATMENT OF 8L000
WITH
MONOCLONAL ANTIBODIES TO THE HOMOLOGOUS REGION OF THE A-CHAIN OF THE HLA-DR,
THE
HOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR , THE TWO MONOCLONAL TOGETHER.
MONOCLONAL TO THE HOMOLOGOUS REGION OF THE B-CHAIN PLUS CYCLOPHOSPHOAMIDE AND
THE
HOMOLOGOUS REGION OF CLASS I ANTIGENS MEASURED WITH TIME.
C019+ CD3+ CD19+CD3+ CD19-CD3-
ID B A A8 A AI B A AB A AI B A AB A AI B A AB A Ai
A BC A BC A BC A BC
5I
6
2H 8691 54 4089 5 4 15 235 1 1 3 2 1 6 4 27 33 5
24 N 88 51 5086 N 4 18 104 N 2 1 2 3 N 4 29 28 7
2H ?7Dt 59 6180 7 N 13 N 7 1 N 1 N 0 14 N 26 N I2
09
24 8 N N N 6 32N N N 38 1 N N N 1 59 N N N 56
43
/B
6H. 0 N 0 0 0 40N 42 4349 0 N 1 0 1 58 N 54 54 47
04
/B
D
6H 0 N 0 0 0 49N 41 4546 0 N 3 1 3 43 N 42 44 41
HI
v+
6H 1 N 0 N ~ 10N 14 N 12 0 N 0 N 0 89 N 86 N 87
~
I9
AI
0
6H 10N 1 N 12 21N 25 N 20 2 N 1 N 3 67 N 71 N 68
B = Before: A = After: AB = after addition to antibody to beta chain: AA =
after
addition of antibody to alpha chain: ABC = after addition of antibody to
either
alpha or beta chain and cycloposphoamide: A1 = after addition of antibody to
Class
I.
WO 96123870 ~ 02395452 2002-08-30 pCT/GB96/00208
56
TABL 15
C08 AND C04
CD8+ CDa+ CD4+CD8+ CD4-CD8-
ID B A AB A AI B A AB A AI B A AB A AI B A AB AI
A
A BC A BC A BC A BC
5/
6
2H 3 2 14 104 2 2 8 8 3 0 0 3 2 1 95 94 74 93
79
24 N 3 9 4 4 N 3 8 4 3 N 0 2 2 0 N 94 81 93
90
~
2H 3 N ~ N 4 4 N . N 3 1 N 2 N 1 91 N 93 92
7 , N
09
2+ 10.N N N 15 21 N N 'J38 2 N NN 2 61 NNN 53
TABLE 16
CD3 AND DR
OR+ CD3+ C03+DR+ CD3-DR-
ID B A AB A AI B A AB A AI B A AB A AI B AAB A AI
A BC A BC A BC ABC
5/
6
2H N 9054 N 87 N a 12 N 4 N 2 10 N 3 N 522 N 5
i0
2H 83 y 63 N 81 4 N 8 N 4 4 N ? N 4 9 N23 N 12
09
24 14 N N N 13 30 N N N 36 3 N N N 3 51 NN N 47
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TABLE 17
CD16& 6 AND CD3
C056+&16+ CD3+ C056+&16+CD3+ CD56-&16-CC3-
ID B A AB AI B A AB A AI B A AB A AI B A AB A AI
A
A BC A BC A BC A BC
5I
6
2u N 0 13 4 N 5 9 N 5 N 1 3 N 1 N 94 74 N 90
N
2H p N 1 N 1 6 N 14 N 6 1 N 2 N 1 92N 65 N 92
09
24 42 N N N 41 36 N "J N 38 2 N N N 2 20N N N. 19
TABLE 18
CD45 AND C014
CD45+L CD45+M CD45+H CD45+C014+
ID B A AB A ATB A AB A AI B A AB A AI B A AB A AI
_. A BC A BC A BC A BC
5/
6
2N 0 0 5 ZO 0 44 4350 50 32 55 4350 3167 1 1 1 2 0
10
ZH 0 N 4 N 0 43 N 54 N 35 54 N 42 N 62 1 N 1 N 0
G9
2s 2 N N N 1 I8 N N N 16 7I N N N 76 7 N N N 5
H:
:l.
6H 4 N 3 N 6 63 N 61 N 41 23 N 27 N 40 7 N 7 N 7
Ig
A;
v
6H 2 N 2 N 4 40 N 31 N 44 47 N 60 N 44 6 N 4 N 6
WO 96123870 ~ 02395452 2002-08-30 pCT/GB96/00208
58
TABLE 19
C08 ANO C028
C08* CD28+ C08+CD28+ CD8-CD28-
ID 8 A AS A AI B A AB A AI B A AB A AI B A AB A AI
A BC A BC A BC A BC
5/
6
2H N 3 6 N 3 N 1 4 N 2 N 1 4 N 1 N 95 86 N 94
8
2H 4 N 6 N N 3 N 5 N N 1 N 3 N N 9 N 86 N N
2
TABLE 20
CD34 AND CD2
CD34+ CD2+ CD34+CD2+ C034-C02-
IO B A AB A A B A AB A AI B A AB A AI B A AB A AI
A BC I A BC A BC A 8C
5/
6
2H N 1 34 N N N 6 I3 N N N 3 30 N N N 90 21 N N
2~ N 1 6 9 N N 7 23 4 N N 3 33 43 N N 87 34 34 N
HI
W+
2H 2 1 I2 13 N 2021ZI I2N 4 5 9 14 N 7 73 64 60 N
3
BB
/S.
T
2H 26 2333 14 N 151415 15N 3 3023 36 N 2 32 28 35 N
1 7
24 N 1129 11 N N 1312 9 N N 279 18 N N 48 49 61 N
CA 02395452 2002-08-30
WO 96123870 PCTIGB96100208
59
CHART 1
IMMUNOPHENOTYPIC CHANGES OF UNTREATED (2.
AND TREATED 3.&
BLOOD SAMPLE
OF PATIENT
4? WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUSREGION S-CHAIN
OF THE OF HLA-DR
ANTIGEN MEASURED WITH TIME.
WITHOUT WITH FL1 FL2 TIME
NOTHING001 WITH002 CD45 CD14 2HR
N0001 WE002 CD45 CD14 6HR
001001 002002 CD45 CDI4 24HR
NOTHING003 WITH004 CD3 CD19 2HR
N0003 WE004 CD3 C019 6HR
001003 002004 CD3 CD19 24HR
NOTHING004 WITH005 CD4 C08 2HR
N0oo4 wEOOS co4 coa 6HR
001004 002005 CD4 C08 24HR
NOTHING005 WITH006 C03 OR 2HR
N0005 WE006 C03 DR 6HR
OOI005 002006 CD3 DR 24HR
NOTHING006 WITH007 CD3 CD56&16 2HR
N0006 WE007 CD3 CD56&16 6HR
001006 002007 CD3 CD56&16 24HR
N003 W004 C03 CD8 2HR
N0007 WE008 C03 CD8 6HR
001007 002008 CD3 CD8 24HR
WO 96123870 ~ 02395452 2002-08-30 pCTIGB96100208
CHART 1A
IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOOD SAMPLE OF PATIENT (2.
3. 4)
WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE ,e-CHAIN OF HLA-DR
ANTIGEN
CONJUGATED TO PE MEASURED WITH TIME.
ID FL1 FL2 TIME
WL003 CD45 CD14 2HR
WEL003 CD45 CD14 6HR
003003 CD45 C014 24HR
WL005 CD3 CDI9 2HR
WEL005 C03 CD19 6HR
003035 CD3 COI9 24HR
WL006 CD4 C08 2HR
WEL006 CD4 C08 6HR
OG3006 CD4 CD8 24HR
WL007 CC3 DR 2HR
W~L 007 C03 DR 6HR
WL008 CD3 CD65&16 2HR
WEL 008 CD3 C056~16 6HR
WL005 CD3 C08 2HR
WEL009 C03 CD8 6HR
CA 02395452 2002-08-30
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CHART 2
IMMUNOPHENOTYPIC CHANGES OF UNTREATED PATIENT (1)
AND TREATED BLOOD OF WITH
MONOCLONAL ANTIBODY TO THE HOMOLOGOUS OF HLA-DR
REGION OF THE ~-CHAIN ANTIGEN.
THIS
ANTIBODY AND CYCLOPHOSPHAMIDE. MONOCLONAL ANTIBODY OMOLOGOUS OF THE
TO THE H REGION
a-CHAIN OF HLA-DR ANTIGEN AND MONOCLONAL ANTIBODY MOLOGOUS
TO THE HO REGION OF
CLASS
I ANTIGEN MEASURED WITH TIME.
WITH WITHOUT FL1 FL2 TIME
' NA001 CD45 CDI4 2HR
A2B001: AB CD45 C014 2HR
A2A : AA CD45 CD14 2HR
DNAAOO1:ABC C045 C014 2HR
A1001: AI C045 C014 2HR
NC001 CD3 C019 2HR
CZBOO1:AB C03 C019 2HR
C2AGO1:AA CD3 C019 2HR
DNACOOI:ABC CD3 CD19 2HR
C1001: AI CD3 CD19 2HR
A124HOO1:AI CD3 CD19 24HR
A2B24H001: AB CD3 C019 24HR
A2A24HOO1:AA CD3 CD19 24HR
A2BX24HOO1:AB CD3 CD19 24HR
C
N0001 C04 CD8 2HR
D2B001: A8 C04 C08 2HR
D2A001: AA CD4 CD8 2HR
ONADOO1:ABC CD4 CD8 2HR
D1001: AI CD4 CD8 2HR
D124HOO1:AI C04 CD8 24HR
028X24H001:A8 CD4 C08 24HR
C
D2B001: AB CD4 CD8 24HR
D2A001: AA CD4 CD8 24HR
E1001: AI CD3 OR 2HR
E28001: AB CD3 DR 2HR
E2A001: AA CD3 DR 2HR
F1001: AI C03 C056&16 2HR
F2B001: AB CD3 C056&16 2HR
F2A001: AA CD3 CD56&16 2HR
61001: AI CD28 CD8 2HR
G2A001: AA CD28 CD8 2HR
628001: AB CD28 CD8 2HR
H1001: AI CD7 CD33&13 2HR
WO 96/23870 ~ 02395452 2002-08-30pCTIGB96100208
62
H2A001: AA CD7 CD33&13 2HR
WITH WITHOUT FL1 FL2 TIME
H2B001: AB CD7 C033&13 2HR
I2A001: AA CD21 CD5 2HR
I2BOO1:AB C021 CD5 2HR
J2A001: AA C034 CD2 2HR
J2B001: AB CD34 CD2 2HR
B2A24HOO1:AA CD34 C02 24HR
B2B24HOO1:AB CD34 CD2 24HR
B2BX24H001: CD34 C02 24HR
ABC
K2B001: AB CD10 CD25 2HR
K2A001: AA CD10 C025 2HR
CA 02395452 2002-08-30
R'O 96123870 PCTlGB96100208
63
CHART 3
IMMUNOPHENOTYPIC CHANGES OF UNTREATED PATIENT (8)
ANO TREATED WITH
BLOOD OF
MONOCLONAL ANTIBODY TO THE HOMOLOGOUS OF THE OF HLA-DR ANTIGEN.
REGION Q-CHAIN
~NITH WITHOUT FLI FL2 TIME
AN001 CD45 CD14 2HR
A2001 CDa5 CDia 2HR
CN001 CD3 C019 2HR
~200i CD3 C019 2HR
DN001 CD4 CD8 2HR
~~200i CD4 CD8 2HR
EN001 CD3 DR 2HR
.2001 C03 OR 2HR
FN001 CD3 CD56&~6 2HR
200i C03 CG56&i6 ZHR
GN001 CD28 CD8 2HR
u2001 C028 CD8 2HR
HN001 C07 CD5 2HR
H2001 ' C07 CD5 2HR
IN001 C013 CD20 2HR
I2001 CD13 CD20 2HR
JN001 CD45RA C025 2NR
.2001 CD45RA CD25 2HR
KN001 CD57 C023 2HR
t?001 CD57 CD23 2HR
WO 96/Z3870 ~ 02395452 2002-08-30 pCTIGB96100208
64
CHART 4
IIMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOOD SAMPLE OF PATIENT
(10)
WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE ~-CHAIN OF HLA-DR
ANTIGEN
AND MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF CLASS I ANTIGENS.
WITH WITHOUT FL1 FL2 TIME
CLL0001 C045 C014 2HR
CLL1001 C045 CD14 2HR
CLL2001 CD45 CD14 2HR
CLL0003 C03 C019 2HR
CD3 CDI9 2HR
CLL1003 C03 CD19 2HR
CLL2003 C03 CD19 2HR.
CLL0004 C04 C08 2HR
CLL1004 CD4 CD8 ZHR
CLL2004 CD4 C08 2HR
CLL005 CD3 OR 2HR
CLL1005 CD3 DR 2HR
CLL2005 CD3 DR 2HR
CLL0006 CD3 C056&16 2HR
CLL1006 C03 CD56&16 2HR
CLL2006 C03 CD56&16 2HR
