Blood Reviews (2004) 18, 137–148

www.elsevierhealth.com/journals/blre

Novel therapies for chronic lymphocytic leukemia

Blanche H. Mavromatis, Bruce D. Cheson*

Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA

Summary New therapies for chronic lymphocytic leukemia (CLL) are moving awayfrom non-specific cytotoxic to more targeted approaches. The monoclonal antibodyalemtuzumab induces responses in 33% to 43% of patients with relapsed or refractorydisease, with 2–5% CR. Side effects include infusional reactions as well asimmunosuppressive effects. Rituximab has limited activity in relapsed refractorypatients, but response rates are comparable to follicular non-Hodgkin’s lymphoma inuntreated patients. Other antibodies in early phases of development include anti-CD23 [IDEC-152], anti-CD22 [epratuzumab], Hu1D10 [apolizumab], and anti-CD80[anti-B7, IDEC-114]. Other agents that are being studied include denileukin diftitoxfusion protein (Ontak), and bcl-2 antisense [G3139, Genasense].

The mechanism of action of the new drugs and their role in CLL, as well as theemergence of new prognostic markers are discussed.

�c 2003 Elsevier Ltd. All rights reserved.

KEYWORDSChronic lymphocytic

leukemia;Alemtuzumab;Anti-CD23;Epratuzumab

Introduction

Chronic lymphocytic leukemia (CLL) is the mostcommon leukemia in adults in Western countrieswith about 7000 new cases being diagnosed in theUS in 2002.1 CLL is being increasingly detected inits earlier stages, as a result of routinely performedautomated blood counts. More than half of patientsare asymptomatic at diagnosis, and may be wat-ched closely until the development of symptoms,or rapid and extensive progression of the disease.Nevertheless, CLL remains incurable with currentlyavailable therapies.

Prognostic factors

At the time of diagnosis, it is often difficult todistinguish those patients who are likely to remain

* Corresponding author. Tel.: +1-202-444-7064; fax: +1-202-444-1229.

E-mail address: bdc4@georgetown.edu (B.D. Cheson).

0268-960X/$ - see front matter �c 2003 Elsevier Ltd. All rights reserdoi:10.1016/S0268-960X(03)00039-0

stable from those who will progress, and requiretreatment. Prognostic factors have traditionallyrelied on disease stage,2 based primarily on tumorbulk. A number of new prognostic factors haverecently been proposed to better identify patientsat increased risk of needing treatment, and even-tually to dissect specific therapies. These new di-rections are based on cytogenetic changes, as wellas molecular and immunophenotypic studies.

CytogeneticsThe presence of certain cytogenetic abnormalities,most reliably detected by fluorescent in situ hy-bridization (FISH) can be associated with differ-ences in median survival. D€ohner et al.3 reportedthat 268 of 325 patients had a recurrent cytoge-netic abnormality by FISH. They were able to groupthese patients into distinct categories dependingon their associated cytogenetic abnormality: 17pdeletion, 11p deletion, trisomy 12, normal, and 13qdeletion. Significant differences in the medianoverall survival were observed. In patients with the17p del, the median survival was only 32 months,

ved.

138 B.H. Mavromatis, B.D. Cheson

compared to 133 months for those with a single 13qdel.

Molecular/geneticMutations of the VH genes are found in 50–75% of B-CLL cells with the percentages varying with the VHfamily.4 In clinical studies, the presence of mutatedVH genes correlates with a longer median survivalcompared to patients with unmutated VH genes. Ina study of 62 patients with early stage CLL, Hamblinand colleagues5 demonstrated a median survival of293 months for the patients with mutated VH genescompared to 95 months for those with unmutatedVH genes. The possible existence of two separatediseases was thus entertained, until the develop-ment of gene expression profiling. cDNA microarrayanalysis, first applied to diffuse large B-cellnon-Hodgkin’s lymphomas, was introduced in CLL.Rosenwald et al.6 were able to identify a gene ex-pression signature common to mutated and unmu-tated CLL, with a pattern that distinguished themfrom normal B-cells or other B-cell malignancies.The expression of hundreds of genes in the unmu-tated VH gene CLL patients however still distin-guished them from those with mutated VH genes.These included tyrosine kinases such as ZAP-70,and FGFR1. Microarray analysis also revealed theinvolvement in CLL of other genes not previouslyknown to be expressed, such as Wnt, titin, andRor1. The Wnt genes express proteins involvedin proliferation and malignant transformation,7

while the Ror1 protein is a tyrosine kinase with anextracellular domain very similar to that of Wnt.

ImmunophenotypeCD38 is a surface glycoprotein expressed on plasmacells and bone marrow precursor cells, and occa-sionally in CLL cells as well. It is involved in calciumregulation, but additionally functions as amodulatorof cell–cell interactions together with CD16 and NK-cells.8 The presence of CD38 on the CLL cell alsoserves as a prognostic marker.9 In a study of 145patients with CLL,10 the expression of CD38 antigenon more than 30% of CLL cells correlated with amedian survival of 8 years, compared to 26 years forthose patients whose CLL cells lacked CD38. CD38expression appears to be independentof theVHgenemutation status, and the role of both as prognosticmarkers is being further tested in clinical trials.11–13

Therapy of CLL

Once the need to treat a patient is established,14

the next step is to chose the most suitable therapy.For many years, standard therapies included the

alkylating agent chlorambucil. Recently, however,nucleoside analogs such as fludarabine becamepreferred because of their higher response ratesand longer response durations than chlorambucil.Nevertheless, no prolongation of overall survivalhas been observed.15;16 Fludarabine has also beencombined with alkylating agents such as cyclo-phosphamide. A study of treated and untreatedpatients who received fludarabine (30 mg/m2/dayfor 3 days), and cyclophosphamide (300–500 mg/m2/day for 3 days) failed to show a clear benefitover fludarabine alone when compared to historiccontrols.17 In another study of 37 patients in whichthese agents were given at a different schedule inpatients with untreated CLL (fludarabine 20 mg/m2/day for 5 days, and cyclophosphamide 600 mg/m2 on day 1 with G-CSF),18 a 42% CR and 64% ORRwas achieved. A phase III intergroup (CALGB andECOG) trial is currently evaluating fludarabinecompared to fludarabine and cyclophosphamide inpreviously untreated CLL patients.

New agents

Newagents are clearly needed to improve the courseof this commonhematologicmalignancy. Treatmentstrategies are now moving away from non-specificcytotoxic agents towards the use of targeted ther-apies. These include monoclonal antibodies, smallmolecules, and molecular therapies.

Targeting surface proteins

Monoclonal antibodiesThe characteristic immunophenotype of CLL cellsmakes them ideal targets for monoclonal antibodytherapy. CLL cells express CD19, CD20, CD5, CD23,CD24, CD26, CD40, CD79b, and occasionallyCD38.9;10;19;20 Antibodies against several of thesesurface proteins are currently being investigated(Table 1).

Alemtuzumab (CAMPATH-1H). Alemtuzumab is ahumanized anti-CD52 monoclonal antibody. TheCampath family of monoclonal antibodies was dis-covered in the 1980s during a search for antibodiesagainst T-cells after activation of human comple-ment.21 Although the function of CD52 is not yetknown, this antigen is expressed on most lymphoidcells at various stages of development, and alsocuriously in the male reproductive tract, as theonly non-lymphoid site. After the initial dose find-ing trials, alemtuzumab is now usually adminis-tered intravenously three times a week, with astarting dose of 3 mg on day 1, then 10 mg on day 2

Table 1 Monoclonal antibodies for CLL

Antibody Antigen Conjugate

CAMPATH-1H CD52 NoneRituximab (Rituxan) CD20 NoneEpratuzumab CD22 NoneHu-1D10 (Apolizumab) HLA-DR NoneIDEC-152 CD23 NoneIDEC-114 CD80 NoneBevacizumab VEG-F NoneDenileukin diftitox (Ontak) CD25 IL-2/diphtheriaBL-22 CD22 Pseudomonas

Novel therapies for chronic lymphocytic leukemia 139

to 30 mg on day 3 as tolerated for 12–18 weeks(Table 2). Because of its immunosuppressive ef-fects, prophylaxis against pneumocystis carinii,and herpes virus are strongly recommended.€Osterborg et al.22 reported the results of a phase IItrial in 29 patients with previously treated CLL.Although only 3 had received prior therapy with anucleoside analog, a 4% CR and 42% RR wasachieved. An impressive clearing of CLL cells fromthe blood was observed in 97%. A CR was achievedin the bone marrow in 36%, with a 32% reduction inspleen size, though little effect was seen on lym-phadenopathy. The median duration of responsewas 12 months. Bone marrow suppression was not

Table 2 CAMPATH-1H in CLL

Investigator Pts Prior

€Osterborg1 29 +Bowen2 6 +Rawstron3 17 +McCune4 13 +Stilgenbauer5 11 +Keating6 92 +€Osterborg7 11 )Lundin8 41 )

1 €Osterborg15 €Osterborg A, Dyer MJS, Bunjes D, et al. Ppreviously treated chronic lymphocytic leukemia. J Clin On

2 Bowen AL, Zomas A, Emmett E, et al. Subcutaneouslymphocytic and B-prolymphocytic leukaemia. Br J Haemat

3 Rawstron AC, Davies FE, Evans P, et al. CAMPATH1H thleukemia (CLL). Blood 1997;90:529a (abstr 2356).

4 McCune SL, Gockerman JP, Moore JO, et al. Alemtuzumkemia and prolymphocytic leukemia. Leuk Lymph 2002;43:

5 Stilgenbauer S, Scherer K, Kr€ober A, et al. Campath-1Hgene mutation. Blood 2001;98:771a (abstr 3211).

6 Keating MJ, Flinn I, Jain V, et al. Therapeutic role of alfludarabine: results of a large international study. Blood 20

7 €Osterborg A, Fassas AS, Anagnostopoulos A, et al. Humantreatment in chronic lymphocytic leukaemia. Br J Haemato

8 Lundin J, Kimby E, Bj€orkholm M, et al. Phase II trialmtuzumab (CAMPATH-1H) as first-line treatment for patieBlood 2002;100:768–73.

very common; however, these encouraging resultswere achieved at the expense of significant im-munosuppression. Opportunistic infections in-cluded 11 cases of localized herpes infection, 5oral candidal infections, and 2 patients withpneumocystis carini pneumonia.

In the pivotal trial,23 alemtuzumab was admin-istered to 93 patients with CLL who were refractoryto or had relapsed after fludarabine and alkylatortherapy. Prophylactic trimethoprim/sulfamethox-azole and famcyclovir were administered. Theplanned 12 weeks of therapy was completed by 70%of patients. The overall RR was 33% including 2%CR. The median duration of response was 32months in the responders. An additional 6 patients(7%) had a clearing of the blood, but remainedthrombocytopenic and anemic. This study confirmsthe activity of alemtuzumab in heavily pretreatedpatients, even in those who never responded tofludarabine. No responses were seen in patientswith nodes greater than 5 cm in diameter and thedrug was less active in those patients with a per-formance status of greater than 2. Infusion relatedevents were experienced by 90% of patients,though most were of grade I or II in intensity anddecreased with time. Such symptoms includedrigors in 90% (grade III in 14%) and fever in 85% (17%

therapy CR (%) RR (%)

4 4250 5050 7031 4618 552 33

33 8919 87

hase II multicenter study of human CD52 antibody incol 1997;15:1567–74.CAMPATH-1H in fludarabine-resistant/relapsed chronicol 1997;96:617–19.erapy for patients with refractory chronic lymphocytic

ab in relapsed or refractory chronic lymphocytic leu-1007–11.in refractory B-CLL – complete remission despite p53

emtuzumab (CAMPATH-1H) in patients who have failed02;99:3554–61.ized CD52 monoclonal antibody Campath-1H as first-linel 1996;93:151–53.of subcutaneous anti-CD52 monoclonal antibody ale-nts with B-cell chronic lymphocytic leukemia (B-CLL).

140 B.H. Mavromatis, B.D. Cheson

grade III, 3% grade IV). Nausea occurred in 53% (allgrade I or II), vomiting in 38% (1% grade III), a rashin one-third, all mild to moderate in severity. Themore serious infusional symptoms included dysp-nea in 28% (26 pts, 14 with grade III severity) andhypotension in 17%. Infections were common, oc-curring in 55% of patients during the study, ofwhich 25 were of grade III or IV in severity. Sepsisoccurred in 14 patients, with two deaths. Fur-thermore, 18 patients developed opportunistic in-fections. These included CMV reactivation in 7patients (3 with grade II and 4 with grade III). Ofthe 9 deaths that occurred within 30 days of initi-ating treatment, 5 were due to an infection. Whilethe development of opportunistic infections was ofconcern, these patients were heavily pretreated,and half had experienced an infection in the monthprior to enrolling on the trial.

In a report of the compassionate alemtuzumabexperience (CAM 511) designed to establish safetyand efficacy in a broader setting, the antibody wasadministered primarily by community based he-matologists–oncologists to 223 patients with CLLand T-PLL. Of the 152 patients with CLL, a CR wasreported in 5%, with an overall RR of 43%, responserates similar to the pivotal trial. Grade III or IVtoxicity included anemia 14%, neutropenia 22%,and thrombocytopenia 29%. CMV infections wereseen in 1.5% (4 patients), with one patient dying asa result. Infections resulted in 5 other deaths.No additional infectious complications werereported.24

Recent reports suggest that the administrationof alemtuzumab via the subcutaneous route allowsfor the attainment of blood levels equivalent to thetrough levels when given intravenously.25 There-fore, in an attempt to decrease the number ofinfusional related toxicities seen with the intra-venous alemtuzumab, Lundin et al.26 evaluatedalemtuzumab administered subcutaneously to 41untreated CLL patients all requiring interventionby standardized criteria.27 Treatment was contin-ued to 18 weeks. An impressive overall responserate of 87% was reported, with 19% complete re-missions. Severe infusional reactions with rigorsoccurred in only 17% of patients, unimpressivelocal injection site reactions were seen in 90% ofpatients.

Neutropenia developed in 21% of patients, CMVreactivation in 4 patients, and PCP pneumonia in apatient who had not received prophylaxis becauseof a sulfa allergy. This trial demonstrates that thisalternative route of administration is associatedwith fewer infusional reactions, while maintainingactivity. A larger confirmatory trial is being plan-ned in Europe.

Attempts to maximize the effect of ale-mtuzumab have included combining it with otherknown active drugs given either concurrently, orsequentially. Kennedy and colleagues reported onthe combination of alemtuzumab and fludarabinein 6 patients, previously shown to be refractory toeach agent used alone. Two months after thecompletion of therapy, 1 patient had a CR and 4had a PR. This combination warrants further in-vestigation, with additional collection of pharma-cokinetic and toxicity data.28

The administration of fludarabine followed byalemtuzumab is another approach of combinationtherapy. A phase II study was conducted by theCancer and Leukemia Group B (CALGB) including 56evaluable, previously untreated patients. Treat-ment involved 4 monthly cycles of fludarabine (25mg/m2 qd� 5) followed by a 2 month break period.Patients who did not progress were treated withalemtuzumab for 6 weeks.29 Prophylactic acyclovirand trimethoprim-sulfammethoxazole therapy wasused in all patients during alemtuzumab therapyand continued for an additional 6 months. Follow-ing the fludarabine period, a 4% CR and 52% PR wasachieved. These response rates were much lowerthan the group’s prior experience with fludarabinealone, possibly because of the administration of 4rather than the more standard 6 cycles of fludara-bine.15 Major infections were observed in 11 pa-tients, and 1 patient died of gram positivesepticemia. Alemtuzumab was subsequently ad-ministered to 36 patients, after which 15 CRs(42%), and an overall RR of 92% were observed. Theaddition of alemtuzumab converted 3 of the 11patients with stable disease post-fludarabine to aCR and 5 to a PR. The CR rate increased from 4% to42%, while the ORR remained essentially un-changed. Toxicity from this approach was sub-stantial: 8 patients developed CMV infection duringor within 4 months of therapy, one of whom died,reinforcing the need for CMV surveillance duringalemtuzumab therapy. The future of this sequenceis unclear. Whether maintenance alemtuzumab canprolong response after initial therapy is also beingexplored in patients with refractory CLL.30

Alemtuzumab has also been combined with rit-uximab. In a phase I study conducted by Faderlet al.,31 patients with relapsed or refractory lym-phoid malignancies expressing both CD20 and CD52(including 32 patients with CLL, 9 with CLL/PLL, 1PLL, 4 mantle cell lymphoma, and 2 with Richter’stransformation) were treated with alemtuzumabtogether with rituximab. Prior regimens rangedfrom 1 to 9.

Approximately half of the patients were refrac-tory to fludarabine therapy. Rituximab was

Novel therapies for chronic lymphocytic leukemia 141

administered weekly for four doses, and ale-mtuzumab was given on days 2, 3, and 4 at 3, 10,and 30 mg, respectively, on week 1, then days 3and 5 of subsequent weeks. The treatment wasgiven for 2–4 weeks. Responses were reported in20 of the 32 CLL patients (ORR 63%), with 2 CR and1 nPR. No responses were seen in the patients withRichter’s transformation, or mantle cell lym-phoma. In evaluating the response rate, one has toconsider the brief course of alemtuzumab com-pared to previous studies (4 weeks instead of12–18 weeks). Infusion-related events were themost frequently observed side effects, and were ofgrade II or less in severity. These included fevers(75%), rigors (67%), skin reactions (38%), GI symp-toms (27%), and dyspnea (25%). Infectious side ef-fects included CMV reactivation (27%), fever ofunknown origin (13%), pneumonia (10%), and si-nusitis (6%). This combination will need to be fur-ther studied.

Rituximab. Rituximab is a chimeric monoclonalantibody directed against the CD20 antigen presenton B-cells. The rituximab pivotal trial showed alower response rate of 13% for the 30 SLL patientscompared to the patient group as a whole, whichincreased to a 45% CR and a 17% PR using consensuscriteria.32 Potential explanations include differ-ences in CD20 expression, shorter half-life of theantibody with high numbers of circulating whitecells, and the presence of soluble CD20.33 Partialresponses using single agent rituximab therapy inpreviously treated CLL/SLL patients have rangedfrom 0% to 45% with no complete responses (Table3). Patients with previously untreated SLL, how-ever appear to have higher response rates. In aseries of 41 untreated patients by Hainsworthet al.,34 the 15 patients with SLL achieved aresponse rate of 57%.

In another study,35 21 early stage CLL patients,at high risk based on an elevated b-2-microglobu-lin, were also treated with single agent rituximab.Overall response rates were 90% with 19% CR. Aslightly different approach was tested by Hains-worth HD et al. in 62 untreated patients with in-dolent lymphomas, including 24 patients with SLL.Rituximab was administered weekly for 4 weeks,followed by four similar courses of rituximab 6months apart in those who had an objective re-sponse or stable disease after the first month. Pa-tients with all stages of disease were included, butthose requiring a rapid response to therapy wereexcluded. Three quarters of the patients weredeemed to require treatment by their physician.Response rates were measured at 6 weeks andagain at the end of therapy. The initial responsesincluded 7% CRs, 40% PRs, and 45% SD which in-

creased to 37% CR and 37% PRs at the end oftherapy. The ORR for the SLL patients was 70%.36

A number of attempts have beenmade to improvethe efficacy of rituximab. Increasing the dose orusing a more dose intensive schedule has been metwith only modest improvements in partial responserates, but a marked increase in cost.37;38 A morepromising direction has been the use of rituximab incombination with nucleoside analogs. Pre-clinicalstudies have suggested a chemosensitizing effect ofantibody therapy when given prior to chemother-apy.39 The cancer and leukemia Group B conducteda randomized phase II trial in which patients re-ceived six cycles of fludarabine concurrent withrituximab followed by a 2 month break. Fludarabinewas given at 25 mg/m2 daily for the first 5 days ofeach cycle, while rituximab was administered onday 1 of each cycle (except for the first cycle whereit was given twice during the first week in 44 pa-tients). Patients randomized to the sequential armreceived 6 cycles of fludarabine, followed by a 2month rest period. Patients in either arm of the trialwith stable or better disease were treated with 4weekly doses of rituximab. The overall responserate in the 51 patients treated with the concurrentregimen was 90% with 47% CR, compared to 77% ORRwith 28% CR in the 53 patients on the sequential arm.The complete response rate for the concurrent armwas much higher than anticipated when comparedto historical controls of fludarabine alone. Whetherthe difference in rituximab treatments in each armmight have impacted on the response rates is notknown.

After a median of 23 months of follow up, 18patients from the concurrent arm and 15 patientsfrom the sequential arm experienced a relapse.The estimated 2 year PFS was 70% for both groups.Among 104 patients, 8 deaths were observed.These included six patients from the concurrentarm (of whom two were in CR, with one dying ofwasting syndrome, and another of a pulmonaryembolism), and two patients who received only aninitial therapy, subsequently discontinued becauseof toxicity. In the sequential arm the 2 deaths weresecondary to progressive disease. Grades III and IVNeutropenia were common in the concurrent arm(76% vs. 39% during the induction phase), althoughwithout a higher number of infections. Grade III orIV pulmonary toxicities were observed in the con-current arm. A longer follow of these studieslooking at this combination will help us determineif the concurrent use of rituximab and fludarabinewill improve overall survival.

The combination of fludarabine, rituximab, andcyclophosphamide40 has also been evaluated in 102patients with previously treated CLL (fludarabine

Table 3 Rituximab in CLL/SLL

Investigator Pts. Prior Tx CR (%) RR (%)

Maloney1 3 Yes 0 0McLaughlin2 33 Yes 0 13Nguyen3 15 Yes 0 7Piro4 ;a 7 Yes 0 14Winkler5 9 Yes 0 11Foran6 29 Yes 0 14Huhn7 28 Yes 0 25Byrd8 ;b 33 Yes/no 3 45O’Brien9 ;c 40 Yes 0 36Hainsworth10 15 No NA 57Thomas11 21 No 19 90Hainsworth12 ;d 44 No 9 58

NA, not available.1Maloney DG, Grillo-L�opez AJ, White CA, et al. IDEC-C2B8 (Rituximab) anti-CD20-monoclonal antibody therapy in

patients with relapsed low-grade non-Hodgkin’s lymphoma. Blood 1997;90:2188–95.2McLaughlin P, Grillo-L�opez AJ, Link BK, et al. Rituximab chimeric anti-CD20 monoclonal antibody therapy of

relapsed indolent lymphoma: half of patients respond to a four-dose treatment program. J Clin Oncol1998;16:2825–33.

3 Nguyen DT, Amess JA, Doughty H, et al. IDEC-C2B8 anti-CD20 (rituximab) immunotherapy in patients with low-grade non-Hodgkin’s lymphoma and lymphoproliferative disorders: evaluation of response on 48 patients. Eur JHaematol 1999;62:76–82.

4 Piro LD, White CA, Grillo-Lopez AJ, et al. Extended rituximab (anti-CD20 monoclonal antibody) therapy forrelapsed or refractory low-grade or follicular non-Hodgkin’s lymphoma. Ann Oncol 1999;10:655–61.

a Eight infusions.5Winkler37 Winkler U, Jensen M, Manzke O, et al. Cytokine-release syndrome in patients with B-cell chronic

lymphocytic leukemia and high lymphocyte counts after treatment with an anti-CD20 monoclonal antibody (Rit-uximab, IDEC-C2B8). Blood 1999;94:2217–24.

6 Foran JM, Rohatiner AZ, Cunningham D, et al. European phase II study of rituximab (Chimeric anti-CD20monoclonal antibody) for patients with newly diagnosed mantle-cell lymphoma and previously treated mantle-celllymphoma, immunocytoma, and small B-cell lymphocytic lymphoma. J Clin Oncol 2000;18:317.

7 Huhn D, von Schilling C, Wilhelm M, et al. Rituximab therapy of patients with B-cell chronic lymphocytic leu-kemia. Blood 2001;98:1326–31.

8 Byrd JC, Murphy T, Howard RS, et al. Rituximab using a thrice weekly dosing schedule in B-cell chronic lym-phocytic leukemia and small lymphocytic lymphoma demonstrates clinical activity and acceptable toxicity. J ClinOncol 2001;19:2153–64.

b Three times weekly.9 O’Brien41 cO’Brien SM, Kantarjian H, Thomas DA, et al. Rituximab dose-escalation trial in chronic lymphocytic

leukemia. J Clin Oncol 2001;19:2165–70.c Phase I dose escalation.10 Hainsworth42 Hainsworth JD, Burris HA, III., Morrissey LH, et al. Rituximab monoclonal antibody as initial sys-

temic therapy for patients with low-grade non-Hodgkin’s lymphoma. Blood 2000;95:3052–56.11 Thomas43 Thomas DA, O’Brien S, Giles FJ, et al. Single agent rituxan in early stage chronic lymphocytic leukemia

(CLL). Blood 2001;98:364a (abstr 1533).12 Hainsworth JD, Litchy S, Barton JH, et al. Single-agent rituximab as first line and maintenance treatment for

patients with chronic lymphocytic leukemia or small lymphocytic lymphoma: a phase II trial of the Minnie PearlCancer Research Network. J Clin Oncol 2003;21(9):1746–51.

dWith maintenance rituximab.

142 B.H. Mavromatis, B.D. Cheson

25 mg/m2, cyclophosphamide 250 mg/m2 days 2–4of cycle 1 and days 2–4 of subsequent cycles, rit-uximab 375 mg/m2 day 1 cycle 1 and 500 mg/m2

day 1 of subsequent cycles). A CR rate of 30% wasseen in the fludarabine sensitive patients, but only7% CR in the fludarabine refractory patients. Tox-icities included pneumonia, neutropenic fevers,

sepsis, and one patient with prolonged myelosup-pression. When this regimen was given as initialtreatment41 in 79 patients, CR rates reached 66%with 14% nodular PR and 15% PR. One-fifth of pa-tients had grade IV neutropenia and 4% had grade IVthrombocytopenia. Whether this combination of-fers any additional benefit to the concurrent use of

Novel therapies for chronic lymphocytic leukemia 143

fludarabine and rituximab, or fludarabine alonestill needs to be established. Other nucleoside an-alogs such as pentostatin are also being studied incombination with rituximab.42

[IDEC-152] (anti-CD23). The CD23 antigen is amembrane glycoprotein expressed on the surfaceof almost all CLL/SLL lymphocytes. Thus, it rep-resents a very rational target. IDEC-152 is a pri-matized monoclonal antibody that targets CD23.Exposure of CLL B-cells in vitro to IDEC-152 leads toinduction of apoptosis, antibody dependent cellmediated cytotoxicity, and complement depen-dent cytotoxicity. Synergy after the addition ofeither rituximab or fludarabine has also been ob-served.43 Phase I studies with single agent IDEC-152and in combination with rituximab are underway.

Epratuzumab (hLL2, E-mab, LymphoCide TM).Epratuzumab is monoclonal antibody directedagainst the CD22 on the surface of normal andlymphoma cells. Found on the surface of normaland lymphoma cells, CD22 differs from other targetantigens in that it becomes internalized whenbound to an antibody. However, CD22 is only dimlyexpressed on CLL cells. This monoclonal antibodyhas demonstrated activity in indolent and aggres-sive NHL,44 and has also been shown to be safewhen combined with rituximab.45 No clinical dataare yet available in CLL.

Apolizumab (Hu1D10, Remitogen). Apolizumab isa humanized IgG1 monoclonal antibody targeting anHLA class II epitope (an HLA-DRb chain variant)present on the cell surface of most B-cell malig-nancies, and some mononuclear cells. This epitopeis commonly expressed on the surface of CLLcells46;47 and does not change configuration afterantibody binding. Apolizomab induces ADCC, com-plement mediated lysis, and apoptosis in vitro. Dosefinding phase I trials were conducted in patientswith non-Hodgkin’s lymphoma. Responses wereseen in the phase I trial, but not in the subsequentphase II study. Dose-limiting toxicity consisted ofinfusional related events.48 A dose escalation studywas recently completed in patients with CLL andacute lymphoblastic leukemia (ALL) with dose-lim-iting toxicities of aseptic meningitis, and an atypicalhemolytic–uremic syndrome. A single PR was notedof the 11 patients with CLL.49

Apolizumab has also been evaluated in combi-nation with rituximab in a phase I trial in previouslytreated B-cell NHL and CLL.50 One of 2 CLL patientshad a complete remission. However the study wasclosed because of toxicity which included arterialthrombosis and hemolytic–uremic syndrome.

TRAIL-R2 (DR5). TNF-related apoptosis-inducingligand (Apo2L or TRAIL) is a member of the tumornecrosis family51 that induces apoptosis in tumor

cells, but not in normal cells. The activity of TRAILis mediated through several membrane-bound re-ceptors including TRAIL-R1 (DR4) and TRAIL-R2(DR5),52 death receptors that induce apoptosis.Monoclonal antibodies targeting DR4 (TRAIL-R1)and DR5 (TRAIL-R2) have recently been developedand tested against five different B-cell lymphomalines in vitro.53

Apoptosis was induced in 4/5 cell lines followingexposure to the antibody targeting DR5 (TRA-8).TRAIL has also been demonstrated to have activityin a mouse xenograft model.

Anti-CD80 (anti-B7, IDEC-114). CD80 is an im-portant co-stimulatory molecule on antigen pre-senting cells that facilitates the binding of thesecells to the CD28 receptor on T-cells. CD80 isexpressed at different antigen densities on thesurface of several lymphoma and leukemia cells.CD80 expression on CLL cells ranges from 0% to50%. IDEC-114 is a primatized anti-CD80 antibody.It binds FccRII and FccRIII I and mediates ADCC. It alsobinds complement and mediates CDC activity, andhas been shown to have anti-tumor effects inlymphoma cell lines. Activity was demonstrated ina phase I/II setting in patients with relapsed orrefractory follicular NHL.54 Since IDEC-114 com-bined with rituximab in a human B-lymphoma/SCIDmouse model led to a prolongation in disease freesurvival, a phase I study of the combination isunderway. A trial in CLL has yet to be conducted.

Radioimmunoconjugates. 90Y-ibritumomab ti-uxetan (Zevalin) is a murine monoclonal antibodythat shares the same Fab structure with rituximab.It is radiolabeled with indium-111 for imaging and90Y (yttrium) for therapeutic purposes in an effortto deliver radiation not only to the lymphocytesthat bind to the antibody, but to neighboring lym-phocytes as well.55 90Y-Ibritumomab tiuxetan wasapproved by the FDA for treatment of relapsedfollicular, and low-grade non-Hodgkin’s lymphoma.I31I-tositumomab (Bexxar) another radioimmuno-conjugate that is radiolabed with iodine-131, iscurrently in clinical trials in low-grade non-Hodg-kin’s lymphomas. Because the major side effects ofthese agents is myelosuppression, their use is re-stricted to patients with less than 25% bone marrowinvolvement. Therefore in CLL, the number of bonemarrow lymphocytes would need to be reducedprior to the administration of the radioimmuncon-jugate using either chemotherapy, or anothermonoclonal antibody such as alemtuzumab.

Lym-1 consists of an 131I-labeled mouse IgGj thatrecognizes a polymorphic variant of HLA-DR foundon normal B-cells, as well as malignant lymphomacells.56 When tested in patients with non-Hodgkin’slymphoma and CLL, responses were observed, but

144 B.H. Mavromatis, B.D. Cheson

the data were difficult to interpret because stan-dardized response criteria were not used.57–59

Fusion proteins and immunotoxins

Denileukin diftitox (Ontak) (DAB389IL2). Denileu-kin diftitox is a fusion protein, composed of anenzymatically active diphtheria toxin peptide se-quence fused to human IL-2 that then binds to theintermediate and high affinity IL-2 receptor com-plex which includes CD25, CD122, and CD132. Thisagent has been approved by the FDA for thetreatment of cutaneous T-cell lymphomas. CLLcells from half of patients studied have beendemonstrated to express the IL-2 receptor.60;61

Frankel et al.,62 reported on 18 patients who weretreated with denileukin diftitox using 9 or 18 lg/kg/day over 1 h� 5 days every 21 days. A decreasein the lymph node diameter was seen in 6/10 pa-tients, with an 80% reduction seen in one patient.Side effects included transaminitis, fevers, asthe-nia, and vascular leak syndrome. No conclusionscan yet be drawn and modification in schedule maydecrease some of the side effects. The addition ofbexarotene (Targretin), an RXR-selective retinoidagonist, is also planned in an effort to up-regulatethe IL-2 receptors.63

BL-22 [Anti-CD22(Fv)-PE38]. BL-22 is an immu-notoxin consisting of a modified pseudomonasexotoxin A which is linked to an antibody targetingthe CD22 antigen on the surface of B-cell neo-plasms. It has demonstrated impressive activity innucleoside analog-resistant hairy cell leukemia.64 Aphase II trial in CLL is underway.

Molecular agents

G3139 (Genasense, Genta). Anti-sense oligonucle-otides consist of single strand DNA that is comple-mentary in sequence to a target RNA. Bcl-2 is ananti-apoptotic oncogene that has been cloned at thebreakpoint of t(14;18). The bcl-2 protein is com-monly over-expressed in a variety of tumors, in-cludingmost low-grade lymphomas, and in up to 95%of CLL,65 and is associated with impaired apoptosisand chemotherapy resistance. The possibility of si-lencing the mRNA and potentially improving theeffect of chemotherapy is very appealing.

The first oligonucleotide anti-sense in clinicaltrials, G3139 consists of 18 modified DNA bases (18-mer), that recognizes the first 6 codons of bcl-2,forming a DNA/RNA duplex that inhibits translationof the protein.66 In vitro data suggested synergywith chemotherapy in CLL.67

In a phase I/II study reported by Rai et al.68

G3139 was administered by continuous intravenousinfusion at 3 mg/kg/day for 5–7 days every 3–4weeks. Patients had either relapsed or refractoryCLL including 2 with Richter’s transfomation, andall had previously received fludarabine. Usingcombined data from the phase I and II studies, atotal of 19 patients were evaluable with 2PRs, 9SD,8PD. 35% of patients also had a decrease in theirlymphadenopathy. Side effects included fatigue,hypotension, thrombocytopenia which were re-versible. A large phase III trial in previously treatedCLL is currently underway in which patients arerandomized to fludarabine/cyclophosphamide withor without G3139.

Bevacizumab. Several growth factors includingfibroblast growth factor and vascular endothelialgrowth factor are involved in tumor angiogenesis.Basic fibroblast growth factor (bFGF) upregulatesthe expression of bcl-2 in B-cell chronic lympho-cytic leukemia cell lines, resulting in delay ofapoptosis.69 VEGF is a pro-angiogenic factor regu-lating angiogenesis and hematopoiesis. Bev-acizumab is an anti-VEGF monoclonal antibodycurrently being studied in a variety of solid tumorsand acute myeloid leukemia.70;71 The SouthwestOncology Group (SWOG) is currently conducting abevacizumab phase II clinical trial in patients withrelapsed aggressive NHL.

Other agents

Bendamustine. Bendamustine is a drug with prop-erties of both an alkylating agent and purine ana-log. In a prospective trial by Henze et al.,72 25patients were treated with bendamustine; 14/25achieved a PR (bone marrow not examined), withan overall response rate of 56%. Toxicities included13% grade IV thrombocytopenia, and 9% neutrope-nia. In a phase I/II study in 33 CLL patients, ben-damustine was combined with mitoxantrone; 11/33pts had a CR with 19/33 PR. Dose-limiting toxicitywere hematologic with infections in 4/9 pts at thehigher bendamustine dose.73 How this agent willcompare to the more conventional chemothera-peutic agents remains to be determined.

Depsipeptide (NSC 630176). Depsipeptide is ahistone deacetylase inhibitor. Although the exactmechanism of action is unclear, it induces G0-1arrest in cell lines, and leads to apoptosis andchanges in proliferation. It has also been shown tohave selective cytotoxicity in vitro against humanCLL cells.74 Pre-clinical models in mice showedthat intermittent dosing was more favorable thandaily therapy. Three phase I studies were subse-

Novel therapies for chronic lymphocytic leukemia 145

quently conducted at different administrationschedules. Side effects included nausea, vomiting,fatigue leading to frequent delay in subsequentcycles of treatment. Transient, asymtpomatic EKGchanges were also observed usually within 24 h oftreatment and resolved by the fifth day. Activityhas been noted in CTCL and peripheral T-cell NHL.Phase II studies in several hematologic malignan-cies including CLL are underway.75

EB1089. EB1089 is a novel vitamin D3 analog withcytotoxic and anti-proliferative properties. In an invivo model,76 cells from all 102 treated and un-treated B-CLL patients exposed to EB 1089 under-went apoptosis with an observed reduction of Bcl-2and Mcl-1. This activity was also associated with adownstream activation of caspase-3. This agent isstill in the pre-clinical phase of development.

Future directions

Prognostic markers in CLL have recently evolved toinclude not only staging, but also cytogenetic ab-normalities, immunophenotyping, and molecularmarkers. Furthermore, new proteins identified viagene expression profiling such as ZAP70 may alsohelp identify patients who may benefit from eitherearlier or later intervention. These new advanceswill hopefully also further our understanding of thepathophysiology of this disease.

New therapeutic strategies are progressivelyrelying more on antibodies and other targetedtherapies, used either alone, or in combinationwith nucleoside analogs. Other new agents withpromise include antisense oligonucleotides and hi-stone deacetylase inhibitors.

Future directions for therapy are likely to includecombinations of multiple agents, each targetedagainst a unique property of the malignant cell,based on its surface or intracellular proteins. Theeffect would result in the silencing of a protein as-sociated with more aggressive disease, antibodydependent cell mediated cytotoxicity, complementdependent cytotoxicity, the induction of apoptosisof themalignant cell, or any composite of the above.

The endpoint of these studies will hopefully re-sult in a prolongation of survival in this very com-mon hematologic malignancy.

Practice points: new prognosticmarkers

Poor prognostic markers in CLL include:• 17p deletion.• Mutation of the VH genes.

• CD38 surface glycoprotein positivity.• ZAP70.

Good prognostic markers in CLL include:• 13q deletion.

Practice points: alemtuzumab

• Humanized anti-CD52 monoclonal antibody.• Trials conducted in relapsed or refractory CLL

with RR 33–43%, CR 2–5%.• Administered initially at 3, 10, 30 mg/day,

then at 30 mg/day IV for 12–18 weeks.• PCP, and herpes prophylaxis are strongly

recommended.• Monitor CMV PCR q 2 weeks while on treat-

ment.

Practice points: rituximab

• A chimeric monoclonal antibody targetingCD20.

• In previously treated CLL/SLL, the RR is0–45%.

• Higher rates in early stage CLL with elevatedb-2 microglobulin.

• When given concurrently with fludarabine theRR was 90% with 47% CR compared to RR 77%with 28% CR when given sequentially However2 year PFS same at 70%.

Practice points: developingantibodies

• Anti-CD23 [IDEC-152]: phase I studies.• Epratuzumab [anti-CD22]: CD22 dimly ex-

pressed in CLL. No CLL data yet.• Apolizumab [anti-Hu1D10]: unacceptable toxi-

city.• Anti-TRAIL-R2(DR5): in vitro data.• Anti-CD80 [anti-B7, IDEC-114]: phase I studies.

Practice points: other agents

• Denileukein-diftitox: phase II in CLL.• G3139, Genasense: phase III in CLL completed.• Bevacizumab: phase II studies in NHL.• Depsipeptide: phase II studies.

Research agenda

• New prognostic markers are evolving in CLL.• New drug approach is that of targeted therapy.• New proteins identified via gene expressionprofiling may further understanding of thedisease pathophysiology.

146 B.H. Mavromatis, B.D. Cheson

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