AU2005209613A1 - Method of isolating CD8+ cells, and related hybridoma cells, antibodies and polypeptides - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant: Actual Inventor/s: Address for Service is: Ortho-McNeil Pharmaceutical, Inc.

Didier J. Leturcq SHELSTON IP Margaret Street SYDNEY NSW 2000 CCN: 3710000352 Attorney Code: SW Telephone No: Facsimile No.

(02) 97771111 (02) 9241 4666 Invention Title: METHOD OF ISOLATING CD8+ CELLS, AND RELATED HYBRIDOMA CELLS, ANTIBODIES AND POLYPEPTIDES Details of Original Application No. 37284/00 dated 08 March 2000 The following statement is a full description of this invention, including the best method of performing it known to us:- File: 33092AUP01 500682678_1 .DOC/5844

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cN METHOD OF ISOLATING CD8 CELLS, AND RELATED HYBRIDOMA §^CELLS, ANTIBODIES AND POLYPEPTIDES 00 o The present application is a divisional application of Australian Application No.

37284/00, which is incorporated in its entirety herein by reference.

Throughout this application, various publications are cited. The disclosure of 0 these publications is hereby incorporated by reference into this application to describe N more fully the state of the art to which this invention pertains.

Field of the Invention This invention relates to a positive selection method for isolating CD8 cells using certain CD8-specific antibodies. The isolated CD8 cells have importance as vehicles for combating viral infections and tumors.

Background of the Invention In humans, CD8 cells play a vital role in the immune system's ability to defend against potentially harmful foreign entities, such as bacteria and viruses CD8+ cells circulate in the blood and possess on their surface the CD8 protein. When necessary, these cells are converted into cytotoxic cells cell-killing cells) which proceed to destroy foreign cells, viruses, and other harmful pathogens present in the subject Because of CD8+ cells' effective role in host defence, they hold great potential in isolated form as therapeutics for treating disorders such as viral infections and malignancies In the past, purification of human CD8 cells has been achieved by negative selection. Specifically, 2 Speripheral blood mononuclear cells ("PBMC's") are incubated with a cocktail of monoclonal antibodies 00 C specific for non-CD8 sub-populations. These subpopulations include, for example, B-cells, CD4* cells, NK 5 cells, macrophages and neutrophils, and each contains D specific, non-CD8 "markers". The sub-populations are then 0 removed from the resulting antibody cocktail using magnetic beads This technique has certain major C disadvantages. The first is that several monoclonal antibodies are required for removing non-CD8 cells. The second is that the resulting CD8* population suffers from contamination from non-CD8* cells that possess relatively low levels of non-CD8 markers. Finally, when a magnetic separation procedure is used to remove all non-CD8* cells, a large number of magnetic beads are needed.

3 (1 Summary of the Invention C This invention provides a method of isolating CD8* cells which comprises the steps of 5 contacting a sample of isolated peripheral 0D mononuclear blood cells with a first antibody D which specifically binds to the sequence n AAEGLDTQRFSG, or portion thereof, on CD8 D molecules present on the surface of CD8* cells but does not activate the CD8' cells once bound thereto, under conditions permitting the formation of a first complex between the CD8S cell and first antibody; separating from the sample any first antibody not present in the resulting first complex; contacting the sample with a second, immobilized antibody which specifically binds to the first antibody in the first complex, under conditions permitting the formation of an immobilized, second complex between the first complex and the second antibody, thereby immobilizing the CD8B cells present in the sample; separating from the resulting immobilized second complex the cells present in the sample which were not immobilized in step contacting the immobilized second complex under suitable conditions with an agent which causes the dissociation of the second complex into CD8* cells and an immobilized third complex between the first antibody and second antibody; and separating the immobilized third complex from the CD8 4 ceils,-thereby isolating the CD8 cells.

n 4 a This invention also provides a hybridoma cell line which produces a monoclonal antibody which specifically 00 0 binds to CD8 molecules present on the surface of CD8* cells but does not activate the CD8 cells. This c 5 invention further provides monoclonal antibodies produced ND by each of the instant hybridoma cell lines. Finally, O this invention provides related polypeptides, isolated n CD8* cells and kits.

E4 5 5 Q^ Detailed Description of the Invention 00 The hybridoma cell lines designated 37B1 and 8G6 were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International q\ Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, O Virginia 2010-2209 under ATCC Accession Nos. HB-12441 and S 10 HB-12657, respectively.

This invention provides a method of isolating CD8* cells by employing an anti-CD8 antibody, along with certain other reagents. Specifically, this invention provides a method of isolating CDS' cells which comprises the steps of contacting a sample of isolated peripheral mononuclear blood cells with a first antibody which specifically binds to the sequence AAEGLDTQRFSG, or portion thereof, on CD8 molecules present on the surface of CD8* cells but does not activate the CD8* cells once bound thereto, under conditions permitting the formation of a first complex between the CD8* cell and first antibody; separating from the sample any first antibody not present in the resulting first complex; contacting the sample with a second, immobilized antibody which specifically binds to the first antibody in the first complex, under conditions permitting the formation of an immobilized, second complex between the first complex and the second antibody, thereby immobilizing the CD8* cells present in the sample; r

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6 separating from the resulting immobilized second o0 complex the cells present in the sample which O were not immobilized in step contacting the immobilized second complex under suitable conditions with an agent which causes

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C the dissociation of the second complex into CD8* C cells and an immobilized third complex between the first antibody and second antibody; and C separating the immobilized third complex from the CD8* cells, thereby isolating the CD8* cells.

As used herein, a "CD8' cell" means a T-cell having on its surface the CD8 protein. In the preferred embodiment, the CD8* cells are human CD8* cells. The CD8* cells can be from any CD8* cell-possessing species.

"Isolating" CD8* cells means obtaining a population of peripheral mononuclear blood cells wherein the ratio of CD8* cells to non-CD8* cells is at least about 7:1. In the preferred embodiment of this invention, this ratio is at least about 9:1.

This invention employs several types of antibodies which specifically bind to given epitopes. More particularly, this invention uses a "first antibody" which specifically binds to the sequence AAEGLDTQRFSG, or portion thereof, on CD8 molecules present on the surface of CD8* cells but does not activate the CD8* cells once bound thereto. Here, CD8* cell "activation" means causing CD8+ cells to express y-interferon This activation can be measured using routine methods such as sandwich ELISA assays, which can be performed using commercially available kits.

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o 7 v i Such first antibodies include, but are not limited 0 to, the monoclonal antibodies produced by the hybridoma cell lines 37B1 (ATCC Accession No. HB-12441) and 8G6 (ATCC Accession No. HB-12657). Conditions which permit ID these antibodies to bind to but not activate CD8* cells D are well known in the art. These conditions are i described, for example, in a suitable buffer such as Ca2* 0 and Mg2*-free Dulbecco's Phosphate Buffer Saline (DPBS) containing 1% Human Serum Albumin (HSA) and 0.2% sodium citrate and gentle mixing by "end over end" rotation on a rotator set at 4 rpm.

As used herein, the term "antibody" includes, but is not limited to, both naturally occurring and non-naturally occurring antibodies. Specifically, the term "antibody" includes polyclonal and monoclonal antibodies, and binding fragments thereof. Furthermore, the term "antibody" includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof. In one embodiment, the antibody is a monoclonal antibody. The monoclonal antibody can be human, or that of another species including, for example, mouse and rabbit. In this invention, an antibody which "specifically" binds to a stated epitope binds to that epitope with a dissociation constant of at least about 10-fold less than the dissociation constant with which it binds to any other epitope. In one embodiment, this dissociation constant ratio is at least about 100. In the preferred embodiment, this dissociation constant ratio is at least about 10 3 The "second antibodv" used in the instant method can be any antibody which specifically recognizes an epitope on any portion of the first antibody. In the preferred embodiment, the second antibody specifically recognizes a

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8 g portion of the constant (Fc) region of the first antibody.

Such anti-Fc antibodies are commercially available and 00 include, for example, sheep anti-mouse antibody immobilized on magnetic beads ND The agent that causes dissociation of the immobilized D second complex into CD8* cells and immobilized antibodies ican be any agent which successfully competes with the CD8 D molecule for specific binding to the first antibody. In the preferred embodiment, this agent is the polypeptide designated CD8-3 having the sequence AAEGLDTQRFSG. In one embodiment, the immobilized second antibody comprises an antibody operably affixed to a magnetic bead.

This invention also provides a hybridoma cell line which produces a monoclonal antibody which specifically binds to CD8 molecules present on the surface of CD8* cells but does not activate the CD8* cells. In one embodiment, the hybridoma cell line is selected from the cell lines designated 37B1 (ATCC Accession No. HB-12441) and 8G6 (ATCC Accession No. HB-12657). This invention further provides the monoclonal antibodies produced by each of the instant hybridoma cell lines.

This invention further provides a polypeptide useful for generating the instant monoclonal antibody that comprises the amino acid sequence AAEGLDTQRFSG. In the preferred embodiment, the polypeptide is the polypeptide designated CD8-3 and having the amino acid sequence AAEGLDTQRFS. The instant polypeptide can optionally comriris one or more additional =minn rAA residues at the C-terminal or N-terminal end. In the preferred embodiment, the polypeptide has the sequence

NKPKAAEGLDTQRFSGKRLG.

9 This invention further provides a population of CD8* 00 cells isolated by the instant method.

Finally, this invention provides a kit for use in isolating CD8' cells which comprises, in separate C compartments, an antibody which specifically binds to CI the sequence AAEGLDTQRFSG, or portion thereof, on CD8 0 molecules present on the surface of CD8* cells, but does CI 10 not activate the CD8* cells once bound thereto; and an agent which causes the dissociation of a CD8* cellantibody complex. In one embodiment, the agent which causes the dissociation of a CDSB cell-antibody complex comprises the polypeptide having the sequence AAEGLDTQRFSG. In the preferred embodiment, the agent is the polypeptide consisting of the sequence AAEGLDTQRFSG.

The instant kit can further comprise reagents useful for performing the binding and dissociation steps of the instant method. The components of the instant kit can either be obtained commercially or made according to well known methods in the art. In addition, the components of the instant kit can be in solution or lyophilized as appropriate. In the preferred embodiment, the kit further comprises instructions for use.

The following procedures relating to the instant invention are routine in the art: isolating peripheral mononuclear blood cells from which the CD8* cells are in turn isolated separating unbound antibodies and cells from a sample containing bound antibodies and/or cells via centrifugation or-spinning membrane; and immobilizing antibodies via polystyrene flasks, columns or beads In 10 This invention will be better understood by reference to the Experimental Details which follow, but those skilled 00 0 in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention 5 as described more fully in the claims which follow NO thereafter.

m 11 O O C Experimental Details 00

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Rationale n 5 Human CD8* cells can be isolated from preparations of \0 peripheral blood mononuclear cells (PBMC's) by either 0 positive or negative selection. Positive selection t results in a highly-purified population of CD8* cells.

Negative selection, while resulting in sufficient numbers of CD8* cells, has low levels of contaminating non-CD8 populations remaining after the selection procedure.

The idea was to generate an antibody which has high affinity for CD8* cells, does not activate the cells during the selection process, and is capable of being easily eluted from the cells. An anti-peptide antibody appeared to meet these criteria. However, it was known that anti-peptide antibodies might be of low affinity and may recognize the linear peptide sequence exclusively, preventing reactivity with native antigen.

It was necessary that the anti-CD8 antibody not activate the cells during the selection process, as it would lessen their ability to effectively act as naive responder cells during in vitro stimulation protocols.

The use of peptide release to selectively isolate a cell population has been show by Tseng-Law, et al. for CD34' cells.

12 g Methods r The CD8 alpha chain was examined for hydrophilic 00 D sequences and four peptides selected. All were coupled to keyhole limpet hemocyanin (KLH) as carrier and used to c 5 immunize mice. A C-terminal amino acid was added to each MD of the peptides coupled to KLH to make the monoclonal D antibodies. Antisera from the mice were evaluated for the V ability to recognize both peptide and native CD8 on the D surface of T cells. Only one of the four peptides was capable of recognition of both antigenic forms of CD8.

Monoclonal antibodies were generated to this peptide and the resulting antibody used to isolate CD8* cells from a PBMC preparation. The antibody was successful in isolating a population of highly-purified CD8* cells (Table 1) which were not activated by the isolation procedure (Table 2).

13 00 Table 1 Purification of CD8+ Cells by Positive Selection Analyzed by Flow Cytometry* CD8 T cells 15 (7-24) 82 (56-95) CD4 T cells 36 (14-52) 2 (0.1-10) CD14 Monocytes 15 (7-26) 0.8 (0.2-2) Neutrophils 12 (8-21) 0.6 (0.1-3) CD19 B cells 2 3 (0.5-9) CD56 NK cells 6 (2-17) *Sumimary of 10 normal donors Table 2 Activation of CDB+ Cells Isolated By Negative or Positive Selection (Assessed by IFNy Production) allo- stimulation 1440 3600 m 14

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CA References 001. Nabholz M. and H.R. MacDonald (1983) Annual Review of Immunology 1:273-306.

0 2. Riddell S.R. and P.D. Greenberg (1994) Current Topics Sin Microbiology and Immunology 189:9-34.

3. Riddell S.R. and P.D. Greenberg (1995) Annual Review of Immunology 13:545-586.

4. Horgan K and S. Shaw (1994) Current Protocols in Immunology 2:7.4.1.

5. Lea T, et al. (1988) Journal of Molecular Recognition 1(1):9-18.

6. Kanof, et al. (1994) Current Protocols in Immunology 2:7.1.1.

7. Kanof M.E. (1994) Current Protocols in Immunology 2:7:3:1.

8. PCT International Publication No. WO 95/34817.

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Claims (6)

1. 3. The method of claim 1, wherein the first antibody is S 5 a monoclonal antibody. \O
2. 4. The method of claim 3, wherein the monoclonal Santibody is produced by a hybridoma cell line selected from the group consisting of the cell line C 10 designated 37B1 (ATCC Accession No. HB-12441) and the cell line designated 8G6 (ATCC Accession No. HB- 12657). The method of claim 1, wherein the immobilized second antibody comprises an antibody operably affixed to a magnetic bead.
3. 6. The method of claim 1, wherein the agent which causes the dissociation of immobilized third complex is the polypeptide designated CD8-3 and having the amino acid sequence AAEGLDTQRFSG.
4. 7. A hybridoma cell line which produces a monoclonal antibody which specifically binds to CD8 molecules present on the surface of CD8* cells but does not activate the CD8* cells.
5. 8. The hybridoma cell line of claim 7, wherein the hybridoma cell line is selected from the group consisting of the cell line designated 37B1 (ATCC Accession No. HB-12441) and the cell line designated 8G6 (ATCC Accession No. HB-12657).
6. 17- o0 9. The monoclonal antibody produced by the hybridoma cell line of claim 7. \D 10. The monoclonal antibody produced by the hybridoma cell line of claim 8. 11. A polypeptide useful for generating the monoclonal C antibody of claim 9 which comprises the amino acid sequence AAEGLDTQRFSG. 12. The polypeptide of claim 11, wherein the polypeptide is the polypeptide designated CD8-3 and having the amino acid sequence AAEGLDTQRFSG. 13. A population of CD8* cells isolated by the method of claim 1. 14. A kit for use in isolating CD8 cells which comprises, in separate compartments, an antibody which specifically binds to the sequence AAEGLDTQRFSG, or portion thereof, on CD8 molecules present on the surface of CDSB cells, but does not activate the CD8* cells once bound thereto; and an agent which causes the dissociation of a CD8* cell-antibody complex. The kit of claim 14, wherein the agent which causes the dissociation of a CD8 cell-antibody complex is the polypeptide having the sequence AAEGLDTQRFSG. DATED this 8 th day of September 2005 Shelston IP Attorneys for: Ortho-McNeil Pharmaceutical, Inc.