192

The recent regulatory approval in the UnitedStates and Europe of imatinib mesylate(Gleevec®) for patients with bcr/abl transloca-tion positive chronic myelogenous leukemia(FIG. 14.1) and the subsequent approval forgastrointestinal stromal tumors featuring anactivating c-kit growth factor receptor muta-tion infused the oncology drug developmentcommunity with enthusiasm for anticancertargeted therapy.1,2 Recent major news maga-zines have headlined the considerable publicinterest in new anticancer drugs that exploitdisease-specific genetic defects as the target oftheir mechanism of action.3,4 Many scientists,clinicians, and pharmaceutical company execu-tives now believe that in the next 5 to 10 years,the integration of molecular oncology and mo-lecular diagnostics will further revolutionizeoncology drug discovery and development; cus-tomize the selection, dosing, and route of

administration of both previously approvedtraditional agents and new therapeutics inclinical trials; and truly personalize medicalcare for the cancer patient.5–8

Targeted Therapies for Cancer: Definitions

During the last several years, multiple defini-tions for the term “targeted therapy” haveemerged. From the regulatory perspective,targeted therapy has been defined as a drug inwhose approval label there is a specific refer-ence to a simultaneously or previously ap-proved diagnostic test that must be performedbefore the patient can be considered eligibleto receive the drug. The classic example ofthis definition for targeted therapy is thecoapprovals of the anti-breast cancer an-tibody trastuzumab (Herceptin®) and the eli-gibility tests (Herceptest®, Pathway®, andPathvysion®) (see Chapter 16 and below). Formany scientists and oncologists, targeted ther-apy is defined as a drug with a focused mecha-nism that specifically acts on a well-definedtarget or biologic pathway that, when inacti-vated, causes regression or destruction of themalignant process. Examples of this type oftargeted therapy include hormonal-basedtherapies (see Chapter 15), inhibitors of theepidermal growth factor receptor (EGFR)pathway (see Chapter 17), blockers of inva-sion and metastasis enabling proteins andenzymes (see Chapter 18), antiangiogene-sis agents (see Chapter 19), proapoptoticdrugs (see Chapter 21), and proteasome in-hibitors (see Chapter 24). In addition, mostscientists and oncologists consider anticancerantibody therapeutics that seek out and killmalignant cells bearing the target antigen asanother type of targeted therapy.

Jeffrey S. Ross, MD

Albany Medical College, Albany, New Yorkand Millennium Pharmaceuticals, Inc.,Cambridge, Massachusetts

Targeted Therapy forCancer: IntegratingDiagnostics andTherapeutics

C H A P T E R

14

FIGURE 14.1 Chronic myelogenous leukemia and imatinib(Gleevec®) therapy. Photomicrograph demonstrates a bcr/abltranslocation detected by FISH in a patient with a packed bonemarrow biopsy diagnostic of chronic myelogenous leukemia(inset). Note the yellow “fusion” gene product, indicating the ap-position of one green (chromosome 22) and one red (chromo-some 9) resulting from the translocation.This patient was treatedwith single agent imatinib (Gleevec®) and achieved completeremission of bone marrow histology and absence of bcr/abl byroutine cytogenetics and FISH assessment. See Plate 30 forcolor image.

Chapter 14 Targeted Therapy for Cancer 193

creating enough fresh tissue to perform theassay.10 The drug tamoxifen (Nolvadex®), whichhas both hormonal and nonhormonal mecha-nisms of action, has been the most widely pre-scribed antiestrogen for the treatment ofmetastatic breast cancer and chemopreventionof the disease in high risk women.11,12 Although,ER and progesterone receptor testing is thefront line for predicting tamoxifen response, ad-ditional biomarkers, including HER-2/neu(HER-2) and cathepsin D testing, have beenused to further refine therapy selection.13 Theintroductions of specific estrogen response mod-ulators and aromatase inhibitors such as anas-trozole (Arimidex®), letrozole (Femara®), andthe combination chemotherapeutic, estramus-tine (Emcyt®)14–18 have added new strategies forevaluating tumors for hormonal therapy.

Leukemia and Lymphoma Lead the Way

The introduction of immunophenotyping forleukemia and lymphoma was followed by thefirst applications of DNA based assays, thepolymerase chain reaction, and RNA basedmolecular technologies in these diseases thatcomplemented continuing advances in tumorcytogenetics.19,20 In addition to the imatinib(Gleevec®) targeted therapy for chronic mye-logenous leukemia, other molecular targetedtherapy in hematologic malignancies includesthe use of all-trans-retinoic acid (ATRA) for thetreatment of acute promyelocytic leukemia21;anti-CD20 antibody therapeutics targetingnon-Hodgkin lymphomas, including rituximab(Rituxan®)22; and the emerging Flt-3 target fora subset of acute myelogenous leukemia pa-tients (see below).23

Thirty Years Later: HER-2 and Trastuzumab(Herceptin®)

After the introduction of hormone receptor test-ing, some 30 years then elapsed before the nextmajor targeted cancer chemotherapy programfor a solid tumor was developed. In the mid-1980s, the discovery of the HER-2 (c-erbB2)gene and protein and subsequent associa-tion with an adverse outcome in breast cancerprovided clinicians with a new biomarkerthat could be used to guide adjuvant chemo-therapy.24 The development of trastuzu-mab (Herceptin®), a humanized monoclonal

The Ideal Target

The ideal cancer target (Table 14.1) can be de-fined as a macromolecule that is crucial to themalignant phenotype and is not significantlyexpressed in vital organs and tissues; that hasbiologic relevance that can be reproduciblymeasured in readily obtained clinical samples;that is definably correlated with clinical out-come; and that interruption, interference, orinhibition of such a macromolecule yields aclinical response in a significant proportion ofpatients whose tumors express the target withminimal to absent responses in patients whosetumors do not express the target. For antibodytherapeutics, additional important criteria in-clude the use of cell surface targets that whencomplexed with the therapeutic naked or con-jugated antibody, internalize the antigen–antibody complex by reverse pinocytosis, thusfacilitating tumor cell killing.

The Original Targeted Therapy:Antiestrogens for Breast Cancer

Arguably the first type of targeted therapyin oncology was the development of antiestro-gen therapies for patients with breast cancerthat expressed the estrogen receptor (ER) pro-tein (see Chapter 15) (FIG. 14.2).9 Originallydeveloped as a competitive binding bioassayperformed on fresh tumor protein extracts andused to select for hormone production ablationby surgery (oophorectomy, adrenalectomy, andhypophysectomy), the ER and progesterone re-ceptor test format converted to an immunohis-tochemistry (IHC) platform when the decreasedsize of primary tumors enabled by mass screen-ing programs produced insufficient material for

Table 14.1 Features of the Ideal Anticancer Target

• Crucial to the malignant phenotype

• Not significantly expressed in vital organs and tissues

• A biologically relevant molecular feature

• Reproducibly measurable in readily obtained clinicalsamples

• Correlated with clinical outcome

• When interrupted, interfered with, or inhibited, the result isa clinical response in a significant proportion of patientswhose tumors express the target

• Responses in patients whose tumors do not express thetarget are minimal

194 MOLECULAR ONCOLOGY OF BREAST C ANCER

antibody designed to treat advanced metastaticbreast cancer that had failed first- and second-line chemotherapy, caused a rapid wide adop-tion of HER-2 testing of the patients’ primarytumors.25 However, soon after its approval,widespread confusion concerning the most ap-propriate diagnostic test to determine HER-2status in formalin-fixed paraffin-embeddedbreast cancer tissues (see Chapter 16) substan-tially impacted trastuzumab use.26–33 Since itslaunch in 1998, trastuzumab has become an im-portant therapeutic option for patients withHER-2–positive breast cancer.34–37

Reports that fluorescence in situ hybrid-ization (FISH) could out-perform IHC inpredicting trastuzumab response38 and the

well-documented lower response rates of in-termediate (2�) IHC staining versus intense(3�) staining tumors39 resulted in a variety ofapproaches, including IHC as a primary screenwith follow-on FISH testing of either 1� cases,2� cases, or both 1� and 2� cases or primaryFISH based testing. In a recently publishedstudy where trastuzumab was used as a singleagent, the response rates in 111 assessablepatients with 3� IHC staining was 35% andthe response rates for 2� cases was 0%; theresponse rates in patients with and withoutHER-2 gene amplification detected by FISHwere 34% and 7%, respectively.39 In anotherstudy of HER-2–positive breast cancer treatedwith trastuzumab plus paclitaxel, overall

Relative E

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ER Status by IHC on Core Needle Biopsies

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Inositol triphosphate receptorMGC17687GATA-binding protein 3Hs 347271DKFZp667D095DKFZp43N2412Tyrosine phosphatase IVA.1Tyrosine phosphatase IVA.2DKFZp564F053Interleukin 6 signal transducer.1Interleukin 6 signal transducer.2Reticulon 1Androgen induced protein260170v-mybN-acetyltransferase 1X-box binding protein 1Adipose specific 2ESTDKFZp586J2118Non-metastatic cell 3Estrogen receptor 1KIAA0632 proteinLIV-1 proteinDuodenal cytochrome BVav 3 oncogenecDNA FLJ10561GDNF family receptor alpha 1

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N-myc down-stream regulated gene 2Homolog mouse quaking OKIProtein kinase X-linked.1Protein kinase X-linked.2UNIGENE - ambiguityGS1999fullGamma aminobutyric acid A receptorPotassium intermediateFLJ20005Ataxaia telangiectasia group D associated proteinCtyochromeb5 reductaseProfilin 2BAF 53Ribosomal protein L44Keratin 6BProteasome sub-unit betaHeterogeneous nuclear ribonucleotprotein DAdenylate kinase 2ESTCellular retinoic acid-binding protein 1BTG family member 3Lymphocyte antigen 6 complexMGC5350PH domain containing protein in retina 128kD interferon responsive proteinKIAA0179 proteinChitinase 3-like 2Amyloid beta (A4) precursor like protein 1ESTFlap structure-specific endonuclease1Stathmin 1CDK-associated protein 1Enolase 1CCAAT/enhancer binding protein

FIGURE 14.2 ER status determination. (A) Comparison of ER messenger RNA expression detected by microarray profiling and correspondingER protein expression measured by IHC.The concordance between ER levels determined by IHC and ER levels determined by gene expressionprofiling was about 95%. (B) Genes expressed in ER-positive cases. (C) Genes expressed in ER-negative cases. See Plate 31 for color image.

A

B C

Chapter 14 Targeted Therapy for Cancer 195

overall potency of naked mouse antibodies asanticancer drugs, (3) antibodies penetratedtumor cells poorly, (4) there was limitedsuccess in producing radioisotope and toxinconjugates, and (5) the development of humanantimouse antibodies (HAMA) prevented theuse of multiple dosing schedules.48

The next advance in antibody therapeuticsbegan in the early 1980s when recombinantDNAtechnology was applied to antibody designto reduce the antigenicity of murine andother rodent-derived monoclonal antibodies.Chimeric antibodies were developed where theconstant domains of the human IgG moleculewere combined with the murine variable re-gions by transgenic fusion of the immunoglobu-lin genes; the chimeric monoclonal antibodieswere produced from engineered hybridomasand CHO cells.49,50 The use of chimeric antibod-ies significantly reduced the HAMA responsesbut did not completely eliminate them.51,52 Al-though several chimeric antibodies achievedregulatory approval, certain targets requiredhumanized antibodies to achieve appropriatedosing. Partially humanized antibodies werethen developed where the six complementaritydetermining regions of the heavy and lightchains and a limited number of structuralamino acids of the murine monoclonal antibodywere grafted by recombinant technology to thecomplementarity determining region depletedhuman IgG scaffold.53 Although this processfurther reduced or eliminated the HAMAresponses, in many cases significant furtherantibody design procedures were needed toreestablish the required specificity and affinityof the original murine antibody.54,55

A second approach to reducing the im-munogenicity of monoclonal antibodies hasbeen to replace immunogenic epitopes in themurine variable domains with benign aminoacid sequences, resulting in a deimmunizedvariable domain. The deimmunized variabledomains are genetically linked to human IgGconstant domains to yield a deimmunizedantibody (Biovation, Aberdeen, Scotland). Ad-ditionally, primatized antibodies were subse-quently developed that featured a chimericantibody structure of human and monkeythat, as a near exact copy of a human antibody,further reduced immunogenicity and enabledthe capability for continuous repeat dosingand chronic therapy.56 Finally, fully humanantibodies have now been developed usingmurine sources and transgenic techniques.56

response rates ranged from 67% to 81% com-pared with 41% to 46% in patients with normalexpression of HER-2.40 Interestingly, althoughFISH based testing is more expensive to per-form and is not as widely available as IHC, arecent published review from New York andItaly suggested that FISH was actually themost cost-effective option.41 With trastuzumabachieving excellent results in the treatment ofHER-2–positive advanced metastatic diseaseand under extensive evaluation in major clini-cal trials for its potential efficacy when used inearlier stages, the potential role(s) for HER-2testing as a predictor(s) of responses to othertherapies being resolved by large prospectiveclinical outcome studies and the more conve-nient gene-based chromogenic in situ hy-bridization technique “waiting in the wings,”the story of HER-2 testing in breast cancer willcontinue to unfold over the next several years.

Targeted Anticancer Therapies Using Antibodies

An unprecedented number and variety of tar-geted small molecule and antibody basedtherapeutics are currently in early develop-ment and clinical trials for the treatment ofcancer. Therapeutic antibodies have become amajor strategy in clinical oncology because oftheir ability to specifically bind to primaryand metastatic cancer cells with high affinityand create antitumor effects by complement-mediated cytolysis and antibody-dependentcell-mediated cytotoxicity (naked antibodies) orby the focused delivery of radiation or cellulartoxins (conjugated antibodies).42–47 Currently,there are six anticancer therapeutic antibodiesapproved by the US Food and Drug Adminis-tration (FDA) for sale in the United States(Table 14.2). Therapeutic monoclonal antibod-ies are typically of the IgG class containing twoheavy and two light chains. The heavy chainsform a fused “Y” structure with two light chainsrunning in parallel to the open portion of theheavy chain. The tips of the heavy–light chainpairs form the antigen binding sites, with theprimary antigen recognition regions known asthe complementarity determining regions.

The early promise of mouse monoclonalantibodies for the treatment of human cancerswas not realized because (1) unfocused targetselection led to the identification of targetantigens that were not critical for cancer cellsurvival and progression, (2) there was a low

196 MOLECULAR ONCOLOGY OF BREAST C ANCER

Using modern antibody design and deim-munization technologies, scientists and clini-cians have attempted to improve the efficacyand reduce the toxicity of anticancer antibodytherapeutics.42,48,57–59 The bacteriophage an-tibody design system has facilitated thedevelopment of high affinity antibodies by in-creasing antigen binding rates and reducingcorresponding detachment rates.59 Increasedantigen binding is also achieved in bivalent an-

tibodies with multiple attachment sites, a fea-ture known as avidity. Modern antibody designhas endeavored to create small antibodies thatcan penetrate to cancerous sites but maintaintheir affinity and avidity. A variety of ap-proaches has been used to increase antibody ef-ficacy.42 Clinical trials have recently combinedanticancer antibodies with conventional cyto-toxic drugs, yielding promising results.42–45 Theapplication of radioisotope, a small molecule

Table 14.2 Targeted Anticancer Antibody Therapeutics

FDA Source and Indication(s) (both approvedName Approval Partners Type Target and investigational)

Alemtuzumab 05/01 BTG Monoclonal antibody, CD52 Cancer, leukemia, chronic(Campath®) ILEX Oncology humanized lymphocytic

Schering AG Anticancer, immunologic Cancer, leukemia,

Multiple sclerosis treatment chronic myelogenous

Immunosuppressant Multiple sclerosis,chronic progressive

Daclizumab 03/02 Protein Design Monoclonal IgG1 Chimeric Transplant rejection, general(Zenapax®) Labs Immunosuppressant Transplant rejection, bone

Hoffmann- Antipsoriasis marrowLa Roche Antidiabetic Uveitis

Ophthalmological Multiple sclerosis,

Multiple sclerosis relapsing-remitting

treatment Multiple sclerosis, chronic progressive

Cancer, leukemia, general

Psoriasis

Diabetes, type I

Asthma

Colitis, ulcerative

Rituximab 11/97 IDEC Monoclonal IgG1 CD20 Cancer, lymphoma, non-Hodgkin(Rituxan®) Genentech Chimeric Cancer, lymphoma, B cell

Hoffmann- Anticancer, immunologic Arthritis, rheumatoid La Roche Antiarthritic, immunologic Cancer, leukemia, chronic Zenyaku Kogyo Immunosuppressant lymphocytic

Thrombocytopenic purpura

Trastuzumab 09/98 Genentech Monoclonal IgG1 p185neu Cancer, breast (Herceptin®) Hoffmann- Humanized Cancer, lung, non–small cell

La Roche Anticancer, immunologic Cancer, pancreatic ImmunoGen

Gemtuzumab 05/00 Wyeth/AHP Monoclonal IgG4 CD33/ Cancer, leukemia, AML(Mylotarg®) Humanized coleacheamycin (patients older than

60 years)

Ibritumomab 02/02 IDEC Monoclonal IgG1 CD20/90Y Cancer, lymphoma, low grade,(Zevalin®) Murine follicular, transformed non-

Anticancer Hodgkin (relapsed or refractory)

Edrecolomab 01/95* Glaxo-Smith- Monoclonal IgG2A Epithelial cell Cancer, colorectal (PanorexTM) Kline Murine adhesion

Anticancer molecule(Ep-CAM)

Tositumomab 06/03 Corixa Anti-CD 20 CD20 Cancer, lymphoma,(Bexxar®) Murine non-Hodgkin

Monocolonal antibodywith 131I conjugation

AML, acute myelogenous leukemia.

Chapter 14 Targeted Therapy for Cancer 197

Unconjugated or naked antibodies includea variety of targeting molecules both on themarket and in early and late clinical develop-ment.Avariety of mechanisms has been cited toexplain the therapeutic benefit of these drugs,including enhanced immune effector functionsand direct inactivation of the targeted path-ways as seen in the antibodies directed atsurface receptors such as HER-1 (EGFR) andHER-2.2–5 Surface receptor targeting can re-duce intracellular signaling, resulting in de-creased cell growth and increased apoptosis.56

As seen in Table 14.2, of the seven anti-cancer antibodies on the market, one is conju-gated with a radioisotope Y90-ibritumomabtiuxetan (Zevalin®) and one is conjugated to acomplex natural product toxin gemtuzumabozogamicin (Mylotarg®). Conjugation proce-dures have been designed to improve antibodytherapy efficacy and have used a variety ofmethods to complex the isotope, toxin, or cyto-toxic agent to the antibody.42,43 Cytotoxic smallmolecule drug conjugates have been widelytested, but enthusiasm for this approach hasbeen limited by the relatively low potency ofthese compounds.42 Fungal derived potent tox-ins have yielded greater success with thecalicheamicin conjugated anti-CD33 antibodygemtuzumab ozogamicin approved for thetreatment of acute myelogenous leukemia anda variety of antibodies conjugated with thefungal toxin maytansanoid (DM-1) in preclini-cal development and early clinical trials. Theinterest in radioimmunotherapy increased sig-nificantly in 2001 with the FDA approvals ofthe 90Y-conjugated anti-CD20 antibody Y90-ibritumomab tiuxetan and the 131I-conjugatedanti-CD20 antibody I131-tositumomab. Avariety of isotopes is under investigation inaddition to 90Y as potential conjugates for an-ticancer antibodies.43 Radioimmunotherapyfeatures the phenomenon of the bystandereffect, in which if antigen expression is hetero-geneous, extensive tumor cell killing can stilltake place, even on nonexpressing cells, butcan also lead to significant toxicity when theneighboring cells are vital non-neoplastic tis-sues such as the bone marrow and liver.

Antibody Therapeutics for HematologicMalignancies

The earliest and most successful clinical use ofantibodies in oncology has been for the treat-ment of hematologic malignancies.42–45,56,60–63

cytotoxic drug, and protein toxin conjugationhas resulted in promising results in clinicaltrials and achieved regulatory approval for sev-eral drugs now on the market (see below). Anti-bodies have also been designed to increase theirenhancement of effector functions of antibody-dependent cellular cytotoxicity. Another causeof toxicity of conjugated antibodies has been thelimitations of the conjugation technology,which can restrict the ratio of the number oftoxin molecules per antibody molecule.42,43,55

Methods designed to overcome the toxicity ofconjugated antibodies include the use of anti-body targeted liposomal small molecule drugconjugates and the use of antibody conjugateswith drugs in nanoparticle formats to enhancebonding strength that enable controlled releaseof the cytotoxic agent. Another technique thatuses site selective prodrug activation to reducebystander tissue toxicity is the antibody di-rected enzyme prodrug therapy. An antibodybound enzyme is targeted to tumor cells. Thisallows for selective activation of a nontoxic pro-drug to a cytotoxic agent at the tumor site forcancer therapy.

A variety of factors can reduce antibody ef-ficacy48: (1) limited penetration of the antibodyinto a large solid tumor or into vital regionssuch as the brain, (2) reduced extravasationsof antibodies into target sites due to decreasedvascular permeability, (3) cross-reactivity andnonspecific binding of antibody to normal tis-sues reduces targeting effect, (4) heteroge-neous tumor uptake results in untreatedzones, (5) increased metabolism of injectedantibodies reduces therapeutic effects, and(6) HAMA and human antihuman antibodiesform rapidly and inactivate the therapeuticantibody.

Toxicity has been a major obstacle in thedevelopment of therapeutic antibodies for can-cer.42–45 Cross-reactivity with normal tissuescan cause significant side effects for unconju-gated (naked) antibodies, which can be en-hanced when the antibodies are conjugatedwith toxins or radioisotopes. Immune mediatedcomplications can include dyspnea from pul-monary toxicity, occasional central and pe-ripheral nervous system complications, anddecreased liver and renal function. On occasion,unexpected toxic complications can be seen,such as the cardiotoxicity associated with theHER-2/neu targeting antibody trastuzumab.Radioimmunotherapy with isotopic-conjugatedantibodies can also cause bone marrow suppres-sion (see below).

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By taking advantage of improved recombinanttechnologies generating more specific andhigher affinity monoclonal antibodies with re-duced immunogenicity after humanization ordeimmunization and the emerging conjuga-tion capabilities, antibody therapeutics havebecome a major weapon in the treatment ofleukemias and lymphomas.60–63

Approved in 1997, rituximab (Rituxan®) isarguably the most commercially successful an-ticancer drug of any type since the introductionof taxanes. Rituximab sales exceeded $700 mil-lion in sales in the United States in 2001.44

Targeting the CD20 surface receptor commonto many B cell non-Hodgkin lymphoma sub-types, rituximab is a chimeric monoclonal IgG1antibody that induces apoptosis, antibody-dependent cell cytotoxicity, and complement-mediated cytotoxicity56 and has achievedsignificantly improved disease-free survivalrates compared with patients receiving cyto-toxic agents alone.64–67

Y90-ibritumomab tiuxetan (Zevalin®) con-sists of the murine version of the anti-CD20chimeric monoclonal antibody, rituximab,which has been covalently linked to the metalchelator, MD-DTPA, permitting stable bind-ing of 111In when used for radionucleotidetumor imaging and 90Y when used to produceenhanced targeted cytotoxicity.68–71 In early2002, Y90-ibritumomab tiuxetan became thefirst radioconjugated antibody therapeutic forcancer approved by the FDA. Since its FDAapproval, numerous patients who have re-ceived Y90-ibritumomab tiuxetan after becom-ing refractory to a rituximab-based regimenhave achieved significant responses.69,70

The approval of gemtuzumab ozogamicin(Mylotarg®) by the FDA in 2000 marked thefirst introduction of a plant toxin conjugatedantibody therapeutic.72–76 Gemtuzumab ozo-gamicin is targeted against CD33, a surfacemarker expressed by 90% of myeloid leukemicblasts but absent from stem cells, armedwith calicheamicin, a potent cytotoxic antibi-otic that inhibits DNA synthesis and inducesapoptosis.72 The current indication for use ofgemtuzumab ozogamicin is in acute myeloge-nous leukemia patients older than 60 yearswith the recommendation that before the initi-ation of therapy, the leukemic blast count isbelow 30,000/mL.73–75

Alemtuzumab (Campath®), a humanizedmonoclonal antibody, was approved in mid-2001for the treatment of B-cell chronic lymphocytic

leukemia in patients who have been treatedwith alkylating agents and who have failed flu-darabine therapy.77,78 Daclizumab (Zenapax®)is a chimeric monoclonal antibody that targetsthe interleukin-2 receptor. This antibody is pri-marily used to prevent and treat patients withorgan transplant rejection but has also beenused in a wide variety of chronic inflammatoryconditions, including psoriasis, multiple sclero-sis, ulcerative colitis, asthma, type I diabetesmellitus, uveitis, and also in a variety ofleukemias.79,80 I131-tositumomab (Bexxar®) is aradiolabeled anti-CD20 murine monoclonal an-tibody approved in 2003 for the treatment of re-lapsed and refractory follicular/ low grade andtransformed non-Hodgkin lymphoma.81,82

Antibody Therapeutics for Solid Tumors

Interest in the development of antibody thera-peutics for solid tumors among many commer-cial organizations and universities has beensignificantly impacted by the technologicadvances in antibody engineering and theapproval and recent clinical and commercialsuccess of trastuzumab, the only therapeuticantibody approved by the FDA for the treat-ment of solid tumors (edrecolomab is approvedin Germany, but not in the United States).Trastuzumab has been described extensivelyabove.

During the 4 years since the FDA approvalof trastuzumab, there have been no additionalantibodies approved for the treatment of solidtumors. Nonetheless, significant progress hasbeen made in this field, and a number of bothlate stage and early stage products show sub-stantial promise.

Cetuximab (Erbitux®)The EGFR (HER-1) is the target of a variety ofsmall molecule drugs and the late stage anti-body cetuximab.83 Cetuximab, a chimeric mono-clonal antibody, binds to the EGFR with highaffinity, blocking growth factor binding, recep-tor activation, and subsequent signal transduc-tion events and leading to cell proliferation.84

Cetuximab enhanced the antitumor effects ofchemotherapy and radiotherapy in preclinicalmodels by inhibiting cell proliferation, angio-genesis, and metastasis and by promotingapoptosis.84 Cetuximab has been evaluatedboth alone and in combination with radiother-apy and various cytotoxic chemotherapeutic

Chapter 14 Targeted Therapy for Cancer 199

an approval by the FDA of bevacizumab forthe treatment of metastatic colorectal cancerin February 2004.

Unlike cetuximab, the development of be-vacizumab has not included a diagnostic eligi-bility test. Neither direct measurement ofVEGF expression or assessment of tumor mi-crovessel density has been incorporated into theclinical trials or linked to the response rates tothe antibody. Thus, bevacizumab cannot be con-sidered a true targeted therapy, and further fo-cusing of this agent for colorectal, breast, lung,and other cancers will likely be inhibited by theinability to individually select patients with adiagnostic test who will be more likely to benefitfrom its use, either alone or in combination withother known and novel drugs.

Edrecolomab (Panorex®)Edrecolomab is a murine IgG2A monoclonalantibody that targets the human tumor-associated antigen Ep-CAM (17-1A). Edre-colomab has been approved in Europe(Germany) since 1995, but to date has not beenapproved by the FDA. In a study of 189 pa-tients with resected stage III colorectal can-cer, treatment with edrecolomab resultedin a 32% increase in overall survival com-pared with no treatment (P � 0.01).96 Edre-colomab’s antitumor effects are mediatedthrough antibody-dependent cellular cytotoxi-city, complement-mediated cytolysis, and theinduction of an anti-idiotypic network.97 Edre-colomab is also currently being tested in largemulticenter adjuvant phase III studies instage II/III rectal cancer and stage II coloncancer. Edrecolomab was well tolerated whenused as monotherapy and added little tochemotherapy-related side effects when usedin combination. Sequential treatment of pa-tients with metastatic breast cancer with edre-colomab after adjuvant chemotherapy reducedlevels of disseminated tumor cells in the bonemarrow and eliminated Ep-CAM–positivemicrometastases.98

huJ-591 (Anti-PSMAEXT)Prostate-specific membrane antigen (PSMA)is a membrane-bound glycoprotein restrictedto normal prostatic epithelial cells, prostatecancer, and the endothelium of the neovascu-lature of a wide variety of nonprostatic carci-nomas and other solid tumors.99–101 PSMAexpression per cell progressively increases inprimary prostate cancer, metastatic hormone

agents in a series of phase II/III studies thatprimarily treated patients with either headand neck or colorectal cancer.84,85 Breast cancertrials are also underway.86 Although the FDAapproval process for cetuximab was initiallyslowed because of concerns over clinical trialdesign and outcome data management,87 theantibody was approved for use in the treat-ment of advanced metastatic colorectal cancerin February 2004. Similar to trastuzumab, thedevelopment of cetuximab also included an im-munohistochemical test for determining EGFRoverexpression to define patient eligibility toreceive the antibody. Thus, cetuximab hasjoined trastuzumab as an FDA-approved tar-geted therapy featuring an unconjugated anti-body. Although anti-EGFR small moleculedrugs (see below) are under continuing clinicaltrial evaluation in breast cancer,88 cetuximab isnot currently projected as a potential therapeu-tic for this disease.

Bevacizumab (Avastin®)Bevacizumab (rhuMAb-VEGF) is a human-ized murine monoclonal antibody target-ing the vascular endothelial growth factor(VEGF) ligand. VEGF regulates both vascularproliferation and permeability and functionsas an antiapoptotic factor for newly formedblood vessels.89,91 Patients treated with beva-cizumab alone have shown significant tumorresponses. Patients treated with bevacizumabin combination with conventional chemother-apy have had greater responses.89,91 In clinicaltrials for advanced metastatic breast cancer,the initial results of the combination treat-ment of bevacizumab and paclitaxel showedantitumor activity,92 but follow-on studieswere not convincing that the targeting ofVEGF in this clinical setting would be effec-tive. Bevacizumab has also been combinedwith trastuzumab in a two antibody therapeu-tic strategy for HER-2 overexpressing breastcancer.93 The phase II study evaluating beva-cizumab in metastatic renal cell carcinomareached its prespecified efficacy end point ear-lier than expected. Finally, late-stage clinicaltrials using bevacizumab with 5-fluorouracil,leucovorin, and CPT-11 (Irinotecan®) in ad-vanced colorectal cancer are currently un-derway,94 with recently released resultsindicating a major prolongation in the time todisease progression and overall survival in pa-tients who received the angiogenesis inhibitorversus those who did not.95 These data led to

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sensitive prostate cancer, and hormone refrac-tory metastatic disease.99–101 PSMA expressionis increased further in association with clini-cally advanced prostatic cancer, particularlyin hormone refractory disease, and is an idealsentinel molecule for use in targeting prostaticcancer cells. Increasing expression levels ofPSMA in resected primary prostate cancer isassociated with increased rates of subsequentdisease recurrence.100 In addition, significantPSMA expression has been identified in thetumor vasculature of a variety of nonprostatetumors, including breast cancer (FIG.14.3).101 Hu-manized and fully human antibodies specificfor the extracellular domain of PSMA havebeen developed. A phase I clinical trial of oneof these antibodies, huJ-591 conjugated with90Y, has yielded promising results.102 Pro-grams using toxin conjugates with anti-PSMAantibodies have completed preclinical devel-opment103 and are currently in early stageclinical trials for hormone-refractory advancedmetastatic prostate cancer. Finally, antibodiesto PSMA have been used as diagnostic imag-ing agents (FIG. 14.3), including the commerciallyavailable Prostascint®.104

Selected Targeted Anticancer TherapiesUsing Small Molecules

Table 14.3 lists selected small molecule drugsdesigned to target specific genetic events andbiologic pathways critical to cancer growthand progression.

ATRAArguably the first truly targeted therapyafter the development of hormonal therapyfor breast cancer was the development ofATRA for the treatment of acute promyelo-cytic leukemia, a subset of acute nonlympho-cytic leukemia featuring a disease-definingretinoic acid receptor activating t(15:17) recip-rocal translocation.105,106 For these selected pa-tients, direct targeting of the retinoic acidreceptor with ATRA has resulted in very highresponse rates, delay in disease progression,and long-term cures for these patients.105,106

Imatinib (Gleevec®)The development of imatinib for patients withchronic myelogenous leukemia in 2001 ush-ered in a new excitement both in the scientificand public communities for targeted anti-cancer therapy. Imatinib received fast-trackapproval by the FDA as an ATP-competitiveselective inhibitor of bcr-abl and has unprece-dented efficacy for the treatment of early stagechronic myelogenous leukemia typicallyachieving durable complete hematologic andcomplete cytogenetic remissions, with mini-mal toxicity.107–109 Imatinib is a true targetedtherapy for leukemia in that a test for thebcr/abl translocation must be performed be-fore a patient will be considered as eligible toreceive the drug.

Imatinib has also achieved regulatory ap-proval for the treatment of relapsed andmetastatic gastrointestinal stromal tumors(GISTs), which characteristically feature anactivating point mutation in the c-kit receptortyrosine kinase gene.110 For GISTs, the re-sponse to imatinib treatment appears to bepredictable based on the location of the c-kitmutation.111 The use of imatinib in GISTis also an example of targeted therapy as ameasurement of c-kit expression usually per-formed by IHC, required to confirm the di-agnosis and render the patient eligible fortreatment. Interestingly, most commerciallyavailable antibodies for c-kit recognize the

FIGURE 14.3 PSMA expression in non-prostate cancer. (Top) Tradi-tional bone scan demonstrating bilateral activity in the femur in-directly indicating the presence of metastatic renal cell carcinoma.(Bottom) 111I-huJ591EXT diagnostic immunoscintiscan of the samepatient showing direct localization of the anti-PSMA antibodyconjugate to the sites of metastatic renal cell carcinoma that fea-ture PSMA expression in the tumor neovasculature.

Chapter 14 Targeted Therapy for Cancer 201

an internal tandem duplication that creates anabnormal FLT-3 receptor that promotes thegrowth and survival of the leukemic cells.113–115

Three small molecule compounds are in clinicaltrials for the treatment of acute myelogenousleukemia by targeting the flt-3 internal tandemduplication (ITD). These drugs are also exam-ples of potential true targeted therapies in thata test for detecting an internal tandem duplica-tion that causes the flt-3 gene activation willlikely be required and incorporated into theFDAdrug approval label should these agents besuccessful in future clinical trials.

Gefitinib (Iressa®)Gefitinib was approved by the FDA in 2003 asmonotherapy for the treatment of patientswith locally advanced or metastatic non–smallcell lung cancer after failure of both platinumbased and docetaxel chemotherapies.116,117

total c-kit and do not distinguish the activatedor phosphorylated version, which is the actualtarget of imatinib.

Currently, the high treatment failure rateis directly linked to the test used to character-ize the patients. It is anticipated that eitherthe use of specific antibodies designed to iden-tify the activated c-kit gene or directed se-quencing of the c-kit gene may be requiredbefore imatinib is prescribed for patients withrecurrent or metastatic GIST. Although c-kitactivation has not been linked to breast can-cer, the platelet derived growth factor �, an-other target of imatinib, has been associatedwith breast cancer biology.112

FLT-3 Targeted TherapyIn approximately 30% of cases of acute mye-logenous leukemia and less frequently in otherforms of leukemia, a flt-3 gene mutation creates

Table 14.3 Selected Small Molecule Drugs Designed to Target Specific Genetic Events and Biologic Pathways Critical to CancerGrowth and Progression

Clinical DevelopmentTarget Drug Source Status Comment

PML-RAR-� ATRA Promega Approved First true targeted therapy sincein PML the introduction of ER testing and

hormonal therapy for breast cancer

bcr/abl Imatinib Novartis Approved Has emerged as standard of carein CML for early stage CML

c-kit in GIST Imatinib Novartis Approved Responses in relapsed/metastaticPDGF-� GIST can be predicted by the location

of the activating c-kit mutation

Flt-3 in AML SU5416 Pfizer Early stage Small molecule drugs that targetPKC412 Novartis clinical trials the flt-3 internal tandem MLN-518 Millennium duplication seen in 30% of AML

EGFR in NSCLC Gefitinib Astra Zeneca Approved Preclinical activity in breast cancer; clinical trials are ongoing

EGFR in Erlotinib Genentech/OSI Pending In late stage trials in NSCLC and glioblastoma recently (ASCO 2003) shown

efficacy in treatment of high grade malignant gliomas

Antiangiogenesis Thalidomide Celgene Pending Thalidomide is approved for SU 5416 Pfizer/Sugen treatment of leprosy and widely ZD6474 Astra Zeneca used to treat multiple myeloma;Endostatin Entremed other agents are in early and Marimastat British Biotech mid-stage clinical trialsOthers Others

Bcl-2 G3139 Genta Pending Antisense oligonucleotide targetsthe antiapoptotic gene, bcl-2

Proteasome in Bortezomib Millennium Approved Proteasome inhibition effectivemultiple myeloma in hematologic malignancies, but

of uncertain potential for thetreatment of solid tumors

AML, acute myelogenous leukemia; CML, chronic myelogenous leukemia; NSCLC, non–small cell lung cancer; PDGF, platelet derived growth factor; PML, acutepromyelocytic leukemia; RAR, retinoic acid receptor.

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Gefitinib is a small molecule drug that tar-gets the EGFR. In contrast with the approvalof trastuzumab, the approval of gefitinib didnot include an eligibility requirement refer-ence to a specific tumor diagnostic test de-signed to select patients that were more likelyto respond to the drug. Overexpression ofEGFR typically identified by IHC is extremelycommon in both lung and breast cancers,116–118

but in contrast with HER-2 overexpression,which is virtually limited to cases with geneamplification, multiple mechanisms of dysreg-ulation of EGFR and associated activation ofsignaling pathways have been described forboth of these tumors (see Chapter 17).116–118

Thus, it has been difficult to develop this drugfor expanded indications or combination thera-pies in the absence of a well-defined efficacytest. However, more recently, two independentgroups reported their similar discovery of aspecific activating mutation in the tyrosine ki-nase domain of the EGFR receptor that was as-sociated with a high response of patients withnon-small cell lung cancer to gefitinib.119–120

The clinical significance of this important find-ing for the development of gefitinib in breastcancer is currently unknown. Preclinical stud-ies of gefitinib have demonstrated activity ionbreast cancer cell lines,118 and clinical trialsusing the drug in combination with standardcytotoxic drugs are underway (see Chapter 17).

Erlotinib (Tarceva®)Erlotinib is another targeted inhibitor ofEGFR currently in late stage clinical trials forthe treatment of non–small cell lung cancerand pancreatic cancer.121,122 Clinical studies oferlotinib in breast cancer are ongoing (seeChapter 17). Erlotinib has shown efficacy inpreclinical models of brain tumors123 and hasrecently shown promising results in the treat-ment of high grade malignant gliomas. Todate, similar to gefitinib, the clinical trials forerlotinib have not included an assessment ofthe EGFR status or other diagnostic test foreligibility to receive the drug.

Antiangiogenesis (SU5416, Thalidomide,Endostatin/Angiostatin, and Marimastat)A variety of small molecule drugs is currentlyin clinical trials for breast cancer and othermalignancies that target the establishmentand growth of tumor blood vessels.124–127

Additional compounds that target matrix me-talloproteases, such as the drug marimastat,are also considered to be angiogenesis in-hibitors.128,129 To date, none of these compoundshas linked a diagnostic test such as tumormicrovessel density or the expression of an an-giogenesis promoting gene or protein in theirclinical development plans. Matrix metallopro-tease inhibitors are discussed in Chapter 18,and antiangiogenesis therapies are discussedin detail in Chapter 19.

G3139 (Genasense®)Another emerging strategy in anticancertherapy is the targeting of chemotherapyresistance by overcoming the antiapoptosismechanisms of cancer cells. An example of thisapproach is the novel antisense oligonu-cleotide G3139, which targets the antiapop-totic gene bcl-2.130,131 This agent has beentested mostly in hematologic malignanciesand has not been used widely in either pre-clinical experiments or clinical trials in breastcancer. Therapeutic strategies targeting apop-tosis are covered in depth in Chapter 21.

Bortezomib (Velcade®)Recently, drugs targeting the proteasome havebeen developed that are designed to impactdownstream pathways regulating angiogene-sis, tumor growth, adhesion, and resistance toapoptosis.132,133 One of these agents, borte-zomib (PS-341), has recently been approvedfor the treatment of advanced refractory mul-tiple myeloma.134 Bortezomib has shown pre-clinical activity in breast cancer as a singleagent and is being tested in combination ther-apy strategies in multiple clinical trials.135

Proteasome biology and proteasome inhibitorsare discussed in detail in Chapter 24.

The Future Is Now: Pharmacogenomicsand Personalized Medicine

Targeted therapy in oncology has been a majorstimulus for the evolving field of pharmacoge-nomics (see Chapter 28). In its broadest defini-tion, pharmacogenomics can encompass bothgermline and somatic (disease) gene and pro-tein measurements used to predict the likeli-hood that a patient will respond to a specificsingle or multiagent chemotherapy regimen

Chapter 14 Targeted Therapy for Cancer 203

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