Methods and compositions based on diphtheria toxin-interleukin-3 conjugates
The present invention provides methods for inhibiting interleukin-3 receptor-expressing cells, and, in particular, inhibiting the growth of such cells by using a diphtheria toxin-human interleukin-3 conjugate (DT-IL3) that is toxic to cells expressing the interleukin-3 receptor. In preferred embodiments, the DT-IL3 conjugate is a fusion protein comprising amino acids 1-388 of diphtheria toxin fused via a peptide linker to full-length, human interleukin-3. In certain embodiments, the methods of the present invention relate to the administration of a DT-IL3 conjugate to inhibit the growth of cancer cells and/or cancer stem cells in humans, which cells express one or more subunits of the interleukin-3 receptor. Exemplary cells include myeloid leukemia cancer stem cells. In other embodiments, the methods of the present invention relate to ex vivo purging of bone marrow or peripheral blood to remove cells that express one or more subunits of the interleukin-3 receptor such that the purged bone marrow or peripheral blood is suitable, e.g., for autologous stem cell transplantation to restore hematopoietic function.
1. A method for inhibiting cancer cells in a human diagnosed with Hodgkin's disease, comprising administering an effective amount of a human interleukin-3 (IL-3)-diphtheria toxin conjugate to the human, wherein the cancer cells express the IL-3 receptor, and the growth of the cancer cells is inhibited, and the conjugate is administered at a dose of about 4 μg/kg to about 20 μg/kg.
2. The method of claim 1 , wherein the inhibition results in a reduction in the proliferation of cancer cells, a stabilization in the amount of cancer cells, and/or a reduction in the amount of cancer cells.
3. The method of claim 2 , wherein the stabilization or reduction is measured by blood tests; physical examination; examination of lymphoid tissue; complete blood count; flow cytometric analyses; bone marrow aspirate; bone marrow analyses; bone marrow biopsy; the amount of normal white blood cells, red blood cells, and/or platelets; histology; computed tomography (CT); X-ray imaging; PET scans; and/or immunohistochemistry.
4. The method of claim 1 , wherein the inhibition reduces the amount of IL-3 receptor expressing cells.
5. The method of claim 1 , wherein the human has abnormal cytogenetics.
6. The method of claim 1 , wherein the conjugate is a chemical conjugate.
7. The method of claim 1 , wherein the conjugate is a recombinantly expressed protein.
8. The method of claim 1 , wherein the conjugate is expressed as a single polypeptide comprising the catalytic and translocation domains of diphtheria toxin and human IL-3.
9. The method of claim 8 , wherein said human IL-3 diphtheria toxin conjugate is expressed in E. coli.
10. The method of claim 8 , wherein said human IL-3 diphtheria toxin conjugate is a fusion protein which is expressed from a fusion protein coding sequence comprising an ATG initiation codon.
11. The method of claim 8 , wherein said human IL-3 diphtheria toxin conjugate comprises the catalytic and translocation domains of diphtheria toxin fused via a peptide linker to full-length, mature human IL-3, wherein the peptide linker is two amino acids in length.
12. The method of claim 11 , wherein said peptide linker consists of a Met and a His.
13. The method of claim 11 , wherein said peptide linker is a Met-His linker.
14. The method of claim 11 , wherein the amino acid sequence of said full-length, mature human IL-3 is the amino acid sequence of the mature form of human IL-3 according to GenBank Accession No. AAC08706, wherein the mature form of human IL-3 lacks the signal peptide.
15. The method of claim 11 , wherein the conjugate comprises amino acid residues 1-388 of diphtheria toxin linked via a peptide bond to human IL-3.
16. The method of claim 1 , wherein the human
(i) is in a state of remission from the Hodgkin's disease;
(ii) has been previously treated with a therapeutic agent and/or has undergone radiation therapy;
(iii) is currently being administered a therapeutic agent other than a human IL-3-diphtheria toxin conjugate and/or is undergoing radiation therapy;
(iv) has relapsed from the Hodgkin's disease;
(v) has failed prior treatment of the Hodgkin's disease;
(vi) is susceptible to adverse reactions from other therapies for Hodgkin's disease;
(vii) is refractory to radiation therapy or chemotherapy; or
(viii) has not been previously treated for Hodgkin's disease.
17. The method of claim 16 , wherein the therapeutic agent, chemotherapy, or prior Hodgkin's disease treatment comprises bleomycin sulfate, carboplatin, carmustine, cisplatin, cyclophosphamide, cytarabine, dacarbazine, dexamethasone, doxorubicin, doxorubicin hydrochloride, etoposide, etoposide phosphate, gemcitabine, gemcitabine hydrochloride, ifosfamide, lenalidomide, mechlorethamine hydrochloride, melphalan, methylprednisolone, mTor inhibitors, prednisone, procarbazine hydrochloride, Rituxan, vinblastine sulfate, vincristine sulfate, and/or vinorelbine tartrate.
18. The method of claim 1 , wherein the conjugate is administered at a dose:
(i) of about 4 μg/kg to about 12.5 μg/kg; or
(ii) of about 5.3 μg/kg, about 7.1 μg/kg, about 9.4 μg/kg, or about 12.5 μg/kg.
19. The method of claim 1 , wherein the conjugate is administered:
(i) once a week;
(ii) at least two times a week or at least three times a week;
(iii) over a period of two weeks or more;
(iv) once every day for three days or once every day for five days; and/or
(v) for multiple cycles.