AU1480500A - A method of preparing an undifferential cell - Google Patents

Description

P/00/011 28/5/91 Regulation 3.2 1/1

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT *u U Name of Applicants: Actual Inventors Address for service is: Ilham Mohamed Saleh Saeed ABULJADAYEL Ghazi Jaswinder DHOOT Ilham Mohamed Saleh Saeed ABULJADAYEL Ghazi Jaswinder DHOOT WRAY ASSOCIATES 239 Adelaide Terrace Perth, WA 6000 V. V.

9 Attorney code: WR Invention Title: "A Method of Preparing an Undifferential Cell" This application is a divisional application by virtue of Section 39 of Australian Patent Application 45451/96.

The following statement is a full description of this invention, including the best method of performing it known to me:- -1/2 A METHOD OF PREPARING AN UNDIFFERENTIATED CELL The present invention relates to a method of preparing an undifferentiated cell.

In particular, the present invention relates to a method of preparing an undifferentiated cell from a more differentiated 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 differentiated cell i.e. a recommitted cell.

The present invention also relates to the use of the undifferentiated cell of the present invention or the recommitted cell of the present invention to have an effect (directly or indirectly via the use of products obtained therefrom) on the immune system such as the alleviation of symptoms associted 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 or differentiated to give rise to 15 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 stem cell.

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

2/1 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. 4 th Edition, Eds, Roitt. Brostoff and Male 1996. Publ. Mosby especially Chapter 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 10 and MSCs are each formed by the differentiation of PSCs. Hence, LSCs and MSCs are more differentiated that PSCs.

Examples of differentiated cells include T cells, B cells, eosinophils, basophils, neutrophils, megakaryoctes, monocytes, erythrocytes, granulocytes, mast cells, and lymphocytes.

T cells and B cells are formed by the differentiation of LSCs. Hence, T cells and B cells are more differentiated than LCSs.

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 differentiated than MSCs.

Antigens are associated with undifferentiated and'differentiated cells. The term "associated" here means the cells expressing or capable of expressing, or presenting or capable of being induced to present, or comprising, the respective antigen(s).

Most undifferentiated cells and differentiated cells comprise Major Histocompatability Complex (MHC) Class I antigens and/or Class II antigens. If 2/2 these antigens are associated With those cells then they are called ClassV and/or Class 11- 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 CD3. If these anigens are present then these particular cells are called CD34-, CD19- and CD3- cells respectively. If these antigens are not present then these cells are called CD34-, CDI9- and CD3 cells respectively.

In more detail, PSCs are CD34- cells. LSCs are DR-, CD34- and TdT- cells.

MSCs are CD34, DR-, CD3-, CD33-, CD7- and TdT cells. B cells are CDI9 CD21', CD22- and DR' cells. T cells are CD2-, CD3', and either CD4or CD8 cells. Immature lymphocytes are CD4- and CD8 cells. Activated T cells are DR' cells. Natural killer cells (NKs) are CD56' and CD16 4 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 20 to identify certain cell types whether or not a cell is an undifferentiated cell or a differentiated cell) and the specialisation of that cell type 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 rerrodifferentiation lose morphological and functional complexity by virtue of a process of self-deletion of cyioplasmic structures and the transition to a more juvenile pattern of gene expression. This results in a progressive uniformization of originally distinct cell phenorvoes and to a decrease of responsiveness to regulatory signals operational in adu!t cells. Retrodifferentiation is normally counterbalanced by a process of reonogeny that tends to restore the terminal phenopvoes where the reversion started. This exolains why rerrodiffereniation remains invariably associated to cell reeneration 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 heparocvtes from regenerating liver, or from established heparomas, it has been difficult to obtain permanent class lines from resting adult heparocyes." Uriel (ibid) also reported on apparent retrodifferentiation in cancer, wherein he stated: "the biochemical phenorypes of many tumours show analogous changes of reversion toward immaturity during the preneoplastic phase of liver carcinogenesis, cells also rerrodifferenriate." More recent findings on retrodifferentiation include the work of Minoru Fukunda (Cancer Research (1981] vol 41, pp 4621-4628). Fukunda induced specific changes in the cell surface glycoprotein profile of K562 human leukaemic cells by use of the tumour-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA).

According to Fukunda TPA appeared to induce the K562 human leukaemic cells into a retrodifferentiated stage.

Also, Hass er a! (Cell Growth Differentiation [1991] vol 2, pp 541-548) reported that long term culture of TPA-differentiated U-937 leukaemia cells in the absence of phorbol ester for 32-36 days resulted in a process of retrodifferentiation and that the retrodifferentiated cells detached from the substrate and reinitiated proliferation.

Another case of retrodifferentiation is the work of Curin and Snell (Br. J. Cancer i [1983] vol 48, pp 495-505]. These workers compared enzymatic changes occurring during diethylnitrosamine-induced hepatocarcinogenesis and liver regeneration after partial hepatectomy to normal liver differentiation. Theses workers found changes in enzyme activities during carcinogenesis that were similar to a step-wise reversal of differentiation. According to these workers, their results suggest that an underlying retrodifferentiation process is common to both the process of hepatocarcinogenesis and liver regeneration.

a 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- **8 Even more recently, Kobayashi er 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 macrophage-like cell by treatment with lipopolysaccharide (LPS).

According to the current understanding, as borne out by the teachings found on page 911 of Molecular Biology of the Cell (pub. Garland Publishers Inc. 1983) and more recently Levitt and Mertelsman (ibid), a stem cell, such as a PSC, has the following four characteristics: 6/1 1. it is an undifferentiated cell i.e. it is not terminally differentiated; II. it has the ability to divide without limit: II. it has the ability to give rise to differentiated progeny. such as the differentiated cells mentioned earlier: and iv. when it divides each daughter has a choice: it can either remain as PSC like its parent or it can embark on a course leading irreversibly to terminal differentiation.

Note should be made of the last qualification, namely that according to the general teachings in the art once an undifferentiated cell has differentiated to a more committed cell it can not then retrodifferentiate. This understanding was even supported by the teachings of Uriel (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 they did not retrodifferentiate into undifferentiated cells.

Therefore, according to the state of the art before the present invention, it was believed that it was not possible to form undifferentiated cells, such as stem cells, from more committed cells. However, the present invention shows that this belief is inaccurate and that it is possible to form undifferentiated cells from more committed cells.

Thus, according to a first aspect of the present invention there is provided a method for producing a stem cell by retrodifferentiating a cell which method comprises the step of: contacting the cell with an agent that causes the cell to retrodifferentiate into a stem cell, wherein the retrodifferentiated stem cell is capable of being recommitted into a more differentiated cell.

6/2 As used herein the term "agent" means a substance, such as a chemical or biological compound or composition which is capable of causing a cell to retrodifferentiate. Examples of agents which achieve this outcome include, but are not limited to: substances like monoclonal or polyclonal antibodies. cAMP CD4 or CD8 molecules, a part or all of a Tcell receptor, a ligand. a peptide. a Tcell receptor which are capable of engaging a receptor, be it internal or external of the cell. found associated with a more differentiated cell: or substances which modulate MHC I and or MHC class II expression.

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 p chain of a MHC class II antigen, the p chain of a MHC HLA-DR antigen, the a chain of a MHC class I or class II antigen, the a chain of HLA-DR antigen, the a and the p chain of MHC class II antigen or of a MHC class I antigen. An example of a suitable antibody is CR3/43 (supplied by Dako).

The more differentiated cell is any cell derived or derivable from an undifferentiated cell.

According to a second aspect of the present invention there is provided a method comprising contacting a more differentiated cell with an agent that causes the more differentiated cell to retrodifferentiate into an undifferentiated cell- and then differentiating the undifferentiated cell to a recommitted cell.

The term "recommitted cell" means a cell derived from the undifferentiated cell i.e. a new more differentiated 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.

il***According to a fourth aspect of the present invention there is provided an 10 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 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 precommitted 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 the present invention in the manufacture of a medicament for the treatment of an immunological disorder or disease.

-8- According to a ninth aspect of the present invention there is provided a more differentiated cell having attached thereto an agent that can cause the more differentiated cell to retrodifferentiate into an undifferentiated cell.

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 differentiated cell.

The present invention is highly advantageous as it is now possible to prepare 10 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 *i 15 as a new differentiated cell, with a view to correcting or removing the original more differentiated cell or for correcting or removing a product thereof.

Preferably, the more differentiated cell is capable of retrodifferentiating into an MHC Class I' and/or an MHC Class II undifferentiated cell.

Preferably, the more differentiated cell is capable of retrodifferentiating into an undifferentiated cell comprising a stem cell antigen.

Preferably, the more differentiated cell is capable of retrodifferentiating into a CD34" undifferentiated cell.

Preferably, the more differentiated cell is capable of retrodifferentiating into a lymphohaematopoietic progenitor cell.

-9- Preferably. the more differentiated 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 recognition. Preferably, the undifferentiated cell is an MHC Class I- and/or an MHC Class II- 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.

S 10 The more differentiated cell may comprise any components that are concerned with antigen presentation, capture or recognition. Preferably, the more differentiated cell is an MHC Class I- and/or an MHC Class II1 cell.

Preferably, the agent acts extracellularly of the more differentiated cell.

Preferably, the more differentiated cell comprises a receptor that is operably engageable by the agent and wherein the agent operably engages the receptor.

Preferably, the receptor is a cell surface receptor.

Preferably, the receptor comprises an a- component and/or a P- component.

Preferably, the receptor comprises a p-chain having homologous regions.

Preferably, the receptor comprises at least the homologous regions of the pchain of HLA-DR.

Preferably, the receptor comprises an a-chain having homologous regions.

Preferably. the receptor comprises at least the homologous regions of the achain 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 p-chain of HLA-DR.

Preferably. the agent is an antibody, preferably a monoclonal antibody, to the homologous regions of the a-chain of HLA-DR.

Preferably, the agent is used in conjunction with a biological response modifier.

Preferably, the biological response modifier is an alkylating agent.

Preferably, the alkylating agent is or comprises a cyclophosphoamide.

In one preferred embodiment, the more differentiated cell is a differentiated cell.

Preferably, the more differentiated cell is any one of a B cell or a T cell.

In an alternative preferred embodiment, the more differentiated cell is a more mature undifferentiated cell.

In one preferred embodiment, when the undifferentiated cell is differentiated to a recommitted cell the recommitted cell is of the same lineage as the more differentiated cell prior to retrodifferentiation.

In another preferred embodiment, when the undifferentiated cell is differentiated to a recommitted cell the recommitted cell is of a different lineage as the more differentiated cell prior to retrodifferentiation.

-11 Preferably, the recommitted cell is any one of a B cell, a T cell or a granulocyte.

Preferably, the method is an in vitro method.

Preferably. the agent modulates MHC gene expression, preferably wherein the agent modulates MHC Class F and/or MHC Class II expression.

The agent operably engages the more differentiated cell in order to retrodifferentiate that cell into an undifferentiated cell. In this regard, the agent for the retrodifferentiation of the more differentiated cell into the undifferentiated cell may act in direct engagement or in indirect engagement with the more differentiated cell.

10 An example of direct engagement is when the more differentiated cell has at least one cell surface receptor on its cell surface, such as p-chain having homologous regions (regions that are commonly found having the same or a similar sequence) such as those that my be found on B cells, and wherein the agent directly engages the cell surface receptor. Another example, is when the more differentiated cell has a cell surface receptor on its cell surface such as an a-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 differentiated cell has at least two cell surface receptors on its cell surface and engagement of the agent with one of the receptors affects the other receptor which then induces retrodifferentiation of the more differentiated cell.

The agent for the retrodifferentiation of the more differentiated 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 differentiated cell. For example, preferred agents include any one or more of 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- -12cell 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 [3 chain of a MHC class II antigen, the 3 chain of a MHC HLA-DR antigen, the a chain of a MHC class I or class II antigen, the a chain of HLA-DR antigen, the a and the p chain of MHC class II antigen or of a MHC class I antigen. An example of a suitable antibody is 10 CR3/43 (supplied by Dako).

The more differentiated cell is any cell derived or derivable from an 9 undifferentiated cell.

Thus, in one preferred embodiment, the more differentiated cell is also an undifferentiated cell. By way of example therefore the undifferentiated cell can 15 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 differentiated 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, 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 differentiated 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 expression class I or class II antigens, a cell that can be induced to express class I or class II antigens or an enucleated cell a cell that does not contain a nucleus such as a red blood cell).

-13- In an 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 differentiated cell. However, preferably, the agent acts extracellularly of the more differentiated cell.

e In a preferred embodiment, agent operably engages a receptor present on the 10 surface of the more differentiated cell which receptor may be expressed by the "more differentiated cell, such as a receptor that is capable of being expressed by the more differentiated cell.

Preferably, the receptor is a Class I or a Class II antigen of the major histocompatibility complex (MHC). In preferred embodiments the cell surface receptor is any one of: an HLA-DR receptor, a DM receptor, a DP receptor, a DQ receptor, an HLA-A receptor, an HLA-B receptor, an HLA-C receptor, an HLA-E receptor, an HLA-F receptor, or an HLA-G receptor.

In more preferred embodiments the cell surface receptor is an HLA-DR receptor.

Preferably the contacting step comprises the agent engaging with any one or more of the following: homologous regions of the a-chain of class I antigens, homologous regions of the a-chain of class II antigens, a CD4 cell surface receptor, a CD8 cell surface receptor, homologous regions of the 3-chain of class II antigens in the presence of lymphocytes, homologous regions of the achain of class I antigens in the presence of lymphocytes, or homologous regions of the a-chain of class II antigens in the presence of lymphocytes.

-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 differentiated into a recommitted cell, such as a differentiated cell.

The recommitted cell may be of the same lineage to the more differentiated cell from which the undifferentiated cell was derived.

S:.i Alternatively, the recommitted cell may be of a different lineage to the more differentiated cell from which the undifferentiated cell was derived.

In addition, the present invention also encompasses the method of the present invention for preparing an undifferentiated cell, wherein the method includes 15 differentiating 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 S etc.

Other aspects of the present invention include: The use of any one of the agents of the present invention for preparing an undifferentiated cell from a more differentiated cell.

The use of an undifferentiated cell produced according to the method of the present invention for producing any one of a monoclonal or a polyclonal or a specific antibody from a B-lymphocyte or a T-lymphocyte; a cell from the macrophage monocyte lineage; a nucleated cell capable of expressing class I or class II antigens: a cell capable of being induced to express class I or class II antigens; an enucleated 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 10 a class I or a class II antigen, a cell capable of being induced to express a class I or a class II antigen, or an enucleated cell.

The present invention also encompasses processes utilising the aforementioned uses and products or compositions prepared from such processes.

The present invention also encompasses a medicament comprising an undifferentiated cell according to the present invention or a product obtained therefrom admixed with a suitable diluent, carrier or excipient.

In one preferred embodiment the medicament comprises an antibody or antigen obtained from an undifferentiated cell according to the present invention admixed with a suitable diluent, carrier or excipient.

Preferably the medicament is for the treatment of any one of: cancer, autoimmune diseases, blood disorders, cellular or tissue regeneration, organ regeneration, the treatment of organ or tissue transplants, or congenital metabolic disorders.

In a preferred embodiment the present invention relates to a process of introducing a gene into the genome of an undifferentiated cell, wherein the process comprises introducing the gene into a more differentiated cell, and then 16preparing an undifferentiated cell by the method according to the present invention, whereby the 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 differentiated 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, 10 wherein the process comprises inserting the gene into the genome of a more differentiated 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 S...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 differentiated cell wherein the method comprises forming an undifferentiated cell by the method according to the present invention, differentiating the undifferentiated cell into a recommitted cell, whereby arrangement or rearrangement of the genome and/or nucleus of the cell causes the mutation to be removed.

-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 differentiated cell that produces an entity that cures any symptoms or conditions brought on by or associated with a more differentiated 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 differentiated cell which 1 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 differentiated 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 oo•.

gene to affect the undifferentiated cell, such as bring about the death thereof.

In one preferred embodiment of the present invention, the more differentiated 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 differentiated cell with an agent that causes the more differentiated cell to retrodifferentiate into the undifferentiated cell, and then optionally differentiating 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 differentiated 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 10 present invention but at a lower magnification; Figure 4 which is a microscope picture of cells before the method of the present invention Figure 5 which is a microscope picture of cells prepared by the method of the *present invention; and 15 Figure 6 which is a microscope picture of cells prepared by the method of the present invention.

A. MATERIALS AND METHODS

PATIENTS

Blood samples were obtained in lavender top tubes containing EDTA from patients with B-cell chronic lymphocytic leukaemia's, patients with antibody deficiency (including IgA deficiency and X-linked infantile hypogammaglobulinaemias), patients with HIV infections and AIDS syndrome, a patient with CMV infection, a patient with Hodgkin's lymphomas, a patient with acute T-cell leukaemia, a 6-days old baby with Hodgkin's lymphomas. a patient with acute T-cell leukaemia, a 6-days old baby with blastcytosis, various patients with various infections and clinical conditions, cord blood, bone marrow's, and enriched B-lymphocyte preparations of healthy blood donors.

CLINICAL AND EXPERIMENTAL CONDITIONS The clinical and experimental treatment conditions of patients, including various types of treatment applied to their blood samples, are described in Table 1. Differential white blood cell (WBC) counts were obtained using a Coulter Counter and these are included in the same Table.

TREATMENT OF BLOOD Blood samples, once obtained, were treated with pure monoclonal antibody to the 15 homologous region of the 0-chain of the HLA-DR antigen (DAKO) and left to mix on a head to head roller at room temperature for a maximum of 24 hours. Some samples were mixed first on a head to head roller for 15 minutes after which they were left to incubate in an incubator at 22 0 C. The concentration of monoclonal antibody added to blood samples varied from 10-50ul/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 J-chain of the HLA- DR antigen.

Other treatments included the simultaneous addition of monoclonal antibodies to the homologous regions of the a and a-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 the a-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 CDS, DR and CD3, CD56 16 and CD3. CD45 and CD14, CD8 and CD3, CDS and CD28, simultest control (IgG1 FITC IgG2a PE), CD34 and CD2, CD7 and CD13 33. CD10 and 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 20 majority of the above panel in order to account for the immunophenotypic changes that accompanied different types of treatments and these were carried out separately on different aliquots of the same blood sample. Untreated samples and other control treatments were stained and analyzed simultaneously.

FLOW CYTOM.ETRY 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.

MORPHOLOGY

Morphology was analyzed using microscopy and Wright's stain.

B. RESULTS CD19 AND CD3 PANEL Treatment of blood samples with monoclonal antibody to the homologous region of the 3-chain of the HLA-DR antigen always decreased the relative number of CD19cells. This marker is a pan B-cell antigen (see Table). This antigen is present on all human B lvmohocytes 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.

S 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 CD3-CD19- cells. CD3 is present on all mature i T-lvmphocvtes and on 65%-85% of thymocytes. This marker is always found in association with or gamma/delta T-cell receptors (TCR) and together these complexes are important in transducing signals to the cell interior. Hence, this is an indication that B cells were retrodifferentiating into undifferentiated cells and then being committed to new differentiated cells, namely T cells.

A novel clone of cells appeared in treated blood of B-CLL patients co-expressing the CD19 and CD3 markers i.e. CD19- and CD3" cells (see Charts 1, patient 2, 3 4 at 2hr. 6hr 24hr of starting treatment). Other patients with different conditions showed an increase in the relative number of these clones of cells. These cells were exceptionally large and heavily granulated and extremely high levels of 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.

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 CD19CD3 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 granulitv was detected in treated samples and these cells had high levels of CDI9 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 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 15 treatment of blood with monoclonal antibody to the homologous region of the 3-chain of the HLA-DR antigen. This finding suggests that this type of treatment gives rise to atypical cells that cannot be detected by Coulter (Table 1) but can be accounted for when measured by flow cytometry which counts cells on the basis of surface markers, size and granulity. Furthermore, these atypical cells were accounted for by analysing morphology using Wright's stain under a microscope. Flow cytometric charts of these phenomena are represented in Charts 2, 3 4) and the immunophenotypic .changes obtained on treatment of blood samples seems to suggest that CD19' and CD3 lymphocytes are an interconnected group of cells but remain distinct on the basis of CD19 and CD3 relative expression compared to stem cells.

In Table 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 II positive cells in the blood of these patients are extremely low compared to those found in the blood of B-CLL patients.

Two brothers both with X-linked infantile hypogammaglobulinemia who were B cell deficient showed different immunophenotypic changes in the relative number of CD3cells on treatment of their blood. The younger brother who was 2 months old and not ill, on treatment of his blood, showed a slight increase in the relative number of CD3- cells which was accompanied by a decrease in the relative number of CD3- CD19- cells. On the other hand, the other brother who was 2 years old and was extremely sick and with a relatively high number of activated T cells expressing the DR antigens showed a decrease in the number of CD3- cells on treatment of his blood. No other markers were used to measure other irmunophentypic changes that might have occurred because the blood samples obtained from these two patients were extremely small (Table 2. ID 43/BD and 04/BD).

Patient 91 in Table 2 shows a decrease in the relative number of CD3" cells o following treatment of blood which was accompanied by an increase in the relative number of CD3CD19- cells. However, on analysis of other surface markers such as CD4 and CD8 (see Table 3) the patient was observed to have a high relative number of CD4'CD8' cells in his blood and this was noted prior to treatment of blood samples with monoclonal antibody to the B-chain of the DR antigen and these double o positive cells decreased appreciably following treatment of blood. Furthermore, when further markers were analyzed the relative number of CD3' cells were seen to have elevated (See Table 4).

An enriched preparation of B-lymphocytes obtained from healthy blood donors when treated with monoclonal antibody to the 0-chain of DR antigens showed a dramatic increase in the relative number of CD3' cells which were always accompanied by a decrease in the relative number of CD19 cells and by an increase in the relative number of CD19-CD3- cells. Further analysis using markers such as CD4 and CD8 show a concomitant increase in the relative number of these markers. However, an enriched preparation of T lymphocytes of the same blood donors when treated with the same monoclonal antibody did not show the same changes.

CD4 AND CDS PANEL The CD4 antigen is the receptor for the human immunodificiencv virus. The CD4 molecule binds MHC class II antigen in the B2 domain, a region which is similar to the CD8 binding sites on class I antigens. Binding of CD4 to class II antigen enhances T cell responsiveness to antigens and so does the binding of CD8 to class I antigens. The CD8 antigens are present on the human supressor/cytotoxic Tlymphocytes subset as well as on a subset of natural killer (NK) lymphocytes and a majority of normal thymocytes. The CD4 and CD8 antigens are coexpressed on thymocytes and these cells lose either markers as they mature into T-lymphocytes.

*o 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.

S' CD4'CD8 cells, which are double positive cells, always appeared following o treatment of blood samples with monoclonal antibody to the homologous region of the 0-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 In the same specimens the relative number of single positive cells such as CD8 and CD4' cells was also noted to increase simultaneously. Furthermore, a decrease in the relative number of CD4- CD8- cells which, at least in the case of B-CLL correspond to B cells was noted to fall dramatically in treated samples when compared to untreated specimens which remained at the same level when measured with time. However, measurement of the relative number of CD4'CD8 cells with time in treated samples showed that there was a concomitant increase in the number of single positive cells with a decrease in the relative number of double positive cells. This type of immunophenotypic change is characteristic of thymic development of progenitor cells of the T-lymphocyte lineage in the thymus (Patient number 2,3 and 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 CD4' low cells was affected differently in different blood samples following treatment. The relative number of this type of cells seems unaffected in blood samples of patients with B-CLL following treatment when compared to untreated samples. Such low levels of CD4 expression is found on monocytes and very early thymocytes.

Patient HIV'25 on treatment showed a substantial increase in the number of double positive cells expressing CD4 and CDS simultaneously. On the other hand, patient 91 on treatment showed a decrease in this subrype 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 2,3 and 4).

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

Treated and untreated samples analysed with this panel showed similar immunophenotypic changes to those obtained when blood samples were analysed with the CD19 and CD3 markers (see Table 2) and these antigens as mentioned earlier are pan B and T-cell markers respectively.

Treatment of blood with monoclonal antibodies seems to affect the relative number of DR' B-lymphocytes so that the level of DR+ cells decrease. In contrast, the relative number of CD3* (T-cells) cells increase significantly (see Table 4 and Chart).

26 Furthermore, the relative number of activated T cells increased in the majoriyv 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- CD34blasts 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 (Tcells) and was inversely proportional to decreases in the relative number of DR+ cells (B-cells).

CD56&16 AND CD3 PANEL The CD56&CD16 markers are found on a heterogeneous group of cells, a subset of lymphocytes known generally as large granular lymphocytes and natural killer (NK) lymphocytes. The CD16 antigen is expressed on virtually all resting NK lymphocytes S• and is weakly expressed on some CD3- T lymphocytes from certain individuals.

o This antigen is found on granulocytes in lower amount and is associated with lymphocytes containing large azurophilic granules. The CD16 antigen is the IgG FC receptor III.

A variable number of CD16* lymphocytes coexpress either the CD57 antigen or lowdensity CDS antigen or both. In most individuals, there is virtually no overlap with other T-lymphocyte antigens such as the CD5, CD4, or CD3 antigens. The CD56 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 coexoressing the CD56&CD16 antigens which were heavily granulated and of medium size (see Table 5 and Charts 1, 2. 3 These observations were also accompanied by a marked increase in the relative number of cells expressing the CD3 antigen only (without the expression of CD56 and CD16 markers) and cells coexpressing the CD56&CD16 and CD3 markers together.

In Table 5. patient numbers 2. 3. and 4 represent the same blood sample but being analysed at 2 hours, 6 hours and 24 hours respectively (before and after treatment).

This sample shows that treatment of blood with monoclonal antibody to the homologous region of the a-chain of DR antigen seems to cause spontaneous production of CD56" and CD16" cells, CD3' cells and CD56- and CDL6- CD3cells and these observations were always accompanied by the disappearance of B-cell markers (CD19. DR. CD56. CD16-CD3-).

Onward analysis of this blood sample before and after treatment showed the levels of 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, CD16 and CD3 antigens when compared to immunophenotypic changes observed in treated and untreated samples and this is because the amount of monoclonal antibody added was extremely low relative to the number of B lymphocytes. However, treatment of this patient's blood sample on a separate occasion with an appropriate amount of monoclonal antibody showed significant increases in the relative number of CD3', CD56' CD16- and CD56' and CD16' CD3- cells.

Blood samples of other patients with other conditions showed variable changes in the 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.

AND CD14 PANEL The CD45 antigen is present on all human leukocytes. including lyrohocytes, 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 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 tvrosine 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 0-chain of the DR antigens in created 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 0-chain of the DR antigen seem to give rise to different types of cells that can be segregated on the basis of the level of CD45 and CD14 expression as well as morphology as determined by forward scatter and side scatter (size and granulity respectively) and these results are presented in Table 6 and Charts 2, 3, 4 On treatment the relative number of CD45 low cells (when compared to untreated samples) increased significantly and so did the relative number of cells co-expressing the 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 On analysis of this panel following treatment with time (see Table patient 2,3 and 4 and charts) the relative number of 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 immunophenotypic changes to those obtained with other samples obtained from other B-CLL patients and this is because the samples were analysed at a much earlier incubation time with the monoclonal antibody.. In fact the sequential analysis of bl.ood 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 im-munophenotypic changes measured at time X is not going to be the same at time X plus (its not fixed once induced).

However, these types of changes must be occurring in a more stringent manner in the body otherwise immunopathology would ensue. The effect of treatment of blood samples from other patients -with no B-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 counts.

o o CD8 AND CD3 PANEL The CD8 antigenic determinant interacts with class I MHC molecules, resulting in increased adhesion between the CDS+ 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 CD19 and 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 Chan 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 chances extremely incriminate B cells in the generation of T lymphocyte progenitors and progenies.

Blood samples from a patient with B-CLL (number 2. 3 and 4 Tables 1, 2, 3, 4, 6, 7) in separate aliquots were treated with nothing, PE conjugated monoclonal antibody to the homologous region of the 3-chain of DR antigen and unconjugated form of the same monoclonal antibody. On comparison of PE conjugated treatment clearly indicates no change in the relative number of CD3 positive cells and associated markers such as CD4 which have been observed in significant levels when the same blood sample was treated with unconjugated form of the antibody. However, an increase in the number of CD45 positive cells with no DR antigen being expressed on their surface was noted when measured with time (see Table A finding that was similar to that noted in untreated samples when immunophenotyped with time (Table Furthermore, the relative number of cells expressing CD45 low decreased in time, a phenomenon which was also noted in the untreated samples (when measured with time) of the same patient (see chart 1A).

31 C. COMPARISON OF THE EFFECT OF OTHER MONOCLONAL ANTIBODIES WITH DIFFERENT SPECIFICITY ON T-

LYMPHOPHOIESIS

CD19 AND CD3 PANEL Treatment of blood samples with monoclonal antibody to the homologous region of the a-chain of the DR antigen and the homologous region of MHC Class I antigens decreased the number of CD3- cells and increased the number of CD19- cells.

Treatment of the same blood with monoclonal antibody to the homologous region of the 3-chain of the DR antigen decreased the number of CD 19 cells and increased the number of CD3- cells. Treatment with the latter monoclonal antibody with cyclophosphoamide revealed the same effect (Table 14 patient 5/6 with B-CLL at 2hr treatment).

Onward analysis of CD19- and CD3- cells in the same samples revealed further increases in the relative number of CD3 cells only in blood treated with monoclonal antibody to the homologous region of the 0-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 CD19and 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 Treatment of blood samples of patient 09 with monoclonal antibody to class I antigens increased the relative number of CD3 j cells and decreased slightly the relative number of CD19 32 and CD19-CD3- cells. However, treatment of an enriched preparation of Blymphocytes obtained from healthy blood donors with monoclonal antibodv to the 3chain or c-chain of DR antigen showed similar inmmunophenopic changes to those obtained with patient with B-CLL.

Treatment of HIV- and IgA deficient patients with monoclonal antibody to the chain of the DR antigen increased the relative number of CD3- cells and decreased the relative number of CD19- cells. However, treatment of the same blood sample with monoclonal antibody to the homologous region of class I antigen did not produce the same effect. Treatment of blood samples obtained from patients (34/BD and 04/BD) with B-cell deficiency showed variable immunophenotypic changes when r treated with monoclonal antibodies to the a-chain of the DR antigen, class I antigens and CD4 antigen.

15 CD4 AND CD8 PANEL Blood samples analysed using the CD19 and CD3 panel (Table 14) were also immunophenotyped with the CD4 and CD8 panel (Table 15). Both panels seem to agree and confirm each other. Incubation for 2 hours of blood samples of patients 20 with B-CLL (Table 15, patients 5/6 and 10, Charts 2, 3 4) with monoclonal antibody to the homologous region of the 0-chain of the DR antigen or with this monoclonal antibody plus cyclophosphoamide increased the relative number of CD8and 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 achain 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 j-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 analysed with the CD19 and CD3 panel (Table 14 the same patient). The later findings indicate that the subsequent differentiation is reversible as the undifferentiated cells can differentiate into T-lymphocytes or B-lymphocytes.

DR AND CD3 PAN-EL The immunophenotypic changes obtained with DR and CD3 (Table 16) panel confirm the findings obtained with CD19 and CD3 panel and CD4 and CD8 panel (Tables 14 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 3chain of the DR antigen plus cyclophosphoamide at 2 hour analysis.

From the results, it would appear that the monoclonal antibody to the homologous 15 region of the f-chain of the DR antigen is extremely capable of driving the production of CD3 positive cells from DR- cells.

Furthermore, treatments such as those involving engagement of the a-chain of DR antigens or enasgement of the f-side of the molecule in conjunction with cyclophosphoamide (prolonged incubation time) promoted increases in the relative number of CD19' cells or DR cells.

CD56&16 AND CD3 PANEL Treatment of blood samples, especially of those of patients with B-CLL with high Blymphocyte counts with monoclonal antibody to the homologous region of the f-chain of the DR antigen increased the relative number of CD56&16 positive cells.

In these patients the relative number of CD3' and CD56' and CD16'CD3 cells also increased following treatment of blood samples with monoclonal antibody to the 0chain. 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 CD4S AND CD14 PANEL Blood samples treated with monoclonal antibodies to the 3- or alpha- chains of the DR antigen or to the 3-chain plus cyclophosphoamide or class I antigens were also analysed with the CD45 and CD14 panel (Table 18). The delineation of CD45 low, high and CD45 medium is arbitrary. Treatment of blood sample 5/6 (at 2 hours) with monoclonal antibodies to the 0-chain of the DR antigen or with this monoclonal antibody plus cyclophosphoamide generated CD45- low cells and increased the relative number of CD45' medium cells. However, the former treatment increased the relative number of CD45 high cells and the latter treatment decreased the relative number of CD45- medium cells and these changes appeared to be time dependent.

Blood samples of patient 5/6 and 10 (B-CLL) on treatment with monoclonal antibody 15 to class I antigens showed a decrease in the relative number of CD45' medium cells and similar observations were noted in blood samples 09 and HIV' following the same treatment when compared to untreated samples. Treatment of blood samples of HIV+ and IgA/D patients with monoclonal antibody to class I antigen increased the relative number of CD45' low cells when compared to untreated samples or samples treated with monoclonal antibody to the 3-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, heavily granular and expressing intense levels of antigen were noted in treated blood samples with monoclonal antibody to the homologous region of the O-chain of DR antigen of MHC class II antigens (see Charts 1, 2, 3, 4 CDS AND CD28 PANEL The CD28 antigen is present on approximately 60% to 80% of peripheral blood T (CD3-) lymphocytes, 50% of CD8- T lymphocytes and 5% of immature CD3thymocytes. During thymocyte maturation, CD28 antigen expression increases from low density on most CD4'CD8 immature thymocytes to a higher density on virtually all mature CD3 CD4' or CD8- thvmocvtes. Ceil 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 cytocoxicity, that is major histocompatibility complex (MHC) class I-restricted.

Suppression of cell proliferation is mediated by the CDS-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 0-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 (TdT)" B- and T-lymphoid precursors in normal bone are CD34-, The CD34 antigen is present on early myeloid cells that express the CD33 antigen but lack the CD14 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 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 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 cechain of the same antigen revealed marked increases 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 Tlymphopoiesis via B lymphocyte regression.

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 1-chain of the HLA-DR antigen plus cylophosphoamide, whereas the latter process was noted at 24 hours incubation time with the same treatment in the same sample (Chart 2).

Treatment of blood samples of HIV patient (Table 20 patient HIV-) with monoclonal antibody to the f-chain of the HLA-DR antigen markedly increased the relative number of CD34- and CD2'CD34' cells and so did treatment of the same blood sample with monoclonal antibody to the F-chain of the HLA-DR antigen and monoclonal antibody to the a-chain of the same antigen when added together.

However, treatment of this blood sample with monoclonal antibody to the a-chain of the HLA-DR antigen did not affect the level of CD34" cells. Treatment of blood samples obtained from a 6-day old baby (BB/ST Table 20) who was investigated at that time for leukaemia and who had very high number of atypical cells (blasts) in his blood with monoclonal antibody to the F-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 immunophenotypic changes.

On analysis of untreated blood samples the relative number of CD34' and DR- cells were markedly increased and on treatment with monoclonal antibody to the 3-chain the relative number of CD34- cells further increased but were noted to decrease on treatment with monoclonal antibody to the a-chain of the HLA-DR antigen or treatment with monoclonal antibodies to the c and f-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 f-chain of the HLA-DR antigen alone. On analysis of treated and untreated blood aliquots of the same patient 24 hours later the relative number of CD34+ decreased with all above mentioned treatments except it was maintained at a much higher level with monoclonal antibody to the 3-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 f-chain promotes the production of more CD34' cells from CD2CD34- pool or from more 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 CD19 beads) on treatment with monoclonal antibodies to homologous regions of the ,3-chain of MHC class II antigens. These were accompanied by a change in the morphology of B-lymphocytes.

B-lymphocytes were observed colonising glass slides in untreated blood smears were substituted by granulocytes, monocytes, large numbers of primitive looking cells and nucleated red blood cells. No mitotic figures or significant cell death were observed 15 in treated or untreated blood smears.

o.

The results of Table 20 also demonstrate a further important finding in that according to the method of the present invention it is possible to prepare an undifferentiated cell by the retrodifferentiation of a more mature undifferentiated cell.

D. MICROSCOPE PICTUES In addition to the antigen testing as mentioned above, the method of the present invention was followed visually using a microscope.

In this regard, Figure 1 is a microscope picture of differentiated B cells before the method of the present invention. Figure 2 is a microscope picture of undifferentiated cells formed by the retrodifferentiation of the B cells in accordance with the present invention wherein the agent was a monoclonal antibody to the homologous regions of the -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.

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 p-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 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 15 different lineage as the original differentiated cells.

The retrodifferentiation of T cells to undifferentiated stem cells by the method of the 0 present invention followed by commitment of the undifferentiated cells to new differentiated cells being of a different lineage as the original differentiated cells was also followed by microscopy.

E. SUMMARY In short, the examples describe in vitro experiments that reveal extremely interesting findings regarding the ontogeny and development of T and B lymphocytes which can be utilised in the generation of stem cells to affect lymphohaematopoiesis in peripheral blood samples in a matter of hours.

Treatment of peripheral blood samples obtained from patients with B-cell chronic lymphocytic leukaemia's (B-CLL) with high B lymphocyte counts, with monoclonal antibody to the homologous region of the 3-chain of class-II antigens gave rise to a marked increase in the relative number of single positive (SP) T-lymphocytes and 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 samoies were treated with monoclonal antibodies to the homologous region of the c-chain of class-II antigens or to the homologous region of class-I antigens.

Treatment of whole blood obtained from patients with B-cell chronic lymphocytic leukaemia (CLL) with monoclonal antibody to the homologous region of the B chain 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 CDS I 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 imrmunophenotypic changes that took place in the generation of such cells were identical to those cited for thymocyte development, especially when measured with time.

The percentages of double positive cells (DP) generated at 2 hour incubation time of whole blood with monoclonal antibody to the homologous region of the 3-chain of the DR antigen. decreased with time and these events were accompanied by increase in the percentages of single positive CD4' CD3- and CD8- CD3 cells simultaneously and at later times too. TCR a and 0 chains were also expressed on these types of cells.

B-lymphocytes were constantly observed to lose markers such as CD19. CD21, CD23, IgM and DR and this coincided with the appearance of CD34' and CD34' CD2- cells, increases in CD7- cells, increases in CD8' CD28- and CD28- cells, increases in CD25 cells, the appearance of CD10- and CD34' cells and CD34- and CD19" cells increases in CD5' cells, and cells expressing low levels of antigen. These changes were due to treatment of blood with monoclonal antibody to the homologous region of the O-chain of HLA-DR antigen.

The immunophenotypic changes associated with such treatment is consistent with retrodifferentiation and subsequent commitment 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 6-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 s-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 CD19 and CD3 and DR and CD3. Investigation of t .the supernatant of treated and untreated samples of patients with HIV infection using 15 nephlometry and immunoelectrophoresis reveals increased levels of IgG indicating that the B-cells must have passed through the plasma cell stage. The increase in the relative number of all above-mentioned cells was also accompanied by the appearance of medium size heavily granulated cells expressing the CD56&16 antigens in extremely high amounts. Other cells which were extremely large and heavily granulated were observed transiently and these were positive for CD34 and double positive for CD4 CD8 markers. Other transient cells were also observed and these were large and granular and positive for the CD3 and CD19 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 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 a-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-lymphocyte markers and without myeloid antigens was observed on a separate population of cells. These 10 particular observations were not seen in untreated samples or in samples treated with S. monoclonal antibodies to class I antigens or the homologous region of the c-chain of HLA-DR antigen (see Charts 2 These final results suggest that B-lymphocytes once triggered via the 3-chain of the HLA-DR antigen are not only able to regress into T-lymphocyte progenitor cells but are also capable of existing into the myeloid and erythroid lineages.

It should be noted that the stem cells that are produced by the method of the present invention may be stem cells of any tissue and are not necessarily limited to lymphohaematopoietic progenitor cells.

Other modifications of the present invention will be apparent to those skilled in the r> art.

TABLE 1 CLINICAL DIAGNOSIS OF PATIENTS AND EXPERIMTH..,AL CONDITIONS OF BLOOD INCLUDING COULTER COUNTS (14BC) FOLLOWING AND PRIOR TRE-,-ATMENT OF BLOOD

SAMPLES

SPEC IMENS PATIENT DI1AGNOSI1S EXPT WBC/L -LYMPH -#LYMPH/L AGENT IDCOND X10 9 1O'X 9MLm B A B AB8 A Lm I 8-CLL 12H2 100 NO 6< 36.1 iNO ANT-?7 C0_ 5 2 B-C 39 9.6 6.3 29.9 qNi A T 22C .150 2H-R AT 39. 1 37. 7 7- -A1 4TIj 29.9 23. 3 P 3 -CL' 01HR 39. 5 9.3 71 0967.2 23.3 ANT' AT 220 6.2 I HP.' 39.5 37. 7 71 .9 7 2.5 ANTi -3 AT 220C 28. 3 27.4 PC 3-OLL2~HR73 6 6.5 28.4 A~- AT 220 39 9.3 6.2 en0s 2 4HP., 7 3 7D. A NT! AT 220 39 36.2 28.4 2 5.5 PE 0 B-CLL 2HR AT 2 20 ANTI -8 ANIlTEI A so ANTE so A NTE -B &TOX ir

AGENT

25+25 PATIENT lAGNOS IS EXPT WBC/L =LYMPH #LYMPH/L ACENT iO COND X10-9 B A 1OX-9 fL/mL B A BA 6 3- CLL 24H 01~' AT 22,C 213 90. d S1.96 ANT 178 94 2 ~li 130 9 Cl A 8 5-CLL 2 R 16 7 19 'C I 3.0 AT-3 .AT 22- I__ 9 8-CLL l21HR 89.5 Q7 25.1 76.2 ANT 3 AT 22C I I PANTI-I

ANTI

sl.9 -A N T io 4 B-CLL 2HR 19.3 NO 86 NO 16.7 NO ANTI-B AT 22C

ANTI-!

92 OUT 2HR 5.4 ND 74.5 N NO ANTi-3 PAT I ENT AT 22C I 87 OUT 2 HR 4.8 NO 59.3 NO NO ANTI-8 ATIENT AT 22C 91 OUT 2HR 4.2 NO 54.0 NO NO ANTI -3 PATIENT I AT 22C 54.0 NO 21 OUT 2HR 3.9 NO d.4 NO NO ANTI -B PATIENT AT 22C I 1 1 PATIENT DIAGNOSIS EXPT WBC/L LYMPH -rLYMPH/L A GENT- ID COND X1O-9 B A 10X-9 MLIML B 8 A CUT 2-HR 7.2 NO 0-N D~T- A-T 22C f 26CMV 1H3R1-a No 7-3 moN 4T- IFANT AT2C 93 HiVe- 5. NO N -,TC BS/ST 40% BLAST AT 2 0. N 0 2. MD A., INM SLCOD 2_2 60.5 NO 6 DAYS OLD 2d4R AT 22C HIf V 2 AIDS 2 H R 7 ND.5 ND-'13 N 2.6 MN0 ANTi- AT 22C AN TE 43/8D B CELL 4H ANTI B DEFI CIEN AT 22C ANT I 03,'BD B CE LL 4HR ANT i -B3 DEFICIENT AT 22C ANT -d 2C PATIENT 0DIAGNOSI1S EXPT WBC/L *YMPH #L1-Y14P H/L AGE--N T ID COND XI.O-9 B A 10X-9 ML/mL B A B A hi V AIDJS H R AT 22C AMNTI IgA-D A 6HR AT- DEFI1C IENT AT 22C ANTI1 EXPT COND EXPERIMENTAL CONDITIONS 3 BEFORE FA AER ANTi -8 monoclcnal antibody to the 'nomoiccgous region of the 3-chair 1 of HLA-DR anice-n Ail)T't- mnocclonal ant-,body to the homologous region of: the L-chain of HLA-DR antigenn ANj1TII monoclonal antibody to th."e homoloccus reclion ff Class iantlQens ;%,,T7I-AB both ANTI-B and ANTI -A added togather ANT7-4 monoclonal antibody to the C04 antigen ANT 1+4 :ANTI1-! and ANTI-B, and AINTI-4 added togatner Cytoxic agent Cyclophophamide ML/,ml micro litre per ml L :litre *i TABLE 2 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONS BEFORE AND AFTER TREATMENT OF BLOO SAMPLES WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE 8 CHAIN OF THE HLA-OR WITH C019 AND CD3 MONOCLONAL ANTIBCDIES.

PATIENT CD19+ CD3+ XCD19+CD3+ C 0 3 C019- CD19+HGCD 3- FC+ SA B AB A AaB A 1 88 40 5 19 I 2 6 26 0 12 2 73 15 10 33 2 7 15 41 0 3 73 11 11 33 2 2 1 52 0 2 4 71 13 11 37 2 2 16 47 0 2 85 40 5 16 1 1 6 26 3 18 6 85 43 5 18 1 1 6 27 3 7 90 72 2 4 0 2 7 8 0 14 8 62 25 7 13 0 1 29 55 2 6 9 90 85 2 3 0 0 2 1 1 4 78 50 7 14 0 0 14 26 0 8 92 12 10 38 49 0 1 49 40 0 0 91 7 3 35 29 0 1 59 67 0 0 87 5 3132 3811 1 63 58 0 0 21 1 1 27 29 1 0 71 70 0 0 34 1 1 13 13 0 2 86 84 0 0 39 10 6 23 25 0 0 67 69 0 0 93 6 3 26 27 1 1 68 70 0 0 BB/ST 1 1 12 13 0 0 87 86 0 0 7 2 26 27 0 0 68 67 0 0 43/B0 0 0 40 42 0 1 58 54 0 0 04/80 0 0 49 41 0 3 43 41 0 0 HIV+ 1 1 10 14 0 0 89 87 0 0 IgA/D 10 1 21 25 2 3 167 71 0 0 B: oeoetetet A fe ramn B: oefore Lreatment A: after treacment a.

a TABLE 3 IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONS BEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITH MONOCLONAL ANTIBODY TO THE 8 CHAIN OF THE HOMOLOGOUS REGION OF THE HLA-DR WITH MONOCLONAL ANTIBODIES TO CD4 AND CD8.

PATIENT .CD8+ CD04+ CD4<CD8+ ;CD4-CD. CD4+LOW I i 0 CD4+LOWa 3 A 8 A B A 3 A 8 A _12. 16 2.9 11.8 0 32 2 3 67 6 0 C 2 .2 132 9.1 24.3 0 9 7- J6 5 .3 3 V.2 13.1 7 23.9 0 8.2 73.8 L3.1 6.3 6.6 10.1 ?a.2 7 6 2 .9 0.3 2 77.5 42K a S2.9 1I.

2 S 7.6 T 293 62 3 0 0 6 N 12 NC 8.1 ND 1.7 ND 73.7 ND 0 .9 2.6 1.9 2.8 O C 95 9.8 4.3 D 0 8 3.2 7 3. .9 0.1 2 87.2 79.8 4.3 6 9 2.8 2.9 3 3 0 0 94 9.1 0 5.7 9.41 4 7 9.1 0.6 0.8 88.7 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 87 16.3 21.8 13.4 20.4 2.9 2.6 59.5 48.9 7 5.6 21 11 24.1 9.1 15.2 1 2.6 69.6 53.2 3.7 4.2 34 9 d 11.9 5.7 4.9 2 3.3 67.6 65.3 14.4 14.5 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 65.5 65.9 3.4 1.8 8B/ST 6.3 13 5.7 7.3 2.2 1.1 3-.7 70.3 50.3 7.6 24.1 24.9 0.8 1.1 1.3 5 70.2 69.3 2.9 3.8 a a. a.

49 TABLE d IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHER CONOITIONS BEFORE AND AFTER TREATMENT OF SAMPLES WITH MONOCLONAL ANTIBODY TO ThE 8 CHAIN OF THE HLA-OR WITH MONOCLONAL ANTIBODIES TO CD3 AND0 DR PATIENT T DR+i CD+ COOR+ DRC03 DR+HCD- _3 AI K IA !a K 5 1 H A -7 3 45. 3 T 1. I 2 7-2 1 19.d 19 [29.2 1 39 8 t 3 36.3Q 91_ 3 77 18.3 134 29.4 i S 8 I l.

-7.8 19.2 29.5 6.2 10.3 3I3.2 KIj 7 N 1. lNO 11.35 INO 9. HO t2 2 NO 0 7 6 IND i I I9 I I NO I 7 8 124 2.5 0.7 0. 7 1 4.2 6 61.8 28.9 6+.5 11.2 2 3.3 22.6 54. 6 1 V NO 82.6 4.7 4.3 9.8 3 9.3 22.2 0 17.9 92 23.8 14.1 39.3 41.9 4. 13. 132.4 1 0 1 13.3 7.9 29.6 32.5 3.4J 2.9 33.4 56.5 0 0 ,Q 7 14.8 12.2 28.4 34.1 5.5 6Vsi;i 46.: 0 0 1, NO 11.9 12.9 10.4 13.7 0.8 006 7-0.7 [72.8 0 0 19 25.6 13.7 24.6 25.2 3 2.8 d5*5 25.2 0 13.3 8.9 18.4 18.9 9.9 10.1 H.2 61.7 IS :Z2.1ST 44.2 132..5 111.7 12.2 0.8 0.8 43 49..4 ,0 4. 6 TABLE iMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTH ER CONDITIONS BEFORE AND AF ER TREATMENT OF BLOOD SAMPLES WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DR WITH MONOCLONAL ANTICOIES TO CD16 56 AND C3.

PATIENTS C056-&16 C03+ I C05 &16 CD3 i C056+&16-C03* 2 2 I 1 10.7 1' 0 3 73 2 113 3E C 12 4 32.6 6. 57 21 1 3 12 36.2 12.1 34.5 07 7 73.5 23 j 1 2.2 32.6 12.4 39.6 0. 74.7 22.2 NC 11 ND 9.1 1\1C 2 .6 ND 73.5 7 0.8 0.8 2.B 2.J 0.3 0.2 96.2 96.4 2a 8 5 J 2.4 0.9 E. 31.1 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 A 3.9 29.8 29.4 3 3.2 63.3 63.3 87 47.9 46.4 28.8 36.5 5.8 3.7 16.9 13 21 9.4l 19.7 23.6 4.2 6.7 66 59.5 p p p -7i:' a.

a a a a a 0* TABLE 6 IMMUNOPHENTYPING OF PATIENTS WITH 8-CLL AND OTHER CCNDITIONS BEFORE AND AFTER OF TREATMENT OF BLOOD WITH MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B CHAD4I OF THE HLA-DR WITH MONOCLONAL ANTIBODIES TO CD45 AND C014.

PATIENTS CD45+H CD45 L 21.9 1.8 2 52.2 8.3 38.3 15.3 3 3 3 2 2 9 3 3 8 79.2 2.1 7 15.7 63.1 84.6 34.9 9.4 5. .2 45 13.9 811 55 71.71 13.3 9 T SE r 1,9.7 47.3 16.3 8 12.1 1.9 92 51.7 64.7 27.4 26 6 5.9 3.6 91 -94 J9.2 40.4 44.3 6.5 3.2 87 52.4 61.5 36.1 28.7 76.

21 -13. 43.3 44.3 47.6 6.2 3.3 3" d 2(.6 54.8 59.6 13.3 9.7 39 -3 7 46.3 30.5 d2.1 i.5 8.8 93 I HIV2 26.0 66.8 63.5 6.8 6 7 1aA/D 274 59.8 41.9 33.3 5.9 4.

0~ 0 00*0@ 00 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 TH-E CHAIN OF THE HLA-DR WITH MONOCLONAL ANTIBODIES TO CD8 AND C03.

PATIENTS CD8+ CD3+ CD8+CD3- CD8-CD3- KB A A A A 0.6 1.3 7.5 19.3 4.2 19.3 37.7 3.

3 1.1 1.4 8.3 20.3 5.6 13.4 34.3 2.9 8.3 27 3.9 16.6 3-.2 53.1 92 3.5 1.9 27.6 25.2 18.4 19 50.3 52.8 91 4 3.1 13.2 19 14.1 12.6 63.6 65.3 87 5.7 3.9 19.9 23.6 15 4 17.4 58.3 21 4.8 7 16.3 17.3 13.7 13 65.2 62 34 3 3.6 5.2 6.7 7.6 7.5 84.1 82.3 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 MONOCLONAL ANTIBODIES TO CD45 AND CD14.

TIME DR+CD45+CD14+r CD45+L 2HR 81.7 8.2 8.2 6HR 80.7 8.1 10.6 24HR 79 1.1 18.4 TABLE 9 IMMUNOPHENOTYPING OF A PATINENT WITH B-CLL WITH TIME AFTER TREATMENT OF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR MEASURED WITH MONOCLONAL ANTIBODIES TO CD19 AND CD3.

TIME CD19+DR+r CD3+ CD3+DR+ 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 TABLE r IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTER TREATMENT OF BLO00 WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-0R MEASURED WITH MONOCLONAL ANTIBODIES TO CO4 AND C08.

TIME CD8+& OR+r CD4+ CD4+&CD8+&DR-r CD4+DR+ CD4-CD8-DR- 2HR 77.6 6.8 5.4 1.3 3.8 6HR 75.8 6.7 6. 1.8 9.3 24HR 77 6.4 4.8 1.9 11 TABLE 11 IMMUNOPHENOTYPING OF A PATIENT WITH 8-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 CD3 AND DR.

TIME DR+ CD3+ CD3DR+ C03+OR- 2HR 75 9.5 L.2 10.9 6HR 74.8 8.8 10.9 24HR N NO N NO NO TABLE 12 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 CD16&56 AND CD3.

TIME CD56+&16+DR+r CO3+ CD56+CD16+&CD3+DR+ CD56-CD16-&CD16-

DR-

2HR 82.5 9.5 4.1 6HR 84.3 7.5 4.1 3.3 24HR NO ND NO NO 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+ CD3+ 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 a 4 *4 4.

9*4* TABLE 14 IMMUNOPHENOTYPING OF PATIENTS WiTH B-CLL BEFORE AND AFTER TREATMENT OF BLOO0 WITH.

MONOCLONAL ANTIBODIES TO THE HOMOLOGOUS REGION OF THE A-CHAIN OF THE HLA-0R, THE HOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-OR THE TO MONOCLONAL TOGETHER.

MONOCLONAL TO THE HOMOLOGOUS REGION OF THE B-CHAIN PLUS CYCLOPHOSPHOAMIDE AND THE HOMOLOGOUS REGION OF CLASS I ANTIGENS MEASURED WITH TIME.

C019 I Co03 COD1 -C3 C0 9-CD3iD 8 A A A A A AS A AI l A A A Al 8 A AS A C 8C A C A 0 2H 86 91 54 40 89 5 6 23 5 1 1 3 2 1 6 4 27 33 2d N 88 51 60 86 N d 1l8 10 d N 2 2 3 N 29 28 7 2U T7 N 59 N 80 7 N 13 N 7 N N 0 N 26 N 12 09 92 8 N N N 32 N N N 8 N N N 1 59 N N N 56 43 /8 6H 0 N 0 0 40 N 42 43 49 0 N 1 0 1 58 N 54 54 e7 04

/IB

0 6H 0 N 0 0 0 49 N 41 45 46 0 N 3 1 3 N 42 4d 41

HI

V+

6H 1 N 0 N 1 10 N 14 N 12 0 N 0 0 89 N 86 N 87

A/

6H 10 N 1 N 12 21 N 25 N 20 2 N 1 N 3 67 N 71 N 68 8 Before: A After: AB after addition to antibody to beta chain: AA after addition of antiooov to alpha chain: ABC after addition of antibody to either alpha or beta chain and cycloposphoamide: Al after addition of antibody to Class

I.

*0994* 0 4 4:: TABLE CD8 AND CD4 0 *5*S

S

OS

A S A B A, A Al 8B A 8 4 A A B C A 5 r 2H 3 2 10 4 2 2 S 8 3 0 3 2 9 9 74 7 93 24 N 3 9 3 3 C 2 0 N 4 3i 90 93 2H 3 N 7 rl 4 N 7 N 3 1 N 2 M 1 9; J 3 92 09 ?4 10 N M N 15 21 N N N '38 2 N N 2 1 IN N M 53 TABLE 16 CD3 AND DR CD3+ C13-DR- CD3-DR- A AS A Ai B A AS A Al B A A3 A A[ 8 A As A A! A C A BC A SC 4 BC 6 2H N 90 Sd N 87IN 4 12 N 4 N 2 10 N 3 NI 1 22 N 5 2H 83 63 N 81 4 N 8 N 4 4 N N 4 9 N 23 N 12 09 24 14 N Nl N 13 JO N N N 36 3 N N N 3 Si N N N 47 TABLE 17 CD16&56 AND CD3 0e 0e S OSO S

-S

'i.

S

1 kCD5,6&i1 C03- 1 CDS63±6iC03 C056-&16-C.> t0 A A3 A AI s A AS A A AS A A! A A SC A SC SC A 2 l NO 13 N 4 N 9 N N1 I~ 94 i SC i' 0 N6 1- N 6 1 M 2 M 1 92 65 M 00 09 2 2 Nr N1 N 4 36 Ni ~J N 382 N N1 21 20 l N N 19 TABLE 18 AND CD14 CD45 L CDd+n CD4+5 CO45 CD1d- 1086 A AB A A[ 8 A AB A AIB A AS A A[ B A AB A A! A BC a BC A SC A BC 6 2H0 0 5 10 0 d4 43 50 50 32 55 3 50 31 67 1 i 1 2 0 ON 0 N 0 43 N 5d N 35 54 N 42 N 62 1i 1 NO 09 N N N 1 18 N N N 16 71 N N N 76 7 N N N SH d N 3 N 6 63 N 6 1N 41 23 N 27 N 40 7 N 7 N 7 foes

S

C

S

40N 31 N 44 2 N N 47 N 60 N 44 6 N 4_ 66 j 2 N? N 4 40 N .31 N 44 47 N 60 N 44 6 N 4 N 6j 58 TABLE 2.9 C08 AND CD28 TABLE C034 AND CD2 -I C034+ CD2+ CD34 CO2+ I iO B A AB A A a AAB A AlI8BA ASA A!B8A AB A A! A BC I A BC A BC A BC 6 2H N 1 34 N N N 6 13 N N N 3 30 N N N 90 21 N N 2A N 1 6 9 N N 7 23 4 N N 3 33 43 N N 87 34 34 N Yr 2H 21 12 13 N20 21 2112N 4 59 14 N 7 7364 60 N 3

BB

/S

T

2H 26 2313 14 N 1114 115N3130 136 N 2 3228 35 N 1 7 24N11 29 11 NIN13 129 N N27 9 18 N N 484961 N CHART 1 UNTREATED AND TREATED BLOOD SAMPLE OF PATIENT 3 TO iThE HOMOLOGOUS REGION OF TIHE fl-CHAIN OF HLA-OR IMMUNOPHENOTYPIC CHANGES OF 4) WITH MONOCLONAL ANTIBODY ANTIGEN MEASURED WITHd TIME.

a

THOUT

NOT-i NGOO I 1,00001 MO 0 "iNG03 001003 NOTH ING00A

NOOG-

001004 MOTH ING 0OS NO1D00 001005 NOTHINGOO6 NOO 06 00 1006 N0O3 NOO007 001007 WI1TH '4rTH002 WE 002 002002 Wi TTH00~' WE 004! 002004

WITHOOS

WE 005 002005 WI THOO 6 W EOO00S 002006 WAITHOO7 WE 007 002007 W004 WEOO8 002008 C04l C D 31 C03 CD4 C03 CD03 CD3 CD3 C03 CD3

COO

CD3 CO3 Fl12 Ccl 1 CD 14 CO 14 CD 19 CDI 9 CD i9

COB

COB

CO8

OR

0 R

OR

CDS60&16 C056& 16 CD56& 16

COB

COB

CD8 T M 2HIR 6HPR 24HR 2H 2 4HR 2HR 6HR 2dHR 2HR 6Hnr 2 4HR 2HR 6HR 24HR 2HR 6HR 24HR CHART !A IMMUNOPHENOTIYPIC CHANGES OF UNTREATED AND TREATED SLCO S AMPLE OF PATIENT 3. '4) WITH MONOCLONAL ANTIBODY TO Th-E HOMOLOGOUS REGION OFTHE 2!-CHAIN OF HLA-DR ANTIGEN CONJUGATED TO PE MEASURED WITH TIME.

fn T IME 'w"L0G C045 C 1- 2 HR C" L 0 3 CDd5 colIGH 003003 C D~l5 2D 2dH WeL 0'05 C03 cm* "-HR C.poDOOS CD 3 COGR WLDGCD4 COB 9H '.A:WL06 Cod Cos 2:-i 003006 C04 CO 2 41; 1,L 0 07 CD3 mH WE L 0 07 C03 0 R 6HR 4 L00Q CD3 C065&i6 2HIR WAEL 008 C03 C056&10' 0HP '4O05 CD3 COa 2HR *WELOO9 rD3 COB 6HR CHART 2 IMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED BLOOD OF PATIENT WI7H MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF TIHE a-CHATN OF HLA.DR ANTIGPI. THITS ANTIBODY AND CYCLOPHOSPHAMIDE. MONOCLONAL ANTIBODY TO TH,-E 'HOMOLOGOUS REGION OF TH'E ci-CHAIN OF HLA-OR ANTIGEN AND MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF CLAkSS I ANTIGEN MEASURED WITH TIME.

WETH WTHOUT cLI FL T A2C: Ao& C~d C0l A2A-'-A CDL, AiD0O!: .47 C D Cl INCHO C03 CD19 2Hm C23001l:AB CO3 C019 C2AOOI: AA CD3 C019 H NAC -IABC COO CDI9 H £1001: CO3 COli9H A12dHOD1:Al C03 CD19 2H A2824HOOI: AS CD3 CD19 2 4 HR A2A2AH001 :A COO CDig 2dHR A2SX24H001:-AS CD3 CD! 2 4HiR

C

NDDD1 'D4 COB 2HR' 028001: AS Cd, CO8 2H R D2AODI: AA C04 COS 2HiR DNA0DO1:ABC CD4 CD8 2HR 01D001: Ai C04 C08 2HR D124HDOI:AE CD4 CD8 24HR *D2BX24H00I:AB C04 CD8 24HR c 028001: AB C04 CD8 24HR D2AOI: AA C04 CO8 2 4HPR_ E1001: A[ C03 DR 2HiR E2B001: AB C03 DR 2H.R E2AOO1:. AA CD3 DR 2 Hr F1001: A[ CD3 CD56&16 2HuR -28001: AB CD3 CDS60&16 2HR AA CD3 C056&16 2 HR G1001: At CD28 COS 2 HR G2ADOI: AA C028 COB 2HR -S28001: AB CD29 CD8 2H P H1O01: At CD7 CD33&13 2W? H 2 A001: AA C07 C0'33,1 3 2,L: WITH W UTT H2C1 AS C D]3 13 AC2 CD- 12500 1: iMk J2AOOI: AA C C3 Ou 2 a2A,2 CO! AL',-CC '0282AH001:A.B C D 3 C02 62S2HOOi 3S CdC C12 2~H

ABC

K2B001: A"U CoiC CC025 21H r K2AOOI: A, C I CG2?S 6 3 CHART 3 IMMUNOPHENOTYPIC CHANGES OF UNTEATED AND TREATED SLIOC OF PATIENT Ca) WTIHI MONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGION OF THE .3CHAIN OF HLA-DR ANTIGEN.

I rIUT F F 1-2 N100 CD~5014 RH r00% C--,CV H CHOG 03c 01 iH iClCC- COB r. 1\1 CIO1CO DR2R FNOC 2 DE1 21HR "t'Co CO rOE&-.6 2HP~ 0 0203 COB8H HNOO1 CD27 CO8 2H.R .01 Co- CD5 2PHR [INOD! 013 CD29 2H.R 2001 CoDiO 0020 2:-R JiNcol C 04 51RA 0025 2HiR *2 0 01 C D 41,5A C-025 2H4R KNOO I 0057 0023 2HR K2001 00ul57 0023 2r-.R

'A

CHART 4 IIMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREATED ELCOD SAIMPLE OF PATIENT (lo) WITH MONOCLONAL ANTfIlBODY TO THE HOMOLOGOUS REGION OF ThiE *CHAIN ON HILA-DR ANTIGEN AND MONOCLCONAL ANTIBODY TO THEc HOMOLOGOUS REGION OF C'Li'-.SS IANTIGEINS.

W iTH IITHOUT 7! TL2 I CLL1DGI C6d CC)A2H CLL2001 CDB C')i4 C-LL1OD3 CD03 CDic92- CLL2003 CD 3 COD 9 2H ::CLLOCO4 CNd CO8B HR ILLID i~dC u C08OBi .ClL2004 CDo CDB c CLLOOS- CD3 DR 2 HR CLL1005 C03 DR2 uI C.LL200 C02 OR 2rHR *CLLDDD6 CD3 C0SG&1S 2HR r LLIDDO 003 CDBG&).CI i6 2H R CLL2006 C03 C056,2,6 2FIR

Claims (17)

1. 2. A method according to claim I wherein the committed cells are hemopoietit; cells.
2. 3. A method according to claim I or claim 2 wherein the agent engages a receptor .that, mediates capture, recognition or presentation of an antigen at the surface of the comitted cells.
3. 4. A niethiodf according to any one of claim I to 3 wherein said incubating is from 2 to 24 hours. A method accordinig to any one of the preceding claims wherein the. committed cells-are non-cancecr cells.
4. 6. A method according to any one of the, preceding claims wherein the :.c.Ormitted cells are differentiated cells.
5. 7. A method according to anyone of the. preceding claims wherein the committed-cells are selected from T-cell coloty-forming cells. (CFC-T cells), B-cell col ony-foriuing eelkI (CFC-B cells), eosihophI colony-forning cells (CFC-Eosi'n cells), basophil. colony-forming cells (CFC-Bas Mels), granulocyte/monocyte colony- forming cells'(C-FC-GM cells), megakaryocyre -colony-forniiig cells (CFC-MEG cells), erythrocyte burst-forminig cells (BFC-LE cells), erythrocyte colony-forming cells (CFC-E cells), T cells aind R cells.
6. 8. A method according to any one of the preceding claims; wherein the CD34* cOAis are M'Aajor 1-fistO'cornpatibility Complex (IHC) lass i( an r MHC classircl.
7. 9. A method qccotding claimn 3 wherein. the receptor is anl MI-IC class I antigen or anl MHC class.II antigen. A method according to claim 9 wherein sa- id class I antigen is a Human- Leukocyte-Associated (lILA) -A receptor, an HLA-B receptor, anl FTLA-C receptor. anl I-LA-E receptor, an HLA-F receptor or an I-LA-G'receptor and said class [I anti geri is an FILA-DM receptor. an*HLA-DP receptor, all 1-LA-DQ receptor or anl HLA-DR receptor. It. A method according to claim 10 whereithiereceptor is an 1-LA-DR roceptor.
8. 12. A method according -to claim I1I wherein the receptor comprises a 0-chain.
9. 13. A method according tolclaii 12 wherein dhe j3-chain hias homologous regious.
10. 14. A method according to claim 13 wherein thc receptor comprises at least the hiomologous regions of the P" chain. of lILA-DR. *too& 15. A mjethod according to claim 3 whierein the agent is ain antibody to the receptor.. *16. A method according to claim 15.Wherein the agent is a monoclonal antibody to the receptor.
11. 17. A method according to claim. 16 -wherein the antibody is selected from modoclonal antibody CR314 and rnonocloial antibody TAL I
12. 18. A method according to.any one of the precding claimns wherein the agent is used ill conjunction, with a biological respqnse nioclifier.
13. 19. A method accord'ig to claim 18 wherein the biological response ruodifier is an alkylating agent. 67 A method according to claim 19 wherein the adkylting agent is or comprises cyclophosphoamide.
14. 21. A method of increasing-the relative number of CD451ow cells in a cell population including commnitted cells, which method comprises: contacting the cell population with an agent that operably engages said committed cells; and (ii) incubati.ug committed cells that are engaged by said agent such that the relative number of CD451ow cells increases as a result of said engaging.
15. 22. A method of increasing the relative number of CD3 negative DR negative cells in a cell population including committed cells, which method comprises: contacting the cell population with an agent that operably engages said conmuitted cells; and (ii) incubating conmmitted cells that are engaged by said agent such that the relative number of CD3 negative DRnegative cells increases as a result of said engaging.
16. 23. A method of increasing the relative number of CD34 cells in a cell population including committed cells substantially as hereinbefore described. i 24. A method of increasing the relative number of CD45 low cells in a cell population including committed cells substantially as hereinbefore described. A method of increasing the relative number of CD3 negative DR negative cells in a cell population including committed cells substantially as hereinbefore described. Dated this THIRTY FIRST day of JANUARY
17. 2000. Ilham Mohamed Saleh Saeed ABULJADAYEL Ghazi Jaswinder DHOOT Wrav Associates Perth. Western Australia Patent Attorneys for the Applicant