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IMPROVING THE EFFICACY OF ANTIBODY-BASED CANCER THERAPIES
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.
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.
For antibody-derived molecules, this refers to the number of binding sites for the cognate antigen(s).
No remaining tumour can be detected by visual inspection or by clinical imaging technologies. This does not mean that the disease has been cured.
≥ 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: email@example.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 hours1–4. 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 VALENCY1,3–5. 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-Hodgkin’s 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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acute myelogenous leukaemia. colorectal cancer. Phase III Phase III Phase III mClone Systems EGFR ch IgG LymphoCide (epratuzumab) MDX-210 CD22 ERBB2 X CD64 (FcγRI) hu IgG mu F(ab’)2 Immunomedics Medarex. Phase III Phase III. EpCam. murine. monoclonal antibody.1 versus 6. Clinical strategies RESPONSE DURATION Time from the first response until disease progression or death. Herceptin has synergistic antitumour activity when used in combination with cisplatin and carboplatin13. These gains included longer median duration of response (9. chronic lymphocytic leukaemia. Phase II Titan Pharmaceuticals Anti-idiotypic mAb. NSCLC. non-Hodgkin’s lymphoma. 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. Centocor EpCam mu IgG2a Dukes’ C CRC CD20 ch IgG1 NHL Approved in US IDEC November 1997 Pharmaceuticals. Federal Drug Administration. and REFS 1. SCLC. mAb. CEA.14. OVERALL RESPONSE RATE Sum of partial and complete responses. Combination with cytotoxic drugs. Biologics License Application. methotrexate. hu. 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. 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. CRC.2). carcino-embryonic antigen. small-cell lung cancer. mu. 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 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. Combining different cytotoxic drugs is a widely and successfully used clinical strategy in oncology that increases the response rate and duration of individual drugs. non-small-cell lung cancer. higher overall response rate (50% versus 32%) and lower death rate at one year (22% versus 33%). GlaxoSmithKline VEGF Anti-idiotypic mAb. epidermal-growth-factor receptor. GD3 ganglioside mimic CD20 hu IgG1 mu IgG Metastatic NSCLC. chimeric. 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.1 months). FDA. immunoglobulin. EGFR. Merck KGaA Corixa. cyclophosphamide. NHL. VEGF. but accuracy cannot be guaranteed. Genentech Phase III. epithelial cellular-adhesion molecule. metastatic CRC SCLC.REVIEWS 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. NATURE REVIEWS | C ANCER VOLUME 1 | NOVEMBER 2001 | 1 1 9 © 2001 Macmillan Magazines Ltd . For example. malignant melanoma NHL Phase III. CLL. Ig. gastric cancer AML NHL Combination with chemotherapy 90 yttrium * Phase III clinical trials or later. celecoxib14–18. Every effort has been made to obtain reliable data from several sources (company and industry web sites. ch. The use of antibodies in conjunction with chemotherapeutics is a natural extension of this approach. Not included are ongoing trials with marketed antibody products. BLA. PEM. AML. and additive benefit when used in conjunction with doxorubicin. humanized. taxol or the selective cyclooxygenase-2 inhibitor. 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. polymorphic epithelial mucin. vascular endothelial growth factor.
cisplatin and doxorubicin23. At present. Several variants of the humanization technology have been developed105. 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. Adding Herceptin to a regimen of an anthracycline drug plus cyclophosphamide. these are created by grafting the antigen-binding loops. C 2 H Clinical experience Chimeric. patients’ disease and immune status. 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. from a mouse mAb into a human IgG102–104. local expertise and commercial considerations. known as complementarity-determining regions (CDRs). By contrast. humanized and human antibody evidence indicates that these types of antibody are less immunogenic than those of mice115. auguring well for ongoing oncology trials.21. Humanized antibodies In the simplest case. www. Ongoing randomized Phase III trials are anticipated to establish the statistical significance of improved response rates or duration from combining Rituxan with chemotherapy. direct routes to human antibodies offer faster preclinical development in cases with no existing mouse mAbs. antigen specificity of the antibody and immune complex formation with antigen115. 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.REVIEWS Box 1 | Key therapeutic antibody technologies Murine antibodies Derived by hybridoma technology99 following immunization of mice or. 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.59. Humanized antibodies contain less foreign sequence than their chimeric counterparts and are presumed to be less immunogenic. Single-arm Phase II clinical studies with Rituxan plus chemotherapy (cyclophosphamide. and the clinical success of combining Herceptin and Rituxan with chemotherapy. established tumours in some Light 10 CL chain preclinical xenograft models . respectively100. although the symptoms generally improved with standard medical care19.20. Moreover. whereas Herceptin plus chemotherapy is associated with additional adverse events that are comparable to. Other factors that affect the immunogenicity of antibodies include the method and frequency of administration. dose.101. these tangible clinical benefits of combining Herceptin with chemotherapy come at the price of greater toxicity. with no additional toxicity. Choice of antibody technology Humanization and human antibodies are now the preferred technologies for developing antibodies as therapeutics. doxorubicin. at least five other anticancer antibodies are being tested in combination with chemotherapy (TABLE 1)1.22. The anti-CD20 antibody Rituxan sensitizes some drug-resistant human B-cell lines to the cytotoxic effects of etoposide.112–114. 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. The increased number of these studies probably reflects several factors. human immunoglobulin genes Heavy and genetically disrupted endogenous immunoglobulin loci. was associated with a significant increase in cardiotoxicity. despite the lack of substantiating clinical data. Unfortunately. In addition to Herceptin and Rituxan. High-affinity human antibodies have also been obtained from transgenic mice that contain some. Human antibodies These have high affinity for their respective antigens and are routinely obtained from very large. less commonly.com/reviews/cancer 120 | NOVEMBER 2001 | VOLUME 1 © 2001 Macmillan Magazines Ltd . Similar arguments have been made about humanized and human antibodies. Herceptin alone is generally very well tolerated and typically associated with only very minor adverse events9. Two key factors are preclinical data showing the benefit of combining the specific antibody in question with chemotherapy.nature. rats. 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. single-chain variable fragments (scFvs) or Fab phage display libraries106–110. vincristine and prednisone) in low-grade24 and high-grade25 B-cell nonHodgkin’s lymphoma indicate that there is an additive24 or at least comparable25 clinical benefit of Rituxan plus chemotherapy versus chemotherapy alone. or worse than. Humanization is a clinically well-validated technology that might be favoured if a well-characterized mouse mAb is available. or preferably many. chemotherapy alone19. A human anti-epidermal growth factor (EGF) receptor mAb VL obtained using transgenic mice eradicates large. 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 CH1–CH2–CH3 for light and heavy chains. The choice of different human antibody technologies will depend on their availability. Immunization elicits the VH chain production of human antibodies recoverable using standard hybridoma CH1 technology10.
These data support the concept of a ‘binding-site’ barrier that can impair penetration of the tumour for very high-affinity antibodies123. 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. CDC is initiated by complement component C1q binding to the Fc region of IgG. An anti-melanoma antibody had potent antitumour activity in a mouse model of metastasis. 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. These opposing factors limit the accrual of scFv within tumours to levels that are better suited for imaging than for therapy125. but involves too few tumour cells to be directly imaged in patients. has been reported to be more efficacious in the treatment of micrometastases and minimal residual disease26. direct killing126. or YEAST DISPLAY with DNA shuffling120. following surgery.139. Tumour targeting by IgG is impaired by poor tumour penetration. 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. which is physically linked to its encoding mRNA. and uptake reached a plateau at 10–9–10–11 M. and the Fcγ receptors on immune effector cells. (scFv′)2 . Nevertheless. Small antibody fragments. such as scFv. Alternatively. It remains to be seen whether Panorex alone. The molecular architecture of antibodies is readily modified to create non-natural antibody formats that vary in size and valency. will prove efficacious in Phase III trials that are ongoing in the United States (TABLE 1). but not to other parts of the body. ADCC is likely to be the mechanism underlying the antitumour effects of the Fc–Fcγ receptor interaction. NATURE REVIEWS | C ANCER VOLUME 1 | NOVEMBER 2001 | 1 2 1 © 2001 Macmillan Magazines Ltd . the concept of treating minimal residual disease warrants evaluation in the context of other antitumour antibodies. 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).139.REVIEWS Box 2 | Engineering antibodies for enhanced antitumour activity Antibodies can be engineered with altered properties. macrophages and natural killer cells. or in combination with chemotherapy. is a strategy26. DUKE’S STAGE C COLORECTAL CANCER Cancer that has spread from the colon to nearby lymph nodes. Subsequent target-cell killing can occur in a cell-dependent or cell-independent manner (FIG. FC REGION For an IgG. 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 affinity of antibodies can profoundly affect their ability to localize to tumours. and diffuse more readily within tumours. this comprises the CH2 and CH3 domains (BOX 1). a dimeric anti-ERBB2 antibody fragment. DNA SHUFFLING Process for creating molecular diversity by homologous recombination of DNA in vitro. Indeed. which is bound to the surface of a tumour cell. Selectivity might be crucial as many tumour-associated antigens are expressed on some normal cells at lower levels. and uptake by Fc receptors on reticulo-endothelial cells5. and presumed similar tumour penetration and pharmacokinetic properties124. which results in more rapid and/or more extensive internalization126. with significant effects on tumour targeting ability. such as antigen-binding affinity. 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. Significantly. This laborious task of affinity maturation has been facilitated through the use of RIBOSOME DISPLAY118 in conjunction with DNA SHUFFLING119.27 that attempts to address the difficulties of poor accessibility and limited penetration of solid tumours by antibodies1. IgG homodimerization might enhance in vivo antitumour activity126.122. RIBOSOME DISPLAY Technology for displaying a nascent protein. Clinical trials with Herceptin are now doing just that22. A threshold affinity of 10–7–10–8 M was required for specific tumour localization of scFv. 2). Second. which reflects their size and their recycling by the neonatal Fc receptor. 17-1A29.139. EXTRAVASATE more efficiently than IgG. Using antibodies to target MINIMAL RESIDUAL DISEASE as well as MICROMETASTATIC DISEASE. accrual of IgG within tumours is favoured by their long halflife. as shown with a panel of single-chain variable fragments (scFvs) that bind to the same epitope on ERBB2 with affinities ranging from 10–7–10–11 M121. follow-up periods of several years are required to track any differences in time to disease progression or death. molecular architecture and dimerization state1. that can enhance their tumour targeting and potency. the anti-epithelial cellular adhesion molecule (EpCam) mAb.27 than bulky metastatic disease30. the large size and long duration of such trials makes them exceedingly expensive to conduct. showed increased tumour uptake compared with a monomeric Fab fragment of similar size (~50 kDa).137. Targeting minimal residual disease. 2). but are also rapidly eliminated through the kidney. The first demonstration that the Fc–Fcγ receptor interaction is important for the antitumour activity of an antibody in vivo came with the development of mice that lack FcγRI and FcγRIII31. Third. EXTRAVASATION Movement out of the vasculature compartment into interstitial spaces. MICROMETASTATIC DISEASE Metastatic disease that can be detected by immunohistochemistry of tissue biopsies. IgG homodimerization can also increase the apparent affinity for binding to the cognate antigen on cells. The impact of the antigen-binding affinity of IgG molecules on their tumour-localization ability remains to be determined. Tumourcell killing by ADCC is triggered by the interaction between the FC REGION of an antibody bound to a tumour cell.117. chemotherapy or radiotherapy. The antigen-binding affinity (Kd values) of antibodies has historically been increased by selection from phage display libraries116. which results from their large size. growth arrest126 and synergy with chemotherapy or immunotoxins138. 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. but this benefit was lost in mice that lack FcγRI and FcγRIII receptors.28. FcRn. trials of many thousands of patients might be needed to allow statistical assessment of treatment outcome. now known as edrecolomab (Panorex). such as neutrophils. 3–5. Enhancement of the in vitro antitumour activity of Rituxan by homodimerization138 should encourage additional preclinical and perhaps clinical assessment of this strategy.138. By contrast. For example. that relies on in vitro transcription and translation. First. MINIMAL RESIDUAL DISEASE Tumour remaining in patients following debulking by surgery and/or chemotherapy and/or radiotherapy.
can significantly affect the resulting antibody glycoforms which. that interfere with CDC and allow them to escape complement attack42. on the cusp of regulatory approval (see below). an antibody-fragment–enzyme fusion protein is typically allowed to localize to a tumour and be cleared from the system. small molecules or proteins). One potential problem with this approach is that many malignant cells. BISECTED COMPLEX OLIGOSACCHARIDES Branched carbohydrate that might include several different kinds of monosaccharides. toxin or immunological effector cell b Direct arming c Indirect arming Figure 1 | Strategies for enhancing the potency of antitumour antibodies. tositumomab (Bexxar) and ibritumomab tituxetan (Zevalin). thereby diminishing the systemic toxicities of these cytotoxic agents. as well as for intracerebral therapy with an anti-EGF receptor antibody in a brain tumour model33. for example. 1). Indeed. Alternatively.REVIEWS 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. 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. but absent or present at much lower levels on normal cells. 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. The www. A prodrug is then administered and ideally converted to an active drug solely within the tumour.nature. A judicious choice of both target antigen and antibody is likely to be crucial to the success of all arming strategies. It remains to be seen if these improvements in CDC in vitro translate into more potent antitumour activity in vivo. 39). Numerous strategies for improving the efficacy of antitumour antibodies are now being tested. scFv. arming antibodies with cytokines is intended to create high intratumour concentrations of cytokines to stimulate the antitumour immune response (T cells. The antitumour activity of Herceptin and Rituxan was greatly reduced in mice that lack the activation receptors FcγRI and FcγRIII. such as CD46. that accommodates sequence insertions and deletions. an antibody–streptavidin conjugate is allowed to accrue within a tumour and is then used to capture a biotin–chelator–radionuclide complex. 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. the target antigen should be universally found on tumour cells of a given type.com/reviews/cancer 122 | NOVEMBER 2001 | VOLUME 1 © 2001 Macmillan Magazines Ltd . in turn.28 (FIG. Ongoing studies will address whether these antibodies have enhanced antitumour activity in vivo. This modification of glycosylation was accomplished by cellular engineering of the production host — Chinese hamster ovary cells — by transfection with β-(1. 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. clinical evaluation of armed antibodies has been beset by unacceptably high levels of toxicity in several clinical trials28. induction of apoptosis by crosslinking of effector and target cells could explain these observations. and is necessary for effector functions37. by previous chemotherapy. The cells producing the antibody (the ‘production host’) and. antibody arming is enjoying a renaissance with the approval of the first armed antibody. For radionuclide pre-targeting. yielded up to a twofold enhancement in ADCC in vitro34. The antitumour activity of Herceptin was also attenuated by a mutation (D265A) that impairs binding to FcγRIII and FcγRIIB. including N-acetylglucosamine between main branches. single-chain variable fragment. The importance of the Fc–Fcγ receptor interaction for antitumour activity was subsequently shown for the clinically important antibodies Herceptin and Rituxan32. For prodrug pretargeting. Glycosylation of IgG molecules at Asn297 (KABAT 35 NUMBERING SCHEME ) helps to maintain the tertiary structure of their CH2 domains36 (BOX1). Effector cell populations might be suppressed or diminished in cancer patients. culture conditions. and with two others. gemtuzumab ozogamicin (Mylotarg). devised by the late Elvin Kabat. whereas disruption of the gene that encodes the inhibitory receptor FcγRIIB substantially enhanced antitumour activity32. Increasing the level of BISECTED COMPLEX OLIGOSACCHARIDES attached to the Fc region of an antibody can enhance its ability to support ADCC39. d | Pre-targeting strategies aim for the selective delivery of radionuclides to tumours or selective intratumour activation of prodrugs. The in vivo and clinical significance of this in vitro improvement is unknown. Ideally. Armed antibodies typically show more potent antitumour activity in preclinical tumour xenograft studies than their ‘naked’ parents. b | Direct arming of antibodies entails their covalent linkage to killing machinery. point mutations in the Fc region. Unfortunately. which could curtail the effector functions of antitumour antibodies.4)-N-acetylglucosaminyltransferase III (REF. CD55 and CD59. B cells or natural killer cells). KABAT NUMBERING SCHEME Immunoglobulin amino-acid residue numbering scheme. to a lesser extent. while avoiding the toxicities associated with systemic cytokine delivery. which result in improved binding to FcγRIII. leading many to abandon this approach. Nevertheless. 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. as well as normal cells. including the representative examples shown here and described in BOX 2. express membrane-bound proteins. can influence the ability of the antibody to participate in ADCC38. such as radionuclides or toxins (for example. Direct arming a Enhancing effector functions d Pre-targeting Biotin–chelator– radionuclide Streptavidin Complement-dependent cytotoxicity Point mutations and/or modified glycosylation Antibody-dependent cellular cytotoxicity Prodrug scFv–enzyme Tumour cell Drug Cytokine scFv fragment Immunocytokine Sterically stabilized immunoliposomes Small molecule or protein toxin Radionuclide Bispecific antibody Radionuclide.
It might be possible to exploit antigens on tumour cells for tumour vasculature targeting by the judicious choice of antibody drugs.REVIEWS Box 3 | Alternative targets for anticancer antibodies Most approved and experimental anticancer antibodies directly target tumour cells. 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. A fundamental problem with this approach is the PANNING Process of separating targetbinding clones from nonbinding clones for phage display library.01% of the injected dose per gram of solid tumour in humans compared with 20% or more in mice48. This approval is based on an overall response rate of 30% with acceptable toxicities52. An antibody that neutralizes the angiogenic factor vascular endothelial growth factor (VEGF) has potent antitumour activity in vivo127. This is typically 0. Encouragingly. Direct selection for antibodies that efficiently internalize is now possible by PANNING on cells using antibody phage display libraries43.134. Small-molecule toxin conjugates.136. unfortunately. The mAb BR96 (anti-sialyl Lewis Y antigen) conjugated with doxorubicin has proved highly efficacious in tumour xenograft studies45 but.44. 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.128. but not radionuclides. NATURE REVIEWS | C ANCER VOLUME 1 | NOVEMBER 2001 | 1 2 3 © 2001 Macmillan Magazines Ltd . release from the antibody is followed by a chemical rearrangement to create diradicals that can cause double-stranded DNA breaks and compromise cell viability. small intestine and pancreas46. The combination of anti-VEGF and anti-ERBB2 (Herceptin) antibodies has proved more efficacious in tumour xenograft models than either antibody alone. as these fragments accumulate primarily on perivascular tumour cells122. Conjugation of these small-molecule toxins to antibodies converts them to inactive prodrugs that can selectively target tumours. Moreover. Fab and scFv) results in de facto vasculature targeting5. dose-limiting gastrointestinal toxicities were observed in the breast cancer trial because the immunoconjugate bound to antigen-positive normal cells in gastric mucosa. 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. In the case of calicheamicin. Unfortunately. The humanized anti-CD33 antibody–calicheamicin 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. plasma cells and nonlymphoid tissue. combination of the low molar toxicity of chemotherapeutics and the miniscule proportion of an injected mAb that usually localizes to a solid tumour target. vasculature damage has a multiplicative effect as many tumour cells are dependent on each capillary. and noninvasive imaging indicates that this is due to angiogenesis inhibition129. Tissue factor has recently been targeted to the ED-B domain of fibronectin. tumour vasculature targeting is limited at present by the lack of blood-vessel-specific target antigens. synergistic antitumour activity was observed for immunotoxins directed to the tumour vasculature and the tumour itself.001–0. CD20 is a tetra-spanning membrane protein that is found on mature B cells. and as vascular endothelial cells are not transformed. tumour regrowth following tumour vasculature targeting might occur from a thin layer of tumour cells close to blood vessels132. Targeting the tumour vasculature has several potential advantages over direct tumour targeting131–133: the vasculature is more accessible to antibodies. Tumour targeting with rapidly clearing antibody fragments (for example. but not on stem cells or nonlymphoid tissue. This has encouraged the humanization130 of an anti-VEGF antibody. personal communication). now known as bevacizumab (Avastin) that is now in Phase III clinical trials for metastatic cancers (TABLE 1).133. respectively. Pegram. Tumours were eradicated by intratumour thrombosis in ≤ 30% of the mice treated with no obvious side effects134. A prerequisite for antibody arming with small molecule toxins. Calicheamicin contains a sugar component that contributes to its potency by binding to the minor groove of DNA. Coagulation at non-tumour sites is a potentially significant safety issue to be addressed with tumour-targeted thrombosis. Arming antibodies with conventional cytotoxic chemotherapeutics has been widely explored28. including >90% of B-cell lymphomas.135. encouraging further preclinical studies. has shown little or no efficacy in Phase II trials for metastatic breast cancer46 and advanced gastric adenocarcinoma47. Angiogenesis is the process by which tumours become vascularized by the proliferation of new blood vessels from existing ones. Recognition of this problem inspired the conjugation of antibodies to small-molecule toxins that are 100–1000-fold more potent than conventional chemotherapeutics. Calicheamicins49–52 and maytansinoids53 are the most extensively evaluated of numerous small-molecule toxins used for antibody arming. as well as myeloid progenitor cells and committed precursors. encouraging clinical evaluation of this combination (M. but is absent from stem cells. thereby allowing the tumours to grow beyond a minimal size. which is a natural marker of angiogenesis that is present in many solid tumours but not most normal blood vessels and tissues134. Unfortunately. 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. is that the antibody should be efficiently internalized. Alternative strategies include inhibiting angiogenesis or directly targeting tumour neovasculature5. CD33 is a sialic-acid-binding Ig-like lectin (siglec) that is found on the surface of virtually all acute myelogenous leukaemia cells. antigens most successfully targeted with armed antibodies so far — CD20 and CD33 — closely match these criteria.
Radioimmunoconjugates. or a bacterial toxin. This enhancement is due to specific targeting to tumour cells plus steric blockade of the interaction with a prevalent inhibitor61. can potentially direct the killing of tumour cells by antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)42. 186rhodium and 188 rhodium) are cytotoxic over many cell diameters.56. One of the main problems was the formation of immune complexes between the immunoconjugate and shed mucin. although not CURES (that is. albeit at doses close to the maximum tolerated dose53.55 was less successful and has been abandoned.com/reviews/cancer Effector cell FcγR 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. 186rhodium and 188rhodium. Most immunotoxins comprise either a plant toxin. But bystander-cell killing by radioimmunoconjugates is a double-edged sword. An additional 124 | NOVEMBER 2001 | VOLUME 1 © 2001 Macmillan Magazines Ltd . 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. the tumours eventually regrew following treatment). conjugated or genetically fused to an antibody or antibody fragment28. were obtained even after treating mice that had very large tumours (mean size of 500 mm3) or heterogeneously expressed the target antigen. A prerequisite first step for CDC is recruitment of the complement component C1q by IgG bound to the tumour cell surface. extravasation of fluids and proteins resulting in weight gain and. Pseudomonas exotoxin. tumour-cell-bound C1q can bind to complement receptors. as well as allowing imaging and quantification of radionuclide uptake63. clinical development of immunotoxins has also been plagued with toxicity problems. particularly IgG1 and IgG3. Yarranton. This can lead to the formation of a membrane attack complex that kills the target cell by disrupting its cell membrane. The recruitment of human proteins as toxins circumvents the immunogenicity of non-human protein toxins. CURE Tumour does not reappear for a prolonged time period — deemed sufficient for regrowth of any residual tumour — following anticancer therapies. kidney damage and pulmonary oedema. as some tumour www. Yarranton. personal communication). VASCULAR LEAK SYNDROME Involves damage to vascular endothelial cells. such as C1qR.64. By contrast. and also decreased its immunogenicity and toxicity59. Excitingly. Unfortunately. clinical development of an efficiently internalizing humanized antimucin antibody–calicheamicin conjugate for treatment of solid tumours54. Complete responses. The tumour cell is eliminated by phagocytosis or lysis. Arming antibodies with radionuclides enables them to kill BYSTANDER CELLS. on effector cells. 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 can trigger cell-mediated tumour-cell lysis or phagocytosis. 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. particularly FcγRI and FcγRIII. 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. The killing of neighbouring tumour cells is particularly beneficial. the mean range of β-particles from 90yttrium is >200 cells62. This triggers a proteolytic cascade to activate complement. fused to Pseudomonas57. such as VASCULAR LEAK SYNDROME. For example.58. depending on the type of effector cell. depending on the type of mediating effector cell. Human antibodies.nature. For example. macrophages and natural killer cells. Protein toxin conjugates. This is because the β-particles emitted by commonly used radionuclides (131iodine. for example. Antibodies conjugated to maytansinoids have cured mice that bear human tumour xenografts. but not 90yttrium) also emit γ-rays that can kill cells over an extended distance. Immunotoxins have occasionally been associated with antitumour responses in patients57. Uptake of these immune complexes by liver and spleen led to dose-limiting toxicities (G. such as neutrophils. personal communication). ADCC is triggered by an interaction between the Fc region of an antibody that has bound. Unlike cells in solid tumours. Site-specific PEGYLATION of one recombinant immunotoxin improved its antitumour activity in animal models. Many commonly used β-emitting radionuclides (131iodine. in its most severe form.REVIEWS limitation of the antimucin conjugate might have been its probable inefficient accumulation in solid tumours that were treated48. to a tumour cell and the Fcγ receptors (FcγRs). CR1 (CD35) and CR3 (CD11b/CD18). these tumour cells are readily accessible by virtue of their location in either the circulation or bone marrow (G. such as ricin A chain. So. These encouraging preclinical data have supported the progress of two antibody–maytansinoid conjugates into Phase I clinical trials. 90yttrium. and by immunogenicity that often precludes multiple dosing. through its antigen-binding region. Alternatively. on immune effector cells such as neutrophils. arming antibodies with smallmolecule toxins is a valuable but not universal approach to increase their antitumour potency. macrophages and natural killer cells.
the cytokine could be fused to the amino and/or carboxyl terminus of the light and/or heavy chains. Radionuclides such as 212bismuth. conjugate solubility or promoting aggregation. Using immunoliposomes to target tumour vasculature (BOX 3). Lower doses of Zevalin were still efficacious but were less toxic. provided that the functions of both antibody and cytokine are preserved. drugs and toxins to tumours80–82 (FIG. and new labelling methodologies are urgently needed. including their inherent complexity and extravasation due to their large size (commonly ~100 nm in diameter) 79. Zevalin and Bexxar ( 131iodine-anti-CD20 mouse mAb) have successfully completed Phase III clinical trials in non-Hodgkin’s lymphoma. In one clinical trial. namely Theragyn (pemtumomab) (TABLE 1).78. Such data bode well for ongoing clinical studies with the immunocytokines huKS-IL2 and hu14. Such α-emitters are particularly attractive for radioimmunotherapy of micrometastatic disease. NATURE REVIEWS | C ANCER VOLUME 1 | NOVEMBER 2001 | 1 2 5 © 2001 Macmillan Magazines Ltd . Liposomal formulations of the chemotherapeutics doxorubicin (Doxil) and daunorubicin (DaunoXome) have been approved in recent years for the treatment of Kaposi’s sarcoma. They are expected to be the first approved radioimmunoconjugates. 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. by virtue of their short range and high linear energy transfer70.77. Delivery of chemotherapeutics using immunoliposomes offers substantial benefits over the use of free drugs. AUTOLOGOUS STEM-CELL SUPPORT Immunoliposomes. BYSTANDER CELLS Cells in the immediate vicinity of a cell that has a bound antibody-based targeting agent. 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. Moreover.05) (REF. and neoplasms spread on the surface of body compartments. as well as avoiding the toxicities associated with systemic cytokine delivery72. Beyond Bexxar and Zevalin. Antibodies armed with these α-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 β. such as ovarian cancer and neoplastic meningitis62.73. 213bismuth and 211 astatine emit α-particles that are highly cytotoxic over very short distances (< 100 µm).67. CEA-cide (rabetuzumab) and Cotara1. Liposomes are self-assembled lipid bilayers that encapsulate some of the surrounding medium during their formation. Bispecific antibodies. Several different cytokines have been evaluated. Despite such encouraging progress with immunoliposomes. at least three radioarmed antibodies are now in Phase I/II or later trials. B cells or natural killer cells). Bispecific antibodies (BsAbs) are non-natural antibodies that bind to two different epitopes. 1). In clinical oncology. Interim analysis of data from the first 90 patients showed an overall response rate of 80% with Zevalin versus 44% with Rituxan (P <0. Such large payloads offer an important potential advantage over direct antibody arming. The dark side of bystander-cell killing is damage to non-tumour cells. the systemic toxicity of the immunoliposome-targeted doxorubicin was much less than that of free doxorubicin. rather than the tumour per se. probably by causing expansion of immune effector cells73. MYELOABLATIVE Elimination of myeloid cells and their progenitors.18-IL2. 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-Hodgkin’s lymphoma66. including interleukin (IL)-2. There are also few facilities capable of producing such radionuclides. Moreover. Fusing antibodies to cytokines is a promising alternative arming strategy to the use of toxins and radionuclides. relatively nontoxic) daughter nuclides. obviates the need for extravasation. patients were randomized to treatment with either Rituxan or Zevalin — a 90 yttrium-labelled version of the mouse parent mAb that begat Rituxan. 68). 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.REVIEWS cells might lack the target antigen and thereby escape antibody therapy65. 68). Zevalin treatment of Rituxan-refractory disease gave an overall response rate of 46% (REF. BsAbs have been used most widely for delivering immune effector cells and. Engineered antibody fragments can be attached to the surface of stealth liposomes for selective tumour targeting of large payloads of drugs76. several underlying difficulties remain. These so-called immunocytokines are designed to create high intratumour concentrations of cytokines to stimulate the antitumour immune response (T cells. particularly those in haematological tissue. For example. IL-12 and granulocyte-macrophage colonystimulating factor (GM-CSF)74. in which only one or a few molar equivalents of the payload are attached per antibody to avoid compromising antigen binding. to a lesser extent. and the improvement of methods for loading and retaining drugs75. Indirect arming PEGYLATION Chemical modification of a protein with one or more molecules of polyethylene glycol. toxins or even DNA for gene therapy.71. In theory. in a Phase I/II dose-escalation study of Zevalin. For example. This immunocytokine eliminated established metastases in a syngeneic mouse tumour model. The clinical investigation of antibodies armed with α-emitters began only in the late 1990s62 because it has been difficult to identify α-emitters that have physical half-lives suitable for radioimmunotherapy and that produce acceptable (that is. Radioimmunoconjugates can cause severe toxicities. for delivery of radionuclides. Immunocytokines. which results in severe toxicity. Significant advances in liposome technology include the development of sterically stabilized (‘stealth’) liposomes that are long-lived in vivo. Harvesting of a patient’s haematological stem cells before myeloablative anticancer therapy.and γ-emitters. Bystander killing also helps address the problem of limited accrual of antibody in poorly vascularized or bulky tumours48. followed by rescue by engraftment of the stem cells back into the patient. almost invariably chosen on two different antigens.
87. 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. is administered and allowed to localize to a tumour. An antibody–enzyme fusion protein. FcαRI (CD89) on macrophages. in principle. The prodrug diffuses widely. Pre-targeting prodrugs. although other triggers have also been used82. FcγRI (CD64) on granulocytes and monocytes. 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. 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. Intratumoral injection of this antibody together with systemic prodrug administration proved more www. as observed for many monospecific antibodies (BOX 1). Approval of a BsAb for therapeutic use remains a distant prospect. This strategy typically requires two or three separate components and treatment steps. despite much progress in both basic and clinical research. In addition. Unfortunately. A significant step towards this goal was the development of a mouse catalytic mAb that activates an etoposide prodrug. The therapeutic benefits of this ADEPT system over free doxorubicin reflect 4–12-fold higher intratumour drug concentrations and up to 5-fold lower extratumour drug concentrations. Such thoughts inspired the development of a humanized anti-carcinoembryonic antigen (CEA) antibody–human-β-glucuronidase fusion protein plus doxorubicin glucuronide prodrug89. binding of monospecific antitumour IgG to Fc receptors can potentially be inhibited by serum IgG binding to these receptors. but regrettably it was found to be ineffective in vivo92. which was refractory to systemic BsAb therapy. 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. but it is exceedingly difficult to translate into clinical practice86.com/reviews/cancer Cytotoxic agents. New applications for BsAbs will probably be needed for this modality to significantly benefit cancer patients. In one approach. The immunogenicity of BsAb evaluated in patients will probably be greatly reduced by the use of humanized or human antibodies. 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. so reducing toxicity compared with systematic administration of cytotoxic chemotherapy. more effective strategies are needed for the targeting of effector cells and selective activation in the context of tumour cells. thereby reducing the risk of tumours evading therapy by antigen loss. ADEPT has proved highly effective in several different tumour xenograft studies. thereby compromising the ability of the antitumour antibody to support ADCC. either unassisted or facilitated by a clearing agent. but many obstacles 126 | NOVEMBER 2001 | VOLUME 1 © 2001 Macmillan Magazines Ltd . or historically a conjugate. The cytotoxic effects of the drug are ideally confined to the tumour target. might be targeted specifically to cancer cells through the technique of pre-targeting. and the immunogenicity of their non-human components. Another common clinical problem associated with BsAb is that the systemic activation of effector cells causes widespread cytokine release. the antibody component is first targeted to the tumour followed by clearance of the residual circulating antibody. The risks of using human enzymes for ADEPT can. The most extensively used trigger molecules are CD3 on T cells. as well as bispecific human IgG85. Pre-targeting strategies have advanced markedly. FcγRIII (CD16) on natural killer cells and. A prodrug is administered after the remaining fusion protein has cleared. By contrast. The ‘Achilles heel’ of pretargeting strategies is their inherent complexity.REVIEWS 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.87. Antibody-dependent enzymemediated prodrug therapy (ADEPT) involves pre-targeting of prodrugs to tumours86. So. which leads to serious side effects. Encouraging local antitumour responses have been seen for BsAb targeting of T cells to ovarian cancer cells in small-scale clinical trials83. more recently. Pre-targeting strategies remain to be overcome if they are to provide significant new treatment options for cancer patients. endogenous substrates or inhibitors could interfere with the desired prodrug activation within a tumour. respectively. Powerful new technologies for the production of a plethora of alternative BsAb fragments84. treatment failure occurred because of metastasis outside the peritoneal cavity. but in the ideal case is activated solely at the tumour following contact with the localized fusion protein. A cytotoxic agent is then administered for selective capture or activation at the tumour site.nature. Human enzymes in conjunction with humanized or human mAb should greatly reduce this immunogenicity problem. human carboxypeptidase A1 was engineered to activate a prodrug that is not a substrate for the wild-type enzyme91. A second benefit is the ability to kill bystander tumour cells. such as radionuclides or prodrugs. The use of human enzymes for ADEPT poses the potential risk of unwanted activation of prodrug by endogenous enzymes. The elevated levels of endogenous β-glucuronidase within some tumours also indicate that glucuronide prodrugs have potential as monotherapy90. To circumvent this problem. have the potential to overcome this problem. be circumvented by using a humanized or human catalytic antibody that is designed for prodrug activation by a non-physiological mechanism. 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.
D. D. C. Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy. Significant advantages of pre-targeted over conventional radioimmunotherapy include the much greater ratios of radioactivity in tumour versus nontumour tissues.. This reflects the fact that antibody localization to tumours in mice is often efficient. Oncogene 17. Monoclonal antibody to HER-2/neu receptor modulates repair of radiation-induced DNA damage and enhances radiosensitivity of human breast cancer cells overexpressing this oncogene. 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. 3. D. G. breast cancer that has progressed after chemotherapy for metastatic disease. 2. Pre-targeted radioimmunotherapy. Crit. Pivotal Phase III trial that led to the approval of Rituxan for the treatment of low-grade or follicular non-Hodgkin’s lymphoma.97. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Perspectives Antibodies have started to fulfil their promise as anticancer therapeutics with the five antibodies now marketed as drugs in the United States (Rituxan. in Lung Biology in Health and Disease (eds Jardieu. Oncol. together with reference 9. M. et al. Cancer Res. M. Proc.. targeting minimal residual and micrometastatic disease is attractive as it obviates the need to penetrate bulky tumours. Halin. 19. Clinical trials of antibody therapy. W. et al. R. Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. Kim.. thereby lowering the whole body exposure to radioactivity. Delivery of a radionuclide such as 90yttrium is accomplished using a biotinylated chelator. 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