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What is 3F8?

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3F8 is a mouse derived monoclonal antibody developed by Dr. Cheung at Case Western Reserve University in the 1980’s, and later tested extensively at Memorial Sloan Kettering Cancer Center.  It is directed toward sugar containing molecules called GD2 found on the surface of neuroblastoma cells.  GD2 is also found on the surface of nerve cells.  This fact is responsible for the great pain it causes while it is being infused each day. Its name, 3F8, refers to the tray, row and column it was found in when the monoclonal antibodies were screened.  3F8 has been used in clinical trials for neuroblastoma for more than 20 years.

 

What kinds of monoclonal antibodies are available? 
Most monoclonal antibodies are produced in mice but they have also been developed in other animals.  Because of problems associated with using animal proteins in humans, researchers using chemical and genetic engineering techniques have developed monoclonal antibodies that are more like human antibodies and in fact can now produce completely human antibodies.

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Murine antibodies are entirely mouse derived and contain no human components. In chimeric antibodies 67% of the antibody has been replaced by human antibody. In humanized antibodies the human portion is increased to 90%-95%.  Human antibodies by definition are 100% human, although near human antibodies can also be generated in a genetically modified mouse.
           
Why are humanized monoclonal antibodies necessary?           
There are two main problems that occur with current monoclonal antibodies.
1. The initial hopes for monoclonal antibody therapy in cancer were dampened when patients began developing their own antibodies against the animal antibodies being given.  These antibodies the patients produced called HAMA (Human anti-mouse antibodies) bind up all of the monoclonal antibodies neutralizing them and preventing them from binding to and killing cancer cells.  As a result patients often must stop therapy after a few treatments even though the treatment may have previously been very effective.
            2. The stem of the Y-shaped antibody or Fc portion binds to receptors (Fc Receptors) on a variety of immune cells. The ability to bind to these immune cells is critical in killing cancer cells. [Cheung] Cells from different patients have slightly different Fc receptors and different abilities to bind different antibodies, whether mouse or human. As a result current antibody therapy may work well for some, but not for all patients.  During the process of humanizing the monoclonal antibody, the Fc portion can be genetically modified to enhance its binding to receptors of more patients. 

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Is HAMA always a bad thing?
Not all patient develop HAMA. The development of HAMA can be delayed by several months by administering certain kinds of chemotherapy in high doses prior to initiating treatment.[Kushner] [Kushner] HAMA forces patients to stop treatment with monoclonal antibodies since it neutralizes the antibodies and the disease is left untreated.  On average patients who develop HAMA actually do better than those who do not as long as they are disease free at the start of treatment and complete at least 40 doses.
The proposed reason for the better outcome in patients who develop HAMA is a complicated mechanism called idiotype networking (see figure above).[Cheung] [Cheung] Simply put HAMA’s (Ab2) are antibodies against mouse derived antibodies (Ab1).  These may bind to any part of the antibody.  A few will bind to the idiotype of Ab1.  In order for this to happen the shape of the idiotype of Ab2 must be very similar though not identical to the protein (GD2) on the cancer cell.  Since this is a new shape to the patient, the patient may make antibodies (Ab3) against that idiotype. The result is that the patient is producing their own antibodies that can bind directly to the GD2 protein on the cancer cell and kill it.  It is likely that this series of events requires time to develop, and if HAMA is made too soon,  this idiotype network cannot materialize. 
            At the present time many children who would benefit from 3F8 had to stop therapy because they developed HAMA early.

 

Would humanizing 3F8 eliminate the HAMA problem?
Yes and No.  It would not eliminate HAMA since 5-10% of the antibody is still derived from mouse genes, but it could eliminate it as a “problem.” The only part of a humanized antibody remaining that is derived from mouse genes is the idiotype.  If the patient did develop HAMA it would more likely be against the idiotype and the chances of developing an idiotype network and their own antibodies against the tumor would be even greater.

Why humanize the antibody rather than create a fully human antibody from scratch?
The strength of binding to the cancer cell is very important and is a function of the shape and electrical charge on the idiotype.  Designing and producing the ideal idiotype is beyond present day technology. Achieving a strongly binding idiotype is often a result of luck in addition to special techniques.   
Humanizing an antibody is achieved by taking a B-cell which produces mouse antibodies and replacing the genes for the entire antibody with the exception of the portion of the genes that produce the idiotype. The result is an antibody that is 95% human protein and the original idiotype is preserved. Alternatively, an entirely human antibody can be achieved by starting with a mouse whose antibody genes have been replaced by human antibody genes.  To create an antibody against neuroblastoma researchers may have to start back where they were 20 years ago and create a new antibody and hope for the same good luck in creating such a strongly binding idiotype as 3F8.

What is the current state of the art regarding antibodies and what evidence is there that antibodies help?
There are no double-blind randomized controlled trials of 3F8. The significant side effect of pain makes it impossible to blind the study to either the patients or the investigating physicians. The team at Sloan Kettering has been using 3F8 for nearly two decades. The team has determined that the objective responses against marrow disease are unequivocal and that a randomized trial would require withholding antibodies from some patients.  They felt that this would be unethical.  Data from studies of 3F8 plus GMCSF show a 3 year progression-free survival of 60% when treating patients who have achieved remission prior to starting therapy.  3F8 treatment has achieved long term remission at rates superior to other more intensive therapies which require bone marrow/stem cell transplant. [Cheung] 
Preliminary studies of a chimeric monoclonal antibody (ch14:18, derived from a B-lymphocyte unrelated to 3F8) [Osenga] in high risk neuroblastoma have not been encouraging. In two retrospective analyses, one among stage 4 infants and one among children with high risk neuroblastoma,  addition of antibody treatment did not benefit patient when compared to standard chemotherapy alone. [Simon] [Simon]

Where is 3F8 being used? 
Currently the only places it is available is in the US at Memorial Sloan Kettering Cancer Center and in Hong Kong.

If humanized would the antibody be available to other medical centers around the country and around the world?
            This is what we expect.

 

Are there any things that would be worse about humanizing antibodies?
3f8As noted above the most important properties about the antibodies is the ability of the idiotype to bind to the cancer and stay bound and the ability of the Fc portion to bind to killer immune cells.  The Y shape of the antibody provides two identical copies of the idiotype to bind to the cancer cell.  Having two copies is an advantage and provides stronger and longer lasting binding.  As one occasionally lets go of the cancer cell the other keeps it in the area long enough to bind again. 
The mouse IgG3 antibody 3F8 has a tendency to cluster (e.g. n = 3 antibodies in figure, now 3 x 2 = 6 identical idiotypes). Together through cooperative binding these clusters have enhanced binding making it extremely unlikely that the antibodies will come off of the cancer cell. Humanized antibodies do not have this clustering property. In spite of this, single molecules of 3F8 still bind more strongly than any other available antibody.  It is theoretically possible to engineer humanized antibodies to form bigger clusters.
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Since the active part of the antibody is the same as what has been used for 20 years will Sloan Kettering have to go back to animal studies before proceeding to human trials?
Yes. Regardless of whether the Food and Drug Administration would require it the institutional review board at Memorial Sloan Kettering would most likely not allow human trials to begin until animal studies are completed.

Why does Sloan Kettering need external funding for this project? 
Funding for cancer research is limited in general.  The vast majority of federal funding and private donations for cancer research are directed toward adult cancers.  Fortunately pediatric cancers are uncommon.  Unfortunately cancer funding tends to be distributed based on the number of people who have the cancer rather than the years of potential life lost. For this reason prostate cancer which does not substantially shorten the lives of the majority of patients who have it receives far more funding than all childhood cancers combined.  Similarly the pharmaceutical industry is generally not interested in pediatric cancers because of the small market size unless the treatment also happens to be effective in adult cancers. Furthermore, liability remains a strong disincentive.  As treatments become more targeted to a specific cancer drugs like monoclonal antibodies are less likely to be generally applicable to other cancers.  It is highly unlikely that investments in humanizing 3F8 will yield any large financial returns. 

Would someone or some organization profit from funds raised for this project?
Memorial Sloan Kettering Cancer Center has no intention of marketing humanized 3F8. Intellectual property issues regarding 3F8 are best addressed by the Industrial Offices at Case Western Reserve University and Memorial Sloan Kettering Cancer Center. 

Will humanized 3F8 be marketable? Would it be a wise investment for the purposes of financial gain? 
“No” to both questions. The main reason to invest in humanizing 3F8 is to find an effective treatment for a terrible disease. Motives of donors should be purely altruistic. While only 300 to 400 children would benefit each year in the US worldwide there are more than 20 times that many cases of stage 4 neuroblastoma.  Unless another more effective therapy is found tens of thousands of children will die of this disease over the next decade.

Is humanized 3F8 going to be a cure for neuroblastoma?
By itself it is not likely to be a cure.  As part of a complex treatment plan we expect it to increase the number of children that survive past 5 or 10 years, so that they can live to see a day when there is a cure. It is difficult to determine how much of an effect it will have until it is produced and tested in clinical trials.

Is the quoted 2-3 million dollars all that is needed? 
No. This is the cost of getting the antibody humanized. Humanizing 3F8 would be done by an outside company on contract.  It is estimated that the cost of humanizing the antibody would be approximately $2 million.  An additional $500,000 would be necessary to manufacture it in sufficient supply for the initial clinical trial.  No one could receive it unless it was available in a trial.  The costs of trials can be significant but funding for the trial is likely to be easier to obtain once the product is developed.

If funded today, when would/could a product be ready? 
Humanized 3F8 will never be ready for the market because of the reasons noted above.  However it could be made available in clinical trials.  Although unlikely, if everything fell into place it could be available in 3-4 months but more realistically it would take 12 months or more.

There is another effective antibody in development called 8H9.  Would it be better to focus efforts on humanizing 8H9? 
Not at this time. 8H9 has been developed to fight disease in the central nervous system (CNS). It has been very successful far beyond anyone’s expectations.  Prior to 8H9 no children survived once neuroblastoma entered the brain or spinal cord.  So far every child who has been treated with 8H9 on the salvage regimen has been disease free although most have been treated only in the last few years.  This antibody has worked well when given directly into spinal fluid.  Antibodies given in this location are shielded from HAMA because HAMA is generally barred from the CNS, so that has not been a problem and this therapy can continue without humanizing it.  Recently trials have begun using 8H9 given through an IV.  It is unknown whether this will be as effective as 3F8 and it will be several years before information on this is available.  If it is shown to be effective humanizing it may be warranted.  For now the focus should be on 3F8.

 

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