REVIEWS

IMPROVING THE EFFICACY OF ANTIBODY-BASED CANCER THERAPIES

Paul Carter
A quarter of a century after their advent, monoclonal antibodies have become the most rapidly expanding class of pharmaceuticals for treating a wide variety of human diseases, including cancer. Although antibodies have yet to achieve the ultimate goal of curing cancer, many innovative approaches stand poised to improve the efficacy of antibody-based therapies.

PHAGE DISPLAY

Technology for displaying a protein (or peptide) on the surface of a bacteriophage, which contains the gene(s) that encodes the displayed protein(s), thereby physically linking the genotype and phenotype.
VALENCY

For antibody-derived molecules, this refers to the number of binding sites for the cognate antigen(s).
COMPLETE RESPONSE

No remaining tumour can be detected by visual inspection or by clinical imaging technologies. This does not mean that the disease has been cured.
PARTIAL RESPONSE

50% reduction in tumour with no new lesions or increase in size of an existing lesion.

Immunex, 51 University Street, Seattle, Washington 98101, USA. e-mail: carterp@immunex.com

Antibodies are finally realizing their potential as anticancer therapeutics: since 1995, five antibodies have been approved for the treatment of cancer (TABLE 1). Additional approvals will surely follow from among the 20 or so antibodies now in oncology trials1,2, including 10 that have advanced to Phase III trials or further (TABLE 1). The emergence of antibodies as therapeutics was made possible by the advent of technologies designed to overcome the main limitations of mouse monoclonal antibodies (mAb) immunogenicity of these foreign proteins in patients, inefficient effector functions (see below) and half-lives that are typically less than 20 hours14. These core technologies, in historical order of development, are chimerization and humanization of mouse antibodies, and direct routes to high-affinity human antibodies using PHAGE DISPLAY libraries or transgenic mice (BOX 1). Beyond these core technologies, remarkable progress has been made in engineering antibodies with modified properties for example, molecular size, antigen-binding affinity, specificity and VALENCY1,35. Tumour targeting by antibodies with engineered properties is in its infancy, but holds much promise for enhancing the antitumour activity of antibodies (BOX 2). Important advances in antibody technologies notwithstanding (BOXES 1 and 2), patient tumourresponse data show the urgent need to enhance the efficacy of the current generation of anticancer antibodies. For example, in a Phase II study of the chimeric antiCD20 antibody6 rituximab (Rituxan), in patients with

relapsed low-grade non-Hodgkins lymphoma, only about half of the patients responded7. This included 6% COMPLETE and 42% PARTIAL RESPONSES from 166 patients, similar results to those obtained with a singleagent cytotoxic chemotherapeutic in this group of patients. These data, combined with the mild toxicity profile of Rituxan, led the United States Food and Drug Administration (FDA) to approve Rituxan for relapsed indolent lymphoma. Unfortunately, the median 7 RESPONSE DURATION was only about 12 months . In a Phase III study of trastuzumab (Herceptin) a humanized mAb against the receptor tyrosine kinase ERBB2 (also known as HER2/NEU)8 in metastatic breast cancer, the OVERALL RESPONSE RATE was only 15%: 8 complete and 26 partial responses were observed in 222 patients9. The median response duration and survival were 9.1 and 13 months, respectively9. All clinically approved and most experimental antibody drugs directly target tumour cells. Several strategies are being explored to increase the efficacy of such antibodies, including enhancement of effector functions, direct and indirect arming, and pre-targeting of prodrugs or radionuclides (FIG. 1). In addition, potent antitumour activity might be achieved with antibodies that prevent soluble growth factors from binding to their cognate receptors, such as the epidermal-growth-factor receptor (EGFR)10 and ERBB211,12. Promising and potentially complementary alternative strategies to direct tumour targeting include targeting tumour vasculature, angiogenic growth factors and their receptors (BOX 3)5.
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Table 1 | Antibodies in advanced oncology trials*

Antibody trade name (generic name) Avastin (bevacizumab) BEC2 (mitumomab) Bexxar (tositumomab) Campath (alemtuzumab) CeaVac Antigen target Antibody type Strategy to enhance activity of naked antibody Combination with chemotherapy Vaccine Tumour target Status Corporate sponsors Genentech ImClone Systems, Merck KGaA Corixa, GlaxoSmithKline

VEGF Anti-idiotypic mAb, GD3 ganglioside mimic CD20

hu IgG1 mu IgG

Metastatic NSCLC, metastatic CRC SCLC, malignant melanoma NHL

Phase III, Phase III Phase III, Phase II BLA filed with US FDA

mu IgG2a

131

iodine

CD52

hu IgG1

None

B-cell CLL

Approved in US Millennium May 2001 and ILEX Partners Phase III, Phase II Titan Pharmaceuticals

Anti-idiotypic mAb, CEA mimic ERBB2

mu IgG

Vaccine in combination CRC with chemotherapy for NSCLC CRC or TriAb for NSCLC Combination with chemotherapy Combination with chemotherapy or external beam radiation None Bispecific Metastatic breast cancer overexpressing ERBB2 CRC, locally advanced or metastatic head and neck NHL Ovarian cancer overexpressing ERBB2 AML

Herceptin (trastuzumab) IMC-C225 (centuximab)

hu IgG1

Approved in US Genentech September 1998 BLA filing in progress, Phase III Phase III Phase III mClone Systems

EGFR

ch IgG

LymphoCide (epratuzumab) MDX-210

CD22 ERBB2 X CD64 (FcRI)

hu IgG mu F(ab)2

Immunomedics Medarex, Immuno Designed Molecules

Mylotarg (gemtuzumab ozogamicin) Panorex (edrecolomab) Rituxan (rituximab)

CD33

hu IgG4

Calicheamicin conjugate Combination with chemotherapy Combination with chemotherapy

Approved in US Wyeth May 2000 Laboratories Approved in Germany January 1995 GlaxoSmithKline, Centocor

EpCam

mu IgG2a

Dukes C CRC

CD20

ch IgG1

NHL

Approved in US IDEC November 1997 Pharmaceuticals, Genentech Phase III, Phase II Phase III BLA filed with US FDA Antisoma Protein Design Labs IDEC Pharma ceuticals

Theragyn (pemtumomab) Zamyl Zevalin (ibritumomab tituxetan)

PEM CD33 CD20

mu IgG1 hu IgG1 mu IgG1

90

yttrium

Ovarian cancer, gastric cancer AML NHL

Combination with chemotherapy

90

yttrium

* Phase III clinical trials or later. Not included are ongoing trials with marketed antibody products. Every effort has been made to obtain reliable data from several sources (company and industry web sites, and REFS 1,2), but accuracy cannot be guaranteed. AML, acute myelogenous leukaemia; BLA, Biologics License Application; CEA, carcino-embryonic antigen; ch, chimeric; CLL, chronic lymphocytic leukaemia; CRC, colorectal cancer; EpCam, epithelial cellular-adhesion molecule; FDA, Federal Drug Administration; EGFR, epidermal-growth-factor receptor; hu, humanized; mAb, monoclonal antibody; Ig, immunoglobulin; mu, murine; NHL, non-Hodgkins lymphoma; NSCLC, non-small-cell lung cancer; PEM, polymorphic epithelial mucin; SCLC, small-cell lung cancer; VEGF, vascular endothelial growth factor.

Clinical strategies

RESPONSE DURATION

Time from the first response until disease progression or death.

OVERALL RESPONSE RATE

Sum of partial and complete responses.

Combination with cytotoxic drugs. Combining different cytotoxic drugs is a widely and successfully used clinical strategy in oncology that increases the response rate and duration of individual drugs. The use of antibodies in conjunction with chemotherapeutics is a natural extension of this approach, and is strongly supported by preclinical TUMOUR XENOGRAFT studies that show improved efficacy of antibody and chemotherapeutic combinations compared with each drug used in isolation. For example, Herceptin has synergistic antitumour activity when

used in combination with cisplatin and carboplatin13,14, and additive benefit when used in conjunction with doxorubicin, cyclophosphamide, methotrexate, taxol or the selective cyclooxygenase-2 inhibitor, celecoxib1418. The addition of Herceptin to a cytotoxic chemotherapy regimen was associated with statistically significant benefits in a Phase III trial in ERBB2-overexpressing metastatic breast cancer19. These gains included longer median duration of response (9.1 versus 6.1 months), higher overall response rate (50% versus 32%) and lower death rate at one year (22% versus 33%).

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Box 1 | Key therapeutic antibody technologies Murine antibodies Derived by hybridoma technology99 following immunization of mice or, less commonly, rats. Chimeric antibodies Obtained by joining the antigen-binding variable domains of a mouse monoclonal antibody (mAb) to human constant domains: mouse VL to human CL and mouse VH to human CH1CH2CH3 for light and heavy chains, respectively100,101. Humanized antibodies In the simplest case, these are created by grafting the antigen-binding loops, known as complementarity-determining regions (CDRs), from a mouse mAb into a human IgG102104. The generation of high-affinity humanized antibodies generally requires the transfer of one or more additional residues from the so-called framework regions (FRs) of the mouse parent mAb. Several variants of the humanization technology have been developed105. Human antibodies These have high affinity for their respective antigens and are routinely obtained from very large, single-chain variable fragments (scFvs) or Fab phage display libraries106110. Moreover, the difficulty in obtaining antibodies to self-antigens that are highly conserved between mouse and humans using hybridoma technology is readily overcome using phage display technology111. High-affinity human antibodies have also been obtained from transgenic mice that contain some, or preferably many, human immunoglobulin genes Heavy and genetically disrupted endogenous immunoglobulin loci. Immunization elicits the VH chain production of human antibodies recoverable using standard hybridoma CH1 technology10,59,112114. A human anti-epidermal growth factor (EGF) receptor mAb VL obtained using transgenic mice eradicates large, established tumours in some Light 10 CL chain preclinical xenograft models , auguring well for ongoing oncology trials. C 2
H

Clinical experience Chimeric, humanized and human antibody evidence indicates that these types of antibody are less immunogenic than those of mice115. Humanized antibodies contain less foreign sequence than their chimeric counterparts and are presumed to be less immunogenic. Similar arguments have been made about humanized and human antibodies, despite the lack of substantiating clinical data. Other factors that affect the immunogenicity of antibodies include the method and frequency of administration, dose, patients disease and immune status, antigen specificity of the antibody and immune complex formation with antigen115. Choice of antibody technology Humanization and human antibodies are now the preferred technologies for developing antibodies as therapeutics. Humanization is a clinically well-validated technology that might be favoured if a well-characterized mouse mAb is available. By contrast, direct routes to human antibodies offer faster preclinical development in cases with no existing mouse mAbs. The choice of different human antibody technologies will depend on their availability, local expertise and commercial considerations.

CH3 Mouse Humanized

Chimeric Mouse sequences Glycosylation

Human Human sequences Complementarity determining regions

TUMOUR XENOGRAFT

Commonly refers to the growth of human tumour cells as tumours in immunocompromised mice.

Unfortunately, these tangible clinical benefits of combining Herceptin with chemotherapy come at the price of greater toxicity. Herceptin alone is generally very well tolerated and typically associated with only very minor adverse events9,20, whereas Herceptin plus chemotherapy is associated with additional adverse events that are comparable to, or worse than, chemotherapy alone19,21,22. Adding Herceptin to a regimen of an anthracycline drug plus cyclophosphamide, was associated with a significant increase in cardiotoxicity, although the symptoms generally improved with standard medical care19. At present, Herceptin is used as a single agent for patients with metastatic breast cancer whose tumours overexpress ERBB2 and who have received at least one regimen of chemotherapy for their metastatic disease. Herceptin is also used in combination with taxol for patients with metastatic breast cancer whose tumours overexpress ERBB2 and who have not received previous chemotherapy for their metastatic disease. The anti-CD20 antibody Rituxan sensitizes some drug-resistant human B-cell lines to the cytotoxic effects of etoposide, cisplatin and doxorubicin23. Single-arm

Phase II clinical studies with Rituxan plus chemotherapy (cyclophosphamide, doxorubicin, vincristine and prednisone) in low-grade24 and high-grade25 B-cell nonHodgkins lymphoma indicate that there is an additive24 or at least comparable25 clinical benefit of Rituxan plus chemotherapy versus chemotherapy alone, with no additional toxicity. Ongoing randomized Phase III trials are anticipated to establish the statistical significance of improved response rates or duration from combining Rituxan with chemotherapy. In addition to Herceptin and Rituxan, at least five other anticancer antibodies are being tested in combination with chemotherapy (TABLE 1)1. The increased number of these studies probably reflects several factors, including the need to test an experimental antibody drug in the context of current standard treatment commonly chemotherapy and the desire to improve on the modest antitumour activity of many naked antibodies. Two key factors are preclinical data showing the benefit of combining the specific antibody in question with chemotherapy, and the clinical success of combining Herceptin and Rituxan with chemotherapy.
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Box 2 | Engineering antibodies for enhanced antitumour activity Antibodies can be engineered with altered properties, such as antigen-binding affinity, molecular architecture and dimerization state1, 35, that can enhance their tumour targeting and potency. The antigen-binding affinity (Kd values) of antibodies has historically been increased by selection from phage display libraries116,117. This laborious task of affinity maturation has been facilitated through the use of RIBOSOME DISPLAY118 in conjunction with DNA SHUFFLING119, or YEAST DISPLAY with DNA shuffling120. The affinity of antibodies can profoundly affect their ability to localize to tumours, as shown with a panel of single-chain variable fragments (scFvs) that bind to the same epitope on ERBB2 with affinities ranging from 1071011 M121,122. A threshold affinity of 107108 M was required for specific tumour localization of scFv, and uptake reached a plateau at 1091011 M. These data support the concept of a binding-site barrier that can impair penetration of the tumour for very high-affinity antibodies123. The impact of the antigen-binding affinity of IgG molecules on their tumour-localization ability remains to be determined. Selectivity might be crucial as many tumour-associated antigens are expressed on some normal cells at lower levels. It might be possible to achieve the greatest targeting selectivity by using a low-affinity IgG that relies on a high surface density of antigen on tumour cells for efficient binding. The molecular architecture of antibodies is readily modified to create non-natural antibody formats that vary in size and valency, with significant effects on tumour targeting ability. For example, a dimeric anti-ERBB2 antibody fragment, (scFv)2 , showed increased tumour uptake compared with a monomeric Fab fragment of similar size (~50 kDa), and presumed similar tumour penetration and pharmacokinetic properties124. Increasing the valency of antibodies is a simple way to increase their avidity for cell-surface antigens and might lessen the need for affinity maturation. Tumour targeting by IgG is impaired by poor tumour penetration, which results from their large size, and uptake by Fc receptors on reticulo-endothelial cells5. By contrast, accrual of IgG within tumours is favoured by their long halflife, which reflects their size and their recycling by the neonatal Fc receptor, FcRn. Small antibody fragments, such as scFv, EXTRAVASATE more efficiently than IgG, and diffuse more readily within tumours, but are also rapidly eliminated through the kidney. These opposing factors limit the accrual of scFv within tumours to levels that are better suited for imaging than for therapy125. IgG homodimerization by chemical coupling increases the antitumour activity of antibodies against several different tumour antigens by mechanisms that include more potent antibody-dependent cellular cytotoxicity or complementdependent cytotoxicity137, direct killing126,138,139, growth arrest126 and synergy with chemotherapy or immunotoxins138. IgG homodimerization can also increase the apparent affinity for binding to the cognate antigen on cells, which results in more rapid and/or more extensive internalization126,137,139. Significantly, IgG homodimerization might enhance in vivo antitumour activity126,139. Enhancement of the in vitro antitumour activity of Rituxan by homodimerization138 should encourage additional preclinical and perhaps clinical assessment of this strategy.

RIBOSOME DISPLAY

Technology for displaying a nascent protein, which is physically linked to its encoding mRNA, that relies on in vitro transcription and translation.
DNA SHUFFLING

Process for creating molecular diversity by homologous recombination of DNA in vitro.

YEAST DISPLAY

Technology for displaying a protein on the surface of a yeast cell that contains the gene(s) that encodes the displayed protein(s).
EXTRAVASATION

Movement out of the vasculature compartment into interstitial spaces.

MINIMAL RESIDUAL DISEASE

Tumour remaining in patients following debulking by surgery and/or chemotherapy and/or radiotherapy.
MICROMETASTATIC DISEASE

Metastatic disease that can be detected by immunohistochemistry of tissue biopsies, but involves too few tumour cells to be directly imaged in patients.
DUKES STAGE C COLORECTAL CANCER

Cancer that has spread from the colon to nearby lymph nodes, but not to other parts of the body.
FC REGION

For an IgG, this comprises the CH2 and CH3 domains (BOX 1).

Targeting minimal residual disease. Using antibodies to target MINIMAL RESIDUAL DISEASE as well as MICROMETASTATIC DISEASE, following surgery, chemotherapy or radiotherapy, is a strategy26,27 that attempts to address the difficulties of poor accessibility and limited penetration of solid tumours by antibodies1,28. Indeed, the anti-epithelial cellular adhesion molecule (EpCam) mAb, 17-1A29, now known as edrecolomab (Panorex), has been reported to be more efficacious in the treatment of micrometastases and minimal residual disease26,27 than bulky metastatic disease30. Panorex has been approved for the treatment of DUKES STAGE C COLORECTAL CANCER in Germany based on tangible patient benefit in a 189-patient Phase III trial26. It remains to be seen whether Panorex alone, or in combination with chemotherapy, will prove efficacious in Phase III trials that are ongoing in the United States (TABLE 1). Nevertheless, the concept of treating minimal residual disease warrants evaluation in the context of other antitumour antibodies. Clinical trials with Herceptin are now doing just that22. Several factors might limit the use of antibodies in treating minimal residual disease to those that have already been approved as anticancer therapeutics, or at least have shown benefit to cancer patients. First, trials of many thousands of patients might be needed to allow statistical assessment of treatment outcome. Second, follow-up periods of several years are required to track

any differences in time to disease progression or death. Third, the large size and long duration of such trials makes them exceedingly expensive to conduct.
Enhancing effector functions

Human antibodies of the IgG1 and IgG3 isotypes can potentially support the effector functions of antibodydependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (FIG. 2). Tumourcell killing by ADCC is triggered by the interaction between the FC REGION of an antibody bound to a tumour cell, and the Fc receptors on immune effector cells, such as neutrophils, macrophages and natural killer cells. CDC is initiated by complement component C1q binding to the Fc region of IgG, which is bound to the surface of a tumour cell. Subsequent target-cell killing can occur in a cell-dependent or cell-independent manner (FIG. 2). The first demonstration that the FcFc receptor interaction is important for the antitumour activity of an antibody in vivo came with the development of mice that lack FcRI and FcRIII31. An anti-melanoma antibody had potent antitumour activity in a mouse model of metastasis, but this benefit was lost in mice that lack FcRI and FcRIII receptors. ADCC is likely to be the mechanism underlying the antitumour effects of the FcFc receptor interaction. Alternatively,

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antitumour activity of an antibody by manipulating the Fc region to increase its affinity for the activation receptor(s) and/or by abrogating its ability to bind to the inhibitory receptor. Indeed, point mutations in the Fc region, which result in improved binding to FcRIII, yielded up to a twofold enhancement in ADCC in vitro34. The in vivo and clinical significance of this in vitro improvement is unknown. Glycosylation of IgG molecules at Asn297 (KABAT 35 NUMBERING SCHEME ) helps to maintain the tertiary structure of their CH2 domains36 (BOX1), and is necessary for effector functions37. The cells producing the antibody (the production host) and, to a lesser extent, culture conditions, can significantly affect the resulting antibody glycoforms which, in turn, can influence the ability of the antibody to participate in ADCC38. Increasing the level of BISECTED COMPLEX OLIGOSACCHARIDES attached to the Fc region of an antibody can enhance its ability to support ADCC39. This modification of glycosylation was accomplished by cellular engineering of the production host Chinese hamster ovary cells by transfection with -(1,4)-N-acetylglucosaminyltransferase III (REF. 39). Ongoing studies will address whether these antibodies have enhanced antitumour activity in vivo. Effector cell populations might be suppressed or diminished in cancer patients, for example, by previous chemotherapy, which could curtail the effector functions of antitumour antibodies. Antibodies with improved ability to support CDC have been created by mapping binding sites for the complement component C1q40 on human IgG1 Fc and then creating site-directed mutants with enhanced C1q binding41. It remains to be seen if these improvements in CDC in vitro translate into more potent antitumour activity in vivo. One potential problem with this approach is that many malignant cells, as well as normal cells, express membrane-bound proteins, such as CD46, CD55 and CD59, that interfere with CDC and allow them to escape complement attack42.
Direct arming

a Enhancing effector
functions

d Pre-targeting
Biotinchelator radionuclide Streptavidin

Complement-dependent cytotoxicity

Point mutations and/or modified glycosylation

Antibody-dependent cellular cytotoxicity

Prodrug scFvenzyme

Tumour cell

Drug

Cytokine

scFv fragment

Immunocytokine
Sterically stabilized immunoliposomes

Small molecule or protein toxin

Radionuclide

Bispecific antibody

Radionuclide, toxin or immunological effector cell

b Direct arming

c Indirect arming

Figure 1 | Strategies for enhancing the potency of antitumour antibodies. Numerous strategies for improving the efficacy of antitumour antibodies are now being tested, including the representative examples shown here and described in BOX 2. a | Enhancing effector functions involve improving antibody-dependent cellular cytotoxicity and/or complement-dependent cytotoxicity by means of site-directed mutations or manipulation of antibody glycosylation. b | Direct arming of antibodies entails their covalent linkage to killing machinery, such as radionuclides or toxins (for example, small molecules or proteins). Alternatively, arming antibodies with cytokines is intended to create high intratumour concentrations of cytokines to stimulate the antitumour immune response (T cells, B cells or natural killer cells), while avoiding the toxicities associated with systemic cytokine delivery. c | Indirect arming of antibodies can be achieved by attaching engineered antibody fragments to the surface of liposomes loaded with drugs or toxins for tumour-specific delivery. Bispecific antibodies that bind to two different antigens can be preloaded with the cytotoxic machinery before administration (indirect arming) or alternatively pre-targeted to the tumour before delivery of the cytotoxic payload. d | Pre-targeting strategies aim for the selective delivery of radionuclides to tumours or selective intratumour activation of prodrugs, thereby diminishing the systemic toxicities of these cytotoxic agents. For prodrug pretargeting, an antibody-fragmentenzyme fusion protein is typically allowed to localize to a tumour and be cleared from the system. A prodrug is then administered and ideally converted to an active drug solely within the tumour. For radionuclide pre-targeting, an antibodystreptavidin conjugate is allowed to accrue within a tumour and is then used to capture a biotinchelatorradionuclide complex. scFv, single-chain variable fragment.

KABAT NUMBERING SCHEME

Immunoglobulin amino-acid residue numbering scheme, devised by the late Elvin Kabat, that accommodates sequence insertions and deletions.
BISECTED COMPLEX OLIGOSACCHARIDES

Branched carbohydrate that might include several different kinds of monosaccharides, including N-acetylglucosamine between main branches.

induction of apoptosis by crosslinking of effector and target cells could explain these observations. The importance of the FcFc receptor interaction for antitumour activity was subsequently shown for the clinically important antibodies Herceptin and Rituxan32, as well as for intracerebral therapy with an anti-EGF receptor antibody in a brain tumour model33. The antitumour activity of Herceptin and Rituxan was greatly reduced in mice that lack the activation receptors FcRI and FcRIII, whereas disruption of the gene that encodes the inhibitory receptor FcRIIB substantially enhanced antitumour activity32. The antitumour activity of Herceptin was also attenuated by a mutation (D265A) that impairs binding to FcRIII and FcRIIB. These studies indicate the potential for increasing the

The most widely explored strategy for enhancing the efficacy of antitumour antibodies is direct arming by covalent linkage to toxins or radionuclides1,28 (FIG. 1). Armed antibodies typically show more potent antitumour activity in preclinical tumour xenograft studies than their naked parents. Unfortunately, clinical evaluation of armed antibodies has been beset by unacceptably high levels of toxicity in several clinical trials28, leading many to abandon this approach. Nevertheless, antibody arming is enjoying a renaissance with the approval of the first armed antibody, gemtuzumab ozogamicin (Mylotarg), and with two others, tositumomab (Bexxar) and ibritumomab tituxetan (Zevalin), on the cusp of regulatory approval (see below). A judicious choice of both target antigen and antibody is likely to be crucial to the success of all arming strategies. Ideally, the target antigen should be universally found on tumour cells of a given type, but absent or present at much lower levels on normal cells. The
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Box 3 | Alternative targets for anticancer antibodies Most approved and experimental anticancer antibodies directly target tumour cells. Alternative strategies include inhibiting angiogenesis or directly targeting tumour neovasculature5. Angiogenesis is the process by which tumours become vascularized by the proliferation of new blood vessels from existing ones, thereby allowing the tumours to grow beyond a minimal size. An antibody that neutralizes the angiogenic factor vascular endothelial growth factor (VEGF) has potent antitumour activity in vivo127,128, and noninvasive imaging indicates that this is due to angiogenesis inhibition129. This has encouraged the humanization130 of an anti-VEGF antibody, now known as bevacizumab (Avastin) that is now in Phase III clinical trials for metastatic cancers (TABLE 1). The alternative strategy targeting VEGF receptors is at present being tested in Phase I trials for patients with colorectal cancer and liver metastases with the IMC-1C11 anti-VEGFR2 antibody. Clinically significant questions include whether anti-VEGF treatment will benefit patients with established tumours and whether tumours will recruit other angiogenic growth factors to escape anti-VEGF blockade. The combination of anti-VEGF and anti-ERBB2 (Herceptin) antibodies has proved more efficacious in tumour xenograft models than either antibody alone, encouraging clinical evaluation of this combination (M. Pegram, personal communication). Targeting the tumour vasculature has several potential advantages over direct tumour targeting131133: the vasculature is more accessible to antibodies; vasculature damage has a multiplicative effect as many tumour cells are dependent on each capillary; and as vascular endothelial cells are not transformed, they seem less likely to become resistant to antibody therapy. The therapeutic potential of targeting the tumour vasculature has been shown in tumour xenograft studies132,133. Tissue factor has recently been targeted to the ED-B domain of fibronectin, which is a natural marker of angiogenesis that is present in many solid tumours but not most normal blood vessels and tissues134. Tumours were eradicated by intratumour thrombosis in 30% of the mice treated with no obvious side effects134, encouraging further preclinical studies. Unfortunately, tumour regrowth following tumour vasculature targeting might occur from a thin layer of tumour cells close to blood vessels132,134. Encouragingly, synergistic antitumour activity was observed for immunotoxins directed to the tumour vasculature and the tumour itself. Coagulation at non-tumour sites is a potentially significant safety issue to be addressed with tumour-targeted thrombosis. Unfortunately, tumour vasculature targeting is limited at present by the lack of blood-vessel-specific target antigens. It might be possible to exploit antigens on tumour cells for tumour vasculature targeting by the judicious choice of antibody drugs. Tumour targeting with rapidly clearing antibody fragments (for example, Fab and scFv) results in de facto vasculature targeting5, as these fragments accumulate primarily on perivascular tumour cells122,135,136.

antigens most successfully targeted with armed antibodies so far CD20 and CD33 closely match these criteria. CD20 is a tetra-spanning membrane protein that is found on mature B cells, including >90% of B-cell lymphomas, but is absent from stem cells, plasma cells and nonlymphoid tissue. CD33 is a sialic-acid-binding Ig-like lectin (siglec) that is found on the surface of virtually all acute myelogenous leukaemia cells, as well as myeloid progenitor cells and committed precursors, but not on stem cells or nonlymphoid tissue. A prerequisite for antibody arming with small molecule toxins, but not radionuclides, is that the antibody should be efficiently internalized. Direct selection for antibodies that efficiently internalize is now possible by PANNING on cells using antibody phage display libraries43,44. Small-molecule toxin conjugates. Arming antibodies with conventional cytotoxic chemotherapeutics has been widely explored28. The mAb BR96 (anti-sialyl Lewis Y antigen) conjugated with doxorubicin has proved highly efficacious in tumour xenograft studies45 but, unfortunately, has shown little or no efficacy in Phase II trials for metastatic breast cancer46 and advanced gastric adenocarcinoma47, respectively. Moreover, dose-limiting gastrointestinal toxicities were observed in the breast cancer trial because the immunoconjugate bound to antigen-positive normal cells in gastric mucosa, small intestine and pancreas46. A fundamental problem with this approach is the

PANNING

Process of separating targetbinding clones from nonbinding clones for phage display library.

combination of the low molar toxicity of chemotherapeutics and the miniscule proportion of an injected mAb that usually localizes to a solid tumour target. This is typically 0.0010.01% of the injected dose per gram of solid tumour in humans compared with 20% or more in mice48. Recognition of this problem inspired the conjugation of antibodies to small-molecule toxins that are 1001000-fold more potent than conventional chemotherapeutics. Calicheamicins4952 and maytansinoids53 are the most extensively evaluated of numerous small-molecule toxins used for antibody arming. Conjugation of these small-molecule toxins to antibodies converts them to inactive prodrugs that can selectively target tumours. Activation of these prodrugs involves release of the drug from the antibody and occurs primarily in the tumour following receptor binding to antigen-positive cells and antibody internalization. In the case of calicheamicin, release from the antibody is followed by a chemical rearrangement to create diradicals that can cause double-stranded DNA breaks and compromise cell viability. Calicheamicin contains a sugar component that contributes to its potency by binding to the minor groove of DNA. The humanized anti-CD33 antibodycalicheamicin conjugate Mylotarg has been approved for treatment of CD33-positive acute myeloid leukaemia in first-relapse patients of 60 years old and who are not candidates for cytotoxic chemotherapy. This approval is based on an overall response rate of 30% with acceptable toxicities52.

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limitation of the antimucin conjugate might have been its probable inefficient accumulation in solid tumours that were treated48. So, arming antibodies with smallmolecule toxins is a valuable but not universal approach to increase their antitumour potency. Antibodies conjugated to maytansinoids have cured mice that bear human tumour xenografts, albeit at doses close to the maximum tolerated dose53. Complete responses, although not CURES (that is, the tumours eventually regrew following treatment), were obtained even after treating mice that had very large tumours (mean size of 500 mm3) or heterogeneously expressed the target antigen. These encouraging preclinical data have supported the progress of two antibodymaytansinoid conjugates into Phase I clinical trials. Protein toxin conjugates. Most immunotoxins comprise either a plant toxin, such as ricin A chain, or a bacterial toxin, for example, Pseudomonas exotoxin, conjugated or genetically fused to an antibody or antibody fragment28,56. Immunotoxins have occasionally been associated with antitumour responses in patients57,58. Excitingly, one complete and seven partial responses were recorded in a 35-patient Phase I trial in haematological malignancies using an immunotoxin composed of the single-chain variable fragment (scFv) of an anti-CD25 antibody, fused to Pseudomonas57. Unfortunately, clinical development of immunotoxins has also been plagued with toxicity problems, such as VASCULAR LEAK SYNDROME, and by immunogenicity that often precludes multiple dosing. Site-specific PEGYLATION of one recombinant immunotoxin improved its antitumour activity in animal models, and also decreased its immunogenicity and toxicity59. The recruitment of human proteins as toxins circumvents the immunogenicity of non-human protein toxins. For example, the human ribonuclease angiogenin has been genetically fused to fragments of an antitransferrin receptor antibody and found to be selectively cytotoxic to cells that bear the transferrin receptor60. Site-specific chemical coupling to a ligand or antibody resulted in a 5000-fold increase in in vitro cytotoxic activity of two other human ribonucleases against target cells. This enhancement is due to specific targeting to tumour cells plus steric blockade of the interaction with a prevalent inhibitor61. Radioimmunoconjugates. Arming antibodies with radionuclides enables them to kill BYSTANDER CELLS. This is because the -particles emitted by commonly used radionuclides (131iodine, 90yttrium, 186rhodium and 188 rhodium) are cytotoxic over many cell diameters. For example, the mean range of -particles from 90yttrium is >200 cells62. Many commonly used -emitting radionuclides (131iodine, 186rhodium and 188rhodium, but not 90yttrium) also emit -rays that can kill cells over an extended distance, as well as allowing imaging and quantification of radionuclide uptake63,64. But bystander-cell killing by radioimmunoconjugates is a double-edged sword. The killing of neighbouring tumour cells is particularly beneficial, as some tumour
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Effector cell

FcR

Antibody-dependent cellular cytotoxicity

Fc

Phagocytosis or lysis

Tumour cell Membrane attack complex lysis

Complement-dependent cytotoxicity Phagocytosis or lysis

C1q

C1qR CR1 CR3

Effector cell

Figure 2 | Antibody effector functions. Human antibodies, particularly IgG1 and IgG3, can potentially direct the killing of tumour cells by antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)42. ADCC is triggered by an interaction between the Fc region of an antibody that has bound, through its antigen-binding region, to a tumour cell and the Fc receptors (FcRs), particularly FcRI and FcRIII, on immune effector cells such as neutrophils, macrophages and natural killer cells. The tumour cell is eliminated by phagocytosis or lysis, depending on the type of mediating effector cell. A prerequisite first step for CDC is recruitment of the complement component C1q by IgG bound to the tumour cell surface. This triggers a proteolytic cascade to activate complement. This can lead to the formation of a membrane attack complex that kills the target cell by disrupting its cell membrane. Alternatively, tumour-cell-bound C1q can bind to complement receptors, such as C1qR, CR1 (CD35) and CR3 (CD11b/CD18), on effector cells, such as neutrophils, macrophages and natural killer cells. This can trigger cell-mediated tumour-cell lysis or phagocytosis, depending on the type of effector cell.

CURE

Tumour does not reappear for a prolonged time period deemed sufficient for regrowth of any residual tumour following anticancer therapies.
VASCULAR LEAK SYNDROME

Involves damage to vascular endothelial cells, extravasation of fluids and proteins resulting in weight gain and, in its most severe form, kidney damage and pulmonary oedema.

An important factor contributing to the efficacy of Mylotarg might be the high proportion (~80%) of this drug that localizes to acute myeloid leukaemia cells. Unlike cells in solid tumours, these tumour cells are readily accessible by virtue of their location in either the circulation or bone marrow (G. Yarranton, personal communication). By contrast, clinical development of an efficiently internalizing humanized antimucin antibodycalicheamicin conjugate for treatment of solid tumours54,55 was less successful and has been abandoned. One of the main problems was the formation of immune complexes between the immunoconjugate and shed mucin. Uptake of these immune complexes by liver and spleen led to dose-limiting toxicities (G. Yarranton, personal communication). An additional

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cells might lack the target antigen and thereby escape antibody therapy65. Bystander killing also helps address the problem of limited accrual of antibody in poorly vascularized or bulky tumours48. The dark side of bystander-cell killing is damage to non-tumour cells, particularly those in haematological tissue, which results in severe toxicity. Arming anti-CD20 antibodies with radionuclides exploits the inherent sensitivity of lymphomas to radiation and has resulted in significant antitumour responses in patients with non-Hodgkins lymphoma66,67. In one clinical trial, patients were randomized to treatment with either Rituxan or Zevalin a 90 yttrium-labelled version of the mouse parent mAb that begat Rituxan. Interim analysis of data from the first 90 patients showed an overall response rate of 80% with Zevalin versus 44% with Rituxan (P <0.05) (REF. 68). Moreover, Zevalin treatment of Rituxan-refractory disease gave an overall response rate of 46% (REF. 68). Radioimmunoconjugates can cause severe toxicities. For example, in a Phase I/II dose-escalation study of Zevalin, severe (grade 3 and 4) haematologic toxicities were common: a dose of Zevalin containing 50 mCi 90yttrium was MYELOABLATIVE the patients required AUTOLOGOUS STEM-CELL SUPPORT69. Lower doses of Zevalin were still efficacious but were less toxic. Zevalin and Bexxar ( 131iodine-anti-CD20 mouse mAb) have successfully completed Phase III clinical trials in non-Hodgkins lymphoma. They are expected to be the first approved radioimmunoconjugates. Beyond Bexxar and Zevalin, at least three radioarmed antibodies are now in Phase I/II or later trials, namely Theragyn (pemtumomab) (TABLE 1), CEA-cide (rabetuzumab) and Cotara1. Radionuclides such as 212bismuth, 213bismuth and 211 astatine emit -particles that are highly cytotoxic over very short distances (< 100 m), by virtue of their short range and high linear energy transfer70,71. Antibodies armed with these -emitters hold the promise of local bystander-cell killing within a tumour and greatly reduce the longer range and systemic bystander-cell killing that contributes to the toxicity of antibodies armed with - and -emitters. Such -emitters are particularly attractive for radioimmunotherapy of micrometastatic disease, and neoplasms spread on the surface of body compartments, such as ovarian cancer and neoplastic meningitis62. The clinical investigation of antibodies armed with -emitters began only in the late 1990s62 because it has been difficult to identify -emitters that have physical half-lives suitable for radioimmunotherapy and that produce acceptable (that is, relatively nontoxic) daughter nuclides. There are also few facilities capable of producing such radionuclides, and new labelling methodologies are urgently needed. Immunocytokines. Fusing antibodies to cytokines is a promising alternative arming strategy to the use of toxins and radionuclides. These so-called immunocytokines are designed to create high intratumour concentrations of cytokines to stimulate the antitumour immune response (T cells, B cells or natural killer cells), as well as avoiding the toxicities associated with systemic cytokine delivery72,73. Several different cytokines have been evaluated, including interleukin (IL)-2, IL-12 and granulocyte-macrophage colonystimulating factor (GM-CSF)74. In theory, the cytokine could be fused to the amino and/or carboxyl terminus of the light and/or heavy chains, provided that the functions of both antibody and cytokine are preserved. The most promising so far is an immunocytokine in which IL-2 is fused to the carboxyl terminus of the heavy chain of an antibody. This immunocytokine eliminated established metastases in a syngeneic mouse tumour model, probably by causing expansion of immune effector cells73. Such data bode well for ongoing clinical studies with the immunocytokines huKS-IL2 and hu14.18-IL2.
Indirect arming

PEGYLATION

Chemical modification of a protein with one or more molecules of polyethylene glycol.

BYSTANDER CELLS

Cells in the immediate vicinity of a cell that has a bound antibody-based targeting agent.
MYELOABLATIVE

Elimination of myeloid cells and their progenitors.

AUTOLOGOUS STEM-CELL SUPPORT

Immunoliposomes. Liposomes are self-assembled lipid bilayers that encapsulate some of the surrounding medium during their formation. Liposomal formulations of the chemotherapeutics doxorubicin (Doxil) and daunorubicin (DaunoXome) have been approved in recent years for the treatment of Kaposis sarcoma. Significant advances in liposome technology include the development of sterically stabilized (stealth) liposomes that are long-lived in vivo, and the improvement of methods for loading and retaining drugs75. Engineered antibody fragments can be attached to the surface of stealth liposomes for selective tumour targeting of large payloads of drugs76,77, toxins or even DNA for gene therapy. Such large payloads offer an important potential advantage over direct antibody arming, in which only one or a few molar equivalents of the payload are attached per antibody to avoid compromising antigen binding, conjugate solubility or promoting aggregation. Delivery of chemotherapeutics using immunoliposomes offers substantial benefits over the use of free drugs. For example, anti-ERBB2 immunoliposomes loaded with doxorubicin show greater antitumour activity than free drug or drug loaded in non-targeted liposomes in several tumour xenograft models77,78. Moreover, the systemic toxicity of the immunoliposome-targeted doxorubicin was much less than that of free doxorubicin. Despite such encouraging progress with immunoliposomes, several underlying difficulties remain, including their inherent complexity and extravasation due to their large size (commonly ~100 nm in diameter) 79. Using immunoliposomes to target tumour vasculature (BOX 3), rather than the tumour per se, obviates the need for extravasation. Bispecific antibodies. Bispecific antibodies (BsAbs) are non-natural antibodies that bind to two different epitopes, almost invariably chosen on two different antigens. In clinical oncology, BsAbs have been used most widely for delivering immune effector cells and, to a lesser extent, for delivery of radionuclides, drugs and toxins to tumours8082 (FIG. 1).

Harvesting of a patients haematological stem cells before myeloablative anticancer therapy, followed by rescue by engraftment of the stem cells back into the patient.

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BsAbs that bind to a tumour-associated antigen and a so-called trigger antigen on an immune effector cell can recruit the effector cell to kill a tumour cell that it would otherwise disregard. The most extensively used trigger molecules are CD3 on T cells, FcRI (CD64) on granulocytes and monocytes, FcRIII (CD16) on natural killer cells and, more recently, FcRI (CD89) on macrophages, although other triggers have also been used82. A BsAb that binds to an Fc receptor outside its Fc-binding site can do so in the presence of a vast molar excess of serum IgG. By contrast, binding of monospecific antitumour IgG to Fc receptors can potentially be inhibited by serum IgG binding to these receptors, thereby compromising the ability of the antitumour antibody to support ADCC. Encouraging local antitumour responses have been seen for BsAb targeting of T cells to ovarian cancer cells in small-scale clinical trials83. Unfortunately, treatment failure occurred because of metastasis outside the peritoneal cavity, which was refractory to systemic BsAb therapy. Another common clinical problem associated with BsAb is that the systemic activation of effector cells causes widespread cytokine release, which leads to serious side effects. So, more effective strategies are needed for the targeting of effector cells and selective activation in the context of tumour cells. The immunogenicity of BsAb evaluated in patients will probably be greatly reduced by the use of humanized or human antibodies, as observed for many monospecific antibodies (BOX 1). A further difficulty in the pharmaceutical development of BsAb is the cost and difficulty of producing these complex multichain molecules in sufficient quantity and purity for clinical trials. Powerful new technologies for the production of a plethora of alternative BsAb fragments84, as well as bispecific human IgG85, have the potential to overcome this problem. Approval of a BsAb for therapeutic use remains a distant prospect, despite much progress in both basic and clinical research. New applications for BsAbs will probably be needed for this modality to significantly benefit cancer patients.
Pre-targeting strategies

remain to be overcome if they are to provide significant new treatment options for cancer patients. Pre-targeting prodrugs. Antibody-dependent enzymemediated prodrug therapy (ADEPT) involves pre-targeting of prodrugs to tumours86,87. An antibodyenzyme fusion protein, or historically a conjugate, is administered and allowed to localize to a tumour. A prodrug is administered after the remaining fusion protein has cleared. The prodrug diffuses widely, but in the ideal case is activated solely at the tumour following contact with the localized fusion protein. One main advantage of ADEPT over naked antitumour antibodies is the amplification of the cytotoxic effect by virtue of the catalytic nature of prodrug activation. A second benefit is the ability to kill bystander tumour cells, thereby reducing the risk of tumours evading therapy by antigen loss. The cytotoxic effects of the drug are ideally confined to the tumour target, so reducing toxicity compared with systematic administration of cytotoxic chemotherapy. ADEPT has proved highly effective in several different tumour xenograft studies, but it is exceedingly difficult to translate into clinical practice86,87. A significant but surmountable obstacle has been the immunogenicity of both the enzymes used for prodrug activation and the targeting mAb which are both derived from non-human sources88. Human enzymes in conjunction with humanized or human mAb should greatly reduce this immunogenicity problem. Such thoughts inspired the development of a humanized anti-carcinoembryonic antigen (CEA) antibodyhuman--glucuronidase fusion protein plus doxorubicin glucuronide prodrug89. This ADEPT system gave superior efficacy and reduced toxicity in tumour xenograft studies in mice compared with free doxorubicin given at its maximum tolerated dose89. The therapeutic benefits of this ADEPT system over free doxorubicin reflect 412-fold higher intratumour drug concentrations and up to 5-fold lower extratumour drug concentrations. The elevated levels of endogenous -glucuronidase within some tumours also indicate that glucuronide prodrugs have potential as monotherapy90. The use of human enzymes for ADEPT poses the potential risk of unwanted activation of prodrug by endogenous enzymes. In addition, endogenous substrates or inhibitors could interfere with the desired prodrug activation within a tumour. To circumvent this problem, human carboxypeptidase A1 was engineered to activate a prodrug that is not a substrate for the wild-type enzyme91, but regrettably it was found to be ineffective in vivo92. The risks of using human enzymes for ADEPT can, in principle, be circumvented by using a humanized or human catalytic antibody that is designed for prodrug activation by a non-physiological mechanism. A significant step towards this goal was the development of a mouse catalytic mAb that activates an etoposide prodrug. Intratumoral injection of this antibody together with systemic prodrug administration proved more
www.nature.com/reviews/cancer

Cytotoxic agents, such as radionuclides or prodrugs, might be targeted specifically to cancer cells through the technique of pre-targeting. This strategy typically requires two or three separate components and treatment steps. In one approach, the antibody component is first targeted to the tumour followed by clearance of the residual circulating antibody, either unassisted or facilitated by a clearing agent. A cytotoxic agent is then administered for selective capture or activation at the tumour site. Pre-targeting of radionuclides and prodrugs to tumours is particularly attractive in that it has the potential to greatly reduce the systemic toxicity of conventional radioimmunotherapy and cytotoxic chemotherapy, respectively. The Achilles heel of pretargeting strategies is their inherent complexity, and the immunogenicity of their non-human components. Pre-targeting strategies have advanced markedly, but many obstacles

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efficacious and less toxic than etoposide given at its maximum tolerated dose93. Antibodies with even very modest catalytic efficiency (kcat/KM) might still be potentially useful for ADEPT if used in conjunction with prodrugs of maytansinoids, calicheamicins or other ferociously toxic molecules. Pre-targeted radioimmunotherapy. Schemes for pretargeted radioimmunotherapy have occasionally used BsAbs with specificities for both tumour and radionuclide chelator, but more commonly have exploited the very high-affinity interaction between streptavidin and biotin94,95. An infused antibodystreptavidin conjugate or fusion protein is first allowed to localize to a tumour target. A clearing agent is then used to remove the remaining circulating conjugate. Delivery of a radionuclide such as 90yttrium is accomplished using a biotinylated chelator. The chelatorradionuclide complex is either captured by the antibodystreptavidin bound to tumour cells or cleared rapidly through the kidney by virtue of its low molecular weight. Significant advantages of pre-targeted over conventional radioimmunotherapy include the much greater ratios of radioactivity in tumour versus nontumour tissues, thereby lowering the whole body exposure to radioactivity. Successful preclinical studies have led to small-scale clinical trials with pre-targeted radioimmunotherapy 96,97. Regrettably, the immunogenicity of streptavidin might preclude the repeated treatment cycles that would probably be necessary for effective therapy. Pegylation of streptavidin might reduce its immunogenicity98, as observed for many other proteins including at least one immunotoxin59. Another option is to devise a minimally immunogenic and high-affinity alternative to the streptavidinbiotin system. A further problem for pre-targeted radioimmunotherapy is the large quantity of radionuclide that must be administered in light of the minute proportion captured at the tumour target site.
Perspectives

Antibodies have started to fulfil their promise as anticancer therapeutics with the five antibodies now marketed as drugs in the United States (Rituxan, Herceptin, Mylotarg and alemtuzumab (Campath)) and Germany (Panorex) (TABLE 1). The current emphasis on antibody therapies for non-solid (for example, lymphomas and leukaemias) over solid tumours probably reflects the much greater accessibility of antibodies to non-solid tumours, resulting in improved antibody localization and enhanced efficacy. For solid tumours, targeting minimal residual and micrometastatic disease is attractive as it obviates the need to penetrate bulky tumours. Unfortunately, this approach might be stymied by the need for very large and costly trials with multiyear follow-up for statistical assessment of treatment outcome. It has proved seductively simple to enhance the antitumour efficacy of many antibodies in xenograft studies. This reflects the fact that antibody localization to tumours in mice is often efficient, and targeting antibodies bind human antigens but commonly not the corresponding mouse antigens. By contrast, clinical experience with the same antibodies has often been marked by a lack of efficacy and/or significant toxicity that probably reflects the limited accrual at the tumour site(s) and binding to antigen-positive non-tumour tissue, respectively46,47. Such disappointing clinical data might provide insights into ways to create potentially more efficacious and safer antitumour antibodies. Certainly, the concept of enhancing the antitumour activity of antibodies in cancer patients has been established, but doing so within the realm of manageable toxicities remains an ongoing challenge.

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Acknowledgements
The author thanks L. Weiner, D. Liebowitz and J. Smothers for critical review of this manuscript and G. Yarranton and M. Pegram for kindly sharing unpublished data.

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Online links
DATABASES The following terms in this article are linked online to: CancerNet: http://cancernet.nci.nih.gov/ breast cancer | colorectal cancer | gastric adenocarcinoma | Kaposis sarcoma | myelogenous leukaemia | non-Hodgkins lymphoma | ovarian cancer LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ angiogenin | CD3 | CD16 | CD20 | CD25 | CD33 | CD46 | CD55 | CD59 | CD64 | CD89 | cyclooxygenase-2 | EGFR | ERBB2 | fibronectin | -glucuronidase | GM-CSF | IgG1 | IgG3 | IL-2 | IL-12 | VEGF | VEGFR2 Medscape DrugInfo: http://promini.medscape.com/drugdb/search.asp biotin | Campath | carboplatin | celecoxib | cisplatin | cyclophosphamide | daunorubicin | doxorubicin | etoposide | Herceptin | methotrexate | Mylotarg | prednisone | Rituxan | taxol | vincristine FURTHER INFORMATION Campath information site: www.Campath.com Immunogen, Inc. web site: www.immunogen.com Information on HER2 and Herceptin: www.Herceptin.com Rituxan information site: www.Rituxan.com Access to this interactive links box is free online.

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