Source: http://www.google.com/patents/US6090625?dq=U.S.+patent+number+7,325,728&ei=Y93TTteOAe702wW6uqi1BQ
Timestamp: 2017-11-24 06:55:27
Document Index: 494851634

Matched Legal Cases: ['arts 1', 'arts 1', 'arts 1', 'art 1', 'arts 2', 'arts 2', 'arts 1', 'arts 2', 'arts 2', 'ART 1', 'ART 1', 'ART 2', 'ART 3', 'ART 4']

Patent US6090625 - Method of increasing the relative number of CD34+ cells in a cell population - Google Patents
A method of increasing the relative number of CD34+ cells in a cell population is described. The method comprises contacting the cell population with an agent that causes the relative number of CD34+ cells to increase....http://www.google.com/patents/US6090625?utm_source=gb-gplus-sharePatent US6090625 - Method of increasing the relative number of CD34+ cells in a cell population
Publication number US6090625 A
Application number US 08/594,164
Also published as CA2211147A1, CA2211147C, CN1180374A, CN1289660C, DE69637534D1, EP0807166A1, EP0807166B1, US20020076812, US20110143431, WO1996023870A1
Publication number 08594164, 594164, US 6090625 A, US 6090625A, US-A-6090625, US6090625 A, US6090625A
Inventors Ilham Abuljadayel
Original Assignee Ghazi Jaswinder Dhoot
Patent Citations (1), Non-Patent Citations (84), Referenced by (58), Classifications (15), Legal Events (4)
Method of increasing the relative number of CD34+ cells in a cell population
US 6090625 A
A method of increasing the relative number of CD34+ cells in a cell population is described. The method comprises contacting the cell population with an agent that causes the relative number of CD34+ cells to increase.
1. A method of increasing the relative number of CD34+ cells in a cell population including committed cells, which method comprises:
(i) contacting the cell population with an agent that operably engages said committed cells; and
(ii) incubating committed cells that are engaged by said agent such that the relative number of CD34+ cells increases as a result of said engaging.
2. The method of claim 1 wherein the committed cells are hemopoietic cell.
3. The method of claim 1 wherein the agent engages a receptor that mediates capture, recognition or presentation of an antigen at the surface of the committed cells.
4. The method according to claim 3 wherein the receptor is an MHC class I antigen or an MHC class II antigen.
5. A method according to claim 4 wherein said class I antigen is a Human-Leukocytc-Associated (HLA)-A receptor, an HLA-B receptor, an HLA-C receptor, an HLA-E receptor, an HLA-F receptor or an HLA-G receptor and said class II antigen is an HLA-DM receptor, an HLA-DP receptor, an HLA-DQ receptor or an HLA-DR receptor.
6. The method according to claim 5 wherein the receptor is an HLA-DR receptor.
7. The method according to claim 6 wherein the receptor comprises a β-chain.
8. The method according to claim 7 wherein the β-chain has homologous regions.
9. The method according to claim 8 wherein the receptor comprises at least the homologous region of the β-chain of HLA-DR.
10. The method according to claim 9 wherein the agent is an antibody to the receptor.
11. A method according to claim 10 wherein the agent is a monoclonal antibody to the receptor.
12. A method according to claim 11 wherein the antibody is selected from the group consisting of monoclonal antibody CR3/43 and monoclonal antibody TAL 1B5.
13. The method according to claim 1 wherein said incubating is from 2 to 24 hours.
14. The method according to claim 1 wherein the committed cells are non-cancer cells.
15. The method according to claim 1 wherein the committed cells are differentiated cells.
16. The method according to claim 1 wherein the committed cells are selected from T-cell colony-forming cells (CFC-T cells), B-cell colony-forming cells (CFC-B cells), cosinophil colony-forming cells (CFC-Eosin cells), basophil colony-forming cells (CFC-Bas cells), granulocyte/monocyte colony-forming cells (CFC-GM cells), megakaryocyte colony-forming cells (CFC-MEG cells), erythrocyte burst-forming cells (BFC-E cells), erythrocyte colony-forming cells (CFC-E cells), T-cells and B cells.
17. The method according to claim 1 wherein the CD34+ cells are Major Histocompatibility Complex (MHC) class I+ and/or MHC class II+ cells.
18. A method according to claim 1 wherein the agent is used in conjunction with a biological response modifier.
19. A method according to claim 18 wherein the biological response modifier is an alkylating agent.
20. A method according to claim 19 wherein the alkylating agent is or comprises cyclophosphoamide.
The present invention relates to a method of preparing an undifferentiated cell.
In particular, the present invention relates to a method of preparing an undifferentiated cell from a more committed cell.
In addition the present invention relates to the use of the undifferentiated cell of the present invention for the preparation of a new more committed cell--i.e. a recommitted cell.
By way of introduction, differentiation is a process whereby structures and functions of cells are progressively committed to give rise to more specialised cells, such as the formation of T cells or B cells. Therefore, as the cells become more committed, they become more specialised.
In contrast, retro-differentiation is a process whereby structures and functions of cells are progressively changed to give rise to less specialised cells.
Undifferentiated cells are capable of multilineage differentiation--i.e. they are capable of differentiating into two or more types of specialised cells. A typical example of an undifferentiated cell is a stem cell.
In contrast, differentiated cells are incapable of multilineage differentiation. A typical example of a differentiated cell is a T cell.
By way of example, reference may be made to inter alia Levitt and Mertelsman 1995 (Haematopoietic Stem Cells, published by Marcel Dekker Inc--especially pages 45-59) and Roitt et al (Immunology, 4th Edition, Eds. Roitt, Brostoff and Male 1996, Publ. Mosby--especially Chapter 10).
In short, however, examples of undifferentiated cells include lymphohaematopoietic progenitor cells (LPCs). LPCs include pluripotent stem cells (PSCs), lymphoid stem cells (LSCs) and myeloid stem cells (MSCs). LSCs and MSCs are each formed by the differentiation of PSCs. Hence, LSCs and MSCs are more committed than PSCs.
Examples of differentiated cells include T cells, B cells, eosinophils, basophils, neutrophils, megakaryocytes, monocytes, erythrocytes, granulocytes, mast cells, and lymphocytes.
T cells and B cells are formed by the differentiation of LSCs. Hence, T cells and B cells are more committed than LSCs.
Eosinophils, basophils, neutrophils, megakaryocytes, monocytes, erythrocytes, granulocytes, mast cells, NKs, and lymphocytes are formed by the differentiation of MSCs. Hence, each of these cells are more committed than MSCs.
Antigens are associated with undifferentiated and differentiated cells. The term "associated" here means the cells expressing or capable of expressing, or presenting or capable of being induced to present, or comprising, the respective antigen(s). Most undifferentiated cells and differentiated cells comprise Major Histocompatability Complex (MHC) Class I antigens and/or Class II antigens. If these antigens are associated with those cells then they are called Class I+ and/or Class II+ cells.
Examples of these marker antigens include the antigens CD34, CD19 and CD3. If these antigens are present then these particular cells are called CD34+, CD19+ and CD3+ cells respectively. If these antigens are not present then these cells are called CD34-, CD19- and CD3- cells respectively.
In more detail, PSCs are CD34+ cells. LSCs are DR+, CD34+ and TdT+ cells. MSCs are CD34+, DR+, CD13+, CD33+, CD7+ and TdT+ cells. B cells are CD19+, CD21+, CD22+ and DR+ cells. T cells are CD2+, CD3+, and either CD4+ or CD8+ cells. Immature lymphocytes are CD4+ and CD8+ cells. Activated T cells are DR+ cells. Natural killer cells (NKs) are CD56+ and CD16+ cells. T lymphocytes are CD7+ cells. Leukocytes are CD45+ cells. Granulocytes are CD13+ and CD33+ cells. Monocyte macrophage cells are CD14+ and DR+ cells.
The general concept of retrodifferentiation is not new. In fact, in 1976 Jose Uriel (Cancer Research 36, 4269-4275. Nov. 1976) presented a review on this topic, in which he said:
"retrodifferentiation appears as a common adaptive process for the maintenance of cell integrity against deleterious agents of varied etiology (physical, chemical, and viral). While preserving the entire information encoded on its genome, cells undergoing retrodifferentiation lose morphological and functional complexity by virtue of a process of self-deletion of cytoplasmic structures and the transition to a more juvenile pattern of gene expression. This results in a progressive uniformization of originally distinct cell phenotypes and to a decrease of responsiveness to regulatory signals operational in adult cells. Retrodifferentiation is normally counterbalanced by a process of reontogeny that tends to restore the terminal phenotypes where the reversion started. This explains why retrodifferentiation remains invariably associated to cell regeneration and tissue repair."
Uriel (ibid) then went on to discuss cases of reported retrodifferentiation--such as the work of Gurdon relating to nuclei from gut epithelial cells of Xenopus tadpoles (Advances in Morphogenesis [1966] vol 4, pp 1-43. New York Academic Press, Eds Abercrombie and Bracher), and the work of Bresnick relating to regeneration of liver (Methods in Cancer Research [1971] vol 6, pp 347-391).
"Contrary to the results with fetal or neonatal hepatocytes, with hepatocytes from regenerating liver, or from established hepatomas, it has been difficult to obtain permanent class lines from resting adult hepatocytes."
Uriel (ibid) also reported on apparent retrodifferentiation in cancer, wherein he stated:
"the biochemical phenotypes of many tumours show analogous changes of reversion toward immaturity . . . during the preneoplastic phase of liver carcinogenesis, cells also retrodifferentiate."
More recent findings on retrodifferentiation include the work of Minoru Fukunda (Cancer Research [1981] vol 41, pp 4621-4628). Fukunda induced specific changes in the cell surface glycoprotein profile of K562 human leukaemic cells by use of the 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 et al (Cell Growth & Differentiation [1991] vol 2, pp 541-548) reported that long term culture of TPA-differentiated U-937 leukaemia cells in the absence of phorbol ester for 32-36 days resulted in a process of retrodifferentiation and that the retrodifferentiated cells detached from the substrate and reinitiated proliferation.
Another case of retrodifferentiation is the work of Curtin and Snell (Br. J. Cancer [1983] vol 48, pp 495-505]. These workers compared enzymatic changes occurring during diethylnitrosamine-induced hepatocarcinogenesis and liver regeneration after partial hepatectomy to normal liver differentiation. Theses workers found changes in enzyme activities during carcinogenesis that were similar to a step-wise reversal of differentiation. According to these workers, their results suggest that an underlying retrodifferentiation process is common to both the process of hepatocarcinogenesis and liver regeneration.
More recently, Chastre et al (FEBS Letters [1985] vol 188, number 2, pp 2810-2811] reported on the retrodifferentiation of the human colonic cancerous subclone HT29-18.
Even more recently, Kobayashi et al (Leukaemia Research [1994] vol 18, no. 12, pp 929-933) have reported on the establishment of a retrodifferentiated cell line (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:
i. it is an undifferentiated cell--i.e. it is not terminally differentiated;
ii. it has the ability to divide without limit;
iii. it has the ability to give rise to differentiated progeny, such as the differentiated cells mentioned earlier; and
iv. when it divides each daughter has a choice: it can either remain as PSC like its parent or it can embark on a course leading irreversibly to terminal differentiation.
Note should be made of the last qualification, namely that according to the general teachings in the art once an undifferentiated cell has differentiated to a more committed cell it can not then retrodifferentiate. This understanding was even supported by the teachings of Uriel (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.
According to a second aspect of the present invention there is provided a method comprising contacting a more committed cell with an agent that causes the more committed cell to retrodifferentiate into an undifferentiated cell; and then committing the undifferentiated cell to a recommitted cell.
The term "recommitted cell" means a cell derived from the undifferentiated cell--i.e. a new more committed cell.
According to a fifth aspect of the present invention there is provided the use of an undifferentiated cell produced according to the method of of the present invention in the manufacture of a medicament for the treatment of an immunological disorder or disease.
According to a sixth aspect of the present invention there is provided a recommitted cell produced according to the method of the present invention.
According to a seventh aspect of the present invention there is provided a recommitted cell produced according to the method of the present invention as or in the preparation of a medicament.
According to an eighth aspect of the present invention there is provided the use of a recommitted cell produced according to the method of of the present invention in the manufacture of a medicament for the treatment of an immunological disorder or disease.
According to a ninth aspect of the present invention there is provided a more committed cell having attached thereto an agent that can cause the more committed cell to retrodifferentiate into an undifferentiated cell.
According to a tenth aspect of the present invention there is provided a CD19+ and CD3+ 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.
Preferably, the agent acts extracelluarly of the more committed cell.
Preferably, the more committed cell comprises a receptor that is operably engageable by the agent and wherein the agent operably engages the receptor.
Preferably, the Preferably, the ll surface receptor.
Preferably, the receptor comprises an α- component and/or a β- component.
Preferably, the receptor comprises a β-chain having homologous regions.
Preferably, the receptor comprises at least the homologous regions of the β-chain of HLA-DR.
Preferably, the receptor comprises an α-chain having homologous regions.
Preferably, the receptor comprises at least the homologous regions of the α-chain of HLA-DR.
Preferably, the agent is an antibody to the receptor.
Preferably, the agent is a monoclonal antibody to the receptor.
Preferably, the agent is an antibody, preferably a monoclonal antibody, to the homologous regions of the β-chain of HLA-DR.
Preferably, the agent is an antibody, preferably a monoclonal antibody, to the homologous regions of the α-chain of HLA-DR.
Preferably, the agent is used in conjunction with a biological response modifier.
Preferably, the biological response modifier is an alkylating agent.
Preferably, the alkylating agent is or comprises cyclophosphoamide.
In one preferred embodiment, the more committed cell is a differentiated cell.
Preferably, the more committed cell is any one of a B cell or a T cell.
In an alternative preferred embodiment, the more committed cell is a more mature undifferentiated cell.
In one preferred embodiment, when the undifferentiated cell is committed to a recommitted cell the recommitted cell is of the same lineage as the more committed cell prior to retrodifferentiation.
In another preferred embodiment, when the undifferentiated cell is committed to a recommitted cell the recommitted cell is of a different lineage as the more committed cell prior to retrodifferentiation.
Preferably, the recommitted cell is any one of a B cell, a T cell or a granulocyte.
Preferably, the agent modulates MHC gene expression, preferably wherein the agent modulates MHC Class I+ and/or MHC Class II+ expression.
The agent for the retrodifferentiation of the more committed cell into an undifferentiated cell may be a chemical compound or composition. Preferably, however, the agent is capable of engaging a cell surface receptor on the surface of the more committed cell. For example, preferred agents include any one or more of cyclic adenosine monophosphate (cAMP), a CD4 molecule, a CD8 molecule, a part or all of a T-cell receptor, a ligand (fixed or free), a peptide, a T-cell receptor (TCR), an antibody, a cross-reactive antibody, a monoclonal antibody, or a polyclonal antibody.
The more committed cell is any cell derived or derivable from an undifferentiated cell.
Thus, in one preferred embodiment, the more committed cell is also an undifferentiated cell. By way of example therefore the undifferentiated cell can be a lymphoid stem cell or a myeloid stem cell, and the undifferentiated cell is a pluripotent stem cell.
In another preferred embodiment, the more committed cell is a differentiated cell, such as a CFC-T cell, a CFC-B cell, a CFC-Eosin cell, a CFC-Bas cell, a CFC-Bas cell, a CFC-GM cell, a CFC-MEG cell, a BFC-E cell, a CFC-E cell, a T cell, a B cell, an eosinophil, a basophil, a neutrophil, a monocyte, a megakaryocyte or an erythrocyte; and the undifferentiated cell is a myeloid stem cell, a lymphoid stem cell or a pluripotent stem cell. As used herein, the acronym "CFC" refers to colony forming cells.
If the more committed cell is a differentiated cell then preferably the differentiated cell is a B lymphocyte (activated or non-activated), a T lymphocyte (activated or non-activated), a cell from the macrophage monocyte lineage, a nucleated cell capable of expressing class I or class II antigens, a cell that can be induced to express class I or class II antigens or an enucleated cell (i.e. a cell that does not contain a nucleus--such as a red blood cell).
The agent may act intracellularly within the more committed cell. However, preferably, the agent acts extracelluarly of the more committed cell.
In a preferred embodiment, agent operably engages a receptor present on the surface of the more committed cell - which receptor may be expressed by the more committed cell, such as a receptor that is capable fo being expressed by the more committed cell.
Preferably, the receptor is a Class I or a Class II antigen of the major histocompatibility complex (MHC). In preferred embodiments the cell surface receptor is any one of: an HLA-DR receptor, a DM receptor, a DP receptor, a DQ receptor, an HLA-A receptor, an HLA-B receptor, an HLA-C receptor, an HLA-E receptor, an HLA-F receptor, or an HLA-G receptor.
In more preferred embodiments the cell surface receptor is an HLA-DR receptor.
Preferably the contacting step comprises the agent engaging with any one or more of the following: homologous regions of the α-chain of class I antigens, homologous regions of the α-chain of class II antigens, a CD4 cell surface receptor, a CD8 cell surface receptor, homologous regions of the β-chain of class II antigens in the presence of lymphocytes, homologous regions of the α-chain of class I antigens in the presence of lymphocytes, or homologous regions of the α-chain of class II antigens in the presence of lymphocytes.
Preferably the contacting step occurs in the presence of the biological response modifier.
Preferably the biological response modifier is any one or more of a modulator, such as an immunomodulator, a growth factor, a cytokine, a cell surface receptor, a hormone, a nucleic acid, a nucleotide sequence, an antigen or a peptide.
In a preferred embodiment of the present invention the undifferentiated cell is then committed into a recommitted cell, such as a differentiated cell.
The recommitted cell may be of the same lineage to the more committed cell from which the undifferentiated cell was derived.
Alternatively, the recommitted cell may be of a different lineage to the more committed cell from which the undifferentiated cell was derived.
In an even more preferred embodiment the present invention relates to a process of introducing the genome of a gene into an undifferentiated cell, wherein the process comprises inserting the gene into the genome of a more committed cell, and then preparing an undifferentiated cell by the method according to the present invention, whereby the gene is present in the genome of the undifferentiated cell.
As already mentioned, the present invention also encompasses a medicament comprising an undifferentiated cell prepared by any one of these processes admixed with a suitable diluent, carrier or excipient. With such a medicament the undifferentiated cell could be used to produce a beneficial more committed cell, such as one having a correct genomic structure, in order to alleviate any symptoms or conditions brought on by or associated with a more committed cell having an incorrect genomic structure.
Thus, the present invention also provides a process of removing an acquired mutation from a more committed cell wherein the method comprises forming an undifferentiated cell by the method according to the present invention, committing the undifferentiated cell into a recommitted cell, whereby arrangement or rearrangement of the genome and/or nucleus of the cell causes the mutation to be removed.
The present invention also covers a method of treating a patient suffering from a disease or a disorder resulting from a defective cell or an unwanted cell, the method comprising preparing an undifferentiated cell by contacting a more committed cell with an agent that causes the more committed cell to retrodifferentiate into the undifferentiated cell, and then optionally committing the undifferentiated cell into a recommitted cell; wherein the undifferentiated cell, or the recommitted cell, affects the defective cell or the unwanted cell to alleviate the symptoms of the disease or disorder or to cure the patient of the disease or condition.
In summation, the present invention relates to the preparation of an undifferentiated cell from a more committed cell.
The present invention will now be described by way of example, in which reference shall be made to the following Figures:
FIG. 1 which is a microscope picture of cells before the method of the present invention;
FIG. 2 which is a microscope picture of cells prepared by the method of the present invention;
FIG. 3 which is a microscope picture of cells prepared by the method of the present invention but at a lower magnification;
FIG. 4 which is a microscope picture of cells before the method of the present invention;
FIG. 5 which is a microscope picture of cells prepared by the method of the present invention; and
FIG. 6 which is a microscope picture of cells prepared by the method of the present invention.
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.
Blood samples, once obtained, were treated with pure monoclonal antibody to the homologous region of the β-chain of the HLA-DR antigen (DAKO) and left to mix on a head to head roller at room temperature for a maximum of 24 hours. Some samples were mixed first on a head to head roller for 15 minutes after which they were left to incubate in an incubator at 22° C. The concentration of monoclonal antibody added to blood samples varied from 10-50 μl/ml of blood.
In addition, other treatments were applied at the same concentrations and these included addition of a monoclonal antibody to the homologous of the α-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 (phycoerythin) conjugated monoclonal antibody to the homologous region of the β-chain of the HLA-DR antigen.
Treatment of blood samples with monoclonal antibody to the homologous region of the β-chain of the HLA-DR antigen always decreased the relative number of CD19+ cells. This marker is a pan B-cell antigen (see Table). This antigen is present on all human B lymphocytes at all stages of maturation but is lost on terminally differentiated plasma cells. Hence, this is an indication that B cells were retrodifferentiating into undifferentiated cells.
The same treatment caused the relative number of CD3+ cells to increase dramatically especially in blood of patients with B-CLL, which was always accompanied by an increase in the relative number in CD3- CD19- cells. CD3 is present on all mature T-lymphocytes and on 65% -85% of thymocytes. This marker is always found in association with α-/β- 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 2 hr, 6 hr & 24 hr 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.
The CD19+ CD3+ clones in treated samples seem to decrease with time, reaching original levels to those determined in untreated sample at 2 hrs, 6 hrs and 24 hrs time.
Another type of cell of the same size and granulity was detected in treated samples and these cells had high levels of CD19 expressed on their surface but were negative for the CD3 marker and rich in FC receptors. However, the relative number of these cells appeared to decrease in time. Of interest, at 24 hours treatment of blood sample (2, 3 and 4) there was a decrease in the relative number of CD19- CD3- cells in a group of cells that were initially observed to increase after 2 and 6 hr's treatment of blood samples. However, Coulter counts of WBC populations were reduced on treatment of blood with monoclonal antibody to the homologous region of the β-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 (1, 2, 3 & 4) and the immunophenotypic changes obtained on treatment of blood samples seems to suggest that CD19+ and CD3+ lymphocytes are an interconnected group of cells but remain distinct on the basis of CD19 and CD3 relative expression compared to stem cells.
Patients blood with no B-cell malignancy showed similar trends of inmiunophenotypic 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 class II positive cells in the blood of these patients are extremely low compared to those found in the blood of B-CLL patients.
Two brothers both with X-linked infantile hypogammaglobulinemia who were B cell deficient showed different immunophenotypic changes in the relative number of CD3+ cells on treatment of their blood. The younger brother who was 2 months old and not ill, on treatment of his blood, showed a slight increase in the relative number of CD3+ cells which was accompanied by a decrease in the relative number of CD3- CD19- cells. On the other hand, the other brother who was 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 immunophenotypic changes that might have occurred because the blood samples obtained from these two patients were extremely small (Table 2, ID 43/BD and 04/BD).
Patient 91 in Table 2 shows a decrease in the relative number of CD3+ cells following treatment of blood which was accompanied by an increase in the relative number of CD3- CD19- cells. However, on analysis of other surface markers such as CD4 and CD8 (see Table 3) the patient was observed to have a high relative number of CD4+ CD8+ cells in his blood and this was noted prior to treatment of blood samples with monoclonal antibody to the β-chain of the DR antigen and these double positive cells decreased appreciably following treatment of blood. Furthermore, when further markers were analyzed the relative number of CD3+ cells were seen to have elevated (See Table 4).
An enriched preparation of B-lymphocytes obtained from healthy blood donors when treated with monoclonal antibody to the β-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.
On analysis of the CD4 and CD8 markers--see below--and from a majority of blood samples presented in Table 2, a pattern of staining emerges which supports the presence of a retrodifferentiation process of B-lymphocytes into undifferentiated cells and the subsequent differentiation into T-lymphocytes.
CD4+ CD8+ cells, which are double positive cells, always appeared following treatment of blood samples with monoclonal antibody to the homologous region of the β-chain and these types of cells were markedly increased in the blood of treated samples of patients with B-CLL and which were absent altogether in untreated samples (See Table 3 and Charts 1, 2 3 & 4). In the same specimens the relative number of single positive cells such as CD8+ and CD4+ cells was also noted to increase simultaneously. Furthermore, a decrease in the relative number of CD4- CD8- cells which, at least in the case of B-CLL correspond to B cells was noted to fall dramatically in treated samples when compared to untreated specimens which remained at the same level when measured with time. However, measurement of the relative number of CD4+ CD8+ cells with time in treated samples showed that there was a concomitant increase in the number of single positive cells with a decrease in the relative number of double positive cells. This type of immunophenotypic change is characteristic of thymic development of progenitor cells of the T-lymphocyte lineage in the thymus (Patient number 2,3 and 4). The CD4 antigen is present on the helper/inducer T-lymphocyte subsets (CD4+ CD3+) and a majority of normal thymocytes. However, this antigen is present in low density on the cell surface of monocytes and in the cytoplasm of monocytes and macrophages (CD3- CD4+).
Patient HIV+ 25 on treatment showed a substantial increase in the number of double positive cells expressing CD4 and CD8 simultaneously. On the other hand, patient 91 on treatment showed a decrease in this subtype of cells and the observation of such phenomenon is time dependent. The relative number of CD8+ cells was observed to increase in untreated blood samples of patients with B-CLL when measured with time whereas the relative number of CD4+ and CD4+ low cells was observed to decrease at the same times (Table 3 patient 2,3 and 4).
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).
Furthermore, the relative number of activated T cells increased in the majority of treated blood samples of patients with B-CLL and these types of cells were affected variably in treated samples of patients with other conditions. Furthermore, the relative number of DR high positive cells appeared in significant numbers in treated samples of patients with B-CLL and a 6 day old baby with increased DR+ CD34+ blasts in his blood. However, it should be noted that the blasts which were present in this patient's blood were negative for T and B-cell markers before and after treatment but became more positive for myeloid lineage antigens following treatment. The relative number of CD3- DR- cells increased in the majority of treated blood samples and was proportional to increases in the relative number of CD3+ cells (T-cells) and was inversely proportional to decreases in the relative number of DR+ cells (B-cells).
The CD56&CD16 markers are found on a heterogeneous group of cells, a subset of lymphocytes known generally as large granular lymphocytes and natural killer (NK) lymphocytes. The CD16 antigen is expressed on virtually all resting NK lymphocytes and is weakly expressed on some CD3+ T lymphocytes from certain individuals. This antigen is found on granulocytes in lower amount and is associated with lymphocytes containing large azurophilic granules. The CD16 antigen is the IgG FC receptor III.
A variable number of CD16+ lymphocytes coexpress either the CD57 antigen or low-density CD8 antigen or both. In most individuals, there is virtually no overlap with other T-lymphocyte antigens such as the CD5, CD4, or CD3 antigens. The CD56 antigen is present on essentially all resting and activated CD 16+ NK lymphocytes and these subsets of cells carry out non-major histocompatibility complex restricted cytotoxicity.
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 β-chain of DR antigen seems to cause spontaneous production of CD56+ and CD16+ cells, CD3+ cells and CD56+ and CD16+ CD3+ cells and these observations were always accompanied by the disappearance of B-cell markers (CD19, DR, CD56, CD16- CD3-).
Engagement of the β-chain of the DR antigens in treated blood samples especially those obtained from patients with B-CLL suggests that such a treatment affects the level of CD45 antigens on B-lymphocytes. The overall immunophenotypic changes that took place on stimulation of the β-chain of the DR antigen seem to give rise to different types of cells that can be segregated on the basis of the level of CD45 and CD14 expression as well as morphology as determined by forward scatter and side scatter (size and granulity respectively) and these results are presented in Table 6 and Charts (1, 2, 3, 4 & 5).
On treatment the relative number of CD45 low cells (when compared to untreated samples) increased significantly and so did the relative number of cells co-expressing the CD45 and CD14 antigens. This type of immunophenotypic changes coincided with a decrease in the relative number of CD45 high cells (compared to untreated samples). However, this latter population of cells can be further divided on the basis of morphology and the degree of CD45 expression. One type was extremely large and had extremely high levels of CD45 antigen when compared to the rest of cells present in the charts (see charts 1, 2, 3 and 4). On analysis of this panel following treatment with time (see Table patient 2,3 and 4 and charts) the relative number of CD45+ cells initially fell drastically with time to give rise to CD45 low cells. However, analysis of blood 24 hours later showed the opposite situation.
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 Chart 1).
The relative number of CD8+ cells increased with time in treated and untreated samples but to a higher extent in untreated samples. On the other hand, the relative number of CD8+ CD3 + cells decreased with time in untreated samples. However, the relative number of CD3+ cells increased in treated blood samples when measured with time and these types of cells highly correspond to CD4+ CD3+ single positive cells; a maturer form of thymocytes. In addition, since these samples were also immunophenotyped with other panels (mentioned above in Tables 3, 4, 5 and 6) the overall changes extremely incriminate B cells in the generation of T lymphocyteprogenitors and progenies.
C. Comparison of the Effect of other Mono Monoclonal Antibodies with Different Specificity on T-Lymphophoiesis
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 β-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 β-chain of DR antigen show reversal in the relative number of CD 19+ and CD3+cells when compared to that observed at 2 hour incubation time under exactly the same condition.
In general, treatment of blood samples of the same patient with monoclonal antibody to the homologous region of the α chain of the DR antigen or monoclonal antibody to the homologous of the α-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 α-chain of DR antigen or treated with monoclonal antibody to class I antigens (see Table 14 & Charts 2, 3 & 4). Treatment of blood samples of patient 09 with monoclonal antibody to class I antigens increased the relative number of CD3+ cells and decreased slightly the relative number of CD19+ and CD19- CD3- cells. However, treatment of an enriched preparation of B-lymphocytes obtained from healthy blood donors with monoclonal antibody to the β-chain or α-chain of DR antigen showed similar immunophenotypic changes to those obtained with patient with B-CLL.
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 with B-CLL (Table 15, patients 5/6 and 10, Charts 2, 3 & 4) with monoclonal antibody to the homologous region of the β-chain of the DR antigen or with this monoclonal antibody plus cyclophosphoamide increased the relative number of CD8+ and CD4+ cells and cells coexpressing both markers. On the other hand, treatment of the same samples with monoclonal antibodies to the homologous region of the α-chain of the DR antigen or the homologous region of the a-chain of class I antigen did not produce the same effects.
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 β-chain, confirming earlier observations noted with the same treatment when the same blood samples were analysed with CD3 and CD19 and DR and CD3 panels.
Blood samples of patient 5/6 and 10 (B-CLL) on treatment with monoclonal antibody to class I antigens showed a decrease in the relative number of CD45+ medium cells and similar observations were noted in blood samples 09 and HIV+ following the same treatment when compared to untreated samples. Treatment of blood samples of HIV+ and IgA/D patients with monoclonal antibody to class I antigen increased the relative number of CD45+ low cells when compared to untreated samples or samples treated with monoclonal antibody to the β-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 β-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 CD45 antigen were noted in treated blood samples with monoclonal antibody to the homologous region of the β-chain of DR antigen of MHC class II antigens (see Charts 1, 2, 3, 4 & 5).
The CD28 antigen is present on approximately 60% to 80% of peripheral blood T (CD3+) lymphocytes, 50% of CD8+ T lymphocytes and 5% of immature CD3-thymocytes. During thymocyte maturation, CD28 antigen expression increases from low density on most CD4+ CD8+ immature thymocytes to a higher density on virtually all mature CD3+, CD4+ or CD8+ thymocytes. Cell activation further augments CD28 antigen density. Expression of the CD28 also divides the CD8+ lymphocytes into two functional groups. CD8+ CD28+ lymphocytes mediate alloantigen-specific cytotoxicity, that is major histocompatibility complex (MHC) class I-restricted. Suppression of cell proliferation is mediated by the CD8+ CD28- subset. The CD28 antigen is a cell adhesion molecule and functions as a ligand for the B7/BB-1 antigen which is present on activated B lymphocytes.
Treatment of blood samples of patients (Table 19, patients 5/6 and 8) with B-CLL with monoclonal antibody to the homologous region of β-chain of the DR antigen increased the relative number of CD8+, CD28+ and CD8+ CD28+ cells and all other types of treatments did not.
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 CD45 RA or CD45RA antigens. Approximately 60% of acute B-lymphoid leukaemia's and acute myeloid leukaemia express the CD34 antigen. The antigen is not expressed on chronic lymphoid leukaemia (B or T lineage) or lymphomas. The CD2 antigen is present on T lymphocytes and a subset of natural killer lymphocytes (NK).
The results are shown in Charts 2, 3 and 5.
Analysis of blood samples of a patient with B-CLL (Table 20, patient 5/6 at 2hours) after treatment with monoclonal antibodies to the β-chain of the DR antigen or the α-chain 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 β-chain of the HLA-DR antigen, are in direct support that the process is giving rise to T-lymphopoiesis via B lymphocyte regression.
Treatment of blood samples of HIV+ patient (Table 20 patient HIV+) with monoclonal antibody to the β-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 β-chain of the HLA-DR antigen and monoclonal antibody to the α-chain of the same antigen when added together. However, treatment of this blood sample with monoclonal antibody to the α-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 β-chain of the HLA-DR antigen, or monoclonal antibody to the α-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 β-chain the relative number of CD34+ cells further increased but were noted to decrease on treatment with monoclonal antibody to the α-chain of the HLA-DR antigen or treatment with monoclonal antibodies to the α and β-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 β-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 β-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 β-chain promotes the production of more CD34+ cells from CD2+ CD34+ 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, immunophenotypic 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 β-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 in treated or untreated blood smears.
The results of Table 20 also demonstrate a further important finding in that according to the method of the present invention it is possible to prepare an undifferentiated cell by the retrodifferentiation of a more mature undifferentiated cell.
In this regard, FIG. 1 is a microscope picture of differentiated B cells stained with Wright's stain before the method of the present invention. FIG. 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 β-chain of HLA-DR antigen. The undifferentiated cells are the dark stained clumps of cells. FIG. 3 is a microscope picture of the same undifferentiated cells but at a lower magnification. Cells shown in these figures were stained with Wright's stain and photographed using 100× magnification.
FIGS. 1 to 3 therefore visually demonstrate the retrodifferentiation of B cells to undifferentiated stem cells by the method of the present invention.
FIG. 4 is a microscope picture of differentiated B cells stained with Wright's stain, before the method of the present invention. FIG. 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 β-chain of HLA-DR antigen. Again, the undifferentiated cells are the dark stained clumps of cells. FIG. 6 is a microscope picture of the formation of differentiated granulocyte cells from the same undifferentiated cells of FIG. 5.
FIGS. 4 to 6 therefore visually demonstrate the retrodifferentiation of B cells to undifferentiated stem cells by the method of the present invention followed by commitment of the undifferentiated cells to new differentiated cells being of a different lineage as the original differentiated cells.
The retrodifferentiation of T cells to undifferentiated stem cells by the method of the present invention followed by commitment of the undifferentiated cells to new differentiated cells being of a different lineage as the original differentiated cells was also followed by microscopy.
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 β-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 samples were treated with monoclonal antibodies to the homologous region of the α-chain of class-II antigens or to the homologous region of class-I antigens.
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 β-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 β-chain of the HLA-DR antigen showed a significant increase in the relative number of cells expressing the myeloid markers such as CD13 and CD33. These markers were coexpressed with the CD56 & 16 or the CD7 antigens. However, the relative number of CD7+ cells with T-lymphocyte markers and without myeloid antigens was observed on a separate population of cells. These particular observations were not seen in untreated samples or in samples treated with monoclonal antibodies to class I antigens or the homologous region of the c-chain of HLA-DR antigen (see Charts 2 & 3). These final results suggest that B-lymphocytes once triggered via the β-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.
TABLE 1__________________________________________________________________________CLINICAL DIAGNOSIS OF PATIENTS AND EXPERIMENTAL CONDITIONSOF BLOOD SAMPLES INCLUDING COULTER COUNTS (WBC)FOLLOWING AND PRIOR TREATMENT OF BLOOD SPECIMENSWITH VARIOUS MONOCLONAL ANTIBODIES AND OTHER AGENTS            WBC/L       # LYMPH/LPATIENT     EXPT X10-9 % LYMPH                        10X-9 AGENTID   DIAGNOSIS       COND B  A  B  A  B  A  ML/mL__________________________________________________________________________ 1   B-CLL  12 HR            100               ND 86.1                     ND 86.1                           ND ANTI-B       AT 22 C.               50 2   B-CLL  2 HR 39.1               9.6                  74.4                     63.3                        29.9  ANTI-B       AT 22 C.         6.1   50       2 HR 39.1               37.7                  74.4                     75.1                        29.9                           28.3                              ANTI-B       AT 22 C.               PE                              50 3   B-CLL  6 HR 39.5               9.3                  71.9                     67.2                        28.3  ANTI-B       AT 22 C.         6.2   50       6 HR 39.5               37.7                  71.9                     72.5                        28.3                           27.4                              ANTI-B       AT 22 C.               PE                              50 4   B-CLL  24 HR            39 9.3                  73 66.5                        28.4  ANTI-B       AT 22 C.         6.2   50       24 HR            39 36.2                  73 70.4                        28.4                           25.5                              ANTI-B       AT 22 C.               PE                              50 5   B-CLL  2 HR                   ANTI-B       AT 22 C.               50                              ANTI-A                              50                              ANTI-I                              50                              ANTI-B                              & TOXIC                              AGENT                              25 + 25 6   B-CLL  24 HR                  ANTI-B       AT 22 C.               50 7   B-CLL  24 HR            170               128                  95.4                     91.1                        16.9                           11.6                              ANTI-B       AT 22 C.               10               178   94.2                        16.8  ANTI-I                              10               130   90.4                        11.9  ANTI-B                              & TOXIC                              AGENT                              10 + 20 8   B-CLL  24 HR            16 7  81.9                     51.2                        14 3.0                              ANTI-B       AT 22 C.               20 9   B-CLL  12 HR            +++               89.5                  87 85.1                        +++                           76.2                              ANTI-B       AT 22 C.               30               +++                  85.4     +++                              ANTI-I                              30               +++   89.4     ANTI-4                              30                     84.9  +++                              ANTI-I + II + 4               95.4           10 + 10 + 1010   B-CLL  2 HR 19.3               ND 86 ND 16.7                           ND ANTI-B       AT 22 C.               30                              ANTI-I                              3092   OUT    2 HR 5.4               ND 74.5                     ND    ND ANTI-BPATIENT       AT 22 C.               2087   OUT    2 HR 4.8               ND 59.3                     ND    ND ANTI-BPATIENT       AT 22 C.               2091   OUT    2 HR 4.2               ND 54.0                     ND    ND ANTI-BPATIENT       AT 22 C.               2021   OUT    2 HR 3.9               ND 47.4                     ND    ND ANTI-BPATIENT       AT 22 C.               2034   OUT    2 HR 7.2               ND 20.0                     ND    ND ANTI-BPATIENT       AT 22 C.               2036   CMV    4 HR 13.4               ND 7.3                     ND    ND ANTI-BINFANT AT 22 C.               2093   HIV+   4 HR 5.6               ND 43.4                     ND    ND ANTI-BINFANT AT 22 C.               20BB/ST40% BLAST       2 HR AT            60.5               ND 20.2                     ND 12.2                           ND ANTI-BIN BLOOD       22 C.                  506 DAYS OLD       24 HR                  ANTI-A       AT 22 C.               50                              ANTI-AB                              25 + 25HIV25AIDS   2 HR 7.5               ND 34.8                     ND 2.6                           ND ANTI-B       AT 22 C.               50                              ANTI-A                              50                              ANTI-AB                              25 + 2543/BDB CELL 4 HR                   ANTI-BDEFICIENT       AT 22 C.               20                              ANTI-I                              20                              ANTI-4                              20OB/BDB CELL 4 HR                   ANTI-BDEFICIENT       AT 22 C.               20                              ANTI-I                              20                              ANTI-4                              20HIV+ AIDS   6 HR                   ANTI-B       AT 22 C.               20                              ANTI-IIgA-DIgA    6 HR                   ANTI-BDEFICIENT       AT 22 C.               20                              ANTI-I                              20__________________________________________________________________________ EXPT COND: EXPERIMENTAL CONDITIONS B: BEFORE A: AFTER ANTIB: monoclonal antibody to the homologous region of the chain of HLADR anigen ANTIA: monoclonal antibody to the homologous region of the chain of HLADR antigen ANTII: monoclonal antibody to the homologous region of Class I antigens ANTIAB: both ANTIB and ANTIA added togather ANTI4: monoclonal antibody to the CD4 antigen ANTII + II + 4: ANTII and ANTIB and ANTI4 added togather Cytoxic agent: Cyclophophamide ML/mL: micro liter per ml L: liter
TABLE 2______________________________________IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL ANDOTHER CONDITIONS BEFORE AND AFTER TREATMENTOF BLOOD SAMPLES WITH MONOCLONAL ANTIBODYTO THE HOMOLOGOUS REGION OF THE B CHAIN OF THEHLA-DR WITH CD19 AND CD3 MONOCLONAL ANTIBODIES                                         %                       %        %        CD19+%                %          CD19+    CD3-     HGCD3-CD19+            CD3+       CD3+     CD19-    FC+PATIENT B     A      B   A    B   A    B   A    B   A______________________________________ 1      88    40      5  19   1   2     6  26   0   12 2      73    15     10  33   2   7    15  41   0    5 3      73    11     11  33   2   2    14  52   0    2 4      71    13     11  37   2   2    16  47   0    2 5      85    40      5  16   1   1     6  26   3   18 6      85    43      5  18   1   1     6  27   3   10 7      90    72      2   4   0   2     7   8   0   14 8      62    25      7  13   0   1    29  55   2    6 9      90    85      2   3   0   0     2   1   1    410      78    50      7  14   0   0    14  26   0    892      12    10     38  49   0   1    49  40   0    091       7     3     35  29   0   1    59  67   0    087       5     3     32  38   1   1    63  58   0    021       1     1     27  29   1   0    71  70   0    034       1     1     13  13   0   2    86  84   0    039      10     6     23  25   0   0    67  69   0    093       6     3     26  27   1   1    68  70   0    0BB/ST    1     1     12  13   0   0    87  86   0    0HIV25    7     2     26  27   0   0    68  67   0    043/BD    0     0     40  42   0   1    58  54   0    004/BD    0     0     49  41   0   3    43  41   0    0HIV+     1     1     10  14   0   0    89  87   0    0IgA/D   10     1     21  25   2   3    67  71   0    0______________________________________ B: before treatment A: after treatment
TABLE 3__________________________________________________________________________IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL AND OTHER CONDITIONSBEFORE AND AFTER TREATMENT OF BLOOD SAMPLES WITHMONOCLONAL ANTIBODY TO THE B CHAIN OF THE HOMOLOGOUS REGIONOF THE HLA-DR WITH MONOCLONAL ANTIBODIES TO CD4 AND CD8               %     %%            %      CD4+  CD4-   CD4+CD8+         CD4+   CD8+  CD8-   LOWPATIENT B  A   B  A   B  A  B  A   B  A__________________________________________________________________________ 1    2.8    16  2.9           11.4               0  3.2                     93.1                        67.6                            0  0 2    6.2    13.2        9.1           24.3               0  9.4                     78.7                        46  5.8                               6.3 3    7.2    13.1        7.4           23.9               0  8.2                     78.8                        48.1                            6.3                               6.6 4    10.1    24.2        7.6           24.9               0.3                  2.8                     77.5                        42  4.6                               5 5    2.9    16.2        1.8           7.6 0  2  95 62.3                            0  0 6    ND 12  ND 8.1 ND 1.7                     ND 75.7                            ND 0 7    1.9    2.6 1.9           2.8 0  0  95.8                        94.3                            0  0 8    3.2    7   3.9           6.9 0.1                  2  87.3                        79.8                            4.3                               6 9    2.8    2.9 3  3   0  0  94 94.1                            0  010    5.7    9.4 4.7           9.1 0.6                  0.8                     88.7                        79.2                            0  092    21 19  21.6           21  0.8                  1.9                     50.5                        52.5                            5.3                               4.891    15.4    18.1        13.6           17.9               6.2                  2.6                     57 57.3                            7.3                               3.587    16.8    21.8        13.4           20.4               2.9                  2.6                     59.5                        48.9                            7  5.621    16 24.1        9.1           15.2               1  2.6                     69.6                        53.2                            3.7                               4.234    9.4    11.9        5.7           4.9 2  3.3                     67.6                        65.3                            14.4                               14.539    12.1    12.6        13.1           14.6               0.4                  1.3                     62.3                        66.7                            11.9                               4.393    18.9    20.3        9.7           10.3               1.8                  1.4                     65.5                        65.9                            3.4                               1.8BB/ST 6.3    13  5.7           7.3 2.2                  1.1                     34.7                        70.3                            50.3                               7.6HIV25 24.1    24.9        0.8           1.1 1.3                  5  70.2                        69.3                            2.9                               3.8__________________________________________________________________________
TABLE 4__________________________________________________________________________IMMUNOPHENOTYPING OF PATIENTS WITH B-CLLAND OTHER CONDITIONS BEFORE AND AFTER TREATMENTOF SAMPLES WITH MONOCLONAL ANTIBODY TO THE B CHAINOF THE HLA-DR WITH MONOCLONAL ANTIBODIES TO CD3 AND DR              CD+    DR-    DR+DR+         CD+    DR+    CD3-   HCD3-PATIENTB  A   B  A   B  A   B  A   B  A__________________________________________________________________________ 1   87 45.5       3.5          20.8              2.5                 4.2 6.9                        21.6                            0  7.6 2   76.2   19.4       9.6          29.2              3.9                 8.7 10.3                        36.8                            0  5.5 3   77.7   18.3       8.4          29.4              4.1                 8.8 9.6                        38.1                            0  4.7 4   76.8   19.2       7.6          29.5              6.2                 10.5                     9.1                        37.2                            0  3.3 5   ND 47.1       ND 11.5              ND 9.9 ND 22.4                            ND 7.3 6   ND 7   91.4   85.8       2.4          2.5 0.7                 0.7 5.1                        4.2 0  6.3 8   61.8   28.9       6.5          11.2              2  3.3 28.6                        54.6                            0  1.5 9   ND10   82.6   44.7       4.3          9.8 3.3                 5   9.8                        22.2                            0  17.992   23.8   14.1       39.3          41.9              4.5                 3.5 32.4                        40.5                            0  091   13.3   7.9 29.6          32.5              3.4                 2.9 53.4                        56.5                            0  087   14.8   12.2       28.4          34.1              5.5                 6.6 51.1                        46.5                            0  021   ND34   11.9   12.9       10.4          13.7              0.8                 0.6 76.7                        72.8                            0  039   25.6   13.7       24.6          25.2              3  2.8 46.5                        25.2                            0. 093   13.3   8.9 18.4          18.9              9.9                 10.1                     58.2                        61.7                            0  0BB/ST44.2   32.5       11.7          12.2              0.8                 0.8 43 49.4                            0  4.6__________________________________________________________________________
TABLE 5______________________________________IMMUNOPHENOTYPING OF PATIENTS WITH B-CLLAND OTHER CONDITIONS BEFORE AND AFTER TREATMENTOF BLOOD SAMPLES WITH MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DRWITH MONOCLONAL ANTIBODIES TO CD16 + 56 AND CD3                        CD56+ &   CD56+ &CD56+ &                      16+       16-16                 CD3+      CD3+      CD3-PATIENTS   B      A       B    A    B    A    B    A______________________________________ 1      2      4.3     5.7  19.7 0.7  1.7  91.3 73 2      11.5   38.9    12.4 32.6 1    6.6  74.5 21 3      12     36.2    12.1 34.5 0.7  6    75.5 23 4      12.2   32.6    12.4 39.6 0.5  5    74.7 22.2 5      ND     13.1    ND   9.4  ND   2.6  ND   73.5 6      ND 7      0.8    0.8     2.8  2.4  0.3  0.2  96.2 96.4 8      24.8   52      5.4  12.4 0.9  4.1  68.3 31.1 9      ND10      1.1    1.3     6.1  13.7 2.1  2.5  90.5 82.492      23.8   34.5    44.3 44.8 2    1.5  29.2 18.691      4.6    3.9     28.8 29.4 3    3.2  63.3 63.387      47.9   46.4    28.8 36.5 5.8  3.7  16.9 1321      9.4    9.4     19.7 23.6 4.2  6.7  66   59.534      21.5   12.8    11.4 13.7 1.8  0.6  64.6 72.839      7      2.7     23.4 26.1 1.1  0.1  68.2 7193      55.8   54.9    26.2 26.3 1.7  2    16.1 16.8BB/ST   28.8   29.9    12   14.3 0.8  1.8  49.4 53.6______________________________________
TABLE 6______________________________________IMMUNOPHENOTYPING OF PATIENTS WITH B-CLLAND OTHER CONDITIONS BEFORE AND AFTER OF TREATMENTOF BLOOD WITH MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DRWITH MONOCLONAL ANTIBODIES TO CD45 AND CD14                              CD45+  CD45+ H       CD45+ L       CD14+PATIENTS B      A        B    A      B    A______________________________________ 1       90.5   70.1     7.5  21.9   0.8  3.3 2       85.8   52.2     8.8  38.3   5.3  9.5 3       84.3   52.2     9.9  33.8   5.1  13.2 4       91.5   79.2     2.1  7      5.7  10.8 5       63.1   84.6     34.9 9.4    0.5  3.6 6       ND 7       52.8   85.2     45.6 13.9   0.5  0.6 8       71.1   55       71.1 34.5   5.3  8.7 9       SEE10       79.7   47.3     16.3 48     2.1  1.992       61.7   64.7     27.4 26.6   5.9  3.691       49.4   49.2     40.4 44.3   6.5  3.287       52.4   61.5     36.1 28.7   7    6.521       45.8   43.3     44.3 47.6   6.2  3.334       24.4   24.6     54.8 59.6   13.3 9.739       48.7   46.3     30.5 42.1   14.5 8.893       SEEHIV+     22.6   26.9     66.8 63.5   6.8  6.7IgA/D    47.4   59.8     41.9 33.3   5.9  4.1______________________________________
TABLE 7______________________________________IMMUNOPHENOTYPING OF PATIENTS WITH B-CLLAND OTHER CONDITIONS BEFORE AND AFTER TREATMENTOF BLOOD WITH MONOCLONAL ANTIBODIES TO THEHOMOLOGOUS REGION OF THE B CHAIN OF THE HLA-DRWITH MONOCLONAL ANTIBODIES TO CD8 AND CD3                         CD8+       CD8-CD8+             CD3+         CD3+       CD3-PATIENT B     A      B    A     B    A     B    A______________________________________ 2      0.6   1.3    7.5  19.3  4.2  19.3  87.7 63.8 3      1.1   1.4    8.3  20.3  5.6  18.4  84.8 59.8 4      3.5   2.9    8.3  27    3.9  16.6  84.2 53.192      3.5   1.9    27.6 25.2  18.4 19    50.3 52.891      4     3.1    18.2 19    14.1 12.6  63.6 65.387      5.7   3.9    19.9 23.6  15.4 17.4  58.8 5521      4.8   7.4    16.3 17.3  13.7 13    65.2 6234      3     3.6    5.2  6.7   7.6  7.5   84.1 82.3______________________________________
TABLE 8______________________________________IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITHTIME AFTER TREATMENT OF BLOOD WITH PE CONJUGATEDMONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGIONOF THE B-CHAIN OF THE HLA-DR MEASURE WITHMONOCLONAL ANTIBODIES TO CD45 AND CD14TIME    DR + CD45 + CD14 + r                    CD45 + L CD45 + H______________________________________2 HR    81.7             8.2      8.26 HR    80.7             8.1      10.624 HR   79               1.1      18.4______________________________________
TABLE 9______________________________________IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITHTIME AFTER TREATMENT OF BLOOD WITH PE CONJUGATEDMONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGIONOF THE B-CHAIN OF THE HLA-DR MEASURE WITHMONOCLONAL ANTIBODIES TO CD19 AND CD3                               CD19 -TIME   CD19 + DR + r              CD3+     CD3 + DR+                               CD3 - DR-______________________________________2 HR   87.4        10.1     1.8     10.76 HR   75.5        10.4     3.1     10.724 HR  74          11.7     2.9     11______________________________________
TABLE 10__________________________________________________________________________IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTER TREATMENTOF BLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THE HOMOLOGOUSREGION OF THE B-CHAIN OF THE HLA-DR MEASURE WITH MONOCLONALANTIBODIES TO CD4 AND CD8TIME    CD8 + & DR + r       CD4+           CD4 + &CD8 + &DR + r                       CD4 + DR+                             CD4 - CD8 - DR-__________________________________________________________________________2 HR    77.6    6.8 5.4         1.3   8.86 HR    75.8    6.7 6.4         1.8   9.324 HR    77      6.4 4.8         1.9   11__________________________________________________________________________
TABLE 11______________________________________IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITHTIME AFTER TREATMENT OF BLOOD WITH PE CONJUGATEDMONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGIONOF THE B-CHAIN OF THE HLA-DR MEASURE WITHMONOCLONAL ANTIBODIES TO CD3 AND DRTIME     DR+    CD3+      CD3 + DR+                             CD3 + DR-______________________________________2 HR     75     9.5       4.2     10.96 HR     74.8   8.8       4.8     10.924 HR    ND     ND        ND      ND______________________________________
TABLE 12__________________________________________________________________________IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITH TIME AFTER TREATMENT OFBLOOD WITH PE CONJUGATED MONOCLONAL ANTIBODY TO THE HOMOLOGOUSREGION OF THE B-CHAIN OF THE HLA-DR MEASURE WITH MONOCLONALANTIBODIES TO CD16&56 AND CD3             CD56 + CD16 + &CD3 + DR+                           CD56 - CD16 - &CD16-TIME    CD56 + &16 + DR + r         CD3+             r             DR-__________________________________________________________________________2 HR    82.5      9.5 4.1           3.56 HR    84.3      7.5 4.1           3.324 HR    ND        ND  ND            ND__________________________________________________________________________
TABLE 13______________________________________IMMUNOPHENOTYPING OF A PATIENT WITH B-CLL WITHTIME AFTER TREATMENT OF BLOOD WITH PE CONJUGATEDMONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGIONOF THE B-CHAIN OF THE HLA-DR MEASURED WITHMONOCLONAL ANTIBODIES TO CD8 AND CD3                    CD8 + CD +TIME   CD8 + DR+ CD3+    3&DR + r                            CD8 - CD3 - DR-______________________________________2 HR   76.2      6.6     6.7     10.66 HR   76.5      6.2     6.2     10.3______________________________________
TABLE 14__________________________________________________________________________IMMUNOPHENOTYPING OF PATIENTS WITH B-CLL BEFORE AND AFTER TREATMENT OFBLOOD WITHMONOCLONAL ANTIBODIES TO THE HOMOLOGOUS REGION OF THE A-CHAIN OF THEHLA-DR,THE HOMOLOGOUS REGION OF THE B-CHAIN OF THE HLA-DR, THE TWO MONOCLONALTOGETHER,MONOCLONAL TO THE HOMOLOGOUS REGION OF THE B-CHAIN PLUSCYCLOPHOSPHOAMIDEAND THE HOMOLOGOUS REGION OF CLASS I ANTIGENS MEASURED WITH TIME                       CD19+       CD19-CD19+           CD3+        CD3+        CD3-ID  B AA AB      ABC         AI           B AA AB                  ABC                     AI                       B AA AB                              ABC                                 AI                                   B AA AB                                          ABC                                             AI__________________________________________________________________________5/62H  86 91 54      40 89            5             4  16                  23  5                       1 1  3 2  1  6                                     4  27    33                                         524   N 88 51      60 86            N             4  18                  10  4                        N                         2  1 2  3  N                                     4  29    28                                         7102H  77  N 59       N 80            7              N 13                   N  7                       1  N 1  N 0 14                                      N 26    120924   8  N  N       N  6           32              N  N                   N 38                       1  N  N                               N 1 59                                      N  N    5643/BD6H   0  N  0       0  0           40              N 42                  43 49                       0  N 1 0  1 58                                      N 54    54                                        4704/BD6H   0  N  0       0  0           49              N 41                  45 46                       0  N 3 1  3 43                                      N 42    44                                        41HIV+6H   1  N  0       N  1           10              N 14                   N 12                       0  N 0  N 0 89                                      N 86    87IgA/D6H  10  N  1       N 12           21              N 25                   N 20                       2  N 1  N 3 67                                      N 71    68__________________________________________________________________________ B = Before; A = After; AB = after addition to antibody to beta chain; AA = after addition of antibody to alpha chain; ABC = after addition of antibody to either alpha or beta chain and cycloposphoamide; AI = after addition of antibody to Class I.
TABLE 15__________________________________________________________________________CD8 AND CD4                       CD4+        CD4-CD8+            CD4+        CD8+        CD8-ID  B AA AB      ABC         AI           B AA AB                  ABC                     AI                       B AA AB                              ABC                                 AI                                   B AA AB                                          ABC                                             AI__________________________________________________________________________5/62H   3 2  14      10  4            2             2  8 8   3                       0 0  3 2  1 95                                     94 74    79                                        9324   N 3   9       4  4            N             3  8 4   3                        N                         0  2 2  0  N                                     94 81    90                                        93102H   3  N  7       N  4           4  N 7  N  3                       1  N 2  N 1 91                                      N 83    920924  10  N  N       N 15           21              N  N                   N 38                       2  N  N                               N 2 61                                      N  N    53__________________________________________________________________________
TABLE 16__________________________________________________________________________CD3 AND DR                       CD3+        CD3-DR+             CD3+        DR+         DR-ID  B AA AB      ABC         AI           B AA AB                  ABC                     AI                       B AA AB                              ABC                                 AI                                   B AA AB                                          ABC                                             AI__________________________________________________________________________5/62H   N 90 54       N 87            N             4  12                   N  4                        N                         2  10                               N 3  N                                     5  22     5102H  83  N 63       N 81            4             N   8                   N  4                       4  N  7                               N 4  9                                      N 23    120924  14  N  N       N 13           36              N  N                   N 36                       3  N  N                               N 3 51                                      N N     47__________________________________________________________________________
TABLE 17__________________________________________________________________________CD16 & 56 AND CD3                       CD56+ & 16+ CD56- & 16-CD56+ & 16+     CD3+        CD3+        CD3-ID  B AA AB      ABC         AI           B AA AB                  ABC                     AI                       B AA AB                              ABC                                 AI                                   B AA AB                                          ABC                                             AI__________________________________________________________________________5/62H   N 0  13       N  4            N             5   9                   N  5                        N                         1  3  N 1  N                                     94 74    90102H   0  N  1       N  1            6              N 14                   N  6                       1  N 2  N 1 92                                      N 65    920924  42  N  N       N 41           36              N  N                   N 38                       2  N  N                               N 2 20                                      N  N    19__________________________________________________________________________
TABLE 18__________________________________________________________________________CD45 AND CD14                                   CD45+CD45+ L         CD45+ M     CD45+ H     CD14+ID  B AA AB      ABC         AI           B AA AB                  ABC                     AI                       B AA AB                              ABC                                 AI                                   B AA AB                                          ABC                                             AI__________________________________________________________________________5/62H  0 0  5 10 0 44             43 50                  50 32                       55                         43 50                              31 67                                   1 1  1     2                                        0102H  0  N 0  N 0 43              N 54                   N 35                       54                          N 42                               N 62                                   1  N 1     0N0924  2  N  N       N 1 18              N  N                   N 16                       71                          N  N                               N 76                                   7  N  N    5NHIV+6H  4  N 3  N 6 63              N 61                   N 41                       23                          N 27                               N 40                                   7  N 7     7NIgA/D6H  2  N 2  N 4 40              N 31                   N 44                       47                          N 60                               N 44                                   6  N 4     6N__________________________________________________________________________
TABLE 19__________________________________________________________________________CD8 AND CD28                       CD8+        CD8-CD8+            CD28+       CD28+       CD28-ID  B AA AB      ABC         AI           B AA AB                  ABC                     AI                       B AA AB                              ABC                                 AI                                   B AA AB                                          ABC                                             AI__________________________________________________________________________5/62H   N 3  6  N 3  N             1  4  N 2  N                         1  4  N 1  N                                     95 86    942H  4  N 6  N  N           3  N 5  N  N                       1  N 3  N  N                                   92                                      N 86     N__________________________________________________________________________
TABLE 20__________________________________________________________________________CD34 AND CD2                       CD34+       CD34-CD34+           CD2+        CD2+        CD2-ID  B AA AB      ABC         AI           B AA AB                  ABC                     AI                       B AA AB                              ABC                                 AI                                   B AA AB                                          ABC                                             AI__________________________________________________________________________5/62H   N  1 34       N  N            N              6 13                   N  N                        N                          3 30                               N  N                                    N                                     90 21     N24   N  1  6       9  N            N              7 23                   4  N                        N                          3 33                              43  N                                    N                                     87 34    34                                         NHIV+2H   2  1 12      13  N           20             21 21                  12  N                        4                          5  9                              14  N                                   73                                     73 64    60                                         NBB/ST2H  26 23 33      14  N           15             14 15                  15  N                       31                         30 23                              36  N                                   27                                     32 28    35                                         N24   N 11 29      11  N            N             13 12                   9  N                        N                         27  9                              18  N                                    N                                     48 49    61                                         N__________________________________________________________________________
CHART 1______________________________________IMMUNOPHENOTYPIC CHANGES OF UNTREATED ANDTREATED BLOOD SAMPLE OF PATIENT (2, 3 & 4) WITHMONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGIONOF THE β-CHAIN OF HLA-DR ANTIGEN MEASUREDWITH TIMEWITHOUT     WITH      FL1     FL2     TIME______________________________________NOTHING001  WITH002   CD45    CD14    2 HRNO001       WE002     CD45    CD14    6 HR001001      002002    CD45    CD14    24 HRNOTHING003  WITH004   CD3     CD19    2 HRNO003       WE004     CD3     CD19    6 HR001003      002004    CD3     CD19    24 HRNOTHING004  WITH005   CD4     CD8     2 HRNO004       WE005     CD4     CD8     6 HR001004      002005    CD4     CD8     24 HRNOTHING005  WITH006   CD3     DR      2 HRNO005       WE006     CD3     DR      6 HR001005      002006    CD3     DR      24 HRNOTHING006  WITH007   CD3     CD56&16 2 HRNO006       WE007     CD3     CD56&16 6 HR001006      002007    CD3     CD56&16 24 HRN003        W004      CD3     CD8     2 HRNO007       WE008     CD3     CD8     6 HR001007      002008    CD3     CD8     24 HR______________________________________
CHART 1A______________________________________IMMUNOPHENOTYPIC CHANGES OF UNTREATED ANDTREATED BLOOD SAMPLE OF PATIENT (2, 3, 4) WITHMONOCLONAL ANTIBODY TO THE HOMOLOGOUS REGIONOF THE β-CHAIN OF HLA-DR ANTIGEN MEASUREDWITH TIMEID           FL1      FL2         TIME______________________________________WL003        CD45     CD14        2 HRWEL003       CD45     CD14        6 HR003003       CD45     CD14        24 HRWL005        CD3      CD19        2 HRWEL005       CD3      CD19        6 HR003005       CD3      CD19        24 HRWL006        CD4      CD8         2 HRWEL006       CD4      CD8         6 HR003006       CD4      CD8         24 HRWL007        CD3      DR          2 HRWEL 007      CD3      DR          6 HRWL008        CD3      CD65&16     2 HRWEL 008      CD3      CD56&16     6 HRWL005        CD3      CD8         2 HRWEL009       CD3      CD8         6 HR______________________________________
CHART 2______________________________________IMMUNOPHENOTYPIC CHANGES OF UNTREATED ANDTREATED BLOOD OF PATIENT (1) WITH MONOCLONALANTIBODY TO THE HOMOLOGOUS REGION OF THEβ-CHAIN OF HLA-DR ANTIGEN, THIS ANTIBODY ANDCYCLOPHOSPHAMIDE, MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF THE α-CHAIN OF HLA-DRANTIGEN AND MONOCLONAL ANTIBODY TO THEHOMOLOGOUS REGION OF CLASS I ANTIGEN MEASUREDWITH TIMEWITH       WITHOUT    FL1     FL2     TIME______________________________________      NA001      CD45    CD14    2 HRA2B001: AB            CD45    CD14    2 HRA2A : AA              CD45    CD14    2 HRDNAA001:ABC           CD45    CD14    2 HRA1001: AI             CD45    CD14    2 HR      NC001      CD3     CD19    2 HRC2B001:AB             CD3     CD19    2 HRC2A001:AA             CD3     CD19    2 HRDNAC001:ABC           CD3     CD19    2 HRC1001: AI             CD3     CD19    2 HRA124H001:AI           CD3     CD19    24 HRA2B24H001: AB         CD3     CD19    24 HRA2A24H001:AA          CD3     CD19    24 HRA2BX24H001:AB         CD3     CD19    24 HR      ND001      CD4     CD8     2 HRD2B001: AB            CD4     CD8     2 HRD2A001: AA            CD4     CD8     2 HRDNAD001:ABC           CD4     CD8     2 HRD1001: AI             CD4     CD8     2 HRD124H001:AI           CD4     CD8     24 HRD2BX24H001:AB         CD4     CD8     24 HRCD2B001: AB            CD4     CD8     24 HRD2A001: AA            CD4     CD8     24 HRE1001: AI             CD3     DR      2 HRE2B001: AB            CD3     DR      2 HRE2A001: AA            CD3     DR      2 HRF1001: AI             CD3     CD56&16 2 HRF2B001: AB            CD3     CD56&16 2 HRF2A001: AA            CD3     CD56&16 2 HRG1001: AI             CD28    CD8     2 HRG2A001: AA            CD28    CD8     2 HRG2B001: AB            CD28    CD8     2 HRH1001: AI             CD7     CD33&13 2 HRH2A001: AA            CD7     CD33&13 2 HRH2B001: AB            CD7     CD33&13 2 HRI2A001: AA            CD21    CD5     2 HRI2B001:AB             CD21    CD5     2 HRJ2A001: AA            CD34    CD2     2 HRJ2B001: AB            CD34    CD2     2 HRB2A24H001:AA          CD34    CD2     24 HRB2B24H001:AB          CD34    CD2     24 HRB2BX24H001:           CD34    CD2     24 HRABCK2B001: AB            CD10    CD25    2 HRK2A001: AA            CD10    CD25    2 HR______________________________________
CHART 3______________________________________IMMUNOPHENOTYPIC CHANGES OF UNTREATED ANDTREATED BLOOD OF PATIENT (8) WITH MONOCLONALANTIBODY TO THE HOMOLOGOUS REGION OF THEβ-CHAIN OF HLA-DR ANTIGENWITH     WITHOUT    FL1       FL2    TIME______________________________________    AN001      CD45      CD14   2 HRA2001               CD45      CD14   2 HR    CN001      CD3       CD19   2 HRC2001               CD3       CD19   2 HR    DN001      CD4       CD8    2 HRD2001               CD4       CD8    2 HR    EN001      CD3       DR     2 HRE2001               CD3       DR     2 HR    FN001      CD3       CD56&16                                2 HRF2001               CD3       CD56&16                                2 HR    GN001      CD28      CD8    2 HRG2001               CD28      CD8    2 HR    HN001      CD7       CD5    2 HRH2001               CD7       CD5    2 HR    IN001      CD13      CD20   2 HRI2001               CD13      CD20   2 HR    JN001      CD45RA    CD25   2 HRJ2001               CD45RA    CD25   2 HR    KN001      CD57      CD23   2 HRK2001               CD57      CD23   2 HR______________________________________
CHART 4______________________________________IIMMUNOPHENOTYPIC CHANGES OF UNTREATED AND TREAT-ED BLOOD SAMPLE OF PATIENT (10) WITH MONOCLONALANTIBODY TO THE HOMOLOGOUS REGION OF THEβ-CHAIN OF HLA-DR ANTIGEN AND MONOCLONAL ANTI-BODY TO THE HOMOLOGOUS REGION OF CLASS I ANTIGENS.WITH      WITHOUT    FL1     FL2      TIME______________________________________     CLL0001    CD45    CD14     2 HRCLL1001              CD45    CD14     2 HRCLL2001              CD45    CD14     2 HR     CLL0003    CD3     CD19     2 HR                CD3     CD19     2 HRCLL1003              CD3     CD19     2 HRCLL2003              CD3     CD19     2 HR     CLL0004    CD4     CD8      2 HRCLL1004              CD4     CD8      2 HRCLL2004              CD4     CD8      2 HR     CLL005     CD3     DR       2 HRCLL1005              CD3     DR       2 HRCLL2005              CD3     DR       2 HR     CLL0006    CD3     CD56 & 16                                 2 HRCLL1006              CD3     CD56 & 16                                 2 HRCLL2006              CD3     CD56 & 16                                 2 HR______________________________________
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U.S. Classification 435/372, 435/372.2, 435/372.3
International Classification C12N5/078, C12N5/02, A61K39/00, A61K39/395, A61P37/04, C12N5/00, C12N5/071
Cooperative Classification C12N2501/06, C12N2506/11, C12N5/0634, C12N2501/50
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