How rituximab kills CLL cells is not known for certain. It could do it by invoking direct apoptotic mechanisms. (Apoptosis = programmed cell death, a means by which cells suicide at the end of their useful life) It could do it by activating complement. (Complement is a cascade of proteins - each one activating the next one in the cascade - that is used to kill cells attacked by the immune system) But most people believe that it does it by antibody-dependent-cellular-cytotoxicity or ADCC. This means that antibody on the surface of the CLL cell invites killer cells to destroy the CLL cell. The killer cell might be an NK cell or a monocyte/macrophage or even a polymorph, all of which have Fc receptors that recognize part of the antibody molecule that sticks out when it binds to the cell. The use of G-CSF or GM-CSF with antibody is to try and activate the killer cell.
Although it is very unlikely that CLL cells are killed by the full activation of complement components from C1 to C9, resulting in the so called membrane-attack-complex punching a hole in the cell, some activation of the complement components is likely with the deposition of part of C3 (known as C3b) on the cell surface. This is quickly converted to iC3b. There is a receptor for iC3b on phagocytes and NK cells, called CR3. Binding of iC3b to CR3 enhances the Fc binding and increases the likelihood of ADCC.
There is another mechanism of killing using CR3, known as CR3-dependent cellular cytotoxicity (CR3-DCC), but it isn't normally used for killing tumor cells, only yeast cells. Yeast cells have beta glucan exposed on their surface. CR3-DCC requires the binding of CR3 by both beta glucan and iC3b. The yeast cell wall beta glucan binds to a part of the CR3 called 'the C-terminal lectin domain of CD11b', and primes the CR3 for efficient cytotoxic degranulation responses after it binds the iC3b by an other site known as 'the N-terminal I domain binding site of CD11b'. But tumor cells lack beta glucan on their surface and cannot trigger CR3-DCC.
However, if you do the experiment in a test tube, and add soluble beta glucan to the reaction mixture, the beta glucan binds to CR3 on monocytes and granulocytes and primes the receptor, enabling the effector cell to kill tumor cells. The next step after showing something works in glass tubes (in vitro = in glass) is to show that it works inside a live animal (in vivo = in life). The late professor Gordon Ross from Louisville, Kentucky looked at this. In mouse models (mouse tumors growing in laboratory mice) monoclonal antibodies were used to treat mice with and without beta glucan given intravenously. The regression of the tumors was much greater when the beta glucan was added than with antibody alone, and the mice lived for significantly longer. These experiments also showed that neutrophil granulocytes were the principle effector cells, that mice deficient in CR3 had no enhancement, nor was there any benefit in mice deficient in complement (C3). The paper is available on Medline Hong et al Cancer Res 2003, 63:9023-31.
But mouse tumors are very different to human tumors. The next step is to look at human tumors. The way this is done is to grow a human cell line in an immunodeficient mouse. These mice with severe combined immunodeficiency (SCID mice) are like test tubes on legs. They have no immune system of their own although they have normal numbers of effector cells. Cells from foreign species can grow well in them when they are injected. They are not rejected the way a normal mouse would reject them. In this case the tumor cells were Daudi cells, a tumor line that is driven to divide by the presence of the EB virus. It carries large amounts of CD20 on its surface, and the monoclonal antibody used was rituximab. The beta glucan was fed to the mice by mouth. The result was that the mice that had beta glucan and rituximab lived nearly twice as long as the mice that only had rituximab. This paper is available on Medline Modak et al Leuk Res 2005, 29:679-83.
These results are sufficiently encouraging for the people at Sloan-Kettering to start a clinical trial in children. These are the details:
Phase I Study of Beta-Glucan and Rituximab in Pediatric Patients With Relapsed or Progressive CD20-Positive Lymphoma or Leukemia or Post-Allogeneic Stem Cell Transplant-Related Lymphoproliferative Disorder
Primary Objectives are to determine the maximum tolerated dose of beta-glucan when given in combination with rituximab in pediatric patients with relapsed or progressive CD20-positive lymphoma or leukemia or post-allogeneic stem cell transplant-related lymphoproliferative disorder; to determine the toxicity of this regimen, with special emphasis on the degree of B-cell depletion and immune suppression, in these patients; and to determine the effects of beta-glucan on leukocyte-mediated cytotoxic effects in patients treated with this regimen.
The secondary objective is to determine the anti-tumor effect of this regimen in these patients.
This is a dose-escalation study of beta-glucan. Patients are assigned to 1 of 2 treatment groups according to diagnosis.
Group I (lymphoma or leukemia): Patients receive rituximab IV on days 1, 8, 15, and 22 and oral beta-glucan once daily on days 1-28 (days 8-28 of course 1). Treatment repeats every 42 days for 4 courses in the absence of disease progression or unacceptable toxicity.
Group II (post-allogeneic stem cell transplant-related lymphoproliferative disorder): Patients receive rituximab IV on days 1, 4, 8, 15, and 22 and oral beta-glucan once daily on days 8-28. Beginning on day 42, patients with responding disease may receive monthly rituximab prophylaxis until their CD4 cell count is > 200/mm^3.
Cohorts of 6 patients receive escalating doses of beta-glucan until the maximum tolerated dose (MTD) is determined. The MTD is defined as the dose preceding that at which 2 of 6 patients experience dose-limiting toxicity. Patients are followed every 3 months for 2 years. A total of 6-24 patients will be accrued for this study within 2 years.
Memorial Sloan-Kettering Cancer Center, New York, New York, 10021, United States; Shakeel Modak, MD 212-639-7623