Stabilized radiophosphate-labeled proteins

Details Stabilized radiophosphate-labeled proteins Stabilized radiophosphate-labeled proteins

1999-07-06

2002-08-27

Caputa, Anthony C. (Department: 1642)

Drug, bio-affecting and body treating compositions

Radionuclide or intended radionuclide containing; adjuvant...

Attached to antibody or antibody fragment or immunoglobulin;...

C424S001690, C424S001770, C424S178100, C424S179100, C530S391300, C530S391500

Reexamination Certificate

active

06440386

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to phosphorus-32 and phosphorus-33 labeled proteins which are useful for radiotherapy of human diseases. In particular, the invention relates to proteins which contain peptide sequences that are substrates for protein kinase enzymes, and that can be radiolabeled with a protein kinase and a
32
P or
33
P-labeled phosphate donor and contain an SH2 domain which serves to protect the phosphorylated protein from in vivo dephosphorylation. This invention also relates to a method of therapy using the radiolabeled proteins.
Many radionuclides have been studied for their suitability for internal administration to patients in radiotherapy. Some radionuclide compounds, containing isotopes such as
131
I, can be given systemically, taking advantage of the fact that these elements tend to localize to particular tissues by virtue of their chemical properties. Other radionuclides, such as
198
Au and
103
Pd have been administered in a localized fashion, for instance to the site of a tumor. Most recent approaches, however, have focused on methods of delivering radionuclides to a preselected tissue by attaching the radionuclide to a targeting protein, usually an antibody, which will then localize to that tissue.
A large number of methods for associating radionuclides to antibodies have been developed. The chemical toxicity of many radionuclides means that complex methods must often be used to stably bind the isotope to an antibody. For example, to use
90
Y, which has many desirable radiochemical properties, a chelate must be synthesized and covalently bound to the antibody to stably link the radioisotope to the antibody.
Currently there are isotopes which have been utilized in radiolabeled agents and include
131
I,
90
y,
188
Re,
186
Re,
67
Cu, and
212
Pb/
212
Bi. Each has significant, sometimes multiple, disadvantages including low-energy particle emissions, such as
131
I and
67
CU; severe organ toxicity, such as
90
Y and
212
Pb/
212
Bi; short half-life for radioimmunotherapy, such as
188
Re and
212
Pb/
212
Bi; high gamma-energy emissions, such as
131
I; low specific activity, such as
186
Re; non-amenability to out-patient procedures, such as
131
I,
67
Cu and
212
Pb/
212
Bi and supply and/or cost concerns, such as
188
Re,
186
Re,
67
Cu, and
212
Pb/
212
Bi.
One isotope which has received little attention, due to the difficulty of the chemistry involved in linking it to antibodies, but otherwise displays desirable properties for radioimmunotherapy is
32
P.
32
P is inexpensive, is readily available in high specific activity in a variety of labeled molecules, and has a therapeutically desirable half-life of 14 days. Additionally, it has been previously used clinically, has no gamma emissions, is carrier-free and has an intense beta-emission. It is absorbed by the body and is not readily excreted, and is therefore amenable to use in outpatient procedures. In addition,
32
P emits only &bgr;-radiation with an excellent depth penetration in tissue of approximately 6 mm. Unlike many other radionuclides under consideration for targeted radiotherapy, it is not inherently toxic, and is currently used clinically in some non-targeted applications, for example, for the treatment of ovarian cancer and polycythemia rubra vera.
Another radioisotope of phosphorus,
33
P, has received even less attention than
32
P.
33
P shares the same chemical properties as
32
P, and has similarly desirable radiochemical characteristics. It is available in high specific activity, and has a 25-day half life with a &bgr;-particle emission energy of 0.25 MeV, approximately 15% of the value of the &bgr;-emission energy of
32
P.
One reason radioactive phosphorus has received relatively little attention for targeted radiotherapy applications has been the difficulty of linking it to targeting proteins. Most of the methods currently known are non-specific and slow, and do not efficiently incorporate radionuclide into the targeting protein.
Another reason that radioactive phosphorus has received relatively little attention for radioimmunotherapy, is the rapid dephosphorylation of
32
P in human serum. It has been proposed that enhanced serum stability of
32
P in, e.g., the kemptide sequence can be achieved by modifying the primary sequence of receptor peptides.
One very general method of labeling proteins with
32
P is simply to incubate the protein with &agr;-
32
P-labeled nucleoside triphosphates. Schmidt et al.,
FEBS Lett.
194:305 (1986). The mechanism for the labeling reaction is unknown. The method is slow and gives only poor incorporation of label (less than 1% of the protein molecules are labeled), and is thus too inefficient for therapeutic use.
A second general method of
32
P labeling is to incubate Proteins with [&ggr;-
32
P]ATP or H
3
32
PO
4
in the Presence of chromium ions. Hwang et al.,
Biochim. BioPhys. Acta
882:331 (1986). This method is relatively rapid, but gives an unknown level of label incorporation and also leaves toxic chromium ions bound to the proteins, which would be therapeutically unacceptable.
A third general method is the use of
32
P-diphenylphosphinothionyl chloride as a reactive labeling compound. De Boer et al.,
Clin. Exp. Immunol
. 3:865 (1968). This reagent is thought to react non-specifically with lysine residues in proteins to form a highly stable conjugate, but approximately 50% of the radioactivity also associates non-covalently with the labeled protein. Although this method allows labeling of proteins to high specific activity, the labeling agent is only poorly water soluble, and to achieve good labeling yields large excesses of reagent must be used, wasting relatively large amounts of hazardous radioactive materials.
A less general method of
32
P labeling is the use of periodate-oxidized [&agr;-
32
P]ATP to affinity-label proteins containing an ATP-binding site. Clertant et al.,
J. Biol. Chem
. 257:6300 (1982). Because many targeting proteins which are of therapeutic interest, in particular antibodies, do not contain ATP-binding sites this method is therefore of little general utility.
A more recent method, intended for labeling antibodies for radiotherapy, involves the chemical conjugation of protein kinase substrate peptides to antibodies. Foxwell et al.,
Brit. J. Cancer
57:489 (1988). The conjugates are labeled by treatment with [&ggr;-
32
P]ATP in the presence of the catalytic subunit of cAMP-dependent protein kinase (protein kinase A, PKA), which transfers
32
P-phosphate to a serine residue in the substrate peptide. This method showed differences in the &bgr;-phase half-life between the
32
P-labeled antibody and a corresponding
131
-I-labeled antibody, and also high
32
P uptake in the bone of animals injected with the labeled antibody. Creighton et al., “The development of
32
P technology for radioimmunotherapy” in MONOCLONAL ANTIBODIES 2. APPLICATIONS IN CLINICAL ONCOLOGY. A.A. Epenetos, ed., Chapman and Hall, (1993) pp. 103-109. These results indicate in vivo instability of the label, presumably due to the action of protein phosphatases which are ubiquitous in eukaryotic cells.
Recently, Leung et al., Cancer Res. 55:5968s (1995) successfully expressed a hMN-14Fab′-kemptide fusion protein that can be enzymatically phosphorylated with
32
P by bovine protein kinase. The kemptide sequence was attached at the C-terminus of the human IgG1 hinge region and phosphorylation of the sequence did not affect the immunoreactivity of the fusion antibody, thus resolving the problem of non-site-specific conjugation associated with the chemically linked kemptides.
While these methods serve well to phosphorylate the protein, the attached
32
P was rapidly dephosphorylated from the kemptide sequence in human serum, probably by serum phosphatases. Although enhanced serum stability of the
32
P in the kemptide sequence can be achieved by modifying the primary sequence of receptor peptides, it is always at the expense of reduced ease of phosphorylation by protein kinases

Affiliated with

Also associated with

Rating

Stabilized radiophosphate-labeled proteins does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Stabilized radiophosphate-labeled proteins, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Stabilized radiophosphate-labeled proteins will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2901008