IL133155A - Method for producing a medicament comprising retrodifferentiated cells - Google Patents

Abstract

A method for producing a medicament which method comprises contacting a cell with an agent that causes the cell to retrodifferentiate into a stem cell and admixing the stem cell with a suitable diluent, carrier or excipient, wherein the retrodifferentiated stem cell is capable of being recommitted into a more differentiated cell. 3674 כ" ז בשבט התשס" ד - February 19, 2004

Description

τιηίΛ ο^ταιη Ο'ΝΙΊ π¾ΌΠ nanri τ\ητϋ τνο·>ν A Method for Producing a Medicament Comprising Retrodifferentiated Ce TriStem Trading (Cyprus), Ltd.

C: 3623 1 A METHOD OF PRODUCING A MEDICAMENT FROM A RETRODIFFERENTIATED CELL NOTICE UNDER REGISTRAR'S CTRCULAR NO. 23(P) OF 5 APRIL 1992 Inasmuch as the invention is defined in the appended claims, it will be apparent that the portions of the present specification which fall outside the scope of the claims do not relate directly to the invention. This Notice is not meant to disclaim any legitimate rights to which the Patentee is legally entitled, especially any rights in accordance with Section 49 of the Israel Patents Law.

SUMMARY OF THE INVENTION The present invention relates to a method of preparing an undifferentiated cell from a more committed cell. More specifically, the present invention provides for a method for producing a medicament comprising a stem cell that has been retrodifferentiated from a more committed cell, and wherein said stem cell is in turn capable of being recommitted into a more differentiated cell.

FURTHER RELATED ASPECTS OF THE INVENTION 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. , la 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 more specialised cells, such as the formation of T cells or B cells. Therefore, as the cells become more committed, they 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 types of specialised cells. A typical example of an undifferentiated cell is a siem cell.

In contrast, differentiated cells are incapable of multilineage differentiation. A typical example of a differentiated cell is a T cell.

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, neutrophils, megakaryocytes, monocytes, erythrocyteSi 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, erythrocytes, granulocytes, mast cells, NKs, 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 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 Π antigens. If these antigens are associated with those cells then they are called Class I+ and/or Class Π+ cells.

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 antigen(s) or on the basis of a particular combination of associated antigens.

Examples of these marker antigens include the antigens CD34, CD19 and CDS. 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", CD19- and CDS" 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÷, CD2V, 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 CD13+ 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 retrodifferentiation 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: "retrodifferentiation 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 retrodifferentiation lose morphological and functional complexity by 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 to 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 parenchymal cells for in vitro cultures. According to Uriel: "Contrary to the results with fetal or neonatal hepatocytes, with hepatocytes 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 phenotype - of many -tumour show-analogous-changes of reversion toward immaturity ... during the preneoplastic phase of liver carcinogenesis, cel also retrodifferentiate. " More recent findings on retrodifferentiation include the work of Minoru Fukunda (Cancer Research [1981] vol 41, pp 4621^628). Fukunda induced specific changes in the cell surface glycoprotein profile of K562 human leukaemic cells by use of the tumour-promoting phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TP A). According to Fukunda TPA appeared to induce the K562 human leukaemic cells into a retrodifferentiated stage.

Also, Hass et al (Cell Growth & Differentiation [1991] vol 2, pp 541-548) reported that long term culture of T A-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 K \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 2810-2811] reported on the retrodifferentiation of the human colonic cancerous subclone HT29-18.

Even more recendy, Kobayashi et al (Leukaemia Research [1994] vol 18, no. 12, pp 929-933) have reported on the establishment of a retrodifferentiated cell line (RD-1) from a single rat myelomonocyticleukemia cell which differentiated into a macrophagerlike celli)y-.treatment.jvimiipop 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: 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 an 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 (ibid), Fukunda (ibid), Hass et al (ibid), Curtin and Snell (ibid), Chastre et al (ibid), and Kobayashi et al (ibid) as these workers retrodifferentiated certain types of differentiated cells but wherein those cells remained committed to the same lineage and -he i -did not retrodifferentiate into undifferentiated cells.

Therefore, according to the state oi* h 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.

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 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. 8 According to a tenth aspect of the present invention there is provided a CD19+ 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 " and/or an MHC Class Π " 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 CD34+ undifferentiated cell.

Preferably, the, more committed ceJl,^ lymphohaematopoietic progenitor cell.

Preferably, the more committed cell is capable of retrodifferentiating into a pluripotent stem cell.

The undifferentiated cell may comprise any components that are concerned with antigen presentation, capture or recogmtion. Preferably, the undifferentiated cell is an MHC Class Γ and/or an MHC Class IT cell.

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 Γ and/or an MHC Class IT 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 - component and/or a β- component.

Preferably, the receptor comprises a β-chain having homologous regions. , Preferably, the receptor comprises at least the homologous regions of the /3-chain of HLA-DR.

Preferably, the receptor comprises an α-chain having homologous regions. 10 Preferably, the receptor comprises at least the homologous regions of the a-chain of HLA-DR.

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 0-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 altylating agent is or cempfises cyclophosphamide.

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 prefe ed 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 11 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 die agent modulates MHC Class I÷ and/or MHC Class Π÷ expression.

The agent operably engages the more committed cell in order to retrodifferentiate 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 committed cell.

An example of direct engagement is whence more committed cell has at least one cell surface receptor on its cell surface, such as a β-chain having homologous regions (regions that are commonly found having the same or a similar sequence) such as those that may be found οη*δ ceils, 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 -chain having homologous 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 ecep.tors_affecjtsjiej3t ei re more_ committed cell.

The agent for the retrodifferentiation of the more committed cell into an undifferentiated cell may be a chemical 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 12 cyclic adenosine monophosphate (cAMP), a CD4 molecule, a CD8 molecule, a part 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 agent 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 β chain .of a MHC class Π antigen, the β chain of a MHC HLA-DR antigen, the chain of a MHC class I or class II antigen, the chain of HLA-DR antigen, the a and the β chain of MHC class Π 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 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 pluripotent 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 Π antigens, a cell that can be induced to express class I or 13 class Π 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 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 fa -being expressed by the more committed cell.

Preferably, the receptor is a Clas^- I Or a Class Π 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.

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 Π antigens, a CD4 cell surface receptor, a CD8 cell surface receptor, homologous regions of the β-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 Π antigens in the presence of lymphocytes. 14 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 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 undifferfentiafed 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 ^differentiated j:eUJrom. 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 Π antigens; a cell capable of being induced to express class I or class Π antigens; an enucleated 15 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 Π antigen, or an enucleated cell.

The present invention also encompasses processes utilising the afore-mentioned uses and products or compositions prepared from such proqesses.

The present invention also encompasses a medicament comprising an undifferentiated cell according to the present invention^ 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 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.

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.

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, conmiitting the undifferentiated cell into a reconimitted cell, whereby arrangement or rearrangement of the genome and/or nucleus of the cell causes the mutation to be removed. 17 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 may 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 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. 18 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 shall 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'ef 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 hypogammaglobulinemias), 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 . . . !9 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 JjeatmenJL applied ^ to^ heir blood s 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 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" or 15 minutes after which they were left to incubate in an incubatof-at 22° C. The concentration of monoclonal antibody added to blood samples varied from ΙΟ ΟμΙ/ml 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 CD8, and a PE conjugated monoclonal antibody to the homologous region of the j3-chain of the HLA- 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 20 the j3-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 24 hour intervals.

LABELLED ANTIBODIES The following monoclonal antibodies were used to detect the following markers on cells by flow cytometry: CD19 and CD3, CD4 and CD8, DR and CDS, CD56 & 16 and CD3, CD45 and CD14, CD8 and CD3, CD8 and CD28, simultest control (IgGl FITC + IgG2a PE), CD34 and CD2, CD7 and CD13 & 33, CD10 and CD25, CD5 and CD10, CD5 and CD21, CD7 and CD5, CD13 and CD20, CD23 and CD57 and CD25 and CD45 RA (Becton & dickenson and DAKO).

Each patient's blood sample, both treated and untreated, was analyzed using the 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® 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. 21 MORPHOLOGY Morphology was analyzed using microscopy and Wright's stain.

∑L RESULTS CD19 AND CD3 PANEL Treatment of blood samples with monoclonal .antibody to the homologous region of the j3-chain of the HLA-DR antigen always decreased the relative number of CD19+ 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.

The same treatment caused the relative number of CD3 + cells to increase dramatically especially in blood of patients with B-CLL, which was always accompanied by an increase in the relative number in CD CD19" cells. CD3 is present on all mature T-lymphocytes and on 65% -85% of thymocytes. This marker is always found in association with α-Ιβ- 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 CD1.9-and.CD3-markers^_.i.e-.CD19^Land 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 CD19 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 lymphocytes. 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 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 (2, 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 2 and 6 hr's treatment of blood samples. However, Coulter Counts of WBC populations were reduced on treatment of blood with monoclonal antibody to the homologous region of the 0-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 whith ctfunts cells on the basis of surface markers, size and granulity. Furthermore, these atypical cells were accounted for by analysing 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 CD 19 and CD3 relative expression compared to stem cells.

In jr,abje_2._patient numbers 5 and 6 represent the same patient but analysis of treated and untreated blood samples were monitored with time and at the same time (see Table 1).

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 23 class Π 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 5 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" - · ' CD 19' cells On the other hand, the other brother who was 2 years old and was 10 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). 15 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. Howeve'r? oifanalysis of other surface markers such as CD4 and CD8 (see Table 3) the patient was observed to have a high relative number 20 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). 5 An-enriched-preparadon-of,B=lymphocyJes_obtained from healthy blood donors when treated with monoclonal antibody to the 3-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 0 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 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 Π antigen in the B2 domain, a region which is similar to the CD8 binding sites on class I antigens. Binding of CD4 to class II antigen enhances T cell responsiveness to antigens and so does the binding of CD8 to class -I- ntigens.- The CD8 antigens are . present on the human._supressor/cytotpxic T- lymphocytes subset as well as on a subset of natural killer (NK) lymphocytes and a majority of normal thymocytes. The CD4 and CD 8 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 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 j3-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" -GD8~cells~wb.ich.--at -least-in-the ase _of _Bd LLj:orrespond_io B cells was notedjo_ 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 25 lineage in the thymus (Patient number 2,3 and 4). The CD4 antigen is present on the helper/inducer T- lymphocyte subsets (CD4+CD3+) and a majority of normal thymocytes. However, this antigen is present in low density on the cell surface of monocytes and in the cytoplasm of monocytes and macrophages (CD3"CD4+).

The relative number of CD4T 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 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,J3*>and* 4).

DR AND CD3 PANEL The DR markers are present on monocytes, dendritic cells, B-cells and activated T- lymphocytes.

Treated and untreated samples analysed with this panel showed similar inHnui_ophenotypic changes to-&os the CD 19 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 significandy (see Table 4 and Chart). 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- ce ls) and was inversely proportional to decreases in the relative number of DR-f 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 CD 16 antigen is exp*resse¾ 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 CD 16 antigen is the IgG FC receptor ΙΠ.

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 Omer.T-lymphocy.te.anti antigen is present on essentially all resting and activated CD16+ NK lymphocytes and these subsets of cells carry out non-major histocompatibility complex restricted cytotoxicity.

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 27 coexpressing the CD56&CD16 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 CD 16 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 monoclonal antibody to the homologous region of the 3-chain of DR antigen seems to cause spontaneous production of CO56 and CD16÷ ceils, CD3÷ cells and CD56÷ and D\6÷ CD3 + cells and these observations were always accompanied by the disappearance of B-cell markers (CD.19, DR, CD56, CD16 CD3 ).

Onward analysis of this blood sample before and after* treatment showed the levels of CD56÷ and CD16÷ cells to decrease with 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, CD 16 and CD 3 antigens when compared to immunophenorypic 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 significant increases in the relative number of CD3 ", CD56+ & CD16+ and CD56+ and^CD.16 _CD.3 _cells. .

Blood samples of other patients with other conditions showed variable changes in the level 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. 28 CD45 AND CD14 PANEL The CD45 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 CD14 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, mitogen- activated T lymphocytes, erythrocytes, or platelets.

The CD45 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 j8-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 jS-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 CD 14 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 , 2, 3, 4 & 5).

On treatment the relative number of CD45 low cells (when compared to untreated samples)-increased-significantlv^and,sojlid the relative number of cells co-expressing the CD45 and CD14 antigens. This type of immunophenotypic changes 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 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 CD45 low cells. However, analysis of blood 24 hours later showed the opposite situation. .

Samples 5 and 7 reveal opposite immunophenoty ic 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 immunophenotypic 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 irnmunopathology would ensue. The effect of treatment of blood samples from other patients with no B-cell 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 immunophenotypic changes to those obtained with B-CLL with high B lymphocyte couats. " 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 in 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 CD 19 and 30 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 CDS" 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 cells; a maturer form of thymocytes. In addition, since these samples were also immunophenotyped 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.

Blood samples from a patient with B-CLL (number 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 -chain of DR antigen (Table 14 patient 5/6 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 /3 -chain of DR antigen show reversal in the relative number of CD19+ 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 CD19"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 CD19+ 32 and CD19'CD3' cells. However, treatment of an enriched preparation of B- lymphocytes obtained from healthy blood donors with monoclonal antibody to the β- 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 β- chain of the DR antigen increased the relative number of CD3÷ cells and decreased the relative number of CD194" 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 CDS PANEL Blood samples analysed using the CD 19 and CD3 panel (Table 14) were also immunophenotyped with the CD4 ana-CDft panel (Table 15). Both panels seem to agree and confirm each other. Incubation for 2 hours of blood samples of patients with 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 cyclophosphamide 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 3-chain of the DR antigen plus cyclophosphoamide revealed reverse changes in the relative number of CD4 and CD8 positive cells (Table 15, 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 33 analysed with the CD 19 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 irnmunophenotypic 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 & 15 & 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 β- 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 β -chain of the DR antigen is extremely capable of driving the production of CD3 positive cells from DR4" cells.

Furthermore, treatments such as thoseSnvo!ving engagement of the α-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.

CO56&16 AND CD3 PANEL Treatment of blood samples, especially of those of patients with B-CLL with high B-lymphocyte-counts with^nonoclonal-antibodyjo 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 CD 16 +CD3 + cells also increased following treatment of blood samples with monoclonal antibody to the β-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. 34 CD45 AND CD14 PANEL Blood samples treated with monoclonal antibodies to the β- or alpha- chains of the DR antigen or to the 0-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 5/6 (at 2 hours) with monoclonal antibodies to the j3-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"1" 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 ΗΓν"1" following the same treatment when compared to untreated samp Fes. Treatment of blood samples of HTV-f and IgA D patients with m<5fiocl6nal 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 j3-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 0-chain of the DR antigen. Medium CD45+ cells increased in blood samples of IgA/D patient following monoclonal antibody to class I antigen treatment.

Cells that were extremely large, heavil granular and expressing intense levels of homologous region of the j3-chain of DR antigen of MHC class Π antigens (see Charts 1, 2, 3, 4 & 5). 35 CD8 AND CO28 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 CD4+CD8't' immature thymocytes to a higher density on virtually all mature CD3 + , CD4÷ or CD8÷ 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 alloantigen- specific 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 B7/BB-1 antigen which is present on activated B lymphocytes.

Treatment of blood samples of patients (Table 19, patients 5/6 and 8) with B-CLL with monoclonal antibody to the homologous region of j3-chain of the DR antigen increased the relative number of CD8+, CD28+ and CD8+CD28÷ cells and all other types of treatments did not.

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 CD34 is also expressed on stromal cell precursors. Terminal deoxynucleotidyl transferase -(-T-dT)~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 CD15 antigens and on early erythroid cells that express the CD71 antigen and dimly express the CD45 antigen. The CD34 antigen is also found on 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 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 CD5, while CD34+- cells that are lineage-committed express the CD38 antigen in high density.

Most CD34+ cells reciprocally express either the CD45RO 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 mirkedlncreases in the relative number of CD34+ and CD34+CD2" 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 CD34+ and CD34+CD2+ cells observed here seems to coincide with increases in the relative number of CD4÷CD8+, 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- ymphopoiesis^ia-B4ymphocyte-regression..i___J On analysing 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 /3-chain of the HLA-DR 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 HTV+) with monoclonal antibody to the β-chain of the HLA-DR antigen markedly increased the relative number of CD34÷ and CD2÷CD34'i' 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 α-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"+ ceils. 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 0-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 immunopheriotypic 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 0-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 and /3-chains of the molecule when added together. However, the latter treatment increased the relative number of CD34÷CD2+ cells and the opposite occurred when the same blood sample was treated with monoclonal antibody to the j3-chain of the HLA-DR antigen alone. On analysis of treated and untreated blood aliquots of the same patient 24 hours later the -relative^umber-^fXD34-^decreased_WA^ treatments except it was maintained at a much higher level with monoclonal antibody to the jS-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 0-chain promotes the production of more CD34+ cells from CD2+CD34÷ pool or from more 38 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 CD13&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 3-chain of MHC class Π 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 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^*- is ossible to prepare an undifferentiated cell by the retrodifferentiation of a more mature undifferentiated cell. a 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, Jugure J„is,j3L_nmrj3sco e pic are 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. 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 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. 4 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.

EL 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 lymphocytic leukaemia's (B-CLL) with high B lymphocyte counts, with monoclonal antibody to the homologous region of the /S-chain of class-Π antigens gave rise to a marked increase in the relative number of single positive (SP) T-lymphocytes and 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-Π 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 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 cefts (DP) generated at 2 hour incubation time of whole blood with monoclonal antibody to the homologous region of the jS-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 and β chains were also expressed on these types of cells.

B-lymphocytes were constantly observed to lose markers such as CD 19, CD21, C-D23-,4gM^nd~DR-and-tliis^ou^ CD2+ cells, increases in CD7+ cells, increases in CD8+ CD28+ and CD28+ cells, increases in CD25+ cells, the appearance of CD 10* and CD34+ cells and CD34* and CD19* cells increases in CD5 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 jS-chain of HLA-DR antigen. 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 0-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 jS-chain of HLA-DR antigen, with CD16&56 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 CD 19 and CD3 and DR and CDS. Investigation of the supernatant of treated and untreated samples of patients with HIV infection using nephlometry and immunoelectrophoresis reveals increased levels of IgG indicating that the B-cells must have passed through the plasma dell 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 CD8 markers. Other transient cells were also observed and these were large and granular and positive for the CD3 and CD 19 receptors. CD25 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 0-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 42 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 significant increase in the relative number of cells expressing the myeloid markers such as CD 13 and CD33. These markers were coexpressed with the CD56 & 16 or the CD7 antigens. However, the relative number of CD7" cells with T-Iymphocyte 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-cha"in 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-Iymphocyte progenitor cells but are also capable of existing into the myeloid and erythroid lineages. t l 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 modifications of the present invention will be apparent to those skilled in the art. 43 TABLE 1 CLINICAL DIAGNOSIS OF PATIENTS AND EXPERIMENTAL CONDITIONS OF 8L00D SAMPLES INCLUDING COULTER COUNTS (WBC) FOLLOWING AND PRIOR TREATMENT OF BLOOD SPECIMENS WITH VARIOUS MONOCLONAL ANTIBODIES AND OTHER AGENTS PATIENT DIAGNOSIS EXPT WBC/L JLYMPH #LYMPH/L AGENT ID COND X10-9 10X-9 ML/mL B A B A B A 1 B-CLL 12HR 100 ND 86.1 ND 86.1 ND ANTI-8 AT 22C 50 2 B-CLL 2HR 39.1 9.6 74.463.3 29.9 ANTI-8 AT 22C 6.1 50 2HR AT 39.137.7 74.475.1 ANTI-8 22C 29.9 28.3 PE 50 3 B-CLL 5HR 39.5 9.3 71.967.2 28.3 ANTI-8 AT 22C 6.2. 50 6 HR 39.537.7 71.972.5 ANTI-8 AT 22C 28.3 27.4 PE 50 4 B-CLL 24HR ■73 66.5 28.4 ANTI-8 AT 22C 39 9.3 6.2 50 24HR 73 .70.4 ANTI-B AT 22C 39 36.2 28.4 25.5 PE 50 5 B-CLL 2HR AT 22C ANTI-5 - 50 ANTI -A 50 ANTI-I 50 ANTI-S &TOXIC AGENT 25+25 44 PATIENT DIAGNOSIS EXPT WBC/L 2LYMPH #LYMPH/L AGENT ID COND X10-9 B A 10X-9 ML/mL B A B A 6 B-CLL 24HR ANTI-S AT 22C 50 7 3-CLL 24HR 170 128 95.491.1 16.9 11.6 ANTI-3 AT 22C 10 178 94.2 16.3 ANTI-I 10 130 90.4 11.9 ANTI-S & TOXIC AGENT 10+20 3-CLL . 24HR 15 7 31.9.51.2 14 3.0 ANTI-S AT 22C 20 3-CLL 12HR +++ 89.5 37 85.1 +÷÷ 76.2 ANTI-S AT 22C 30 35.4 ANTI-I ++ +++ 30 39.4 ANTI-4 30 ■>-++ 84.9 A N T [ - Ι+Π+4 95.4 10+10+10 0 B-CLL 2HR 19.3 NO & 'v ND 16.7 ND ANTI-S AT 22C 30 ANTI-I 30 2 OUT 2HR 5.4 ND 74.5 ND ND ATI-S PATIENT AT 2C' 20 7 OUT 2HR 4.8 ND. 59.3 NO NO ANTI-B PATIENT AT 22C 20 1 OUT 2HR 4.2 ND 54.0 NO NO ANTI-B PATIENT AT 22C 20 1 OUT 2HR 3.9 ND 47.4 NO NO ANTI-B PATIENT AT 22C 20 45 PATIENT OIAGNOSIS EXPT WBC/L JLYMPH #LYMPH/L AGENT ID COND XlO-9 B A 10X-9 MLJmL B A B A 34 OUT 2HR 7.2 NO 20.0 NO NO ΆΝΤΙ-Β PATIENT AT 22C 20 36 CMV 4HR 13.4 NO 7.3 NO NO ANTI-B INFANT AT 22C 20 93 HIV+ 4HR 5.6 NO 43.4 NO NO ANTI-B INFANT AT 22C 20 B8/ST 43S BLAST 2HR AT 20.2 NO 12.2 NO ANTI-8 IN 3L000 22C 60.5 NO 50 6 DAYS OLD 24HR ANTI -A AT 22C 50 ANTI-AB · 25-25 .,·-.

HIV25 AIOS 2HR 7.5 NO 34.8 MD 2.-6 NO ANTI-8 AT 22C 50 ANTI-A 50 ANTI-AB 25+25 43/80 8 CELL 4HR ANTI-B DEFICIENT AT 22C 20 ANTI-I 20 ANTI-4 20 06/80 B CELL 4HR ANTI-8 DEFICIENT AT 22C j 20 ANTI-I 20 ANTI -4 20 46 EXPT COND : EXPERIMENTAL CONDITIONS B : BEFORE ' . ..

A : AFTER ANTI-B : monoclonal antibody to the homologous. region of the ?-chain of HLA-OR ariigen ANTI-A : monoclonal antibody to the homologous region of the a-chain of HLA-OR antigen ANTI-I : monoclonal antibody to the homologous region of Class I antigens ANTI-AB : both ANTI-B and ANTI-A added togather ANTI-4 : monoclonal antibody to the CD4 antigen - ANTI-I+II+4 : ANTI-I and ANTI-8 and ANTI-4 added togather Cytoxic agent : Cyclophophamide *\ MLIrn] : micro litre per ml L : litre 47 TABLE 2 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONS BEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B CHAIN OF THE HU-DR WITH CD19 AND CD3 MONOCLONAL ANTIBOOIES. : e ore treatment . : a ter treatment 48 TABLE 3 I MUNOPHENOTYPI G 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 HU-DR WITH MONOCLONAL ANTIBODIES TO CD4 AND CD8.

PATIENT 2CD8+ :CD4+ CD4+CD8+ JCD4-CD8- CD4+L0W B A B A B A B A 8 A 1 2.8 16 2.9 11.4 0 3.2 93.1 67.6 0 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 48.1 6.3 6.6 4 10.1 24.2 7.6 24.9 0.3 2.8 77.5 42 4.6 5 5 2.9 16.2 1.8 7.6 0 2 95 62.3 0 0 6 NO 12 ND 8.1 ND 1.7 ND 75.7 ND 0 7 1.9 2.6 1.9 2.8 0 0 95.8 94.3 0 0 8 3.2 7 3.9 6.9 0.1 2 87.3 79.8 4.3 6 9 2.8 2.9 3 3 0 0 94 94.1 0 0 10 5.7 9.4 4.7 9.1 0.6 0.8 33 J 79.2 0 0 92 21 19 21.6 21 0.8 1.9 50.5 52.5 5.3 4.8 91 15.4 18.1 13.6 17.9 6.2 2.6 57 57.3 7.3 3.5 87 16.8 21.8 13.4 20.4 Y9 2.6 59.5 48.9 7 ■ 5.6 21 16 24.1 9.1 15.2 1 2.6 69.6 53.2 3.7 4.2 34 9.4 11.9 5.7 4.9 2 3.3 67.6 65.3 14.414.5 39 12.1 12.6 13.1 14.6 0.4 1.3 62.3 66.7 11.9 4.3 93 18.9 20.3 9.7 10.3 1.8 1.4 65.5 65.9 3.4 1.8 B8/ST 6.3 13 5.7 7.3 2.2 1.1 34.7 70.3 50.3 7.6 HIV25 ■24.1 24.9 0.8 1.1 1.3 5 70.2 69.3 2.9 3.8 49 TABLE 4 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND 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+OR+ DR-CD3- DR+HCD3 - B A B A B A 3 A 3 A 1 87 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 4.1 8.8 9.6 38.1 0 4.7 4 76.8 19.2 7.6 29.5 6.2 10.5 9.1 37.2 0 3.3 5 NO 47.1 ND 11.5 ND 9.9 NO 22.4 ND 7.3 6 NO 7 91.4 85.8 2.4 2.5 0.7 0.7 5.1 4.2 0 6. 3 8 61.8 28.9 6.5 11.2 2 3.3 28.6 54.6 0 1.5 9 NO 10 82.6 44.7 4.3 9.8 3.3 5 9.8 22.2 0 17.9 92 23.9 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 87 14.8 12.2 23.4 34.1 5.5 6.6 51.1 46.5 0 0 21 NO 34 11.9 12.9 10.4 13.7 0.8 0.6 76.7 72.8 0 0 39 25.6 13.7 24.6 25.2 3 2.8 46.5 25.2 0 0 93 13.3 8.9 18.4 18.9 9.9 10.1 58.2 61.7 0 0 BB/ST 44.2 32.5 11.7 12.2 0.8 0.8 43 49.4 0 4.6 50 TABLE 5 IMMUN0PHENOTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONS BEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-OR WITH MONOCLONAL ANTIBOOIES TO CD16+56 AND CD3.

PATIENTS CD56+&16 C03+ CD56+&16+CD3+ CD56+&16-CD3- 8 A B' A B A B A 1 2 4.3 5.7 19.7 0.7 1.7 91.3 73 2 11.5 38.9 12.4 32.6 1 6.6 74.5 21 3 12 36.2 12.1 34.5 0.7 6 75.5 23 4 12.2 32.6 12.4 39.6 0.5 5 74.7 22.2 5 NO 13.1 NO 9.4 NO 2.6 NO 73.5 6 NO 7 0.8 0.8 2.8 2.4 0.3 .0.2 96.2 96.4 8 24.8 52 5.4 12.4 0.9 4.1 68.3 31.1 9 NO 10 1.1 1.3 6.1 13.7 2.1 2.5 90.5 82.4 92 23.8 34.5 44.3 44.8 2 1.5 29.2 18.6 91 4.6 3.9 28.8 29.4 3 3.2 63.3 63.3 87 47.9 46.4 28.8 36.5 5.3 3.7 16.9 13 21 9.4 9.4 19.7 23.6 4.2 6.7 66 59.5 51 52 TABLE 6 IMMUNOPHENTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONS BEFORE AND AFTER OF TREATMENT OF BLOOD WITH MONOCLONAL ANTIBOUY TO THE HOMOLOGOUS REGION OF THE B CHAIN OF THE HU-DR WITH MONOCLONAL ANTIBODIES TO CD45 AND CD14.

PATIENTS CD45+H CD45+L CD45+CD14+ 3 A 8 A 8 A 1 90.5 70.1 7.5 21.9 0.8 3.3 2 85.8 52.2 8.8 38.3 5.3 9.5 3 84.3 52.2 9.9 33.8 5.1 13.2 4 91.5 79.2 2.1 7 5.7 10.8 r 3 63.1 84.6 34.9 9.4 0.5 3.6 6 ND 7 52.8 85.2 45.6 13.9 0.5 0.6 8 71.1 - 55 71.1 34.5 5.3 8.7 9 SEE 10 79.7 47.3 16.3 48 2.1 1.9 92 61.7 64.7 27.4 26.6 5.9 3.6 91 49.4 49.2 40.4 44.3 _ 6.5 3.2 87 52.4 61.5 36.1 28.7 7 6.5 ^ r 21 45.8 43.3 44.3 47.6 6.2 3.3 34 24.4 24.6 54.8 59.6 13.3 9.7 39 43.7 46.3 30.5 42.1 14.5 8.8 93 SEE HIV+ 22.6 ■ 26.9 66.8 63.5 6.8 6.7 IgA/D .47.4 59.8 41.9 33.3 5.9 4.1 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-DR WITH MONOCLONAL ANTIBODIES TO CD8 AND CD3.

TABLE 8 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 MEASURE WITH MONOCLONL ANTIBODIES TO CD45 AND CD14.

TABLE 9 IMMUNOPHENOTYPING OF A PATINENT WITH Β-CLL· WITH TIME AFTER TREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-OR MEASURED WITH MONOCLONAL ANTIBOOIES TO CD19 AND CD3.

TIME C019+0R+r CD3+ C03+OR+ CD19-CD3-DR- 2HR 87.4 10.1 1.8 10.7 6HR 75.5 10.4 3.1 10.7 24HR 74 11.7 2.9 11 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 CD8.

TABLE 11 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTER TREATMENT OF BLOOO WITH PE CONJUGATED MONOCLONAL ANTI3O0Y TO THE HOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONAL ANTIBODIES TO CD3 AND DR.

TABLE 12 IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTER TREATMENT OF BLOOO WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THE HOMOLOGOUS- REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONAL ANTIBODIES TO CD16&56 AND CD3.

TABLE 13 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 CD8 AND CD3.

TIME CD8+DR+ C03+ CD8+CD+3&DR+r CD8-CD3-DR- 2HR 76.2 6.6 6.7 10.6 6HR 76.5 6.2 6.2 10.3 55 TABLE 14 IMMUNOPHENOTYPING OF PATIENTS WITH 8-CLL BEFORE AND AFTER TREATMENT OF BLOOD 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.

CD19 C03+ C019+CD3+ C019-CD3- 10 B A AB A AI 8 A AB A AI 8 n Λ, A6 A AI B A AB A AI A 3C A BC A 8C A BC 5/ 6 2H 86 91 54 40 89 5 4 16 23 5 1 I 3 2 1 6 4 27 33 5 24 N 88 51 60 86 4 18 10 4 N 2 1 2 3 N 4 29 28 7 10 2H 77 N 59 N 80 1 N 13 N 7 1 N 1 N 0 14 N 26 N 12 09 24 8 N N N 6 32 N N ' N 38 1 N N N 1 59 N N N 56 43 /B 0 6H 0 N 0 0 0 40 N 42 43 49 0 N Ί 0 1 58 N 54 54 47 04 /B 0 6H 0 N 0 0 0 49 N 41 45 46 0 N 3 1 3 43 N 42 44 41 HI V+ 6H 1 N 0 N 1 10 N 14 N 12 0 N 0 N 0 89 N 86 N 87 ig A/ 0 6H 10 N 1 N 12 21 N 25 N 20 2 N 1 N 3 67 N 71 N 68 56 TABLE 15 CD8 AND CD4 TABLE 16 CD3 AND DR DR+ CD3+ CD3÷DR+ CD3-DR- ID B A AB A AI 8 A AB A AI 8 A A3 A AI 8 A AB A AI A 8C A BC A BC A BC 5/ 6 2H N 90 54 N 87 N 4 12 N 4 N 2 10 N 3 N 5 22 N 5 10 H 83 N 63 N 81 4 N 8 N 4 4 N 7 N 4 9 N 23 N 12 9 4 14 N N N 13 30 N N N 36- 3 N N N 3 51 N N N 47 57 TABLE 17 CD16&56 AND CD3 TABLE 13 CD45 AND CD14 CD45+L C045+M C045+H CD45+C014+ ID B A AB A Ai B A AB A AI B A AH \ A AI 8 A AB A AI A 8C A BC A BC A BC 5/ 6 2H 0 0 5 10 0 44 43 50 50 ,32 55 43 50 31 67 1 1 1 2 0 10 2H 0 N 0 N 0 43 N 54 N 35 54 42 N 62 1 N 1 N 0 09 24 2 N N N 1 18 N N N 16 71 N N 76 7 N N N 5 HI V+ 6H 4 N 3 N 6 63 N 61 N 41 23 N 27 N 40 7 N 7 N - 7 ig A/ D -6H- 2 N 2 N 4 40 N 31 N 44 47 N 6Ω N 44 6 N 4 N 6 58 TABLE 19 CD8 AND CD28 TABLE 20 CD34 AND CD2 59 CHART 1 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOOD SAMPLE OF PATIENT (2. 3 & 4) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE 0-CHAIN OF HU-DR ANTIGEN MEASURED WITH TIME.

WITHOUT WITH FL1 FL2 TIME NOTHI G001 WITH002 CD45 C014 2HR NO001 WE002 CD45 C014 6HR 001001 002002 CD45 C014 24HR NOTHING003 ETH004 CD3 C019 2HR NO003 E004 C03 CD19 6HR 001003 002004 C03 C019 24HR NOTHING004 ITH005 CD4 C08 2HR N0004 WE005 CD4 C08 6HR 001004 002005 CD4 C08 24HR NOTHING005 WITH006 CD3 DR 2HR NO005 WE006 C03 OR 6HR 001005 002006 CD3 DR 24HR NOTHIG006 WITH007 CD3 CD56&16 2HR NO006 WE007 C03 CD56&16 6HR 001006 002007 CD3 C056&16 24HR N003 W004 C03 CD8 2HR NO007 WE008 CD3 CD8 6HR 001007 002008 C03 CD8 24HR 60 CHART 1A IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOOO SAMPLE OF PATIENT (2. 3. 4) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE ?-CHAIN OF HLA-DR ANTIGEN CONJUGATED TO PE MEASURED WITH TIME. 10 FL1 FL2 TIME WL003 CD45 CD14 2HR WEL003 CD45 CD14 6HR 003003 CD45 CD14 24HR WL005 CD3 CD19 2HR WEL005 CD3 CD19 6HR 003005 CD3 CD19 24HR WL006 CD4 CD8 ■ 2HR WEL006 CD4 C08 6HR 003006 CD4 CD8 24HR WL007 CD3 OR 2HR WEL 007 CD3 DR 6HR WL008 CD3 CD65&16 2HR WEL 008 CD3 CD56&16. 6HR WL005 CD3 CD8 2HR WEL009 CD3 CD8 6HR 61 CHART 2 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOOD OF PATIENT (1) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE CHAIN OF HU-DR ANTIGEN, THIS ANTIBODY AND CYCLOPHOSPHAMIDE. MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE a-CHAIN OF HLA-DR ANTIGEN AND MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF CLASS I ANTIGEN MEASURED WITH TIME.

WITH WITHOUT FL1 FL2 TIME A001 C045 C014 2HR A2B001: AB C045 CD14 2HR A2A : AA CD45 CD14 2HR DNAA001:ABC C045 CD14 2HR A1001: AI C045 C014 2HR CD3 C019 2HR C2B001:AB CD3 C019 2HR C2A001:AA C03 CD19 2HR DNAC001:ASC CD3 -C019 2HR ClOOl: AI C03 CD19 2HR A12H001:AI C03 ' CD19 24HR A2B24H001: AB CD3 CD19 24HR A2A24H001:AA C03 CD19 24HR A28X24H001:AB CD3 CD19 24HR C O001 CD4 C08 2HR D2B001: AB C04 CD8 2HR D2A00I: AA C04 CD8 2HR ONA0001:ABC CD4 CD8 2HR D1001: AI C04 C08 '2HR 0124H001:AI C04 C08 24HR D2BX24H001:A8 C04 CD8 24HR C D2B001: AB CD4 CD8 24HR 02A001: AA CD4 CD8 24HR E1001: AI CD3 DR 2HR E28001: AB CD3 OR 2HR Γ2Α0'ΟΤΓ~ΑΑ W~ OR " ~2HR— F1001: AI CD3 CD56&16 2HR F2B001: AB C03 CD56&16 2HR F2A001: AA CD3 CD56&16 2HR G1001: AI C028 CD8 2HR G2A001: AA CD28 CD8 2HR G2B001: AB CD28 CD8 2HR H1001: AI CD7 C033&13 2HR 62 CD7 CD33&13 2HR FL1 . FL2 . TIME C07 CD33&I3 2HR CD21 CDS 2H CD21 CD5 2HR C034 C02 2HR CD34 CD2 2HR CD34 CD2 24HR CD34 C02 24HR C034 CD2 24HR CD10 CD25 2HR COLO CD25 2HR 63 CHART 3 IMMUNOPHENOTYPTC CHANGES OF UNTREATED AND TREATED BLOOO OF PATIENT (8) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE ?-CHAIN OF HLA-DR ANTIGEN. · WITH WITHOUT FL1 FL2 TIME AN001 CD45 CD14 2HR A2001 CD45 C014 2HR CNOOl C03 CD19 2HR C2001 CD3 C019 2HR ON001 CD4 CD8 2HR 02001 CD4 C08 2HR ENOOl ■ C03 OR 2HR E20Q1 C03 OR 2HR FN001 CD3 C056&16 2HR F2001 C03 C056&16 2HR GN001 CD23 ' CD8 2HR G200I C023 CD8 2HR HN001 CD7 ■ C05 2HR H2001 CD7 * "* CD5 2HR IN001 CO13 C020 2HR 12001 C013" C020 2HR JNOOl ^ , C045RA C025 2HR J2001 CD45RA C025 2HR N001 C057 C023 2HR K2001 C057 CD23 2HR 64 CHART 4 IIMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOOD SAMPLE OF PATIENT (10) WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE 0-CHAIN OF HLA-DR ANTIGEN AND MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF CLASS I ANTIGENS.

WITH WITHOUT FL1 FL2 TIME CLLOOOl CD45 CD14 2HR CLLIOOI C045 CD14 2HR CLL2001 C045 CD14 2HR CLL0003 CD3 CD19 2HR CD3 CD19 2HR CLL1003 CD3 CD19 2HR CLL2003 CD3 C019 2HR CLL0004 CD4 C08 2HR CLL1004 CD4 CDS 2HR CLL2004 C04 CD8 2HR CLL005 C03 ' DR 2HR CLL1005 C03 OR 2HR CLL2005 CD3 OR 2HR CLL0C06 CD3 CD56&16 2HR CLL1006 CD3 CD56&16 2HR CLL2006 CD3 C056&16 2HR 65

Claims (27)

1. A method for producing a medicament which method comprises contacting a cell with an agent that causes the cell to retrodifferentiate into a stem cell and admixing the stem cell with a suitable dilue^ carrier or excipient, wherein the retrodifferentiated stem cell is capable of being recommitted into a more differentiated cell.

2. A method according to claim 1 which further comprises recommitting the stem cell into a more differentiated cell prior to admixing with a suitable diluent, carrier or excipient.

3. A method according to claim 1 or 2 wherein the cell is a non-cancer cell.

4. A method according to any one of claims 1 to 3 wherein the cell is a differentiated cell. *■ *

5. A method according to claim 4 wherein the cell is selected from a CFC-T cell and a CFC-B cell, a CFC-Eosin cell, a CFC-Fias cell, a CFC-GM cell, a CFC-GM cell, a CFC-MEG cell, a BFC-E cell, a CFC-E cell, a T cell and a B cell.

6. A method according to any one of the preceding claims wherein the stem cell is a pluripotent stem cell.

7. A method according to claim 6 wherein the pluripotent stem cell is selected from a lymphohaemapoietic progenitor cell, a lymphoid stem cell and a myeloid stem cell.

8. A method according to any one of the preceding claims wherein the stem cell and/or the cell is an MHC class Γ and/or MHC class IT cell.

9. A method according to any one of the preceding claims wherein the stem cell and/or the cell is a CD34+ cell. 66

10. A method according to any one of the preceding claims wherein the agent acts extracellularly of the more committed cell.

11. A method according to any one of the preceding claims wherein the cell comprises a receptor that is operably engageable by the agent and wherein the agent operably engages the receptor.

12. A method according to claim 11 wherein the receptor is a cell surface receptor.

13. A method according to claim 11 wherein the receptor is an MHC class I antigen or an MHC class Π antigen.

14. A method according to claim 13 wherein the receptor is selected from an HLA-A receptor, , an HLA-B receptor, an HLA-C receptor, an HLA-DR receptor, an HLA-E receptor, an HLA-F receptor, a DM receptor, a DP receptor and a DQ receptor.

15. A method according to claim 14 wherein the receptor is an HLA-DR receptor. -

16. A method according to any one of claims 11 to 14 wherein the receptor comprises an a-chain and/or a β-chain.

17. A method according to claim 16 wherein the a-chain and/or β -chain has homologous regions.

18. A method according to claim 17 wherein the receptor comprises at least the homologous regions of the β-chain of HLA-DR.

19. A method according to claim 17 wherein the receptor comprises at least the homologous regions of the a-chain of HLA-DR.

20. A method according to any one of claims 11 to 19 wherein the agent is an antibody to the receptor. 133, 155/2 67

21. A method according to Claim 20 wherein the agent is a monoclonal antibody to the receptor.

22. A method according to any one of the preceding claims wherein the agent modulates MHC gene expression.

23. A method according to Claim 22 wherein the agent modulates MHC Class Γ and/or MHC Class IT expression.

24. A method according to any one of the preceding claims wherein the agent is used in conjunction with a biological response modifier.

25. A method according to Claim 24 wherein the biological response modifier is an alkylating agent.

26. A method according to Claim 24 wherein the alkylating agent is or comprises cylophosphoamide.

27. A method according to any of the above claims substantially as described herein. For the Applicant,