Antibodies with immune effector activity and that internalize in folate receptor alpha-positive cells

an antibody and antibody technology, applied in the field of monoclonal antibodies, can solve the problems of cytotoxicity toxicity to normal tissues as well as cancerous tissues, the survival term of 5 years is still less than 40%, and the ability to effectively kill antigen-bearing tumor cells is difficult for many cancers, so as to achieve enhanced antibody-dependent cellular cytotoxicity, anti-tumor activity, and potent therapeutic activity for t-cell leukemia and lymphoma

Inactive Publication Date: 2006-10-26
EISAI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] Provided herein are FRA-specific antibodies that alternatively elicit a robust immune-effector function yet are able to internalize in FRA-positive cells, referred to here as in-out anti-FRA antibodies. As used herein, “in-out antibodies” (“in-out Abs”) refer to antibodies that can alternatively elicit an immune effector activity and internalize within an antigen-presenting cell by binding to target antigen. Without wishing to be bound by any particular theory, it is believed that in-out Abs bind to the cell surface of an antigen-bearing cell and internalize after a period of time unless engaged by immune-effector cells or biochemicals that are recruited to the antigen-antibody-bearing cell. Antibodies that are able to elicit an immune effector effect such ADCC or CDC and internalize have been previously described (Wolff et al. Monoclonal antibody homodimers: enhanced antitumor activity in nude mice. Cancer Res. Jun. 1, 1993;53:2560-5), however, it is not obvious that in-out antibodies can be developed against any antigen or epitope (Kusano et al. Immunocytochemical study on internalization of anti-carbohydrate monoclonal antibodies. Anticancer Res. November-December 1993;13(6A):2207-12). In-out antibodies that can target FRA have not been described previously. FRA-specific antibodies have been previously described but such antibodies are not known to internalize upon binding to the antigen (Cogliati et al. Preparation and biological characterization of conjugates consisting of ricin and a tumor-specific non-internalizing MAb. Anticancer Res. 11:417-21, 1991). Antibodies that can target cell surface antigens do not always elicit an immune effector function upon binding to the cell surface antigen (Niwa et al. Defucosylated chimeric anti-CC chemokine receptor 4 IgG1 with enhanced antibody-dependent cellular cytotoxicity shows potent therapeutic activity to T-cell leukemia and lymphoma. Cancer Res. 64:2127-33, 2004; Kikuchi et al. Apoptosis inducing bivalent single-chain antibody fragments against CD47 showed antitumor potency for multiple myeloma. Leuk Res. 29:445-50, 2005; Scott et al. Immunological effects of chimeric anti-GD3 monoclonal antibody KM871 in patients with metastatic melanoma. Cancer Immun. February 22;5:3, 2005). Provided herein are antibodies that bind to the cell surface antigen FRA and, in the alternative, elicit an immune effector activity (such as ADCC or CDC) and internalize within antigen-positive cells. These antibodies and derivatives thereof are useful for cancer therapy.

Problems solved by technology

Although chemotherapy is the recommended treatment and has enjoyed some success, the 5-year survival term is still less than 40%.
A difficult problem in treating ovarian cancer as well as other cancers with cytotoxic drugs is that often the cytotoxin causes toxicity to normal tissues as well as cancerous tissues.
Finding antibodies that can specifically bind to and effectively kill antigen-bearing tumor cells has proven difficult for many cancers.
This has been due in part to the inability to obtain robust killing due to lack of immune-effector function or to lack of efficient internalization of antibodies carrying immunotoxins.

Method used

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  • Antibodies with immune effector activity and that internalize in folate receptor alpha-positive cells
  • Antibodies with immune effector activity and that internalize in folate receptor alpha-positive cells
  • Antibodies with immune effector activity and that internalize in folate receptor alpha-positive cells

Examples

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Effect test

example 1

In-Out Antibodies That Can Bind to FRA

[0126] The monoclonal antibody ML-1 was developed by grafting the CDRs of the variable domain of a murine antibody specific to FRA onto a human IgG1 constant region. The antibody was shown to bind specifically to FRA protein and cancer cells expressing FRA and was found to have a binding constant of about 5 nM using Biacore®. To demonstrate FRA-specific binding, antigen-specific ELISA were performed using recombinant FRA in 96-well format following methods used by those skilled in the art (FIG. 1A). Antibodies found to react by ELISA were further analyzed for FRA binding using FACS analysis following the manufacturer's protocol. Shown in FIG. 1B are representative data of the FACS analysis whereby FRA-expressing ovarian tumor cells were positive for ML-1 binding in contrast to null cells. Antigen-specific ELISA can be also formatted using whole cells expressing FRA, membrane preparations obtained from such FRA expressing cells, or synthetic, ov...

example 2

[0127] Activity of ML-1 antibody for immune effector activity was assessed by standard antibody-dependent cellular cytotoxicity (ADCC) assays on the FRA-expressing OVCAR-3 cell line. Briefly, OVCAR-3 target cells are seeded in flat-bottom 96-well microplates in complete growth medium (RPMI-1640 containing 10% FBS, 2 mM L-glutamine). The following day, the complete medium is replaced with 100 ul of CHO-CD serum-free medium (Sigma) and 50 ul of antibody-containing conditioned medium is added to target cells and incubated for 20 minutes at 37° C. Subsequently, 100 ul of serum-free medium containing 2×105 effector cells are added to each well and cells are incubated for 5-6 hours at 37° C., 5% CO2. Effector cells are derived from human peripheral blood mononuclear cells (PBMCs), isolated from healthy donors (purchased from Interstate Blood Bank). Prior to use in ADCC, PBMCs are activated by seeding PBMCs at 2.5×106 / ml in complete RPMI containing about 10 ng / ml human recombinant interleu...

example 3

[0129] ML-1 internalizes when bound to FRA-expressing cells. This finding is shown in FIG. 3 using the Hum-ZAP assay. Second immunotoxins are conjugations of a secondary antibody to the ribosome inactivating protein saporin. If the primary antibody being tested is internalized, the saporin is transported into the cell via its binding to the secondary antibody. Once internalized saporin separates from its IgG conjugate, it inhibits protein synthesis and ultimately causes cell death. Hum-ZAP (Advanced Targeting Systems, cat# IT-22) is a secondary chemical conjugate of affinity purified goat anti-human IgG, (mw 210 kDa) that recognizes human monoclonal antibodies. The control molecule, Goat IgG-SAP (Advanced Targeting Systems cat#IT-19) is a conjugate of normal goat IgG and saporin. Briefly, cells are plated into flat-bottom 96-well tissue culture plates at 2500 / well in 80 ul of RPMI 1640 with 10% FCS, 2.0 mM glutamine, 1.0 mM sodium pyruvate, and 0.1 mM MEM non-essential amino acids. ...

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Abstract

This invention relates to the use of monoclonal and polyclonal antibodies that specifically bind to and have the ability in the alternative to become internalized by cells expressing folate receptor alpha (FRA) and to induce an immune effector activity such as antibody-dependent cellular cytotoxicity. The antibodies are useful in specific delivery of pharmacologic agents to FRA-expressing cells as well as in eliciting an immune-effector activity particularly on tumor cells and precursors. The invention is also related to nucleotides encoding the antibodies of the invention, cells expressing the antibodies; methods of detecting cancer cells; and methods of treating cancer using the antibodies.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This claims benefit of U.S. Provisional Application 60 / 674,185, filed Apr. 22, 2005, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION [0002] This invention relates to the use of monoclonal and polyclonal antibodies that specifically bind to and alternatively become internalized by cells expressing or bearing folate receptor alpha (FRA) (“FRA-positive cells”) and induce an immune effector activity such as antibody dependent cellular cytotoxicity. The antibodies are useful in specific delivery of pharmacologic agents to FRA-positive cells as well as in eliciting an immune-effector activity particularly on tumor and dysplastic cells. The invention is also related to cells expressing the monoclonal antibodies, polyclonal antibodies, antibody derivatives, such as chimeric and humanized monoclonal antibodies, antibody fragments, methods of detecting FRA-positive cells, and methods of treating cancer using the an...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K51/00G01N33/53C07H21/04C12P21/06A61K39/395C12N5/06C07K16/28C07K16/46
CPCA61K47/48469A61K47/48746B82Y5/00C07K16/28C07K2317/77C07K16/3069C07K2317/24C07K2317/732C07K16/30A61K31/704A61K31/7068A61K47/6825A61K47/6897A61P35/00A61K47/6851A61K47/6869A61K39/39558C07K16/22C07K2317/21C07K2317/76
Inventor NICOLAIDES, NICHOLASGRASSO, LUIGISASS, PHILIP
Owner EISAI INC
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