1

Radioimmunotherapy of Metastatic Melanoma

Introduction
Melanoma is an increasing health problem1. Roughly 40,000 patients in the United States
and 100,000 people worldwide suffer from this condition. Primary melanoma is on the skin and
can be treated with surgical removal. Unfortunately, there is no widely accepted treatment for
metastatic melanoma. The metastatic melanoma has a five-year mortality rate of 94%. This poor
rate of survivability has been relatively consistent for more than two decades2.
Melanoma is particularly problematic with regard to economics as it disproportionately
affects younger individuals2. Traditional immune approaches to treating metastatic melanoma
have included nonspecific immune stimulants combined with tumor-associated antigens.
Treatment has also included vaccinations with a variety of compounds including gene construct
encoding proteins, carbohydrates, peptides, tumor cell lysates, and tumor cells. However, even
with these treatments the survival rate remains at around 6% for five years with an average
survival time of only 8.5 months2.
A variety of therapeutic approaches have been attempted to treat metastatic melanoma3.
These include biological therapies, chemotherapy, and combination therapies. However, the
majority of these treatments show little promise for improving the bleak outlook for patients
suffering from this condition. Traditionally, systemic chemotherapy has been the treatment of
choice for patients with stage IV melanoma3.
The most common chemotherapy for metastatic melanoma includes using Dacarbazine3.
Unfortunately, the response rate to Dacarbazine is only positive in 20% or less of the cases. Even

when this chemotherapy is successful, it is rarely sustained. The cancerous cells tend to build up
resistance to the Dacarbazine3.
A study4 by Speer in 2003 determined how the melanoma cells begin escaping the
cytotoxic effects of the Dacarbazine. It was discovered that the Dacarbazine induces the
melanoma cells to produce Interleukin 8 and over expression and secretion of Vascular
Endothelial Growth Factor. It was discovered that the melanoma cells that add higher levels of
Vascular Endothelial Growth Factor and Interleukin 8 were more resistant to the Dacarbazine4.
A relatively new approach proposed for the treatment of metastatic melanoma is
radioimmunotherapy3. This approach involves labeling antibodies with a radionuclide. The
radionuclide is carried by the antibody to the target cell and the cytotoxic radiation is delivered.
The antibody will specifically bind with the antigen of the tumor and allow the radiation to be
delivered directly with a higher dose than would be allowed with non-targeted treatment. It
should be noted that it is important that the tumor cell express a unique antigen so that it can be
specifically targeted by the antibodies carrying the radiation3.

Research Hypothesis
The hypothesis is that radioimmunotherapy using melanin-binding antibodies in
combination with chemotherapy will be more effective than chemotherapy alone for treating
metastatic melanoma.

Literature Review

Benzamides
It has been known for some time that radiolabeled benzamides differentially target
melanoma tissue in people5. A number of attempts have been made to make refinements to the
tracers to the increase uptake within the melanoma tissue and increase the excretion rate from the
normal tissues. Something that is common to the benzamides and their derivatives is their rapid
uptake into the melanoma tissue. The mechanism of uptake remains mysterious. However,
several processes have been proposed including a direct bind with the melanin molecule
resulting in incorporation within the melanin biosynthesis pathway6.
One type of benzamides is labeled with I7. This benzamide binds with melanin within
mice that have xenografts of human melanoma tumors. The tumor growth was significantly
reduced. When multiple doses were given, the tumors regressed and the therapeutic response was
durable over 125 days. The compound used had a fluoro-benzoate element was potential for
radio labeling with F. This would allow monitoring the uptake of the agent by using positron
emission tomography (PET). The benzamide labeled with I is a molecule that is relatively small.
This allows it to pass the blood-brain barrier. This means the treatment is promising for those
suffering from melanoma that has metastasized into the cerebral tissues8.
Synthesized heteroaromatic benzamide analogs have been synthesized that incorporate
the heteroaromatic structure and this replaces the benzene ring9. These synthetic analogs tend to
have stronger affinities for melanin than the original compound. This means they had increased
uptake when in the melanoma tumor and a longer time of retention6. This is not an ideal situation
for radionuclide therapy.

Another research study investigating the use of benzamides for treating metastatic
melanoma used a derivative of quinoxaline5. This molecule binds to melanin with a high
specificity as well as an uptake that is long lasting. The derivative clears from normal tissues
rapidly. A preclinical study in mice discovered that this derivative inhibited tumor growth. The
tumor cells remaining were less aggressive and led to an increased survival time. The untreated
mice in the study developed metastases, while those treated with the derivative were healthy10.

Receptors and Peptides

Melanoma lesions exhibit overexpression of the melanocortin-1 receptor8. The melanocyte-stimulating hormone (-MSH) is a peptide that binds to the melanocortin-1 receptor.
Once this was discovered an investigation was done regarding the analogs of -MSH. The
possibility that or emitting radionuclides of -MSH might be used to deliver therapeutic
doses of radiation to metastases melanoma. The first studies done labeling -MSH peptide were
not promising. There was a low uptake for the receptor versus background. There was also a
relatively poor radionuclide stability3.
Recent developments for peptide analogs of non-natural amino acids containing MSH
and peptide cyclization have revealed better receptor affinity and increased stability of the
radionuclide10. This increases the likelihood that working with the -MSH peptide will
eventually yield a therapeutic agent that is effective6.
A Melanoma targeting peptide has been labeled with Re and shown positive therapeutic
effects in mice bearing human melanomas9. The treatment extended life in the mice with tumors
and there were no toxic effects observed. The analog of the peptide was labeled with Pb. This
peptide decreased tumor growth as well as survival time in melanoma bearing mice with flank

tumors. The peptide exhibited rapid uptake by the tumor as well as retention. This peptide was
cleared rapidly in the remainder of the body. It should be noted that the isotope of Pb decays into
Bi. The Bi decays through both and decay resulting in the release of high doses of radiation
within the uptake area. The survival time of the mice increased as the dose was increased.
Roughly, 45% of the mice that received the highest dose survived disease-free throughout the
study1.

Monoclonal Antibodies
For more than three decades there have been numerous animal and laboratory studies
done regarding treating metastatic melanoma with radionuclide therapy6. While some of the
results of been encouraging, there have been few human trials of the methods. One of the first
attempts to use radionuclides to treat metastatic melanoma was with monoclonal antibodies.
During the 1980s, the requirements for using radioimmunotherapy with monoclonal antibodies
were outlined2. These requirements provided the necessary framework for human testing.
Studies were done that reported shrinkage of the melanoma metastases in patients treated
with radioactive iodine labeled fragments of monoclonal antibodies7. These antibodies were for a
high molecular weight antigen that was melanoma associated. Therapy using the radiolabeled
monoclonal and bodies is now done in clinical practice to treat B-cell lymphoma. Unfortunately,
the progress toward treating melanoma with this approach has progressed more slowly9.
There have been promising results from preclinical studies using nude mice carrying
human melanomas5. A melanin binding monoclonal antibody labeled with radioactive Rhenium
was used to treat the mice. It was discovered that there was a slowing of the tumor growth and

the effects on platelet counts and white blood cell counts were transient. These problems with the
blood clots resolved within two weeks of completing the therapy1.
There is a potential problem with treating metastatic melanoma with radioactively tagged
monoclonal antibodies8. Melanin is located within the melanoma cells. This means that necrotic
cells may be more effective targets than the viable tumor cells. Melanin can also be found within
the extracellular space as well as in the melanophages3.
Cetuximab is a chimeric monoclonal antibody that targets the epidermal growth factor
receptors7. This antibody has been shown to bind to the melanoma xenografts averaging more
than 7% of the dose/g found in the tumor at 72 hours following injection. The antigenic target
offers a possible avenue for future research into using monoclonal antibodies to treat metastatic
melanoma5.
It is possible that monoclonal antibodies that act against the surface antigens on the
melanoma cells will have therapeutic benefits2. These antigens include ganglioside GD3 and
glycoprotein gp57. Early studies have indicated that using the monoclonal antibodies against the
GD3 antigen with the radiolabeled -emitter Bismuth may be effective4.
The undesirable irradiation of healthy tissues has limited radioimmunotherapy in people7.
There have been a number of strategies suggested to overcome this limitation. For example, it
has been suggested that enzymatic linkers be used to cleave the radioisotope from a monoclonal
antibody in normal tissues and blood. This would be done after the binding with the tumor cells.
This allows for a reduction of radiation reaching normal tissues while there is still enough
radiation reaching the tumor to have a therapeutic effect. The timing for this treatment is crucial
and is determined by factors related to the pharmacokinetics of the particular

radiopharmaceutical being used. Computer modeling studies have indicated that many
radiopharmaceuticals can be three times as effective when this approach is used1.
Having accurate dosage measurements lead to the radio immunotherapy being more
effective and having fewer side effects8. Hybrid imaging techniques such as PET/CT and
SPECT/CT can be used to measure the dose reaching specific areas. An accurate attenuation is
possible when using the CT data simultaneously with PET or SPECT tomographic data. This
leads to an accurate quantification of the radionuclide uptake in the target cells as well as the
normal tissues. This technique provides the opportunity for allowing the maximum therapeutic
dose to be delivered to the tumor cells while the dose reaching the normal tissues is not lethal4.
In order to reduce the unwanted effects on healthy tissues, radiolabeled monoclonal
antibodies have been injected directly into the melanoma metastases5. The monoclonal antibody
targeting the melanoma tissue was labeled with Bismuth. The Bismuth emits radiation that can
be used in doses between 5.5 and 50 MBq. Significant cell death was observed in the metastases.
This was not accompanied by changes in electrolytes for blood proteins. Therefore, injecting the
therapeutic substance directly into the melanoma tissue is especially promising. This is yet to be
explored in human trials2.

Melanin-Binding Monoclonal Antibodies Combined with Dacarbazine

A study done by Revskaya et al. in 2009 used radioactively tagged melanin-binding
antibodies combined with dacarbazine. The authors point out that melanin is an attractive target
for radio immunotherapy when treating melanoma. Radiolabeled monoclonal antibodies for
melanin are presently in clinical evaluation.

Two approaches to improving the targeting of tumors for radiation treatment using
melanin-binding monoclonal antibodies have been pursued9. One approach is to use an additional
monoclonal antibody to the melanin that will provide information on whether antibodies to
melanin are a promising approach for developing therapeutic applications. The second approach
involves using the melanin binding monoclonal antibodies for melanin targeting. This involves
the antibody being bound to extracellular melanin that has been released from the necrotic
melanoma cells. If the chemotherapeutic agent, in this case dacarbazine, is administered
followed by the radioimmunotherapy the treatment may be more effective. This is due to more
radiation being delivered with the additional free melanin. The dacarbazine will kill melanoma
cells that release extracellular melanin. This is beneficial for assisting with targeting the
remaining melanoma cells for radiation treatment9.
An experiment was done with nude mice bearing human metastatic melanoma9. The
therapeutic efficacy of two melanin binding monoclonal antibodies that were labeled with Re
was explored. The efficacy of radioimmunotherapy was compared to chemotherapy with
dacarbazine alone as well as chemotherapy with radioimmunotherapy. The researcher speculated
that the chemotherapy followed by radioimmunotherapy would be more effective than the other
approaches.
There were two different melanin-binding monoclonal antibodies tested9. These were
6D2 and 11B11. The therapeutic efficacy of these two types of antibodies was found to be
similar. Applying the radioimmunotherapy following the chemotherapy with dacarbazine was
found to be more effective than chemotherapy or radioimmunotherapy alone.
It should be noted that the study done by Revskaya et al. had results that were very
encouraging. Using the radioactively labeled melanin binding monoclonal antibodies following

treatment with chemotherapy was highly effective with the mice. The results of the study clearly
indicated that following chemotherapy with radio immunotherapy using radioactively tagged
monoclonal antibodies is especially useful for halting the early stages of the tumor growth9. This
is often the most important time of treatment for helping an individual suffering from metastatic
melanoma. The next logical step in this progression of research is to try this approach with
human subjects.

Methods
The study involves 70 patients diagnosed with metastatic melanoma. Other inclusion
criteria for the study include being 18 years of age or older, having a life expectancy of four
months or more, and still being able to carry out light office or housework. Exclusion criteria
will include subjects who have been previously treated for cancer and have been disease free for
a span of less than five years. Another exclusion criterion will be primary ocular melanoma or
previously receiving treatment with monoclonal antibodies. Individuals will be excluded who are
lactating, pregnant, have untreated metastases within their central nervous system, suffer from
autoimmune diseases, or have received a cancer vaccine. The subjects should also not be
receiving another type of treatment for their cancer for treatment for another disorder with an
immunosuppressive agent. People who have used systemic corticosteroids for an extended period
must also be excluded.
While no specific stage of the disease will be required of the participants, their stage will
be noted and used in the data analysis. The participants should be randomly assigned to Group 1
receiving chemotherapy with dacarbazine and Group 2 who are treated with chemotherapy
followed by radioimmunotherapy. All participants in the study will sign an informed consent

10

form. Prior to any part of the study with human subjects, the experiment will be approved by the
appropriate institutional review board.
The study will be a double blind, randomized study. The participants will be randomly
assigned to one of the two study groups and stratified according to their baseline stage of
metastasis. The participants will be assigned to the treatment groups on a 1:1 ratio, which is
expected to yield two groups with a relatively equal number of participants. All participants in
both groups will be treated with chemotherapy followed by radioimmunotherapy. For both
groups, the chemotherapy will be administered for five days. After 24 hours, the treatment group
will receive an injection of radiolabeled monoclonal antibodies.
The primary endpoint of this experiment is the optimal response rate. This is determined
by the number of patients who have a complete or partial remission of their cancer. The endpoint
will also include mortality rates. Metastatic melanoma is often fatal and it is deemed important to
include this data. Notes will be taken of all findings including remission rates, spreading, or
decline of the cancer, survival, and tumor size.

Possible Results
The hypothesis of this study is that radioimmunotherapy using monoclonal antibodies
that bind to melanin combined with chemotherapy will be a more effective treatment than
chemotherapy alone with regard to metastatic melanoma. This means that the hypothesis will be
supported if the treatment using both chemotherapy and radioimmunotherapy is most effective.
The results of the treatment will be measured in a number of different ways. The patients will be
checked for remission or partial remission of their cancer. There will also be an analysis done of
whether the cancer has become more localized, or spread to other areas of the body. The size and

11

total number of the tumors will be recorded as well. Also of importance is the survival rate of the
subject.
Support for the hypothesis that radio immunotherapy combined with chemotherapy is
more efficacious includes the combined treatment having higher rates of remission than the
chemotherapy only. . Further support for the combination treatment will be provided by the
cancer becoming more localized. In other words, the cancer will not have spread, but may have
completely disappeared from locations peripheral to the area with the most lesions. The size and
total numbers of tumors will be recorded and it is expected that this number will be smaller for
those who have been treated with the combined approach. Finally, support for the hypothesis of
this study will be improved if the survival rate is greater for the subjects receiving the combined
treatment relative to those only having chemotherapy.
References

1. Dadachova E, Revskaya E, Sesay M et al. Pre-clinical evaluation and efficacy studies of a

melanin-binding IgM antibody labeled with 188Re against experimental human
metastatic melanoma in nude mice. Cancer Biology & Therapy. 2008;7(7):1116-1127.
doi:10.4161/cbt.7.7.6197.
2. Dadachova E, Nosanchuk J, Shi L et al. Dead cells in melanoma tumors provide abundant
antigen for targeted delivery of ionizing radiation by a mAb to melanin. Proceedings of
the National Academy of Sciences. 2004;101(41):14865-14870.
doi:10.1073/pnas.0406180101.
3. Speer T. Targeted radionuclide therapy. Philadelphia: Wolters Kluwer Health/Lippincott
Williams & Wilkins; 2011.
4. Lev, D. C., Ruiz, M., Mills, L., McGary, E. C., Price, J. E., & Bar-Eli, M. (2003).
Dacarbazine Causes Transcriptional Up-Regulation of Interleukin 8 and Vascular
Endothelial Growth Factor in Melanoma Cells: A Possible Escape Mechanism from
Chemotherapy1. Molecular cancer therapeutics. 2003; 2(8), 753-763.
5. Sauer, S., Erba, P. A., Petrini, M., Menrad, A., Giovannoni, L., Grana, C., ... & Menssen,
H. D.. Expression of the oncofetal ED-Bcontaining fibronectin isoform in hematologic

12

tumors enables ED-Btargeted 131I-L19SIP radioimmunotherapy in Hodgkin lymphoma

patients. Blood. 2009; 113(10), 2265-2274.
http://www.bloodjournal.org/content/113/10/2265?sso-checked=true

6. Frey, K., Fiechter, M., Schwager, K., Belloni, B., Barysch, M. J., Neri, D., & Dummer,
R.. Different patterns of fibronectin and tenascinC splice variants expression in primary
and metastatic melanoma lesions. Experimental dermatology. 2011; 20(8), 685-688.
7. Scott, A. M., Liu, Z., Murone, C., Johns, T. G., MacGregor, D., Smyth, F. E., ... & Old, L.
J. Immunological effects of chimeric anti-GD3 monoclonal antibody KM871 in patients
with metastatic melanoma. Cancer Immunity Archive.2005; 5(1), 3.
http://archive.cancerimmunity.org/v5p3/041221.htm
8. Miao, Y., Hylarides, M., Fisher, D. R., Shelton, T., Moore, H., Wester, D. W., ... & Quinn,
T. P.. Melanoma therapy via peptide-targeted -radiation. Clinical cancer research. 2005;
11(15), 5616-5621.
9. Revskaya E, Jongco A, Sellers R et al. Radioimmunotherapy of Experimental Human
Metastatic Melanoma with Melanin-Binding Antibodies and in Combination with
Dacarbazine. Clinical Cancer Research. 2009;15(7):2373-2379. doi:10.1158/10780432.ccr-08-2376.
10. Raja C, Graham P, Abbas Rizvi S et al. Interim Analysis of Toxicity and Response in
Phase 1 Trial of Systemic Targeted Alpha Therapy for Metastatic Melanoma. Cancer
Biology & Therapy. 2007;6(6):846-852.