WO1996040041A2

WO1996040041A2 - Antibodies to fas antigen capable of inhibiting apoptosis

Info

Publication number: WO1996040041A2
Application number: PCT/US1996/009153
Authority: WO
Inventors: Thomas Gesner
Original assignee: Chiron Corp
Current assignee: Novartis Vaccines and Diagnostics Inc
Priority date: 1995-06-07
Filing date: 1996-06-05
Publication date: 1996-12-19
Anticipated expiration: 1997-12-07
Also published as: WO1996040041A3; AU6091496A

Abstract

Antibodies capable of recognizing and binding Fas antigen and further capable of inhibiting apoptosis, as well as hybridomas capable of producing the antibodies are disclosed. Methods for use of the antibodies to treat disease wherein apoptosis is implicated are also disclosed.

Description

ANTIBODIES TO FAS ANTIGEN CAPABLE OF INHIBITING APOPTOSIS

Technical Field

The invention relates to the field of immunology. More specifically, the invention relates to antibodies capable of binding Fas antigen and preventing apoptosis, to hybndomas capable of producing the antibodies and to methods of using the antibodies to treat and prevent diseases and symptoms of diseases caused by apoptosis. Background of the Invention

The presence of Fas antigen on cell surfaces was first described by Yonehara et al, (1989) J. Exp. Med. 169:1747-1756. Fas antigen was described as a cell surface component with a molecular weight of approximately 200,000 daltons. Yonehara et al identifed Fas antigen by means a monoclonal antibody capable of binding to Fas antigen. The monoclonal antibody had an activity the authors described as "indistinguishable from the cytolytic activity of TNF." However, it was pointed out that the molecular weight of Fas antigen is different from that of the TNF receptor. Fas antigen appears on cells that do not express the TNF receptor and on cells that do express the TNF receptor. On those cells positive for Fas antigen and TNF receptor, Fas antigen is co-downregulated with the TNF receptor when the cells are incubated with TNF or with the anti-Fas antigen antibody. Subsequently, the Fas antigen has been detected on the cell surface of myeloid cells, T lymphoblastoid cells and diploid fibroblasts. The cDNA for human Fas has been cloned and sequenced. Itoh et al, (1991) Cell 66:233-243. The sequence was also used to express Fas on the surface of murine T lymphoma WR19L and fibroblast L929 cells. Binding of an anti-Fas antibody resulted in the death of these cells by apoptosis accompanied by fragmentation of chromosomal DNA and fragmentation of nuclei.

The Fas Ligand has been cloned by Suda et al, (1993) Cell 75: 1169-1178 and the

protein sequence determined from the nucleotide sequence reveals that Fas Ligand is a type π integral membrane protein with homology to tumor necrosis factor. Interaction of Fas antigen and Fas Ligand leads to apoptosis and is involved in T-cell mediated cytotoxicity.

Antibodies to Fas antigen are known in the art to induce apoptosis and have been administered to mice with resulting fulminating hepatitis due to apoptosis in liver cells. Ogaswara et al, (1993) Nature 364:806-809. Further, it is known that autoimmune diseases such as Sjόgren syndrome, type I diabetes and viral hepatitis are associated with T-cell migration and infiltration of the affected area. Therefore, a need exists in the art for molecules capable of binding Fas and inhibiting apoptosis onset. Brief Description of the Drawings Figure 1 shows the results of experiments utilizing the antibodies of the invention.

SKW 6.4 cells (2 x 10⁴ cells) were preincubated with C33 (open triangle); C40 (closed square); C28 (closed triangle); or C42 (closed circle) for 1 hour at 37°C. CHI 1 was added to 100 ng/ml and incubated for 20 hours at 37°C. The last two hours in the presence of 1 μCi ³H-thymidine. The results show that C28 and C42 antibodies are potent inhibitors of CHI 1 induced cell death across the range of concentrations tested. C33 and C40 were more effective at higher concentrations of antibody. The results of measurements of incorporation of ³H-thymidine by SKW 6.4 cells with (lower arrow) and without (upper arrow) treatment with CHI 1 are shown on the left axis of the figure. Figure 2 shows the inhibition of Fas ligand killing of SKW 6.4 cells by monoclonal antibodies. SKW 6.4 cells (2.86 x 10⁴ cells) were preincubated with C33 (open square); C40 (open diamond); C28 (closed diamond); or C42 (closed square) for 1 hour at 37°C. SF9 cells expressing Fas ligand on the cell surface (5.72 x 103) were added to the mixture resulting in E:T = 0.2: 1. The cells were then co-cultured for 6 hours at 37°C. The last 2.5 hours in the presence of 1 μCi ³H-thymidine. The results show that that all of the antibodies tested are capable of inhibiting apoptosis.

Figure 3 shows that the antibodies of the invention are capable of blocking the interaction of cells expressing Fas antigen with cells expressing Fas ligand on their respective cell surfaces. SKW 6.4 cells were preincubated with and without antibody and co-cultured with SF9 cells expressing Fas ligand on the cell surface, essentially as describe in Figure 2. Figure 3 A is a photomicrograph of a co-culture without antibody. Multiple rosettes of SF9 (larger) and SKW 6.4 (smaller) cells may be seen indicating cell-cell interactions. Figure 3B is a photomicrograph of a co-culture with antibody and no rosettes are seen.

Figure 4 shows the inhibition of Fas ligand killing of SKW 6.4 cells cells were preincubated with C42 or ZB.4 (Immunotech, Westbrook ME) and co-cultured with Sf9 cells expressing Fas ligand on the cell surface essentially as described above. Thymidine incorporation is shown for C42 (open squares) and ZB.4 (closed squares). Incorporation of thymidine for SKW 6.4 cells alone (upper arrow) and for SKW 6.4 cells co-cultured with Sf9-Ligand cells (lower arrow) are shown on the right vertical axis. Brief Description of the Invention

The invention relates to hybridomas capable of producing antibodies capable of recognizing and bind Fas antigen and to inhibit apoptosis upon binding. The invention also relates to the antibodies produced by the hybridomas. Finally, the invention relates to methods of treatment using the antibodies. Detailed Description of the Invention

As used herein, the term "Fas antigen" refers to the cell surface antigen cloned by Itoh et al, (1991) Cell 66:233-243. The Fas antigen is approximately 200,000 MW and can mediate cell death by apoptosis. As used herein, the term "Fas Ligand" refers to the cell surface protein cloned by

Suda et al, Cell (1993) 75: 1169-1178. Fas Ligand interacts with Fas antigen and induces apoptosis in the cell having Fas antigen on its surface.

As used herein, the term antibody encompasses monoclonal antibodies and fragments thereof. Such fragments will include Fab, Fab2, Fv. Antibodies should also be understood to include humanized antibodies. Such antibodies are designed to reduce immunogenicity of the antibodies in human patients and therefore mitigate any HAMA response. Humanization may be accomplished, for example, by a CDR grafting approach in which CDR regions are inserted within a set of human framework regions, or by a veneering approach in which amino acids exposed at the surface of the folded antibody protein are replaced with consensus amino acids from the human framework regions at the same positions (see EP 519,596, published 23 December 1992).

Example A. Cloning / Expression / Purification of Soluble Fas antigen (sFas) 1. Total RNA isolation from Fas⁺ cells

Cells expressing Fas antigen on their cell surface (Fas⁺ cells) (e.g. U937 cells) are

solubilized in guanidine isothiocyanate and fractionated over cesium chloride. The RNA pellet may then be solubilized in water and the total RNA preparation incubated with reverse transcriptase to synthesize cDNA. Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989). Maniatis et al. Molecular Cloning: A Laboratory Manual (1982).

2. PCR of cDN A: Engineering of sFas

Using PCR primers based on the human Fas published sequence (Itoh et al, (1991) Cell 66:235-243), cDNA encoding the extracellular region of Fas with a C-terminus epitope tag (glu-glu) (sFas) may be constructed. The 5' primer may utilize a sequence 5' of the initiating ATG codon preceded by an insertion of a sequence encoding for a Pst I

restriction enzyme site. The 3¹ primer may consist of a coding region 5' of ASN^,

including the ASN^7 sequence, followed by the glu-glu sequence, a stop codon and a Not I restriction enzyme site sequence. The sequence of the 5' and 3' primers may be: 5'- GTACCTGCAGGGAAGCTCTTTCACTTCGGAGG-3 and 5'-

GTACGCGGCCGCTTATTCCATTGGCATGTATTCGTTAGATCTGGATCCTTCCT CTTTGC-3¹, respectively. The PCR reaction is carried out under appropriate conditions.

3. Ligation into p AcC 13 expression vector Both the baculovirus expression plasmid pAcC13 and PCR product are digested with Pst I and Not I. The plasmid may be further treated with calf intestine alkaline phosphatase. The digested plasmid and PCR product are purified (Glassmilk, BIO-101), and subsequently ligated with T4 ligase.

Escherichia coli (E. coli) are transformed with the products of the ligase reaction. Positive colonies (i.e. those colonies containing plasmid with insert) are further screened by PCR using the cloning primers.

4. Baculovirus/Sf9 expression

Using standard protocols and/or commercially available expression systems, the cDNA for sFas may be incorporated into baculovirus for expression of sFas in Sf9 cells (Spodopterafrugiperda). Expression in insect cells has also been described by Summers and Smith ( 1987) Texas Agricultural Experiment Station Bulletin No. 1555.

Commercially available kits include the MaxBac kit (Invitrogen, San Diego, CA). The baculovirus infected Sf9 cells lead to the expression and secretion of sFas into culture medium.

5. Affinity purification of sFas sFas contained in Sf9 medium is concentrated over a YM10 membrane (Amicon), then passed over an anti-glu-glu antibody column (anti-glu-glu monoclonal antibody coupled to protein G Sepharose). Bound sFas is eluted with the glu-glu hexapeptide. After separation of the hexapeptide from sFas, the homogeneous sFas is used as antigen for monoclonal antibody production. 6. Production of Sf9 cells expressing Fas-Ligand on the cell surface

Sf9 cells capable of expressing full length Fas-Ligand on the cell surface may be developed essentially as described above, using the published sequence of Fas-Ligand (Suda et al, (1993) Cell 75:1169-1178). It will be appreciated that the addition of a glu- glu epitope tag is unnecessary.

B. Production of Hybridomas Capable of Producing Fas Monoclonal Antibody sFas antigen produced by expression in SF9 cells was used as an immunogen in mice to produce hybridomas capable of producing antibodies which recognize and bind Fas antigen. The method used was essentially that of Kohler and Milstein, (1975) Nature 256:495-497 with a standard PEG fusion modification. Although Fas antigen produced by in SF9 cells was used as an immunogen, Fas antigen from other sources may also be used. Such sources include recombinant Fas antigen produced in E. coli and yeast such as Saccharomyces cerevisiae. Cells expressing Fas antigen on their surface may also be used as immunogen, including SKW 6.4 and U937 cells. SF9 cells capable of expressing Fas antigen on the cell surface may also be prepared as described in U.S. 5,397,703, the disclosure of which is herein incorporated by reference.

Primary screens of hybridomas for antibodies capable of binding Fas antigen may be conducted by Fas ELIS A. The Fas ELIS A consists essentially of coating Fas antigen (100 ng or more/well) onto a 96 well plate, blocking said wells with an irrelevant protein (e.g. bovine serum albumin) and incubating with hybridoma supernatants. The wells are then incubated with an enzyme conjugated anti-mouse antibody (e.g. alkaline phosphatase or horse radish peroxidase - coupled to goat, rabbit, donkey or sheep anti mouse immunoglobulin) and develeoped with an appropriate substrate. The wells are read on an ELISA reader (e.g. Dynatech MRX plate reader).

C. Further Screening of Monoclonal Antibodies for Anti-Apoptosis Activity Using the methods described above, ten hybridomas were identified and subjected

to further analysis.

1. Inhibition of Apoptosis in a Fas⁺ cell line

SKW6-4 cells (human B cell line, Fas⁺) were pre-incubated with ELIS A⁺

monoclonal antibodies. Cells expressing Fas ligand on the cell surface (Fas-L⁺ cells) or an apoptosis- inducing antibody (e.g. CHI 1, Immunotech) were added and co-cultured

for 5-24 hrs at 37°C, the last two in the presence of ^H-thymidine (1 μCi). Cells were lysed and DNA collected using a SKATRON cell harvester. Radioactivity was

enumerated in a liquid scintillation counter. Enhanced ^H-thymidine incorporation caused by the monoclonal antibody induced suppression of Fas-L mediated cell death identified which monoclonal antibodies had anti-apoptosis activity.

Figure 1 shows the results of experiments utilizing four antibodies of the invention. SKW 6.4 cells (2 x 10⁴ cells) were preincubated with C33-1.6.1 (C33) (open triangle); C40-1.3.3 (C40) (closed square); C28-3.7.1 (C28) (closed triangle); or C42- 7.4.5 (C42) (closed circle) for 1 hour at 37°C. CHI 1 (apoptosis - inducing antibody) was added to 100 ng/ml and incubated for 20 hours at 37°C. The last two hours in the presence of 1 μCi ³H-thymidine. The results show that C28 and C42 antibodies are potent inhibitors of CHI 1 induced cell death across the range of concentrations tested. C33 and C40 were more effective at higher concentrations of antibody. Their respective isotypes of the four antibodies are C28:IgGι, C33:IgGι, C40:IgG2b and C42:IgGι. The

results of measurements of incorporation of ³H-thymidine by SKW 6.4 cells with (lower arrow) and without (upper arrow) treatment with CHI 1 are shown on the left axis of the figure.

2. Prevention of Fas-Fas-L Interaction

Figure 2 shows the inhibition of Fas ligand killing of SKW 6.4 cells by the monoclonal antibodies of the invention. SKW 6.4 cells (2.86 x 10⁴ cells) were preincubated with C33 (open square); C40 (open diamond); C28 (closed diamond); or C42 (closed square) for 1 hour at 37°C. SF9 cells expressing Fas ligand on the cell surface (5.72 x 103) were added to the mixture resulting in E:T = 0.2: 1. The cells were then co-cultured for 6 hours at 37°C. The last 2.5 hours in the presence of 1 μCi ³H- thymidine. The results show that that all of the antibodies tested are capable of inhibiting apoptosis.

Figure 4 shows the inhibition of Fas ligand killing of SKW 6.4 cells cells were preincubated with C42 or ZB.4 (Immunotech, Westbrook ME) and co-cultured with Sf9 cells expressing Fas ligand on the cell surface essentially as described above. Thymidine incorporation is shown for C42 (open squares) and ZB.4 (closed squares). Incorporation of thymidine for SKW 6.4 cells alone (upper arrow) and for SKW 6.4 cells co-cultured with Sf9-Ligand cells (lower arrow) are shown on the right vertical axis.

Figure 3 shows that the antibodies of the invention are capable of blocking the interaction of cells expressing Fas antigen with cells expressing Fas ligand on their respective cell surfaces. SKW 6.4 cells were preincubated with and without antibody and co-cultured with SF9 cells expressing Fas ligand on the cell surface, essentially as describe in Figure 2. Sf9/Fas-L expressing cells were added and the mixture was centrifuged (1500 rpm, 2 minutes) and incubated 1-3 hrs at 25°C-27°C. The cell pellet was gently resuspended and resolved by photomicroscopy. Figure 3 A is a photomicrograph of a co- culture without antibody. Multiple rosettes of SF9 (larger) and SKW 6.4 (smaller) cells

may be seen indicating cell-cell interactions. Figure 3B is a photomicrograph of a co-

culture with antibody and no rosettes are seen.

Deposit Information

The following materials were deposited with the American Type Culture

Collection:

Hybridoma Deposit Date CMCC Ace. No. A.T.C.C. Ace. No.

C33-1-6-1, June 07, 1995 CMCC #11263; C40-1.3.3, June 07, 1995 CMCC #11264; C28-3-7-1, June 07, 1995 CMCC #11266; C42-7-4-5, June 07, 1995 CMCC #11267

The above materials were deposited by Chiron Corporation, an assignee of the present invention with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for purposes of Patent Procedure. The accession number is available from the ATCC at telephone number (301) 881-2600. The Chiron Master Culture Collection Accession Number is also provided. These deposits are provided as convenience to those of skill in the art, and are not an admission that a deposit is required under 35 U.S.C. §112. The nucleic acid sequence of

these deposits, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and should be referred to in the event of an error in - li ¬

the sequence described herein. A license may be required to make, use, or sell the deposited materials, and no such license is granted hereby.

Claims

Claims I claim:

1. A murine x murine hybridoma selected from the group consisting of:

(a) C42-7-4-5;

(b) C28-3-7-1;

(c) C40-1.3.3; and

(d) C33-1-6-1.

2. An antibody or fragment thereof, said antibody or fragment thereof capable of recognizing and binding Fas antigen, and further capable blocking the binding of a monoclonal antibody, said monoclonal antibody produced by a murine x murine hybridoma selected from the group consisting of:

(a) C42-7-4-5;

(b) C28-3-7-1;

(c) C40-1.3.3; and

(d) C33-1-6-1.

3. The antibody or fragment thereof of claim 2 wherein the antibody is a humanized antibody.

4. A method of treating a disease, said disease characterized by increased apoptosis, comprising administration of a monoclonal antibody to a patient in need of such treatment, said monoclonal antibody produced by a murine x murine hybridoma selected from the group consisting of:

(a) C42-7-4-5;

(b) C28-3-7-1; (c) C40-1.3.3; and

(d) C33-1-6-1.

5. The method of claim 4, wherein the disease is rheumatoid arthritis.

6. The method of claim 4, wherein the disease is Sjόgren syndrome.

7. The method of claim 4, wherein the disease is type I diabetes or diabetes mellitus.

8. The method of claim 4, wherein the disease is viral hepatitis