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Title:
RADIOLABELED MONOCLONAL ANTIBODIES, METHODS OF PREPARATION USING TCEP, AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2009/058203
Kind Code:
A1
Abstract:
This application discloses methods for producing radiolabeled IgM or IgA monoclonal antibodies using tris(2-carboxyethyl) phosphine hydrochloride (TCEP) to reduce disulfide bonds to generate sulfhydryl (-SH) groups and treating the monoclonal antibody with a radioisotope that binds to -SH groups, radiolabeled antibodies produced by the methods, monoclonal anti-melanin IgM antibodies containing about 42 to about 45 -SH groups, and methods of treating and imaging cancer, such an melanoma, and infections using the radiolabeled antibodies.

Inventors:
DADACHOVA EKATERINA (US)
CASADEVALL ARTURO (US)
SESAY MUCTARR A (US)
DAMANIA HEMA (US)
SMARIGA PAULETTE E (US)
Application Number:
PCT/US2008/011956
Publication Date:
May 07, 2009
Filing Date:
October 21, 2008
Export Citation:
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Assignee:
EINSTEIN COLL MED (US)
GOODWIN BIOTECHNOLOGY INC (US)
PAIN THERAPEUTICS INC (US)
DADACHOVA EKATERINA (US)
CASADEVALL ARTURO (US)
SESAY MUCTARR A (US)
DAMANIA HEMA (US)
SMARIGA PAULETTE E (US)
International Classes:
C07K16/00
Foreign References:
US20040156780A12004-08-12
US20040241748A12004-12-02
US6080384A2000-06-27
US20040018586A12004-01-29
US20060120959A12006-06-08
Attorney, Agent or Firm:
MILLER, Alan, D. et al. (ROTHSTEIN & EBENSTEIN LLP90 Park Avenu, New York New York, US)
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Claims:

What is claimed is:

1. A method of producing a radiolabeled IgM or IgA monoclonal antibody, the method comprising the steps of:

(a) treating the IgM or IgA monoclonal antibody with tris(2-carboxyethyl) phosphine hydrochloride (TCEP) to reduce disulfide (S-S) bonds to generate sulfhydryl (-SH) groups, and

(b) treating the reduced IgM or IgA monoclonal antibody of step (a) with a radioisotope that binds to -SH groups, thereby producing a radiolabeled IgM or IgA monoclonal antibody.

2. The method of Claim 1, wherein in step (a), the monoclonal antibody is treated with TCEP at a molar ratio of TCEP HCl:antibody of 20-80:1.

3. The method of Claim 1, wherein in step (a), the monoclonal antibody is treated with TCEP at a molar ratio of TCEP HCl:antibody of 50: 1.

4. The method of any of Claims 1-3, wherein in step (a), the monoclonal antibody is treated with TCEP for 20-80 minutes.

5. The method of any of Claims 1-3, wherein in step (a), the monoclonal antibody is treated with TCEP for 30-60 minutes.

6. The method of any of Claims 1-3, wherein in step (a), the monoclonal antibody is treated with TCEP for 30 minutes.

7. The method of any of Claims 1-6, which further comprises adding L-ascorbic acid to buffer used for purification of the radiolabeled IgM or IgA monoclonal antibody and purifying the radiolabeled IgM or IgA monoclonal antibody to remove TCEP and un- reacted radioisotope.

8. The method of any of Claims 1-7, wherein the radioisotope is selected from the group consisting of 94m-Tc, 99m-Tc, 188-Re, 186-Re, 118m-Sb, 122-Sb, 70-As, 71-As and 72-As.

9. The method of any of Claims 1-7, wherein the radioisotope is 94m-Tc, 99m-Tc, 188- Re or 186-Re, and wherein the radioisotope is reduced prior to treating the monoclonal antibody in step (b).

10. The method of any of Claims 1 -9, wherein the radioisotope is 188-Re.

11. The method of any of Claims 1-10, wherein the radiolabeled monoclonal antibody is a radiolabeled IgA monoclonal antibody.

12. The method of any of Claims 1-10, wherein the radiolabeled monoclonal antibody is a radiolabeled IgM monoclonal antibody.

13. The method of Claim 11 or 12, wherein the radiolabeled monoclonal antibody is an anti-melanin radiolabeled monoclonal antibody.

14. The method of Claim 13, wherein radiolabeled anti-melanin monoclonal antibody binds to both eumelanin and pheomelanin.

15. A radiolabeled monoclonal antibody produced by the method of any of Claims 1-12.

16. A radiolabeled monoclonal antibody produced by the method of Claims 13 or 14.

17. A radiolabeled anti-melanin IgM monoclonal antibody, wherein the antibody is radiolabeled with a radioisotope that binds to -SH groups, wherein the number of -SH groups on the radiolabeled monoclonal antibody is between 42-45, and wherein at least one atom of the radioisotope is bound to at least one -S.

18. The radiolabeled anti-melanin IgM monoclonal antibody of Claim 17, wherein the number of -SH groups on the radiolabeled monoclonal antibody is between 43-44, and wherein at least one atom of the radioisotope is bound to at least one -S.

19. The radiolabeled monoclonal antibody of Claim 17 or 18, wherein the antibody is radiolabeled with an isotope selected from the group consisting of 94m-Tc, 99m-Tc, 188-Re, 186-Re, 118m-Sb, 122-Sb, 70-As, 71-As and 72-As.

20. The radiolabeled monoclonal antibody of Claim 17 or 18, wherein the antibody is radiolabeled with 188-Re.

21. The radiolabeled monoclonal antibody of any of Claims 17-20, wherein the radiolabeled monoclonal antibody binds to both eumelanin and pheomelanin.

22. A method for treating a melanin-containing melanoma in a subject which comprises administering to the subject an amount of a radiolabeled monoclonal antibody of any of Claims 16-21 effective to treat the melanoma, wherein the radiolabeled monoclonal antibody specifically binds to melanin.

23. The method of Claim 22, which comprises multiple administrations of the radiolabeled antibody to the subject.

24. The method of Claim 22 or 23, wherein the amount effective to treat the melanoma is a dose of 0.5-100O mCi.

25. The method of any of Claims 22-24, wherein administration of the radiolabeled monoclonal antibody to the subject inhibits growth of the melanoma.

26. A method for imaging a melanin-containing melanoma in a subject which comprises administering to the subject an amount of a radiolabeled monoclonal antibody of any of Claims 16-21 effective to image the melanoma, wherein the radiolabeled monoclonal antibody specifically binds to melanin.

27. The method of any of Claims 22-26, wherein the radiolabeled monoclonal antibody is not taken up by non-cancerous melanin-containing tissue.

28. The method of Claim 27, wherein the non-cancerous melanin-containing tissue is hair, eyes, skin, brain, spinal cord, and/or peripheral neurons.

29. The method of any of Claims 22-28, wherein where the radiolabeled monoclonal antibody binds to melanin from a dead or dying melanoma cell.

30. A method for treating an infection in a subject which comprises administering to the subject an amount of a radiolabeled monoclonal antibody of Claim 15 effective to treat the infection, wherein the antibody specifically binds to an agent causing the infection.

31. An anti-melanin IgM monoclonal antibody that contains about 42 to about 45 -SH groups.

32. The anti-melanin IgM monoclonal antibody of Claim 31, wherein the number of -SH groups on the monoclonal antibody is about 43 to about 44.

Description:

RADIOLABELED MONOCLONAL ANTIBODIES, METHODS OF PREPARATION USING TCEP, AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

61/000,795, filed on October 29, 2007, the content of which is hereby incorporated by reference into the subject application.

STATEMENT OF GOVERNMENT SUPPORT

[0002] The invention disclosed herein was made with U.S. Government (National

Institutes of Health) support under Albert Einstein College of Medicine (AECOM) Comprehensive Cancer Center grant number 3P30CA013330. Accordingly, the U.S. Government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to methods for producing radiolabeled IgM or IgA monoclonal antibodies using Tris(2-Carboxyethyl) Phosphine Hydrochloride (TCEP), radiolabeled antibodies produced by the methods, and methods of treating and imaging cancers, in particular melanomas, and infections using the radiolabeled antibodies.

BACKGROUND OF THE INVENTION

[0004] Throughout this application various publications are referred to in parenthesis.

Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.

[0005] Cancer and infectious diseases continue to be major health threats in the developing and western world. As an example, melanoma poses an increasing health problem that affects about 40,000 patients each year in the United States and an estimated 100,000 world-wide. While primary melanomas that are localized to the skin can be successfully treated by surgical removal, there is no satisfactory treatment for metastatic melanoma, a

condition that currently has an estimated 5-year survival rate of 6%. Targeted radionuclide therapy has evolved into an efficient modality for cancer patients in whom standard antineoplastic therapies have failed (1). One type of targeted radionuclide therapy - radioimmunotherapy (RIT) takes advantage of the specificity of the antigen-antibody interaction to deliver localized lethal doses of radiation to target cells using radiolabeled antibodies (2,3). The clinical success of FDA-approved drugs such as Zevalin® and Bexxar® (anti-CD20 monoclonal antibodies labeled with 90- Yttrium ( 90 Y) and 131-Iodine ( 131 I), respectively), for the treatment of relapsed or refractory B-cell non-Hodgkin's leukemia (NHL) is evidence of the potential of RIT as an anti-neoplastic strategy. Encouraging reports on the use of RIT as an initial treatment for follicular lymphoma (4) have recently led to the recommendation that RIT be used as therapy of first choice for this malignancy. Hence, the increasing acceptance of RIT for certain lymphomas combined with the development of a technical infrastructure to support this type of therapy have created a favorable environment for the development of radionuclide therapy for metastatic melanoma in the clinical setting provided that suitable targets can be identified.

[0006] Melanoma owes its name to the presence of the pigment melanin. Given that even amelanotic melanomas contain some melanin, this pigment presents a potential target for development of radionuclide therapy of metastatic melanoma. Historically, melanin was not considered a target for RIT because it is an intracellular pigment outside the reach of a specific antibody. Because melanomas are rapidly growing, cell turnover releases melanin pigment into the extracellular space that can be targeted for delivery of cytotoxic radiation by radiolabeled melanin-binding antibodies. Experimental results have established the feasibility of targeting melanin released from dead melanoma cells in tumors with radiolabeled antibodies (5) and peptides (6). Furthermore, this strategy is attractive because melanin in normal tissues is not accessible to the antibody by virtue of its intracellular location.

[0007] To test this hypothesis, a murine IgM monoclonal antibody (mAb) known as

6D2 was used. 6D2 was generated from mice immunized with melanin produced by the fungus, Cryptococcus neoformans (7). This antibody also binds human melanin since both fungal and human melanins have structural similarities (8), and are negatively charged. Nude mice bearing MNTl pigmented human melanoma tumors were treated with mAb 6D2 labeled with 1.5 mCi of the beta-emitter 188-Rhenium ( Re). Mice treated with radiolabeled mAb 6D2 manifested inhibition of tumor growth and prolonged survival. MAb 6D2 bound tumor

melanin but did not bind to normal melanized tissues in C57BL6 black mice. The mechanism of melanoma targeting with mAb 6D2 involved antibody binding to extracellular melanin released during tumor cell turnover or to dying tumor cells with damaged or permeable membranes. These results provided the basis for the pre-clinical development of radiolabeling of 188 Re-6D2 mAb. However, the development of 188 Re-6D2 for clinical use highlighted the desirability of the development of a robust and reproducible radiolabeling procedure with quality control for the clinical setting.

SUMMARY OF THE INVENTION

[0008] The present invention provides methods of producing a radiolabeled IgM or IgA monoclonal antibody, the method comprising the steps of: (a) treating the IgM or IgA monoclonal antibody with tris(2-Carboxyethyl) Phosphine Hydrochloride (TCEP) to reduce disulfide (S-S) bonds to generate sulfhydryl (-SH) groups, and (b) treating the reduced IgM or IgA monoclonal antibody of step (a) with a radioisotope that binds to -SH groups, thereby producing a radiolabeled IgM or IgA monoclonal antibody. Preferably, the radiolabeled antibody is purified using buffer containing L-ascorbic acid.

[0009] The invention provides methods for treating and/or imaging an infection in a subject which comprises administering to the subject any of the radiolabeled monoclonal antibodies produced by any of the methods disclosed herein in an amount effective to treat or image the infection, wherein the antibody specifically binds to the agent causing the infection.

[0010] The invention further provides radiolabeled anti-melanin IgM monoclonal antibodies, wherein the antibody is radiolabeled with a radioisotope that binds to -SH groups, wherein the number of -SH groups on the radiolabeled monoclonal antibody is about 42 to about 45, and wherein at least one atom of the radioisotope is bound to at least one -S. [0011] The invention also provides methods for treating and/or imaging melanin- containing melanomas in a subject, where the methods comprise administering to the subject any of the radiolabeled anti-melanin monoclonal antibodies disclosed herein, and/or produced by any of the methods disclosed herein, in an amount effective to treat or image the melanoma, wherein the radiolabeled monoclonal antibody specifically binds to melanin. [0012] The invention also provides pharmaceutical compositions formulated in dosage form, comprising any of the radiolabeled monoclonal antibodies disclosed herein or produced

by any of the methods disclosed herein dissolved or dispersed in a pharmaceutically acceptable diluent or carrier.

BRIEF DESCRIPTION OF THE FIGURES

[0013] Figure 1. Structural integrity of 6D2 mAb after treatment with TCEP. For comparative purposes samples of 6D2 treated with dithiothreitol (DTT) as in (5) are also shown. Non-reducing SDS-PAGE (4-20% tris-glycine gel) was used. Lane # 1 : empty; Lane # 2: Standards: Novex, SeeBlue Pre-Stained Standards: Myosin 250 kDa, BSA 98 kDa, Glutamic Dehydrogenase, 64 kDa, Alcohol Dehydrogenase 50 kDa [also used but not illustrated in this view of gel - 36 kDa carbonic anhydrase, 30 kDa myoglobin, 16 kDa lysozyme, 6 kDa aprotinin, 4 kDa insulin b chain]; Lane # 3: affinity purified 6D2 standard; Lane # 4: 6D2:TCEP, 1 :10 molar ratio; Lane # 5: 6D2:TCEP, 1 :100 molar ratio; Lane # 6: 6D2 treated with DTT; Lane # 7: the same; Lane # 8: mouse myeloma IgM standard; Lane # 9: 6D2:TCEP, 1 :50 molar ratio; Lane # 10: empty.

[0014] Figure 2A-2E. Structural integrity of 6D2 mAb after treatment with TCEP at a

TCEP:mAb ratio of 50:1 for various periods of time. The samples were subsequently labeled with "cold" rhenium. Non-reducing SDS-PAGE (4-20% Tris-Glycine gel) was used. A) SDS-PAGE: Lane # 1 : Pre-stained MW marker, Myosin 200 kDa, B glactosides 116.3 kDa, Phosphorylase b 97.4 kDa, Bovine Albumin 66.3 kDa, Glutamic Dehydrogenase, 55.4 kDa; Lane # 2: 6D2, 5 minute treatment; Lane # 3: 6D2, 15 minute treatment; Lane # 4: 6D2, 30 minute treatment; Lane # 5: 6D2, 120 minute treatment; Lane # 6: 6D2, 30 minute treatment; Lane # 7: 6D2, 60 minute treatment; Lane # 8: Sigma Std. IgM; Lane # 9: 6D2 reference standard. (B-E) size exclusion HPLC of 6D2, wavelength 280 nm: B) 6D2 reference standard; C) 6D2, 5 minute treatment; D) 6D2, 30 minute treatment; E) 6D2, 120 minute treatment.

[0015] Figure 3A-3D. Radiochromatographic profiles of 188 Re-6D2 preparations: A) eluted from HiPrep column and stabilized with 0.2 mg/mL L-ascorbic acid; B) the same preparation as in (A) but frozen for 24 hours in a resin vial; C) stabilized with 0.2 mg/mL L- ascorbic acid and "cold" 6D2, stored at 4 0 C for 6 hours and passed through infusion set; D) eluted from HiPrep column with 0.2 mg/mL L-ascorbic acid in saline.

[0016] Figure 4A-4C. Binding of 188 Re-6D2 to melanin by ELISA: A) immediately after preparation; B) after passing through infusion set 6 hours after preparation; C) after overnight storage at 4 0 C and -8O 0 C in glass and resin vials.

[0017] Figure 5A-5B. Biodistribution of 188 Re-6D2 in nude mice bearing A2058- derived melanoma tumors after IV administration: A) blood, whole body and carcass clearance; B) distribution in major organs and tumors.

[0018] Figure 6A-6B. Whole body autoradiography of 188 Re-6D2 in A2058 human melanoma-bearing nude mice after IV administration: A) 4 hours; B) 24 hours. [0019] Figure 7A-7C. Therapy of A2058 human melanoma-bearing nude mice with various doses of I88 Re-6D2: A) tumor volumes; (B, C) histology of the tumors: B) tumor from untreated mouse; C) tumor from a mouse treated with 1.5 mCi. Tissues were stained with hematoxylin and eosin. Melanin granules are marked with black arrows. Left panels in B and C - 25 X magnification; right panel - 400 X magnification.

[0020] Figure 8A-8B. Platelet and white blood counts in A2058 human melanoma- bearing nude mice treated with various doses of 188 Re-6D2: A) platelet; B) white blood count.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The subject invention is directed to a method of producing a radiolabeled IgM or

IgA monoclonal antibody, the method comprising the steps of: (a) treating the IgM or IgA monoclonal antibody with tris(2-Carboxyethyl) Phosphine Hydrochloride (TCEP) to reduce disulfide (S-S) bonds to generate sulfhydryl (-SH) groups, and (b) treating the reduced IgM or IgA monoclonal antibody of step (a) with a radioisotope that binds to -SH groups, thereby producing a radiolabeled IgM or IgA monoclonal antibody.

[0022] Preferably, in step (a) of the method, the monoclonal antibody is treated with

TCEP at a molar ratio of TCEP HCl:antibody of about 20:1 to about 80:1. More preferably, the monoclonal antibody is treated with TCEP at a molar ratio of TCEP HCl: antibody of 50:1. Preferably, in step (a), the monoclonal antibody is treated with TCEP for about 20 to about 80 minutes, more preferably for 30-60 minutes, and most preferably for 30 minutes. [0023] Preferably, the method additionally comprises adding L-ascorbic acid to the buffer used for purification of the radiolabeled IgM or IgA monoclonal antibody and purifying the radiolabeled IgM or IgA monoclonal antibody to remove TCEP and un-reacted radioisotope into the buffer containing the L-ascorbic acid.

[0024] The antibody can be a human antibody or a non-human antibody such as a goat antibody or a mouse antibody. Antibodies can be "humanized" using standard recombinant DNA techniques. By transferring the mouse antibody binding site coding region into a human antibody gene, a "human antibody" can be engineered that retains the specificity and biological effects of the original mouse antibody but has the potential to be nonimmunogenic

in humans. Additionally, antibody effector functions can be improved through manipulation of the antibody constant region genes (e.g., 25-27).

[0025] The methods disclosed herein can be used with any radioisotope that binds to -

SH groups. Examples of radioisotopes that bind to -SH groups include, but are not limited to, Technetium-94m (94m-Tc, 52 minute half-life, positron emitter), Technetium-99m (99m-Tc, 6 hour half-life, gamma emitter), 188-Rhenium (188-Re, 16.9 hour half-life, beta- and gamma-emitter), 186-Rhenium (186-Re, 90.6 hour half-life, beta- and gamma-emitter), 118m- Antimony (118m-Sb, 5 hour half-life, positron- and gamma-emitter), 122- Antimony (122-Sb, 2.7 day half-life, beta- and gamma-emitter), 70-Arsenic (70-As, 53 minute half-life, positron- and gamma-emitter), 71-Arsenic (71-As, 64.8 hour half-life, positron- and gamma- emitter) and 72-Arsenic (72-As, 26 hour half-life, positron- and gamma-emitter). The high- energy β-emitter 188-Rhenium (E max = 2.12 MeV) is a preferred radioisotope. Re has the additional advantage that it emits γ-rays which can be used for imaging studies. [0026] When the radioisotope is 94m-Tc, 99m-Tc, 188-Re or 186-Re, preferably the radioisotope is reduced prior to treating the monoclonal antibody in step (b) of the method. Reducing agents that can be used for reduction of the Tc and Re radioisotopes include, but are not limited to, salts of tin (II) (e.g., stannous chloride, stannous tartrate, stannous citrate), SO 2 -releasing compounds such as sodium dithionite, concentrated HCl, and sodium borohydride.

[0027] Preferred radiolabeled monoclonal antibodies include an anti-melanin IgM antibody radiolabeled with 188-Re. Preferably, the radiolabeled anti-melanin monoclonal antibody binds to both eumelanin and pheomelanin.

[0028] The invention also provides radiolabeled monoclonal antibodies produced by any of the methods disclosed herein.

[0029] The invention provides an anti-melanin IgM monoclonal antibody that contains about 42 to about 45-SH groups, and preferably about 43 to about 44 -SH groups. [0030] The invention further provides a radiolabeled anti-melanin IgM monoclonal antibody, wherein the antibody is radiolabeled with a radioisotope that binds to -SH groups, wherein the number of -SH groups on the radiolabeled monoclonal antibody is about 42 to about 45, and wherein at least one atom of the radioisotope is bound to at least one -S. Preferably, the number of -SH groups on the radiolabeled monoclonal antibody is between 43-44, and at least one atom of the radioisotope is bound to at least one -S. Preferably, the antibody is radiolabeled with an isotope selected from the group consisting of 94m-Tc, 99m-

Tc, 188-Re, 186-Re, 118m-Sb, 122-Sb, 70-As, 71-As and 72-As. More preferably, the radiolabeled monoclonal antibody is radiolabeled with 188-Re. Preferably, the radiolabeled anti-melanin monoclonal antibody binds to both eumelanin and pheomelanin. [0031] The invention also provides a method for treating and/or imaging a melanin- containing melanoma in a subject which comprises administering to the subject any of the radiolabeled anti-melanin monoclonal antibodies disclosed herein, and/or produced by any of the methods disclosed herein, in an amount effective to treat or image the melanoma, wherein the radiolabeled monoclonal antibody specifically binds to melanin. As used herein, the term "treat" a melanoma means to eradicate the melanoma, to reduce the size of the melanoma, to stabilize the melanoma so that it does not increase in size, or to inhibit the growth and/or spread of the melanoma.

[0032] Preferably, the radiolabeled monoclonal anti-melanin antibody is not taken up by non-cancerous melanin-containing tissue, such as, for example, hair, eyes, skin, brain, spinal cord, and/or peripheral neurons. Rather, the radiolabeled monoclonal anti-melanin antibody binds to melanin from dead or dying melanoma cells.

[0033] The invention also provides a method for treating and/or imaging an infection in a subject, where the method comprises administering to the subject any of the radiolabeled monoclonal antibodies produced by any of the methods disclosed herein in an amount effective to treat or image the infection, wherein the antibody specifically binds to the agent causing the infection. As used herein, the term "treat" an infection means to eliminate the infection, to reduce the number of the microorganisms causing the infection in the subject, to prevent the infection from spreading in the subject, or to reduce the further spread of the infection in the subject.

[0034] The subject can be any mammal and is preferably a human.

[0035] The invention also provides a pharmaceutical composition formulated in dosage form, comprising any of the radiolabeled antibodies disclosed herein, or any of the radiolabeled antibodies produced by any of the methods disclosed herein, dissolved or dispersed in a pharmaceutically acceptable carrier or diluent, wherein the dosage is appropriate to treat or image an infection or a cancer such as melanoma. As used herein, the term "carrier" or "diluent" encompasses any of the standard pharmaceutical carriers or diluents, such as a sterile isotonic saline, phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsions.

[0036] In order to calculate the dose of the radioisotope that can be used in treatment without radiotoxicity to vital organs, a diagnostic scan of the patient with the antibody

radiolabeled with a diagnostic radioisotope or with low activity therapeutic radioisotope can be performed prior to therapy, as is customary in nuclear medicine. The dosimetry calculations can be performed using the data from the diagnostic scan (28).

[0037] Clinical data (29, 30) indicate that fractionated doses of radiolabeled antibodies are more effective than single doses against tumors and are less radiotoxic to normal organs.

Depending on the status of a patient and the effectiveness of the first treatment with RIT, the treatment may consist of one dose or several subsequent fractionated doses.

[0038] The dose of the radioisotope for treatment of humans is typically about 0.5 mCi to about 500 mCi.

[0039] The radiolabeled antibody or agent can be delivered to the subject by a variety of means. Preferably, the radiolabeled antibody or agent is administered parenterally. The radiolabeled antibody can be injected, for example, into the bloodstream, into a muscle or into an organ.

[0040] This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

EXPERIMENTAL DETAILS Materials and Methods

[0041] Antibody, melanoma cell line and radioisotope. Fungal melanin binding MAb

6D2 previously described in (7) was produced by Goodwin Biotechnology Inc. (Plantation, FL). The mAb 6D2 was purified via either a 1 -column affinity purification (for studies involving the generation of sulfhydryl groups with TCEP) or a multi-column purification (for all other studies). Purity of the 6D2 from these two processes was >95% via HPLC-SEC. A2058 certified cell line derived from a lymph node metastasis from a patient with malignant melanoma was obtained from the American Type Culture Collection (10801 University Boulevard, Manassas, Virginia 21110-2209). The cells were maintained as monolayers in Dulbecco's Modified Eagle's Medium with 4 mM L-glutamine, 4.5 g/L glucose, 1.5g/L sodium bicarbonate, supplemented with 10% fetal bovine serum and 5% penicillin- streptomycin solution at 37 0 C and 5% carbon dioxide, and harvested using 0.25% (w/v) trypsin-EDTA solution. The cells were washed in serum-free Dulbecco's Modified Eagle's Medium before inoculation into nude mice.

[0042] 188 Re as sodium perrhenate Na 188 ReO 4 was eluted from a 188 W/ 188 Re generator

(Oak Ridge National Laboratory, Oak Ridge, TN) by passing 15 mL 150 mM NaCl solution through a generator with a 20 or 30 mL syringe at a rate of 2 - 5 mL/minute. The eluate was

1 δδ collected into a sterile vial and Re activity was measured in the dose calibrator using calibration-setting number 496x10.

[0043] Generation of sulfhydryl (SH) groups on 6D2 via reduction of disulfide bonds with TCEP. Tris(2-Carboxyethyl) Phosphine Hydrochloride (TCEP HCl, Pierce) was evaluated as a reducing agent for generating -SH groups on the proteins via reduction of disulfide bonds. For this purpose, the influence of TCEP molar excess over 6D2 mAb on the mAb structural integrity and on radiolabeling yields was initially evaluated. Four hundred μg (140 μL) samples of 6D2 mAb (2.857 mg/mL initial concentration, in 20 mM sodium phosphate buffer + 150 mM NaCl, pH=7.5) were incubated for 1 hour at room temperature with 0, 2, 10, 50 and 100 molar excess of TCEP (1.5 mM solution in PBS) over 6D2 mAb. After incubation, each sample was split into two 200 μg aliquots. One aliquot was treated with 15 mM N-ethylmaleimide (Pierce) in PBS to protect the generated -SH groups from recombining with each other, and analyzed by non-reducing SDS-PAGE (4% and 4-20% tris- glycine non-reducing gels). Another aliquot was radiolabeled with 188 Re as described below and dependence of radiolabeling yields on the TCEP molar excess over 6D2 was determined. [0044] The kinetics of generating -SH groups on 6D2 via TCEP reduction at constant

TCEP to 6D2 molar ratio was studied by incubating 6D2 mAb at the above concentration with 50 molar excess of TCEP (1.5 mM solution in PBS) over 6D2 for 5 minutes - 4 hours at room temperature. Each sample was then split into two aliquots. The first aliquot was labeled with "cold" sodium perrhenate following the radiolabeling procedure described below and analyzed by non-reducing SDS-PAGE and size exclusion HPLC on TSK4000 column (TosoHaas, Japan) eluted with PBS at 1 mL/minute. The eluted protein was analyzed by UV

1 δδ absorption at 280 nm wavelength. The second aliquot was radiolabeled with Re as described below and dependence of radiolabeling yields on the reduction time with TCEP was determined.

[0045] Radiolabeling with 188 Re, quality control and purification and stabilization of the final product. Reduction of perrhenate Na 188 ReO^ A volume of 1.5 mL of 0.5 g/mL sodium gluconate solution was added to 15 mL saline containing Na 188 ReO 4 eluted from the

1 δδ i δδ

W/ Re generator followed by addition of 0.1 mL 150 mg/mL stannous chloride in 1 M HCl. The reaction mixture was then incubated at 37 0 C for 1 hour and the percentage of 188 Re

reduction was determined with 10 cm SG-ITLC strips (silica gel instant tin layer chromatography, Gelman Sciences) developed with acetone. In this system reduced Re stays at the point of application while unreduced perrhenate moves with the solvent front. The strips were cut in half and counted in a scintillation counter.

[0046] Radiolabeling of TCEP -treated 6D2 mAb with 188-Re. Thirty minutes after the start of Re reduction, 0.1 mL of 5 mM TCEP (50:1 TCEP to 6D2 molar ratio) was added to the vial containing 10 mg (5 mg/mL) 6D2 and incubated at room temperature for 30 minutes. Then 6D2 treated with TCEP was mixed with reduced 188 Re and the mixture was incubated at 37 0 C for 1 hour. Percentage of incorporation of 188 Re into 6D2 was determined using SG- ITLC developed with saline where radiolabeled 6D2 stays at the point of application, while small molecular 188 Re-containing species travel with the solvent front. The amount of radiocolloids in the preparation was quantified by using SG-ITLC strips pre-saturated with BSA. When such strips are developed in ethanol:NH 4 OH:H 2 θ (2:1 :5), the radiolabeled proteins move with the solvent front, whereas radiocolloids stay at the point of application. The strips were cut in half and counted in a scintillation counter.

[0047] Purification of 188 Re -6D2 on a size exclusion column, melanin-binding ELISA and stabilization with L-ascorbic and "cold" 6D2. Radiolabeled 188 Re-6D2 was loaded onto the pre-equilibrated with saline HiPrep size exclusion column (GE Healthcare, Sweden) with

1 RS a peristaltic pump and eluted with approximately 25 mL saline. The purified Re-6D2 was immediately analyzed for: 1) percentage of 188 Re incorporation into 6D2 by SG-ITLC developed with saline and by size exclusion HPLC on TSK4000 column with the eluted protein detected at 280 run wavelength and radioactivity by Bioscan Flow Count detector, and 2) ability to bind to melanin by melanin-binding ELISA on 96-well plates covered with synthetic melanin (Huntington, NJ). The purified 188 Re-6D2 was stabilized by addition of 20 mg/mL L-ascorbic acid for the final concentration of 0.2 mg/mL. In some experiments the L- ascorbic acid was added directly to the saline used for eluting the 188 Re-6D2 from the HiPrep column; in other experiments after addition of L-ascorbic acid to the purified Re-6D2, "cold" 6D2 was added as well in quantities needed to make the total amount of 10 mg 6D2 in the preparation. The influence of overnight (about 18 hours) freezing at -8O 0 C at radiochemical purity and immunoreactivity of radiolabeled mAb was evaluated by ITLC, HPLC and melanin-binding ELISA.

[0048] Animal model of human metastatic melanoma. All animal studies were carried out in accordance with the guidelines of the Institutes for Animal Studies at the Albert Einstein College of Medicine and of the University of California, Davis. For

pharmacokinetics and whole body autoradiography (WBAR) studies, forty 11 -week old female nude mice were implanted subcutaneously with 5 x 10 6 A2058 cells in each of two abdominal sites and used for experiments 14 days later. For therapy/acute hematologic toxicity evaluation studies, 5-6 week-old female nude mice were implanted subcutaneously with 8 x 10 6 A2058 cells into the left flank and used for therapeutic experiments 12 days after tumors reached the size of approximately 0.15 cm 3 (0.02-0.4 cm 3 ).

[0049] Pharmacokinetics and whole body autoradiography (WBAR). For pharmacokinetic experiments thirty-six nude mice bearing A2058 cell-derived melanoma tumors were given either 30 μCi 188 Re-6D2 (total amount of 6D2 150 μg/lOOμL) and sacrificed at 5 minutes, 2 hours, 4 hours, and 24 hours; or 120 μCi 188 Re-6D2 (total amount of 6D2 150 μg/lOOμL) and sacrificed at 48 hours. Blood clearance samples (2 μL) were collected at 5 minutes, 1 hour, 2, 4, 24 and 48 hours from the dorsal tail vein and counted in a sodium iodide gamma well counter (Packard, Downers Grove, IL). Decay-corrected radioactivity in the blood was expressed as %ID, using a weight-based theoretical blood volume. Whole body activity was measured at the time of injection, and at 2, 4, 24 and 48 hrs using the iso-responsive sodium iodide detector system (Picker Nuclear, North Haven, CT); the counts were decay corrected and expressed as % ID. Pharmacokinetic data for other tissues were obtained by removing and weighing the tissues, and counting them in the same gamma well counter. The concentration of radioactivity in each organ was expressed as % ID/g.

[0050] For WBAR, four mice were given 120 μCi 188 Re-6D2 (total amount of 6D2 150 μg/300μL) and 2 mice were sacrificed for WBAR at each time point of 4 and 24 hours. The mice were anesthetized by intravenous injection of 60 mg/100 μL aqueous solution of sodium pentobarbital, and flash frozen in a hexane-dry ice bath. The frozen mice were embedded in frozen 4% carboxymethylcellulose, and sagittal sections were taken at -2O 0 C with a Leica Polycut. Sections of 50 μm thickness were taken to show tumors, spleen, kidney, liver and the midline of the vertebral column. The sections then were desiccated, and autoradiograms were prepared by exposing the sections to x-ray film (Kodak BioMax MS, Rochester, N. Y.).

[0051] Treatment ofA2058 tumor-bearing mice with increasing doses of Re-6D2 and evaluation of acute hematologic toxicity. The nude mice bearing A2058 cell-derived melanoma tumors were randomized into 5 groups of 6 mice. Groups 1-4 received IV injections of 0.15, 0.5, 1.0 and 1.5 mCi 188 Re-6D2. Control group was left untreated. Mice were weighed and the volume of the tumors was measured immediately before administration

of radiolabeled mAb and weekly thereafter. Tumors were measured in three dimensions with calipers, and tumor volume was calculated by multiplying the product of the three perpendicular diameters by 0.5, assuming an elliptical geometry. To evaluate the acute hematologic toxicity of RIT, platelet and white blood cell (WBC) counts were measured on the day of therapy and Days 3, 7, 14, and 28 (9) after administration of 188 Re-6D2. Blood samples from each animal were collected from the tail vein and individually diluted 1 :200 into 25% (v/v) ammonium oxalate for platelet counts, and 1 :20 into 2% (v/v) acetic acid for WBC counts. The diluted blood cells were counted using a hemocytometer and light microscopy at magnification XlOO (WBCs) or X450 (platelets).

[0052] Tumor histology. To assess the effects of radiolabeled mAb, A2058 tumors from the treated and control mice were removed at the end of the therapy study and fixed in 10% neutral buffered formalin. Tissues were routinely processed, paraffin embedded, cut to 5 μm, and stained with hematoxylin and eosin (H&E) for histological evaluation. [0053] Statistical analysis. The Wilcoxon rank sum test was used to compare tumor sizes and platelet and WBC counts between different treatment groups in therapy studies. Differences were considered statistically significant when P values were < 0.05.

Results

[0054] Influence of TCEP concentration and incubation time on mAb 6D2 SH group generation. The efficiency of TCEP in generating -SH groups was estimated from the radiolabeling yields with 188 Re while the structural integrity of TCEP -treated mAb 6D2 was assessed with non-reducing SDS-PAGE and size exclusion HPLC. Lower concentrations of TCEP (2:1 and 10:1 TCEP to 6D2 molar ratios) were inefficient at generating -SH groups and this resulted in low radiolabeling yields with 188 Re of 25% (Table 1). In contrast, TCEP to 6D2 molar ratios of 50:1 and 100:1 led to 70-72% radiolabeling yields (Table 1). However, at the 100:1 molar ratio there was some fragmentation of 6D2 mAb relative to that observed for lower concentrations (Fig. 1). Consequently, a 50 molar excess of TCEP over 6D2 mAb was used for all subsequent experiments.

[0055] In evaluating the effect of incubation time, it was noted that incubation times of

5 and 15 minutes resulted in 41-42% radiolabeling yields with 188 Re, longer times of 30 and 60 minutes increased the yield to 71-72%, whereas additional time up to 240 minutes did not produce further yield (Table 2). SDS-PAGE analysis of mAb 6D2 samples treated with TCEP for different times and then labeled with "cold" Re demonstrated that after short incubation times with TCEP (up to 30 minutes) most of 6D2 remained as an intact IgM molecule,

whereas 120 minutes incubation caused significantly more fragmentation of IgM (Fig. 2A). Based on these results, incubation time of 30 minutes and a 50:1 molar ratio of TCEP to mAb 6D2 were used as preferred conditions for generation of -SH groups on 6D2 mAb. [0056] Interestingly, however, although non-reducing SDS-PAGE showed fragments in labeled with "cold" Re antibody preparations (Fig. 2A), the antibody was eluted from the size exclusion HPLC column as a single peak at 6.7 minutes similar to the one for intact native 6D2 (Fig. 2B) for all preparations (Fig. 2C-F). Blaunstein et al. (10) observed the same inconsistency between SDS-PAGE and size exclusion HPLC of TCEP-treated murine IgG radiolabeled with 99m Tc, while Michaelsen et al. (11) reported similar findings for human IgGl. In both reports the discordance between SDS-PAGE and HPLC did not affect the immunoreactivity of mAbs towards their respective antigens. The fragmentation apparent by SDS-PAGE might reflect electrophoretic forces pulling apart the parts of an antibody held together by fewer disulfide bridges as a result of TCEP treatment in comparison to native mAb. The size exclusion HPLC, on the other hand, permits detection of the antibody in the form in which it is present in solution.

[0057] Radiochemical purity, stabilization of 188 Re-6D2 with L-ascorbic acid and

"cold" 6D2 and melanin-binding ELISA. Since radiolabeling yields with 188 Re of TCEP- treated 6D2 were 71-72%, post-radiolabeling purification was necessary. This was accomplished by passing 188 Re-6D2 through size exclusion HiPrep column, which can be loaded with up to 25 mL volumes of protein solutions. Purification of 188 Re-6D2 resulted in radiochemical purity of 92-93% with < 1% radiocolloids as determined by SG-ITLC. Approximately 70% of the total amount of the antibody was recovered from the HiPrep column. The purified antibody was immediately stabilized with 0.2 mg/mL L-ascorbic acid, which is widely used as a radioprotector for radiopharmaceuticals. In addition to its radioprotective properties, L-ascorbic acid also decreased the pH of 188 Re-6D2 in saline from 5.3 to 4.8; the lower pH could have further stabilized the 188 Re radiolabel on the antibody (12).

[0058] The radiochromatographic profile of the purified 188 Re-6D2 stabilized with L- ascorbic acid is shown in Fig. 3 A. The stability of 188 Re radiolabel on the antibody was evaluated after freezing the radiolabeled mAb at -8O 0 C immediately after preparation and thawing 24 hours later. Freezing the radiolabeled mAb might be necessary if the clinical procedure has to be postponed or because of the transportation problems. Both SG-ITLC (91% radiochemical purity) and radiochromatography (Fig. 3B) showed the 188 Re radiolabel remained attached to the antibody.

[0059] The addition of "cold" 6D2 to 188 Re-6D2 immediately after purification on

HiPrep column increased the overall amount of mAb in the preparation to 10 mg and improved the radiochromatographic profile (Fig. 3C), possibly due to more complete recovery of the antibody fraction from the HPLC column because of resulting higher concentration (0.9 mg/mL versus 0.25 mg/mL in preparations without addition of "cold" 6D2). In an attempt to further increase the radiochemical purity of the final product, 0.2 mg/mL L-ascorbic acid was added to the saline used in elution of the HiPrep column. This modification of purification procedure further increased the radiochemical purity of Re- 6D2 as per ITLC (97%) and radiochromatography (Fig. 3D).

[0060] Immunoreactivity of radiolabeled mAb is an important quality control parameter to ensure the fidelity of radiolabeled mAb to bind to its respective antigen. The immunoreactivity of 188 Re-6D2 was evaluated by melanin-binding ELISA using 96- well plates coated with synthetic melanin. 188 Re-6D2 bound to melanin to the same degree as native 6D2 (Fig. 4A). Storage of 188 Re-6D2 for 6 hours at 4 0 C (proposed shelf-life of 188 Re- 6D2 in the clinical trial) followed by passing it through the infusion set did not cause any significant decrease in its immunoreactivity (Fig. 4B). Likewise, overnight (about 18 hours) freezing of 188 Re-6D2 at -8O 0 C in a resin vial did not affect its ability to bind to melanin (Fig. 4C).

[0061] Biodistribution and WBAR of' 88 Re-6D2. The biodistribution of 188 Re-6D2 was evaluated in nude mice bearing tumors derived from A2058 human metastatic melanoma cell

1 BS line. Re-6D2 was quickly cleared from the blood with a half-life of approximately 5 hours and from the body with a half-life of approximately 10 hours (Fig. 5A). The kidney uptake was significant at 2 hours post-IV injection (18% ID/g); however, it rapidly decreased to 5.6% at 24 hours and to 3% ID/g at 48 hours (Fig. 5B). Overall, the clearance of 188 Re-6D2 from all major organs was rapid and mirrored the clearance from the blood (Fig. 5B and Fig. 6).

[0062] The WBAR at 4 hours showed some uptake in the thyroid (Fig. 6A), which is due to 8% of free 188 Re-perrhenate in preparation and which disappeared at 24 hours images (Fig. 6B) due to inability of 188 Re-perrhenate to accumulate in thyroid tissue (13). Tumor uptake was modest with a maximum uptake of 1.94 % ID/g reached at 4 hours, which decreased to 0.51 and 0.21%, at 24 and 48 hours, respectively (Fig. 5B and Fig. 6). However, this was clearly melanin-specific uptake as tumor-to-muscle ratio for these tumors, which are located in a muscle bed, grew progressively from 2 at 5 minutes post- injection to 5 at 4 hours, and stayed around 5 at 24 and 48 hours.

[0063] Therapy for A2058 human metastatic melanoma tumors in nude mice, tumor histology and evaluation of acute hematologic toxicity of Re-6D2. The ability of Re- 6D2 to affect the growth of human metastatic melanoma tumors was evaluated in nude mice by administering increased amounts of 188 Re-6D2. Although the lowest dose of 0.15 mCi had no effect on tumor progression relative to untreated mice, the doses of 0.5, 1.0 and 1.5 mCi significantly (P<0.05) slowed down the tumor growth (Fig. 7A). The radiation treatment caused widespread necrosis of the tumors in the groups treated with higher doses. This effect was especially pronounced in the group receiving 0.5 mCi. On Day 35 post-treatment, the tumors of the untreated mice and mice treated with 1.0 and 1.5 mCi were removed and analyzed histologically.

[0064] In untreated mice, the tumors were large and had extensive central necrosis.

The tumor cells had a high mitotic index (5 per 400X field) and there were low numbers of infiltrating lymphocytes and macrophages around the tumor (Fig. 7B). In contrast, the tumors from mice treated with 188 Re-6D2 had more extensive central necrosis than the tumors from control mice and very few viable tumor cells remaining (Fig. 7C). The neoplastic cells present within the tumors from treated animals had far fewer mitotic figures (0.4/400X field) and more individual cell apoptosis and necrosis than did the tumors from control animals. In addition, one tumor from a treated mouse had extensive fibrovascular granulation tissue with infiltrates of lymphocytes, macrophage, plasma cells, and a few neutrophils inside the tumor itself (not seen in control tumors).

[0065] Interestingly, although only few granules of melanin were visible in control tumors (Fig. 7B), abundant melanin deposits were found in treated tumors (Fig. 7C). The better visibility of melanin in treated tumors was due to most of it becoming extracellular, presumably, as a result of the treatment with radiolabeled mAb.

[0066] The hematologic toxicity of the radiolabeled mAb treatment was evaluated by measuring the numbers of platelets and WBCs in blood of treated and control mice. The drop in platelet and WBC count in groups treated with high doses was detected on days 3 and 7 post-treatment (Fig. 8); however, the counts normalized by Day 14. The body weight of mice in all groups was stable (results not shown).

[0067] Radiolabeling of monoclonal antibody using TCEP versus DTT. The number of

-SH groups generated on 6D2 by TCEP reduction was determined under optimal conditions, i.e., using 50 molar excess of TCEP over 6D2 mAb and 30 minutes incubation of mAb with TCEP at room temperature. For comparison, the same amount (0.5 mg) of 6D2 was reduced with dithiothreitol (DTT) according to (Dadachova et al. 2004 (5)). The antibody samples

were purified from unreacted reducing agents on Centricon-50 microconcentrators by washing with 3 x 1.5 mL of ammonium acetate buffer, pH = 6.5-7.0. The determination of - SH groups on the antibody was carried out using Ellman's reagent with spectrophotometric detection according to Ellman's reagent manufacturer instructions (Pierce, USA) and techniques described in Dadachova and Mirzadeh 1997 (24).

[0068] The number of -SH groups per 6D2 molecule was determined to be 44 and 56 for TCEP and DTT reduction, respectively. Thus, there is 1.27 times more -SH groups on the mAb post-DTT reduction than post-TCEP reduction. This coincides with the radiolabeling with 188-Re yields. Typical radiolabeling yield post-TCEP reduction is 70%, versus post- DTT reduction being 90%, which gives a ratio of 1.28 (90/70). This result shows that there are chemical differences between 6D2 mAb reduced with DTT versus TCEP, which distinguish this new product from anything previously described.

[0069] Processing the mAb with DTT results in more fragmentation of the antibody than does reduction using TCEP (Figure 1); thus processing with TCEP better maintains the integrity of the antibody.

188

Table 1. Influence of TCEP molar excess over 6D2 mAb on radiolabeling yields with Re. Incubation was carried out at room temperature for 1 hour.

TCEP molar excess over 6D2 Radiolabeling yield, %

0 10

2 25

10 25

50 72

100 70

Table 2. Influence of incubation time of 6D2 mAb with 50 molar excess of TCEP on radiolabeling yields with Re. Incubation at room temperature.

Time of reduction with TCEP, minutes Radiolabeling yield, %

5 42

15 41

30 72

60 71

120 66

240 33

Discussion

[0070] The present invention provides methods for the preparation of full length radiolabeled monoclonal antibodies with multiple IgGs (i.e., IgMs and slgAs) for use in radioimmunotherapy (RIT) and radioimaging. The utilization of a non-sulfur containing reducing agent allows the generation of -SH group(s) on the antibody and subsequent reaction with the radiolabeled reagent without prior purification of the reduced antibody. Thus, the method allows the preparation of a simple, fast, efficient, and cost effective radiolabeled drug product via a one step production. Because the production process is simplified and shortened, the process allows for the production of radiolabeled drug products with short half-lives. The process provides a radiolabeled product that is radiochemically stable and immunoreactive for several hours after final purification. The radiolabeled product can be used for imaging and/or therapy of cancer and infectious diseases. [0071] An efficient, direct and one step radiolabelling of a monoclonal antibody using

Tris(2-Carboxyethyl) Phosphine (TCEP) was developed and exemplified by radiolabeling of a melanin binding IgM monoclonal antibody with 188-Re methodology. TCEP. HCl was evaluated as a reducing agent for generating -SH groups on the proteins via reduction of disulfide bonds. For this purpose, conditions on the influence of TCEP molar excess over 6D2-IgM monoclonal antibody on the structural integrity and radiolabelling yields were determined. Also, conditions for the kinetics of generation of -SH groups on the 6D2-IgM via TCEP. HCl reduction at constant TCEP to 6D2-IgM molar ratio were obtained. A simple, efficient and fast one-step production of radiolabeled 188-Re-6D2-IgM was developed

following the initial reduction of the 62D-IgM with TCEP, reduction of the perrhenate Nal88-ReO4, subsequent coupling of the reduced 188-Re and the -SH groups on the reduced IgM-6D2 and purification of the radiolabeled drug product.

[0072] TCEP has been used to reduce IgG antibody (10). IgG antibodies have a monomer structure compared to the larger IgA and IgM antibodies, which have dimer (IgA) and pentamer or hexamer (IgM) structures, respectively. Prior to the present study, it was not obvious that TCEP would be effective to reduce large IgA and IgM antibodies due to steric hindrance. Furthermore, it was not obvious whether or not TCEP would interfere with radiolabeling of reduced monoclonal antibody because TCEP contains phosphorous that could potentially chelate radioisotopes such as rhenium. IgA and IgM antibodies have the advantage over IgG antibodies in that IgA and IgM antibodies are more rapidly cleared from the patient (31, 32).

[0073] The lack of effective clinical treatment for metastatic melanoma and positive results from treatment of human melanoma in a mouse model with 188 Re-labeled melanin- binding mAb 6D2 (5) encouraged the development of this mAb for clinical evaluation in patients with metastatic melanoma. RIT has experienced a renaissance, and in the last 5 years there have been reports of renewed effort in developing RIT for the treatment of melanoma (reviewed in 14). In addition, there is clinical experience with Re-labeled anti- NCA and anti-CD66 mAbs which proved to be safe and effective in leukemia patients (12, 15).

[0074] The development of 188 Re-6D2 for clinical use required the development of a robust and reproducible radiolabeling procedure and quality control techniques. Since the mAb is radiolabeled with 188 Re by attachment to -SH groups, it was important to identify a suitable reducing agent and to develop conditions that maximized labeling and minimized damage to the immunoglobulin molecule. In earlier studies dithiothreitol (DTT) was utilized for preparation of -SH groups on 6D2 mAb (5); however, DTT competes with mAb for binding 188 Re and separation of reduced mAb from excess of DTT is required before the radiolabeling step is initiated. Since radiolabeled mAbs usually require a final purification step to reach a >90% level of radiochemical purity, the incorporation of an additional purification step into the manufacturing protocol was potentially cumbersome. To avoid this problem, it was necessary to identify a reducing agent that would not compete for 188 Re with the antibody. Consequently, an evaluation was carried out of a phosphine-based agent tris(2- Carboxyethyl) phosphine hydrochloride (TCEP HCl), which is a soluble salt that is stable during prolonged storage in solution and does not react with Re. Importantly, the present

studies demonstrated that TCEP is both effective in generating a sufficient number of -SH groups on 6D2 mAb and facile towards preserving its structural integrity. [0075] The biodistribution and WBAR of 188 Re-6D2 radiolabeled by the present procedure showed that it behaved essentially the same as 188 Re-6D2 mAb radiolabeled after reduction with DTT (5). For example, in the present study I88 Re-6D2 was cleared from the blood with a half-life of 5 hours, which compared favorably with the half life of 6.5 hours determined for DTT-reduced mAb (5). The patterns of clearance from the kidneys and all other major organs were also very similar for TCEP- and DTT-reduced mAb 6D2. [0076] The major difference between the two studies was in the tumor type induced in nude mice. The prior study (5) had used the highly pigmented primary human melanoma cell line MNTl, whereas the current study used the lightly pigmented metastatic human melanoma cell line A2058. Although the tumor uptake in the earlier study (5) was estimated by scintigraphic imaging, it was higher (approximately 15 and 5% ID/g at 3 and 24 hours, respectively) than in the current study (approximately 2 and 0.5 % ID/g at 4 and 24 hours, respectively).

[0077] The difference in tumor uptake was presumably due to the type of melanin contained in the tumor - MNTl tumors contain black eumelanin (8), which is very close in structure to the fungal melanin 6D2 mAb was originally developed against (7). In contrast, A2058 tumor cells are pigmented with pheomelanin, a yellow or reddish-brown melanin-type pigment (8). Both pheomelanin and eumelanin are negatively charged and share some structural similarity (8), with both types found in melanomas where eumelanin is the predominant pigment in primary tumors, whereas pheomelanin is associated with metastatic melanomas and progression of the disease (16). The predominance of pheomelanin in A2058 cells resulted in less binding of 188 Re-6D2 as compared to MNTl tumors. Melanin in a patient's tumors will most likely be represented by both eumelanin and pheomelanin, and even so called "amelanotic" melanomas do contain some melanin to ensure targeting with 188 Re-6D2 (17, 18).

[0078] Despite a modest uptake of 188 Re-6D2 in tumor tissue, compared to the previous study with MNTI, there was significant retardation in A2058 tumor growth not only for the highest dose of 1.5 mCi 188 Re-6D2 used in the previous study (5), but also for the lower doses of 1.0 and 0.5 mCi. This is encouraging for using lower doses in patients. [0079] The ability of low amounts of melanin to provide sufficient target for melanin

RIT was recently predicted by computer modeling, which showed that the doses delivered to the melanoma tumor in a patient will be remarkably similar within 1, 000-fold range of tumor

melanin concentration of 76-0.076 μM with 76 μM melanin concentration being experimentally determined for highly melanized MNTl tumors (19). Interestingly, Epstein and colleagues, one of the first groups to consider extravasated intracellular antigens as targets for RIT (20), did not observe specific uptake of radiolabeled TNT-I mAb in human cervical carcinomas in mice when compared to control mAb, yet observed therapeutic results and preferential targeting within the tumor. Melanin is a unique intracellular antigen because it is a chemically resistant pigment that can accumulate in tumor tissues to provide more target material for the repeated treatments. In fact, histological evaluation of A2058 tumors in control and RIT-treated mice showed that most of the melanin in the treated tumors became extracellular (Fig. 7C), thus providing abundant target for the second round of RIT. The treatment modalities that can increase the amount of accessible melanin in the tumors by killing cells and releasing their melanin contents can be fractionated RIT (21), chemotherapy with dacarbazine or similar agents (22), or alternating magnetic field cancer therapy (23). [0080] Radiolabeling and quality control procedures have been developed for Relabeled melanin-binding 6D2 mAb for use in Phase I clinical trial in patients with metastatic melanoma. The radiolabeled antibody had the immunoreactivity of the native mAb, was stable over time, had fast clearance from the blood and major organs and manifested preferential tumor uptake in the tumor. Furthermore, the efficacy was established of RIT with mAb 6D2 against an aggressive, lightly pigmented melanoma model derived from a metastatic tumor. The doses employed were associated with only limited and transient hematological toxicity. Extension of results showing efficacy to pheomelanin-pigmented cells derived from an aggressive tumor is important given the variability of melanoma tumors and the likelihood that metastatic lesions will be less pigmented than primary lesions. The results provide critically useful information for the manufacture of a clinical lot of the mAb and additional demonstration of the usefulness of this approach in RIT.

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