APRIL stands for a proliferation inducing ligand. It is a TNF family ligand and has a physiological role in B-cell immunity. It is also believed to have a role in several B-cell malignancies including CLL. High levels of APRIL are correlated with poor prognosis. A group in the Netherlands has developed a pair of monoclonal antibodies that block the binding of APRIL to its receptors.
They have shown that blocking APRIL binding to its receptors has an inhibitory effect on known effects of APRIL both in vitro and in vivo. Importantly, mice treated with APRIL antagonistic antibodies display significant reduction in APRIL serum levels after 2 injections and undetectable levels after 4 weeks of antibody treatment. To demonstrate therapeutic capacity, they extended their study to examine the effect of APRIL antagonism on the survival of lymphoma cells. They confirmed that APRIL binding and stimulation of lymphoma cells, as well as APRIL-induced survival of malignant CLL cells in vitro could be blocked by the antibodies. Their observations confirm the survival benefit APRIL provides to CLL cells, and illustrate the activity of the anti-APRIL antibodies in blocking this.
There has been a recent observation that megakaryocytes, which produce significant amounts of APRIL, are a crucial constituent of the bone marrow niche for plasma cells. Such a selective local production of APRIL may be crucial for the survival of lymphoid malignancies in the bone marrow too. A similar situation appears to exist for CLL where the role of the microenvironment is particularly well established. T-cells and Nurse-Like Cells are described to deliver important survival stimuli. In vitro APRIL, either produced by NLCs or delivered as recombinant proteins can promote survival of CLL cells. These data indicate a significant role for APRIL in the survival of CLL cells in patients and thus support therapeutic intervention with targeting this survival benefit. Similarly, diffuse large B-cell lymphoma patients also appear to have a poor prognosis when their serum level of APRIL is high. Perhaps there will be a role in Richter syndrome.
The toxicity of APRIL antagonistic antibodies should be relatively marginal and thus a valuable option in the selective blocking of lymphoma survival signals and thus the treatment of lymphoma patients. The mouse model for CLL provides evidence that this approach could work. Although this is clearly not a complete model for the disease CLL, they have previously provided compelling evidence that a CLL-like phenotype occurs in this mouse model and the selective B-cell expansion that is reminiscent of CLL is prevented by treatment with the antibody.
Showing posts with label CLL monoclonal antibody. Show all posts
Showing posts with label CLL monoclonal antibody. Show all posts
Tuesday, July 05, 2011
More on alemtuzumab
Most people would agree that the alemtuzumab experience has been less that anticipated. We have seen unexpected deaths in some clinical trials and failure of response in patients with bulky disease. A paper in Haematologica addresses this.
They have studied pharmakokinetics in responders and non-responders and lo and behold the non-responders are under-dosed. Maximal concentration of alemtuzumab in responders was 1.69 micrograms per ml and in non-responders was 0.44 micrograms per ml.
I have previously written that the deaths from alemtuzumab, which occurred in an attempt to remove residual disease were caused by giving too large a dose too soon after FCR; I would venture to suggest that failure of bulky disease to respond is due to too small a dose being given. After all the original Lancet paper on Campath had responses in bulky disease.
They have studied pharmakokinetics in responders and non-responders and lo and behold the non-responders are under-dosed. Maximal concentration of alemtuzumab in responders was 1.69 micrograms per ml and in non-responders was 0.44 micrograms per ml.
I have previously written that the deaths from alemtuzumab, which occurred in an attempt to remove residual disease were caused by giving too large a dose too soon after FCR; I would venture to suggest that failure of bulky disease to respond is due to too small a dose being given. After all the original Lancet paper on Campath had responses in bulky disease.
Saturday, June 25, 2011
Blinotumamab 3. Adult ALL
I want to concentrate on two papers which describe the treatment of acute lymphoblastic leukemia with blinatumomab. The first is published in J Clin Oncol this week (June 20th 2011) and descrbes a study of treatment of patients with minimal residual disease detected by PCR following their induction and consolidation treatment. These were all adult patients who carry a bad prognosis. I have a few remaining patients with this disease, but most have quite bad GVHD having required a transplant to salvage them. Most who have not have really bee young adults with what amounts to the same disease that occurs in childhood, which has a much better prognosis.
Although about 80% of adult ALLs have a CR after induction chemotherapy, 50% experience relapse and chemoresistant disease. In this trial patients were treated with Blinatumomab 15 microgm/sq m/24 hours by iv infusion for 4 weeks if they had MRD by PCR following the completion of their induction and consolidation treatment. In patients with an allogeneic donor transplant was offered at any time after the first course of blinatumomab. Responders were permitted to receive three further cycles of blinatumomab.
21 patients entered the trial of whom 20 were evaluable for response and 16 converted to MRD negative, all at the end of the first cycle of treatment. At a median of 405 days follow-up 16/20 patients eligible for follow up remained in ongoing hematological remission. 8 patients had allografts and remain in remission with no treatment associated mortality. 4 patients who did not have allografts have had a clinical relapse in the first 200 days post blinatumomab. In 2 cases the relapse was extramedullary (CSF, testis) and two had marrow relapse (one an initial responder to blinatumomab).
81% had transient grade 3 or 4 adverse events most commonly lymphopenia (this was really an intended event) and hypogammaglobulinemia. There were no drug associated deaths. There were 4 cases of infection. There was no cytokine storm and although there were transient increases in serum levels of some inflammatory cytokins, these increases were of short duration.
This was trial of patients with a more than 90% risk of relapse whose only chance of cure was an allograft (about a 30% success rate).
The second paper was an abstract presented at the recent meeting of EHA (abstract 552) This was a phase 2 study in adult patients with relapsed/refractory ALL of the same treatment detailed above for a first cohort of 5 patients and a second cohort had a dose reduction for the first 7 days to 5 microgm/sq m/24 hours, presumably while there were large numbers of circulating blasts.
Of the first 5 evaluable patients, 2 had CRs and 2 CRis within the first cycle. 3 had become MRD negative. One responder had an extramedullary relapse during cycle 3.
The commonest adverse events were fever and chills and one patient with a high leucocyte count had reversible cytokine storm. the second non-evaluable patient had a reversible event of encephalopathy and disorientation. Despite discontinuation of treatment, he reached MRD negativity. Recruitment of the second cohort is ongoing.
In essense this is the same study as the first, only given after relapse, not before it. I am sure there is a lot of impatience among CLL patients to see this drug used in CLL, but for now adult ALL, although less common, is a more urgent problem. The company involved here is Micromet Inc. of Munich and I guess if there were patient power to get this drug there might be a result.
Although about 80% of adult ALLs have a CR after induction chemotherapy, 50% experience relapse and chemoresistant disease. In this trial patients were treated with Blinatumomab 15 microgm/sq m/24 hours by iv infusion for 4 weeks if they had MRD by PCR following the completion of their induction and consolidation treatment. In patients with an allogeneic donor transplant was offered at any time after the first course of blinatumomab. Responders were permitted to receive three further cycles of blinatumomab.
21 patients entered the trial of whom 20 were evaluable for response and 16 converted to MRD negative, all at the end of the first cycle of treatment. At a median of 405 days follow-up 16/20 patients eligible for follow up remained in ongoing hematological remission. 8 patients had allografts and remain in remission with no treatment associated mortality. 4 patients who did not have allografts have had a clinical relapse in the first 200 days post blinatumomab. In 2 cases the relapse was extramedullary (CSF, testis) and two had marrow relapse (one an initial responder to blinatumomab).
81% had transient grade 3 or 4 adverse events most commonly lymphopenia (this was really an intended event) and hypogammaglobulinemia. There were no drug associated deaths. There were 4 cases of infection. There was no cytokine storm and although there were transient increases in serum levels of some inflammatory cytokins, these increases were of short duration.
This was trial of patients with a more than 90% risk of relapse whose only chance of cure was an allograft (about a 30% success rate).
The second paper was an abstract presented at the recent meeting of EHA (abstract 552) This was a phase 2 study in adult patients with relapsed/refractory ALL of the same treatment detailed above for a first cohort of 5 patients and a second cohort had a dose reduction for the first 7 days to 5 microgm/sq m/24 hours, presumably while there were large numbers of circulating blasts.
Of the first 5 evaluable patients, 2 had CRs and 2 CRis within the first cycle. 3 had become MRD negative. One responder had an extramedullary relapse during cycle 3.
The commonest adverse events were fever and chills and one patient with a high leucocyte count had reversible cytokine storm. the second non-evaluable patient had a reversible event of encephalopathy and disorientation. Despite discontinuation of treatment, he reached MRD negativity. Recruitment of the second cohort is ongoing.
In essense this is the same study as the first, only given after relapse, not before it. I am sure there is a lot of impatience among CLL patients to see this drug used in CLL, but for now adult ALL, although less common, is a more urgent problem. The company involved here is Micromet Inc. of Munich and I guess if there were patient power to get this drug there might be a result.
Tuesday, May 24, 2011
How does GA101 work?
My old colleagues Tim Illidge, Mark Cragg and Martin Glennie have published their recent work on the new anti-CD20 monoclonal antibody, GA101, in a recent edition of Blood. http://bloodjournal.hematologylibrary.org/content/117/17/4519.abstract
GA101 is a fully humanized, type II, properly glycosolated monoclonal, which has been enginered to produce better killing than rituximab.
Anti-CD20 monoclonals invoke a wide range of killing methods, chief among them being antibody dependent cellular cytotoxicity (ADCC), but also phagocytosis mediated by macrophages and/or NK cells through the Fc gamma receptor link, complement dependent cytotoxicity (CDC) and sometimes direct induction of programmed cell death (PCD). PCD certainly does not play a significant part in rituximab killing, but is it important for other anti-CD20 monoclonals?
They have compared GA101 with rituximab against a panel of B-lymphoma cell lines. This shows that GA101 is much better at PCD than rituximab, even when the Fc part of teh molecule is removed. PCD seems to require what they call homotypic adhesion, which simply means that the lymphoma cells aggregate together when exposed to the antibody. The aggregation of the lymphoma cells causes alterations to the actin cytoskeleton of the cells. The actin molecules move to the points of cell to cell contact and if this is chemically inhibited then PCD is deiminished.
Next they showed that the actin-dependent death was independent of apoptosis, and could still work in apoptosis-resistent cells. Lysosymes are involved in PCD. It appears that GA101 induces lysosomal membrane permeabilization and cathepsin-mediated cell death. Cathepsin B is a classic lysosomal protease.
So how does GA101 fit into the pantheon of monoclonal anti-CD20s? It binds to a very sinilar site on the larger loop of the CD20 molecule (in contrast to ofatumumab which binds to the smaller loop). However, recent crystallographic evidence shows that it binds with a completely different orientation to rituximab. How exactly the aggregation of lymphoma cells in response to antibody coating occurs is not clear. It certainly requires bivalency, but the FC portion of the antibody is not required.
Another unexplained phenomnon is the fact that not all cells are killed by PCD, yet when the surviving cells are washed and retested they are killed in the same proportion; they have not acquired resistance.
GA101 is not the only monoclonal capable of triggering such severe actin relocalization; again it is not clear why it does so.
Lysosome function as cellular recycling and waste didposal units by degrading organelles and macromolecules delivered to the lysosomal compartment by autophagy, endocytosis and phagocytosis. They carry over 50 different degradation enzymes. If they leak they destroy the cell that conatins them in a non-apoptotic way. Again the link between actin repositioning and lysosomal permeabilization is missing.
All this work should not detract from the fact that GA101 has been glycoengineered to produce enhanced ADCC, but it does appear that it has a second string to its bow; one that should overcome the need for a TP53 pathway and therefore GA101 may prove to be effective in drug resistant CLL.
GA101 is a fully humanized, type II, properly glycosolated monoclonal, which has been enginered to produce better killing than rituximab.
Anti-CD20 monoclonals invoke a wide range of killing methods, chief among them being antibody dependent cellular cytotoxicity (ADCC), but also phagocytosis mediated by macrophages and/or NK cells through the Fc gamma receptor link, complement dependent cytotoxicity (CDC) and sometimes direct induction of programmed cell death (PCD). PCD certainly does not play a significant part in rituximab killing, but is it important for other anti-CD20 monoclonals?
They have compared GA101 with rituximab against a panel of B-lymphoma cell lines. This shows that GA101 is much better at PCD than rituximab, even when the Fc part of teh molecule is removed. PCD seems to require what they call homotypic adhesion, which simply means that the lymphoma cells aggregate together when exposed to the antibody. The aggregation of the lymphoma cells causes alterations to the actin cytoskeleton of the cells. The actin molecules move to the points of cell to cell contact and if this is chemically inhibited then PCD is deiminished.
Next they showed that the actin-dependent death was independent of apoptosis, and could still work in apoptosis-resistent cells. Lysosymes are involved in PCD. It appears that GA101 induces lysosomal membrane permeabilization and cathepsin-mediated cell death. Cathepsin B is a classic lysosomal protease.
So how does GA101 fit into the pantheon of monoclonal anti-CD20s? It binds to a very sinilar site on the larger loop of the CD20 molecule (in contrast to ofatumumab which binds to the smaller loop). However, recent crystallographic evidence shows that it binds with a completely different orientation to rituximab. How exactly the aggregation of lymphoma cells in response to antibody coating occurs is not clear. It certainly requires bivalency, but the FC portion of the antibody is not required.
Another unexplained phenomnon is the fact that not all cells are killed by PCD, yet when the surviving cells are washed and retested they are killed in the same proportion; they have not acquired resistance.
GA101 is not the only monoclonal capable of triggering such severe actin relocalization; again it is not clear why it does so.
Lysosome function as cellular recycling and waste didposal units by degrading organelles and macromolecules delivered to the lysosomal compartment by autophagy, endocytosis and phagocytosis. They carry over 50 different degradation enzymes. If they leak they destroy the cell that conatins them in a non-apoptotic way. Again the link between actin repositioning and lysosomal permeabilization is missing.
All this work should not detract from the fact that GA101 has been glycoengineered to produce enhanced ADCC, but it does appear that it has a second string to its bow; one that should overcome the need for a TP53 pathway and therefore GA101 may prove to be effective in drug resistant CLL.
Friday, August 27, 2010
How does rituximab work?
There is no doubt that rituximab was the drug of the decade as far as sufferers from lymphoid malignancies are concerned, but the big question is why does it work? After all, I spent nearly thirty years testing monoclonal antibodies as treatments for lymphoma with very disappointing results. Studies using anti-idiotype, anti-CD5, anti-CD19, anti-CD22, anti-CD23 and anti-CD37 were all very disappointing, but by a lucky chance the anti-CD20s were effective. Why?
We know that the CD20 molecule is an unusual one, but there is one loop that stands out from the cell surface, though it never gets far from the surface. It is against this loop that most of the antibodies (including rituximab) react. There is a much smaller loop (on the left in the diagram) that hardly protrudes from the surface at all and it is against this that the antibody ofatumumab reacts.
My ex-colleagues in Southampton, Mark Cragg and Martin Glennie have been beavering away at this topic for years and in three recent papers that have described what they think is going on (Lim et al Haematologica 2010; 95:135-143; Beers et al Blood 2010; 115:5191-5201; and Beers et al Semin Hematol 2010; 47:107-114.). In this article I will try to distill what they have discovered into simple language.
Simply binding an antibody to a leukemia cell isn't enough to kill that cell. Usually some sort of effector mechanism is necessary to kill the cell. When we experiment with B cells and antibody in a test tube, it becomes apparent that there are a number of means of killing those cells. The easiest to demonstrate is Complement Dependent Cytotoxicity (CDC). Complement is a series of proteins labelled C1 to C9 which form a cascade of reactions eventually ending up by punching holes in the target cell. When you have a blood transfusion and get the wrong blood group it is CDC that destroys your blood cells (and may kill you), but it is not clear that this is an important mechanism inside the body even if it does work in the test tube.
You can destroy Complement by heating serum to 56 degrees Celsius or in an animal by injecting Cobra Venom Factor. In this way you can determine whether CDC is an important mechanism. In general terms, most investigators do not think it is an important mechanism in human antibody therapy.
Not all anti-CD20 antibodies permit CDC and Cragg and Glennie have divided monoclonal anti-CD20s into those that do (Type I) and those that don't (Type II). Rituximab is a Type I antibody. Type I antibodies have other characteristics, chief of which is to redistribute CD20 into lipid rafts which are then internalized, thus clearing the antigen from the surface. This process is known as antigenic modulation, which I have written about earlier.
There was much confusion about antigenic modulation, and it has always been said that CD20 is one of the few antigens that doesn't modulate, which is why antibodies against it work so well. That turns out to be true for some types of lymphocytes and not for others. Cells from diffuse large B-cell lymphoma and from follicular lymphoma and from some lymphoblastoid cell lines are poor at antigenic modulation, whereas other cell lines and cells from mantle cell lymphoma are much better at it. Best of all are CLL cells. Normal B cells are also good modulators.
Antigenic modulation is distinct from antigen shaving in which the antigen/antibody complex is skimmed from the surface of the malignant B cell by macrophages, usually in the spleen. With antigenic modulation the antibody is internalized by the malignant B cells. Note that both these mechanisms consume the monoclonal antibody and may account for the more rapid than expected clearance of antibody from the circulation.
Most people believe that the most important method of killing that rituximab uses is ADCC which stands for Antibody Dependent Cellular Cytoxicity. This process uses other cells (either NK cells or macrophages) to kill the tumor cell. When the antibody latches on to the CD20, the other end of the molecule sticks out away from the cell surface. This is known as the Fc portion of the molecule (the name comes from the 1950s when immunoglobulin molecules were broken up by various chemicals; the Fc portion was the fraction that could be crystalized). NK cells and macrophages have receptors that recognize Fc (called Fc receptors and labelled CD16, CD32 and CD64). Certain individuals have minor molecular variations (polymorphisms) of CD16 and CD32 which make their Fc receptors less effective. If these people get follicular lymphoma then rituximab works less well than in people without the polymorphisms. However these polymorphisms do not affect the outcome of CLL patients treated with FCR, suggesting that rituximab may act differently in CLL.
However, Complement activation may still be important. It seems likely that activation of Complement is the cause of the acute 'allergic' reaction caused by the first dose of rituximab, which is especially bad where there is a high white count. It is my opinion that the anaphylactoid fragments of Complement, C3a and C5a are responsible. Furthermore, there is evidence that both C3b and C5b inhibit ADCC.
What about Type II antibodies? There aren't very many. The best known is tositumomab, the antibody that is labelled with radioactive iodine in Bexxar. These antibodies do not activate complement and do not push CD20 into lipid rafts. Neither do they cause antigenic modulation. But do they kill tumor cells? They can certainly activate ADCC, but they also have another mechanism of cytotoxicity. This form of killing involves homotypic adhesion, which simply means that two tumor cells stick together. It is known that the process requires cholesterol and is energy dependent. It involves mitochondria moving within the cells to the area where they are in contact. Cell death is caspase independent, which means that it doesn't use apoptosis and is therefore likely to be effective in p53 deficient tumors. The killing requires lysosomes which swell and release their contents into the cytoplasm and extracellular space. Lysosomes are spherical organelles that contain enzymes (acid hydrolases) that break up endocytized materials and cellular debris. If they release their enzymes into the cytoplasm, it's like letting a tiger out of its cage. Something's going to die - in this case, the cell.
Of the new antibodies available in the clinic or in trials, ofatumumab is a Type I antibody whose ability to stay on the surface (because it goes for the little loop, not the big one) for a long time negates the negative effect of antigenic modulation. On the other hand, GA101 is a type II antibody. It has good direct cell killing and its Fc has been optimized by adding the correct sugars to the amino acids so that ADCC has been improved.
We know that the CD20 molecule is an unusual one, but there is one loop that stands out from the cell surface, though it never gets far from the surface. It is against this loop that most of the antibodies (including rituximab) react. There is a much smaller loop (on the left in the diagram) that hardly protrudes from the surface at all and it is against this that the antibody ofatumumab reacts.My ex-colleagues in Southampton, Mark Cragg and Martin Glennie have been beavering away at this topic for years and in three recent papers that have described what they think is going on (Lim et al Haematologica 2010; 95:135-143; Beers et al Blood 2010; 115:5191-5201; and Beers et al Semin Hematol 2010; 47:107-114.). In this article I will try to distill what they have discovered into simple language.
Simply binding an antibody to a leukemia cell isn't enough to kill that cell. Usually some sort of effector mechanism is necessary to kill the cell. When we experiment with B cells and antibody in a test tube, it becomes apparent that there are a number of means of killing those cells. The easiest to demonstrate is Complement Dependent Cytotoxicity (CDC). Complement is a series of proteins labelled C1 to C9 which form a cascade of reactions eventually ending up by punching holes in the target cell. When you have a blood transfusion and get the wrong blood group it is CDC that destroys your blood cells (and may kill you), but it is not clear that this is an important mechanism inside the body even if it does work in the test tube.
You can destroy Complement by heating serum to 56 degrees Celsius or in an animal by injecting Cobra Venom Factor. In this way you can determine whether CDC is an important mechanism. In general terms, most investigators do not think it is an important mechanism in human antibody therapy.
Not all anti-CD20 antibodies permit CDC and Cragg and Glennie have divided monoclonal anti-CD20s into those that do (Type I) and those that don't (Type II). Rituximab is a Type I antibody. Type I antibodies have other characteristics, chief of which is to redistribute CD20 into lipid rafts which are then internalized, thus clearing the antigen from the surface. This process is known as antigenic modulation, which I have written about earlier.
There was much confusion about antigenic modulation, and it has always been said that CD20 is one of the few antigens that doesn't modulate, which is why antibodies against it work so well. That turns out to be true for some types of lymphocytes and not for others. Cells from diffuse large B-cell lymphoma and from follicular lymphoma and from some lymphoblastoid cell lines are poor at antigenic modulation, whereas other cell lines and cells from mantle cell lymphoma are much better at it. Best of all are CLL cells. Normal B cells are also good modulators.
Antigenic modulation is distinct from antigen shaving in which the antigen/antibody complex is skimmed from the surface of the malignant B cell by macrophages, usually in the spleen. With antigenic modulation the antibody is internalized by the malignant B cells. Note that both these mechanisms consume the monoclonal antibody and may account for the more rapid than expected clearance of antibody from the circulation.
Most people believe that the most important method of killing that rituximab uses is ADCC which stands for Antibody Dependent Cellular Cytoxicity. This process uses other cells (either NK cells or macrophages) to kill the tumor cell. When the antibody latches on to the CD20, the other end of the molecule sticks out away from the cell surface. This is known as the Fc portion of the molecule (the name comes from the 1950s when immunoglobulin molecules were broken up by various chemicals; the Fc portion was the fraction that could be crystalized). NK cells and macrophages have receptors that recognize Fc (called Fc receptors and labelled CD16, CD32 and CD64). Certain individuals have minor molecular variations (polymorphisms) of CD16 and CD32 which make their Fc receptors less effective. If these people get follicular lymphoma then rituximab works less well than in people without the polymorphisms. However these polymorphisms do not affect the outcome of CLL patients treated with FCR, suggesting that rituximab may act differently in CLL.
However, Complement activation may still be important. It seems likely that activation of Complement is the cause of the acute 'allergic' reaction caused by the first dose of rituximab, which is especially bad where there is a high white count. It is my opinion that the anaphylactoid fragments of Complement, C3a and C5a are responsible. Furthermore, there is evidence that both C3b and C5b inhibit ADCC.
What about Type II antibodies? There aren't very many. The best known is tositumomab, the antibody that is labelled with radioactive iodine in Bexxar. These antibodies do not activate complement and do not push CD20 into lipid rafts. Neither do they cause antigenic modulation. But do they kill tumor cells? They can certainly activate ADCC, but they also have another mechanism of cytotoxicity. This form of killing involves homotypic adhesion, which simply means that two tumor cells stick together. It is known that the process requires cholesterol and is energy dependent. It involves mitochondria moving within the cells to the area where they are in contact. Cell death is caspase independent, which means that it doesn't use apoptosis and is therefore likely to be effective in p53 deficient tumors. The killing requires lysosomes which swell and release their contents into the cytoplasm and extracellular space. Lysosomes are spherical organelles that contain enzymes (acid hydrolases) that break up endocytized materials and cellular debris. If they release their enzymes into the cytoplasm, it's like letting a tiger out of its cage. Something's going to die - in this case, the cell.
Of the new antibodies available in the clinic or in trials, ofatumumab is a Type I antibody whose ability to stay on the surface (because it goes for the little loop, not the big one) for a long time negates the negative effect of antigenic modulation. On the other hand, GA101 is a type II antibody. It has good direct cell killing and its Fc has been optimized by adding the correct sugars to the amino acids so that ADCC has been improved.
Tuesday, July 13, 2010
Antigenic modulation
28 years ago I wrote this editorial for the Lancet. It stll has relevance today as you will see in a subsequent post.
Antigenic modulation
The passive serotherapy of cancer has had a long gestation and little success. However, the advent of monoclonal antibodies with their high specificity has awakened interest in the subject. Lately there have been several reports of attempts to treat lymphoid malignant disease with murine monoclonal antibodies, and antibodies to melanoma, neuroblastoma, leiomyosarcoma, teratoma, and colonic and breast carcinomas have been raised. It has also been proposed that monoclonal antibodies might be used to remove T cells before bone-marrow transplantation to prevent graft-versus-host disease, and to remove tumour cells from bone-marrow autografts after high-dose chemotherapy.
All these applications are threatened by the phenomenon of antigenic modulation. This effect was first described by Boyse et al. for the TL antigen of murine thymic leukaemia, and involves the temporary disappearance of the target antigen from the surface of the tumour cell in the presence of antibody. When antibody is removed from the system the cell re-expresses the antigen. The process begins with the complexing of antibody with antigen on the cell surface. One divalent molecule of antibody links with adjacent molecules of antigen, so that the antigen is rearranged into patches and caps before internalisation of the whole complex by pinocytosis.
The speed of the reaction is remarkable. Incubation of the target cell with antibody at 37 deg C for as little as two minutes can prevent complement-dependent cytolysis. Complete clearance of the antigen is not, apparently, necessary to render the antibody ineffective, and mere persistence of the antigen on the surface of the cell does not mean that that cell will be susceptible to antibody-induced killing or clearing. Simple rearrangement of antigen and antibody in the lipid bilayer seems to hinder the deposition of complement components sufficiently to protect the cell surface. In some systems antigenic modulation takes place so quickly that it appreciably protects the cell even against simultaneous attack by antibody and complement.
A further contribution to chronic antigenic modulation in vivo arises from the
metabolic response to the combination of antibody with cell surface antigen. A surge of intracellular cyclic AMP is succeeded by reduced delivery of antigen to the cell surface. Antibodies kill tumour cells by invoking various effector mechanisms such as complement dependent cytolysis, K cell killing, and binding to Fc or C3 receptors on macrophages. All these mechanisms depend on the antibody remaining on the cell surface in an accessible form for a finite period, and all are susceptible to antigenic modulation.
Most current attempts to bypass this mechanism involve giving the antibody a "warhead" so that these effector mechanisms need not be invoked. It has proved difficult to link cytotoxic drugs to antibody in such a way that the link remains stable in vivo. Labelling the antibody with radioactive isotopes 7 carries the risk of obliterating the antibody combining site. Some groups have tried coupling the biological toxins abrin and ricin to antibody but their safety in vivo remains in doubt.
A more attractive solution has been offered by Glennie and Stevenson. They have constructed a univalent antibody by papain cleavage of one Fab fragment from each immunoglobulin molecule. The resulting antibody cannot cross-link with adjacent antigen molecules and therefore does not cause antigenic modulation. However, it does retain the ability to fix complement. To show its efficacy they have treated the guinea pig prolymphocytic leukaemia L2C with a polyclonal, univalent rabbit antibody against the idiotypic determinants of the surface immunoglobulin. Univalent antibody was more effective in vitro at inducing complement-dependent cytolysis and in vivo it prolonged the life of the guinea pig. In this it was three times as effective as whole IgG. This particular biochemical manoeuvre is effective only for
rabbit IgG but other procedures are available to produce univalent antibody from the immunoglobulin of other species. When they are applied to murine monoclonal antibodies there are immense prospects for successful immunotherapy.
Antigenic modulation
The passive serotherapy of cancer has had a long gestation and little success. However, the advent of monoclonal antibodies with their high specificity has awakened interest in the subject. Lately there have been several reports of attempts to treat lymphoid malignant disease with murine monoclonal antibodies, and antibodies to melanoma, neuroblastoma, leiomyosarcoma, teratoma, and colonic and breast carcinomas have been raised. It has also been proposed that monoclonal antibodies might be used to remove T cells before bone-marrow transplantation to prevent graft-versus-host disease, and to remove tumour cells from bone-marrow autografts after high-dose chemotherapy.
All these applications are threatened by the phenomenon of antigenic modulation. This effect was first described by Boyse et al. for the TL antigen of murine thymic leukaemia, and involves the temporary disappearance of the target antigen from the surface of the tumour cell in the presence of antibody. When antibody is removed from the system the cell re-expresses the antigen. The process begins with the complexing of antibody with antigen on the cell surface. One divalent molecule of antibody links with adjacent molecules of antigen, so that the antigen is rearranged into patches and caps before internalisation of the whole complex by pinocytosis.
The speed of the reaction is remarkable. Incubation of the target cell with antibody at 37 deg C for as little as two minutes can prevent complement-dependent cytolysis. Complete clearance of the antigen is not, apparently, necessary to render the antibody ineffective, and mere persistence of the antigen on the surface of the cell does not mean that that cell will be susceptible to antibody-induced killing or clearing. Simple rearrangement of antigen and antibody in the lipid bilayer seems to hinder the deposition of complement components sufficiently to protect the cell surface. In some systems antigenic modulation takes place so quickly that it appreciably protects the cell even against simultaneous attack by antibody and complement.
A further contribution to chronic antigenic modulation in vivo arises from the
metabolic response to the combination of antibody with cell surface antigen. A surge of intracellular cyclic AMP is succeeded by reduced delivery of antigen to the cell surface. Antibodies kill tumour cells by invoking various effector mechanisms such as complement dependent cytolysis, K cell killing, and binding to Fc or C3 receptors on macrophages. All these mechanisms depend on the antibody remaining on the cell surface in an accessible form for a finite period, and all are susceptible to antigenic modulation.
Most current attempts to bypass this mechanism involve giving the antibody a "warhead" so that these effector mechanisms need not be invoked. It has proved difficult to link cytotoxic drugs to antibody in such a way that the link remains stable in vivo. Labelling the antibody with radioactive isotopes 7 carries the risk of obliterating the antibody combining site. Some groups have tried coupling the biological toxins abrin and ricin to antibody but their safety in vivo remains in doubt.
A more attractive solution has been offered by Glennie and Stevenson. They have constructed a univalent antibody by papain cleavage of one Fab fragment from each immunoglobulin molecule. The resulting antibody cannot cross-link with adjacent antigen molecules and therefore does not cause antigenic modulation. However, it does retain the ability to fix complement. To show its efficacy they have treated the guinea pig prolymphocytic leukaemia L2C with a polyclonal, univalent rabbit antibody against the idiotypic determinants of the surface immunoglobulin. Univalent antibody was more effective in vitro at inducing complement-dependent cytolysis and in vivo it prolonged the life of the guinea pig. In this it was three times as effective as whole IgG. This particular biochemical manoeuvre is effective only for
rabbit IgG but other procedures are available to produce univalent antibody from the immunoglobulin of other species. When they are applied to murine monoclonal antibodies there are immense prospects for successful immunotherapy.
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