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Role of Complement in Rituxan Therapy

Relative Efficacy of CDC in Different Leukemias



Role of Complement in Rituxan Therapy


by Chaya Venkat

This post, and the ASH2002 abstract attached below may be a hard slog to get through, but I strongly recommend you read it, especially if Rituxan is in your future.

We have discussed in previous posts that Rituxan does not do any large percentage of the killing of the b-cells by itself. All it does (mostly) is to "finger" the B-cells, by virtue of locking on to the CD20 marker they exhibit. We also know that the actual killing is done by two functions of the immune system: ADCC, antibody dependent cellular cytotoxicity, is carried out by other cells of the immune system, such as T-cells, NK-cells etc. We have talked in previous posts of ways of boosting the T-cell and NK cell counts, to make this part more efficient.

The second part, CDC, or Complement Dependent Cytotoxicity, is the topic of interest in this post. A little bit of background: We know that once you remove all the red blood cells and white blood cells etc from whole blood, what is left behind is called "plasma". It is a clear, amber colored liquid, that is more than 90% water. It has in it about 6% proteins, some lipids and salts. Some of these proteins form the "Complement" of the immune system. Unlike whole blood, plasma transfusions do not require blood type matching, since there are no live cells in it.

What is Complement? Complement is a set of proteins in the plasma, that form a complex and inter-linked chain that work together in fighting infections. When an "antigen" on the surface of a bacteria is tagged by an antibody (in our case, the CD20 marker on B-cells is the "antigen", and it is tagged by the Rituxan acting as the "antibody"), this combination of **antigen-antibody** pair triggers a response from the Complement system in the blood plasma. There are more than 20 proteins involved in the complement system. They work together to start the process of destroying and taking apart the antigen-antibody complex, tearing it into pieces and attracting macrophages etc to the site that actually cart away and gobble up the debris. If this bit of biology 101 has you cross-eyed, just remember this sound bite: Complement in your blood is what kills B-cells, once they are tagged by Rituxan. The abstract below describes cell studies, animal studies, etc. But the interesting and telling part deals with a live CLL patient, who was administered Rituxan at the standard dose, once a week for four weeks. His blood was analyzed for Complement proteins before and after each infusion of Rituxan. Prior to the first administration, his Complement level was normal. During infusion, it was clearly seen that Rituxan homed in on the CD20 positive B-cells, and that Complement proteins in turn zeroed in on the **B-cell/Rituxan** pair, as they are supposed to, to begin the process of destroying the whole shebang. At the end of the infusion, much of the available supply of the Complement proteins in the blood had been used up, there was a **five fold decrease** in the concentration of these essential proteins by the end of the first infusion. The level recovered somewhat by the next week, in time for the second infusion, but not all the way. By the time the fourth infusion rolled around, the crucial Complement protein concentrations were down as much as ten-fold. Given time, the body produced more of these proteins. Three weeks after the last dose of Rituxan, the Complement levels were almost back up to normal.

What does this say about how Rituxan should be administered? Well, for starters, treating with mega doses of Rituxan, or standard doses but three or more times a week is not likely to get you much more by way of cell kill, you will specifically and literally be pissing away money. Without giving the body time to recover Complement levels depleted after initial therapy does not accomplish tumor lysis (cell kill). I am inclined to think the once a week, four week approach is better for this reason than either the mega doses or much more frequent infusions, or even more prolonged (8 weeks instead of 4 weeks) approaches. Also to think about, is it a good idea to wring out the last bit of Complement proteins in your body, by over taxing the available resources? Seems to me that if you do so, until these proteins levels are replenished by your body, you are vulnerable to opportunistic bacterial infections.

What are the implications for early stage versus late stage patients? There has been enough evidence now to show that if the tumor burden is high to begin with, Rituxan does not seem to be able to handle it as well as if the number of tumor cells was small to begin with, the remissions are poorer, and more of the cancer cells are left behind. But perhaps we have it backwards, it is not that the Rituxan cannot handle the larger number of tumor cells, but that the Complement proteins are depleted before the job is done, hence the large amount of residual cancer cells left un-killed?? Wow. This has really got me thinking.

Last but not least, can we give mother nature a hand here? Why can't we make sure there is sufficient concentration of Complement before each Rituxan infusion, heck why not make it a little bit better than normal? Plasma transfusions are no big deal, and maybe rather than the whole plasma, it might be possible to transfuse just the complement proteins. I understood both are readily available commercial products. This is not a toxic drug, nor a complex  cytokine like Interleukin-2 with potential for unforeseen consequences and complex autocrine loop feedback effects. Unlike whole blood transfusions, there is no need to match blood types, or worry about iron overload type of issues down the road. This seems to be such a simple and yet elegant way of improving the efficacy of this very important drug, I am surprised it is not being done routinely right now. By the way, this is not my idea, the authors of the abstract below from ASH2002 spell it out clearly enough in their concluding remark:

**"We suggest that if an anti-tumor monoclonal antibody such as Rituxan requires robust Complement activation for therapeutic efficacy, then insuring an adequate level of Complement activity in a patient, by supplementation with either fresh plasma or a purified Complement component such as C2, may provide an important approach for improving the therapeutic efficacy"**

OK, guys. Let's get some discussion going on this. Let us find out the holes in this argument, and if it still sounds like a good idea after we kick it around, let us go out there and ask our individual health care experts why this cannot be done. Even if a small fraction of you print out this abstract, and discuss it with your experts next time you see them, we might get some action on this front. Patient advocacy does not stop with me writing these long posts. Each and every one of you can participate in getting the message out, being pro-active in communicating with your health care professionals. We can learn a thing or two from the AIDs and Breast Cancer and Prostate Cancer patient groups, in how to leverage our self-interest and commitment to where it makes a difference in drug development, research funding, and public policy.


[2256] Complement (C) Activation Is Required for Rituximab (RTX) Mediated Killing of CD20 Positive Cells, and a High Tumor Burden May Decrease Therapeutic Efficacy Due to C Depletion as a Consequence of Therapy. In Vitro and In Vivo Studies.

Ronald Taylor, Adam Kennedy, Michael Solga, Paul Beum, Patricia Foley, Margaret Lindorfer, Charles Hess, John Densmore.

Biochemistry, Universtiy of Virginia School of Medicine, Charlottesville, VA, USA; Comparative Medicine, Universtiy of Virginia School of Medicine, Charlottesville, VA, USA; Hematology/Oncology, Universtiy of Virginia School of Medicine, Charlottesville, VA, USA

We investigated C activation, C3bi deposition, and cell killing when RTX was bound to Raji or DB cells, in serum (NHS) as a C source. In the presence of RTX and C, large numbers of C3bi molecules deposit per cell, and fluorescence microscopy revealed that C3bi co-localized with bound RTX. Both cell types are killed by RTX in the presence of NHS, and use of mAb 3E7, specific for C3bi, enhanced killing. However, in the absence of NHS, little or no killing was demonstrable. RTX was infused into monkeys and we found that it rapidly bound to circulating B cells and activated C; 2 min after RTX infusion, C3bi was co-localized with RTX bound to the B cells. A similar pattern of co-localization of C3bi and RTX was obtained in vitro in opsonization experiments with blood samples taken from patients with B cell lymphomas.

A patient with CLL was treated with the standard 4 week course of RTX therapy. Analyses of blood samples taken during this time revealed
the following: Immediately after the first infusion, flow cytometry measurements and fluorescence microscopy indicated that RTX and deposited C3bi were associated with both B cells and cellular debris, and a high degree of co-localization of C3bi with RTX was evident. After the first infusion, CH50 assays demonstrated that the patient's C titer, normal before treatment, had been substantially reduced (~ 5 fold). Although C levels were partially restored before the second infusion, the same pattern of C depletion occurred after this infusion, and by week three and four the patient's C levels were reduced ~ 10-fold, but returned to baseline 3 weeks later. We found, however, that in vitro supplementation of the C-depleted sera with C component C2 markedly increased C activity, leading to levels that were at least 50% of the pre-therapy baseline. In vitro studies with RTX, B cell lines, and NHS gave rise to similar findings. That is, at high cell concentrations RTX-mediated C3bi deposition and killing is limited by serum C, and both of these activities can be enhanced by supplementation with purified human C2. Our results indicate that the primary mechanism of action of RTX in killing CD20 positive cells is mediated through C, and it is likely that the in vivo form of this mAb bound to target cells has covalently incorporated C3bi. We suggest that if an anti-tumor mAb such as RTX requires robust C activation for therapeutic efficacy, then insuring an adequate level of C activity in a patient, by supplementation with either fresh plasma or a purified C component such as C2, may provide an important approach for improving the therapeutic efficacy of a C-fixing mAb.

Keywords: Rituximab\ Complement\ Immunotherapy

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Relative Efficacy of CDC in Different Leukemias


by Chaya Venkat

A member observed that the abstract above indicated differential CDC efficacy - with high CDC killing of follicular lymphoma cells, whereas mantle cell lymphoma and diffuse large cell lymphoma cells were moderately sensible to CDC, and small lymphocytic lymphoma cells were almost all resistant. Hid interpretation was that Rituximab did a good job of killing the FLL cells, a moderate job on MCL cells and a terrible job on SLL cells.

His interpretation is quite correct.

The consensus seems to be emerging that one, **repeat one**, of the major mechanisms of cell kill during Rituxan therapy is CDC, (complement dependent cytotoxicity), which is poor in SLL/CLL, moderate in MCL, and quite good in FLL. This is attributed to the lower expression of CD20 in CLL cells, as well as the higher expression of complement inhibitory markers CD55, CD59 etc in CLL cells, compared to FLL and MCL. This may explain why the overall response rate is so much better using Rituxan in FLL and even MCL, and significantly poorer for CLL. 

There are other mechanisms for cell kill during Rituxan therapy, namely antibody dependent cellular cytotoxicity (ADCC) and direct apoptosis due to the Rituxan all by itself. The former works a lot better when T-cells, NK cells, neutrophils etc are in good shape, hence the greater efficacy of Rituxan even in CLL patients, if  these are early stage patients. 

It seems clear that response rates will increase in CLL as well, to the nice high levels obtained in say FLL, if we can get around the inhibition of the complement dependent cytotoxicity (CDC) mechanisms. This is especially important to previously treated patients, whose ADCC mechanisms may not work as well, whereas CDC mechanisms are more robust, and less likely to be compromised as a result of prior chemotherapy. 

It is a complex picture, and I am not sure we have all the pieces of the puzzle yet, but we surely know more now than we did even a couple of years ago. I am willing to bet folks at IDEC, Genentech and other companies are busy trying to find ways around these road blocks, to further increase the efficacy of Rituxan.

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