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Autoantigenic fragments, methods and assays

a technology of autoantigen and autoantigen, applied in the direction of genetic material ingredients, instruments, antibody medical ingredients, etc., can solve the problems of non-caspase substrates for granzyme and granule-induced cell death that have not been defined, and achieve the effect of lessening the impact of lessening at least one symptom of the diseas

Inactive Publication Date: 2005-05-26
ROSEN ANTONY +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach effectively generates unique autoantigenic fragments that can tolerize patients to prevent autoimmune diseases and stimulate immune responses against malignant cells, offering a therapeutic and prophylactic solution by using granzyme B-derived fragments to modulate the immune system's response.

Problems solved by technology

However, non-caspase substrates for granzyme B (and potentially other granule proteases) during granule-induced cell death have not previously been defined.

Method used

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  • Autoantigenic fragments, methods and assays
  • Autoantigenic fragments, methods and assays
  • Autoantigenic fragments, methods and assays

Examples

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example 1

General Materials and Methods

[0062] Materials. Purified DNA-dependent protein kinase (DNA-PK) and SP1 were purchased from Promega (Madison, Wis.). ATP was purchased from Fluka (Ronkonkoma, N.Y.), and 32P-ATP was from Du Pont / NEN (Wilmington, Del.). Ac-DEVD-CHO and Ac-YVAD-CHO were manufactured by Merck (Rahway, N.J.). Caspase-3 was purified as described (Nicholson et al., 1995). Patient sera were used to immunoblot the nuclear mitotic apparatus protein (NuMA), poly(ADP-ribose) polymerase (PARP) and DNA-PKCS (Casciola-Rosen et al., 1995; Greidinger et al., 1996). Monoclonal antibodies can be made by methods known in the art. Two different monoclonal antibodies, designated 18-2 and 25-4 (kind gifts from Dr. Tim Carter, St. Johns University, Jamaica, N.Y.) were also used to detect DNA-PKCS by immunoblotting (see Table II for a summary of the antibodies used to detect DNA-PKCS and its cleaved fragments). Rabbit polyclonal antibodies to caspases were raised against the large subunits o...

example 2

DNA-PKCS and NuMA are Very Efficient Substrates for Purified Granzyme B.

[0071] Granzyme B has previously been reported to cleave the precursors of several caspases (including caspases 3, 7 and 10), resulting in activation of their proteolytic activity. The catalytic efficiency of cleavage of these substrates by granzyme B serves as a useful standard against which granzyme B-mediated cleavages of other substrates can be compared. Purified [35S]methionine-labeled precursors of caspase-3 and caspase-7, or THP.1 cytosols (containing these precursor proteases) were incubated in vitro with increasing concentrations of purified granzyme B. The dose-response data obtained (FIG. 1) was used to calculate catalytic constant (kcat / Km) values of 1.8±0.6×105M−1s−1 (radiolabeled substrate) and 1.9±0.1×105 M−1s−1 (immunoblotting) for caspase-7, and 3.6±1.0×104 M−1s−1 (radiolabeled substrate) and 2.3±0.4×104M−1s−1 (immunoblotting) for caspase-3 (Table I). Thus, granzyme B cleaves caspase-7 approxi...

example 3

Different Substrate Fragments are Detected After Cleaving Autoantigens in Vitro with Granzyme B or Caspase-3.

[0074] To directly compare the fragments generated by granzyme B and caspase-3, purified DNA-PKCS, in vitro translated [35S]methionine-labeled PARP, and endogenous substrates (NuMA and DNA-PKCS in HeLa cell lysates) were incubated with protease and electrophoresed in adjacent lanes. When granzyme B was used to cleave DNA-PKCS, fragments of 100 kDa and 250 kDa were generated, (detected by immunoblotting using antibodies recognizing the C-terminus or N-terminus of DNA-PKCS, respectively) (FIG. 2, lanes 2 & 5; and Table II). In contrast, caspase-3 cleavage yielded a 150 kDa C-terminal fragment (FIG. 2, lane 3) and a 250 kDa N-terminal fragment (FIG. 2, lane 6).

[0075] Granzyme B-mediated cleavage of NuMA generated a novel fragment migrating at 175 kDa on SDS-PAGE, which was distinct from the 185 kDa fragment detected after cleavage with caspase-3 (FIG. 2, lanes 7-9). Similarly...

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Abstract

The present invention provides a method of producing autoantigens, compositions comprising autoantigenic fragments and methods of using autoantigenic fragments in the treatment of a condition associated with an autoimmune response. Also provided are assays for the detection or assessment of an autoimmune response.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional application of U.S. application Ser. No. 09 / 296,662, filed Apr. 22, 1999, now allowed.STATEMENT REGARDING FEDERALLY-SPONSORED R&D [0002] This invention was made in part under Federally Sponsored Research. The U.S. Government may have certain rights in this invention.REFERENCE TO MICROFICHE APPENDIX [0003] Not applicable. FIELD OF THE INVENTION [0004] This invention relates to the production of autoantigenic fragments from autoantigens and the uses of the autoantigenic fragments. BACKGROUND OF THE INVENTION [0005] The mature immune system of animals differentiates between self-molecules and non-self-molecules and mounts an immune response only against the latter. The immune system learns which molecules are self through constant exposure to those molecules that are normally a part of the animal. Thus, the mature immune system is tolerized to the presence of molecules that are self. However, the immune sys...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/00C12P21/06
CPCG01N2800/24C12P21/06
Inventor ROSEN, ANTONYNICHOLSON, DONALDCASCIOLA-ROSEN, LIVIAANDRADE, FELIPEROY, SOPHIETHORNBERRY, NANCY
Owner ROSEN ANTONY
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