Therapies, Targets, Drug Development and Cancer

with a focus on the treatment of B cell malignancies

Richard F Ambinder
This lecture is focused on four classes of agents that have had a profound effect on the treatment of B
lineage malignancies. In some cases, similar agents or the same agents also have application to other
malignancies. The focus of the lecture is on the very different approaches to drug discovery and clinical
translation that led to the use of these agents in the clinic as first line treatments for B lineage tumors.

B lineage tumors refers to malignancies that arise from cells that have taken at least the initial step in
differentiation to become B cells, they have rearranged their immunoglobulin genes. Included in the list
of malignancies are leukemia, lymphoma and myeloma.
The lecture proceeds in the order of discovery. The first class of agents to be considered are the
microtubule inhibitors.
The vinca alkaloids came to medical attention when two Canadian brothers (Noble) interested in an oral
hypoglycemic for the treatment of diabetes received an envelope from a patient traveling in Jamaica
with the dried leaves of a periwinkle (1953). The patient reported that periwinkle tea was used for this
purpose in Jamaica. When mice were treated with periwinkle extract, no effect on blood glucose was
found but the WBC fell. A chemist (Beer) extracted vinblastine. The National Cancer Institute of Canada
named its most prestigious prize (the Noble Prize) after Robert Noble. The Eli Lilly (drug company) in
Indiana followed up with further investigation and isolated vincristine from the same plant (1958). In the
meantime the US NCI recognized the value of screening potential drug libraries. In 1958 the NCI screen
began to include natural products. In 1964 an extract from the bark of the Pacific Yew tree was found to
have cytotoxic properties against cancer cell lines. Ultimately, the taxanes were identified. These include
paclitaxel and docetaxel. Because enormous amounts of tree bark were required for isolation of the
drug and the tree was an endangered species, drug supply was a huge issue for many years.

The vinca alkaloids inhibit microtubule polymerization. These drugs bind to tubulin dimers and prevent
the assembly of microtubules. Microtubules are important in many cellular processes including mitosis
and axonal transport. Formation of the mitotic spindle requires microtubules. Microtubules are
important for axonal transport. Vinblastine and vincristine remain important drugs for the treatment of
many malignancies but especially lymphoid malignancies. They are administered intravenously. A
common feature of all the vinca alkaloids is neurotoxicityespecially paresthesias (pins and needles) in
the toes and finger tips. Patients often have impaired vibration sense after treatment that may last
indefinitely. Constipation associated with autonomic neuropathy is also a side effect.
The taxanes stabilize microtubules preventing their disassembly. These drugs bind to GDP-bound tubulin
and inhibit depolymerization.
The taxanes are used mainly in the treatment of solid tumors including breast, ovarian and lung cancer
among others.
The initial discovery of the microtubule inhibitors illustrates a classic drug discovery paradigm, chance
observation followed by systematic screening.
The next set of drugs also involves chance, but this time the story has many more twists and turns
before systematic screening.
IMiDs. These are structural and functional analogues of thalidomide. Thalidomide was first used to treat
morning sickness associated with pregnancy and as a sedative was discovered to cause horrendous birth
defects involving the limbs. The drug was largely abandoned but a few stocks hadnt been cleared from
shelves and cabinets when a physician caring for a patient dying with a form of leprosy acute erythema
nodosum leprosum treated his patient and discovered a remarkable improvement in her clinical status.
This observation was followed up and confirmed and ultimately thalidomide was licensed to treat this
rare disease. Researchers investigating the role of angiogenesis in supporting tumor growth noted that
thalidomide inhibited the development of blood vessels in several model systems. The possible anti
tumor effect was evaluated in myeloma patients who had failed bone marrow transplantation.
Thalidomide induced complete remissions in some patients, partial remissions in others and was of no
benefit in others. Its side effect profile is very different than many commonly used anticancer drugs in
that it does not lead to cytopenias (neutropenia, thrombocytopenia, anemia). Rather its side effects are
substantially neurologic (constipation, lethargy, neuropathy).
IMiDs inhibit the production of proinflammatory cytokines by monocytes, macrophages and some other
cell types. Among these proinflammatory cytokines are TNF alpha, cyclooxygenase 2, TGF beta, IL1beta,
IL6, IL12. The molecular mechanism is not well understood. These drugs also result in down modulation
of leukocyte adhesion molecules including integrins and ICAM1. IMiDs also have T-cell co-stimulatory
activity. IMiDs may substitute for or potentiate second signals required for T cell activation leading to
T cell proliferation and NK activity. IMiDs also inhibit secretion of VEGF and bFGF. IMiDs also have direct
apoptotic effects on some tumor cells. The mechanisms responsible for birth defects and for anti-tumor
responses remain contentious.

Chemical modifications of the structural backbone of the molecule were made with the goal of
increasing the potency of TNF alpha inhibition. Lenoalidomide is one such analogue and has a 2000-fold
greater effect on TNF alpha secretion than thalidomide. Its side effect profile is very different from
thalidomide. In rabbit models that predict teratogenicity for thalidomide, lenalidomide is not
teratogenic. It does lead to cytopenias. Like thalidomide, it is active against myeloma. It shows activity
even in people who have failed thalidomide. It is also active in the treatment of another malignancy
myelodysplasia. Other IMiDs are in clinical trials in myeloma and other cancers. The target remains
contentious but there is no debate about efficacy. The name for the class of drugs (IMiDs,
immunomodulatory) hints that we understand how they work or that they work through immune
mediated mechanisms. It remains possible that they work through immune mediated mechanisms. Both
thalidomide and lenalidomide are approved upfront and are commonly used as the first treatment
(usually in combination with another agent or agents) for multiple myeloma.

The second class of agents to be considered are monoclonal antibodies. Many B cell lymphomas express
surface Ig. Ig molecules differ in their idiotypes i.e. their antigen binding domains. However, at a first
approximation, the cells in a particular B cell tumor will all share the same idiotype. Thus an antiidiotype antibody should be entirely tumor specific. Early attempts at therapy with anti-idiotype
antibody met with some success. However, the approach was limited by the time and labor required to
synthesize and manufacture individualized antibodies, by immune responses to the mouse antibodies,
and by escape mutations (the idiotype sequence evolved in patients who relapsed).
An antibody that targeted a pan-B cell antigen (CD20) rather than a particular idiotype has been much
more successful. Rituximab is a chimeric molecule with the variable regions being mouse in origin and
the constant regions being human. Treatment with rituximab in patients with follicular lymphoma often
leads to partial or complete remissions. Adverse effects are modest. Although normal B cells are
targeted and disappear from the blood, Ig levels are not much affected. This is because most Ig is made
by plasma cells. Plasma cells do not express CD20, are long lived and continue to produce Ig. Rituximab
likely inhibits the ability to generate B cell responses to new antigens but has little impact on extant
responses. Because CD20 is not expressed on B cell precursors, with time new B cells will be produced.
Three mechanisms seem to be important for rituximab-mediated killing. One mechanism is a direct
apoptotic effect i.e. rituximab binds to CD20 and signaling pathways are activated that lead directly to
cell death. A second mechanism is antibody dependent cellular cytotoxicity. The Fc portion of the

molecule binds monocytes or NK cells leading to tumor killing. Several studies have shown that patients
with follicular lymphoma with the best response to rituximab have a particular FC receptor
polymorphism that may allow effector cells to bind rituximab with higher affinity. A third mechanism is
complement mediated cell lysis. The relative contribution of these mechanisms may differ patient to
patient and tumor type to tumor type.

Chimeric vs Humanized
Chimeric antibodies have a mouse variable

region spliced to a human constant region.

human

mouse

A variety of manipulations can yield

antibodies whose variable regions are also

partially human (humanized).
human

humanized

Side effects of rituximab are generally mild. There is some infusional toxicity in early treatment related
to killing of normal B cells but these diminish with time. The development of human anti-chimeric
antibodies (HACA) or human anti-mouse antibodies (HAMA) is distinctly unusual, perhaps because
rituximab leads to the death of cells that would produce these antibodies.
CD20 doesnt commonly either down-regulate or mutate. Resistance to rituximab seems to reflect other
changes in cells and is still poorly understood. However, it may be overcome in some instances by
arming CD20 targeted antibodies with radio-isotopes such as 90Y or 131I.
Monoclonal antibodies to treat lymphoma is one of the best examples of a rationale approach to
targeted therapyalthough insisting on tumor specificity was abandoned along the way. The target of
CD20 antibodies is not at all tumor specificbut has proven to be a very safe target nonetheless.
Rituximab is now incorporated into virtually all front line therapies for B cell lymphoma.
Other surface molecules are targeted by other antibody-based pharmaceuticals. For example, tumor
cells in Hodgkin lymphoma express CD30. In contrast to antibodies that target CD20, antibodies that
target CD30 are not generally effective in the treatment of Hodgkin lymphoma. However, an interesting
conjugate of anti-CD30 (brentuximab) and a microtubule inhibitor (vedotin) has recently been approved
for the treatment of Hodgkin lymphoma. The drug is called brentuximab vedotin. The antibody binds to
CD30, the antibody-CD30 complex is internalized and travels to the lysozome where a protease present
in the lyzosome releases and activates the microtubule inhibitor which then leads to cell cycle arrest. If
CD30 didn't internalize upon antibody binding, the approach wouldn't work because the microtuble
agent would not be released and activated. Note that CD20 is not internalized when it binds antibody.

Review the powerpoint slides about antibody nomenclature. Be able to "translate" anitbody drug names
in the manner illustrated.
The third and newest class of agents are the proteasome inhibitors. Only one is presently licensed.
Bortezomib is a competitive inhibitor of the proteasome. The proteasome is a major waste disposal
system for the cell. It degrades midfolded proteins and other proteins that are marked by ubiquitination.
In phase 1 trials designed to evaluate toxicity of agents thought to be potentially promising in the
treatment of cancer, bortezomib proved to be effective against multiple myeloma. When the
proteasome is inhibited, misfolded proteins accumulate. Multiple myeloma is a tumor of plasma cells,
cells that are immunoglobulin factories. It appears that these cells may be particularly sensitive to
proteasome inhibition because as their protein production is so high their misfolded proteins are also
very high. Without a way to dispose of misfolded proteins, alarms are triggered and apoptosis ensues.
Other ideas regarding the relationship between proteasome inhibition and drug efficacy in myeloma
have also been advanced.

Bortezomib