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Title:  Laser inactivation of inhibitory molecules in central nervous system myelin

United States Patent:  6,479,053

Issued:  November 12, 2002

Inventors:  Jay; Daniel G. (Brighton, MA)

Assignee:  President and Fellows of Harvard College ()

Appl. No.:  529843

Filed:  June 12, 2000

PCT Filed:  October 23, 1998

PCT NO:  PCT/US98/22387

371 Date:  June 12, 2000

102(e) Date:  June 12, 2000

PCT PUB.NO.:  WO99/20190

PCT PUB. Date:  April 29, 1999

Abstract

Methods for specifically inactivating myelin proteins which inhibit nerve regeneration are described. These methods are useful to promote axon regeneration.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The antibodies used in the present invention are prepared by a variety of known methods. Monoclonal antibodies are preferred for use, and such monoclonal antibodies can be prepared in various ways using techniques well understood by those having ordinary skill in the art. For example, monoclonal antibodies can be prepared using hybridoma technology (Kohler, et al., Nature, 256:495 (1975); Hammerling, et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)).

Either function blocking or non-function blocking monoclonal antibodies can with the invention, however, use of non-function blocking monoclonal antibodies is preferred. Non-function blocking antibodies facilitate the selectivity of the treatment according to the invention by providing for the inactivation of only those MAG proteins which are at the site of the laser irradiation rather than all MAG proteins in the tissue to which the antibodies bind. In addition in the method of the invention, the antibodies are applied acutely. In a clinical setting, this short exposure is less likely to lead to an inflammatory response against the antibodies, which inflammatory response may disrupt the treatment or otherwise interfere with the treatment of an individual.

Of special interest to the present invention are antibodies which are produced in humans, or a "humanized" (i.e. non-immunogenic in a human) by recombinant or other technology such that they will not be antigenic in humans. Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion, i.e., chimeric antibodies) as is well understood by those having ordinary skill in the art. General reviews of "humanized" chimeric antibodies are provided by Morrison, S. L. (Science, 229:1202-1207 (1985)) and by Oi, et al., (BioTeclmiques, 4:214-ff. (1986)).

The antibodies to MAG are bound to a laser activated chromophore tag. This chromophore tag in turn, when later exposed to laser activation will react in such a manner as to produce free radicals that will inactivate MAG. An example of a chromophore that can be used in the invention is malachite green. The binding of the chromophore tag to the antibodies can be done using standard techniques commonly known to those of ordinary skill in the art. For example, antibodies and other ligands reagents can be labeled with malachite green isothiocyanate (Molecular Probes Inc.) as described in Jay (1988).

The tagged MAG antibodies are administered to a patient at the desired site of treatment according to techniques commonly know n to those of ordinary skill in the art. For example, the tagged MAG antibodies may be infused into the area of the nerve to be treated using a catheter.

In a clinical application the MAG of CNS nerves may be inactivated by introduction of the laser via an optic fiber to the site of lesion according to generally accepted surgical procedures. For example, to treat the optic nerve the laser would be focused on the optic nerve by introduction of the laser light via an optic fiber into the eye cavity or the eyeball itself, depending on the location of the lesion. To treat spinal cord nerves, the laser light would be introduced into the spinal column via the interstices between the vertebrae. The nerve to be treated would then be subjected to laser light at a wavelength that is not absorbed by cellular components and for a time to activate the chromophore tag, e.g. two to five minutes. Inactivation of MAG by CALI results in the specific and irreversible inactivation of each MAG protein that is bound to a labeled antibody when irradiated. Since MAG protein in myelin is replaced, or turns over, approximately every 40 days, the method of the invention would be repeated as many times as is necessary to permit complete axon regeneration.

The method of chromophore-assisted laser inactivation (CALI) will inactivate specific proteins with high spatial and temporal resolution Jay, 1988). CALI can convert "binding reagents" such as antibodies or ligands into function-blockers. It takes advantage of both the specificity inherent in the ligand-receptor interaction and the spatial specificity of the laser activated chromophore tag. For example, a "probe" (which can be a ligand or antibody) is tagged with the dye malachite green (MG) or other chromophore which produces free radicals upon exposure to laser light having a wavelength that is not significantly absorbed by cellular components. The probe is then incubated with the sample of interest. The excess unbound probe need not be washed away due to the spatial specificity of malachite green (half-maximal effect radius =15 .ANG.; Liao et al., 1995). An area of inactivation is selected, and irradiated with a laser beam at a wavelength of 620 nm. This light is absorbed by the malachite green to generate short-lived free radicals that selectively inactivate proteins bound to the MG-labeled probe based on proximity within the laser spot. The laser beam does not cause nonspecific light damage to tissues because the wavelength of light used is not significantly absorbed by cellular components (Jay, 1988; Liao et al., 1994).

As a direct demonstration of its spatial specificity, CALI can inactivate a single subunit of the T cell receptor in living T cells, with only slight effects on nearest neighbors and no effect on other subunits of the multiprotein complex (Liao et al., 1995). CALI has been successfully used to selectively inactivate 40 different proteins (reviewed in Wang and Jay, 1996). Inactivation of proteins by CALI has phenocopied genetic loss of function mutations precisely in all cases tested (n=4) (Schmucker et al., 1994; Schroeder et al. 1996). Further, CALI has been frequently employed with whole embryos to address in vivo function and caused no nonspecific damage to cells or tissue (Jay and Keshishian, 1990; Diamond et al., 1993; Schinucker et al., 1994; Schroeder et al. 1996).

In the specific examples set forth below, CALI is done as described (Jay and Keshishian, 1990; Diamond et al., 1993). CALI is performed with a Nd:YAG driven dye laser using the fluorescent laser dye DCM (Spectra Physics Corp.). It generates a 2 mm spot with 15 mJ per 3 nsec. pulse at a frequency of 10 Hz. Samples are subjected to 2 minutes of laser pulses which has been previously shown to have no nonspecific effect on cells or tissues (Beermann and Jay, 1994).

As used herein, the term "axon regeneration" is defined to mean the growth of an axon from the nerve cell body (soma) through the site of lesion (which lesion can occur by any well known means, e.g., break, cut or crush) to the site innervation thus re-innervating the original target tissue of such nerve. The term "promote" is defined to mean that axon regeneration (i) occurs in an environment where otherwise no axon regeneration would be observed, or (ii) is enhanced, as evidenced by a greater number of axons or greater length of axons.

Claim 1 of 6 Claims

We claim:

1. A method of inactivating myelin associated glycoprotein (MAG) in mammalian tissue using chromophore-assisted laser inactivation (CALI) comprising:

(a) exposing MAG to an anti-MAG antibody with a laser activated chromophore tag for a time and under conditions to form a MAG/antibody complex; and

(b) irradiating the MAG/antibody complex with a laser beam for a time and under conditions to irradiate the chromophore tag, wherein the irradiated tag selectively inactivates the MAG in the MAG/antibody complex.
 


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