REVIEW ARTICLE
`
`BioDrugs 2000 Oct; 14 (4): 221-246
`1173-8804/00/0010-0221/$20.00/0
`
`© Adis International Limited. All rights reserved.
`
`Biological Therapy of Breast Cancer
`John W. Park, Debasish Tripathy, Michael J. Campbell and Laura J. Esserman
`Carol Franc Buck Breast Care Center, UCSF Cancer Center, San Francisco, California, USA
`
`Contents
` . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
`Abstract
`1. Monoclonal Antibody-Based Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
`1.1 Anti-HER2 Monoclonal Antibody-Based Therapies . . . . . . . . . . . . . . . . . . . . . . . . . 222
`1.1.1 HER2 as a Target for Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
`1.1.2 Trastuzumab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
`1.1.3 Anti-HER2 Bispecific Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
`1.1.4 Anti-HER2 Immunotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
`1.1.5 Anti-HER2 Immunoliposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
`1.2 Monoclonal Antibody-Based Therapies Against Other Antigens . . . . . . . . . . . . . . . . . 227
`2. Active Specific Immunotherapy/Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
`2.1 Breast Cancer Vaccines: General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . 228
`2.2 Anti-HER2 Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
`2.2.1 HER2 as a Target for Vaccine Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
`2.2.2 Anti-HER2 Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
`2.2.3 Anti-HER2 Vaccines for Breast Ductal Carcinoma in Situ . . . . . . . . . . . . . . . . . . 230
`2.3 Antimucin Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
`2.4 Anticarcinoembryonic Antigen Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
`2.5 Anti-p53 Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
`2.6 Other Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
`3. Differentiation Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
`3.1 Retinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
`4. Antimetastasis Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
`5. Antiangiogenesis Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
`6. Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
`6.1 Immunological Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
`6.2 Tumour Suppressor Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
`6.3 Anti-Oncogene Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
`7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
`
`Abstract
`
`Breast cancer treatment has now entered a new era in which biological thera-
`pies, based on a rapidly expanding cellular and molecular understanding of breast
`cancer pathogenesis, have joined the standard armamentarium of surgery, radia-
`tion, chemotherapy, and hormone therapy. In 1998, the anti-HER2 humanised
`monoclonal antibody trastuzumab became the first biological therapy to receive
`US Food and Drug Administration (FDA) approval for the treatment of breast
`cancer, thus marking a milestone that almost certainly will be repeated with other
`new agents. HER2 (ErbB2) has been the focus of many therapeutic strategies because
`of its frequent gene amplification and overexpression in breast cancer, its role in
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`tumourigenesis and cancer progression, and its prognostic and predictive signif-
`icance in clinical studies.
`In preclinical studies, trastuzumab showed antiproliferative activity against
`HER2-overexpressing breast cancers in vitro and in tumour xenograft models. In
`a phase II clinical trial of 222 stage IV patients, trastuzumab was associated with
`an objective response rate of 15%. A randomised phase III clinical trial demon-
`strated that first-line chemotherapy for stage IV patients in combination with
`trastuzumab was significantly superior to chemotherapy alone. Chemotherapy
`plus trastuzumab was associated with a median time to progression of 7.2 months,
`versus 4.5 months for chemotherapy alone (p < 0.001), and a response rate of
`45% versus 29% for chemotherapy alone (p = 0.001).
`Other novel therapies involving antibody targeting of HER2 are under devel-
`opment, including bispecific antibodies, immunotoxins, and immunoliposomes.
`Vaccine approaches are also under active investigation, including those directed
`against HER2 and mucin antigens. Gene therapy strategies under development
`include gene transfer of immunomodulatory genes and of anti-oncogene con-
`structs. Other biological therapies include agents designed to induce differentia-
`tion or inhibit invasion, angiogenesis and metastasis.
`
`For many years, the only effective treatment for
`breast cancer was mastectomy, epitomised by the
`development of the Halsted procedure in 1894. The
`subsequent introduction of radiation therapy and
`the advent of improved surgical techniques, includ-
`ing breast conserving approaches, have greatly im-
`proved local-regional management of breast can-
`cer. In a parallel development, systemic treatment of
`breast cancer has also undergone tremendous prog-
`ress, and now includes an array of active cytotoxic
`agents for chemotherapy and hormone-directed
`agents for endocrine therapy. However, further pro-
`gress is clearly required, as the treatment of breast
`cancer remains incomplete or ineffective for many
`patients, and treatment-related toxicities are con-
`siderable.
`Breast cancer treatment now appears on the
`threshold of another major revolution, in which
`these established modalities can be complemented
`by novel biological therapies based on a rapidly
`expanding cellular and molecular understanding of
`breast cancer pathogenesis. In 1998, the anti-HER2
`monoclonal antibody trastuzumab became the first
`biological therapy to receive US Food and Drug
`Administration (FDA) approval for the treatment
`of breast cancer, thus marking a milestone that al-
`most certainly will be repeated with other new
`
`agents. These new biotherapeutic strategies include
`other monoclonal antibody (MAb)-based agents,
`vaccine-based therapies, prodifferentiation agents,
`antimetastatic agents, antiangiogenesis agents and
`gene therapies.
`This review discusses representative examples
`of this new wave of biological therapies for breast
`cancer, with a focus on agents that have been re-
`cently approved or are currently in or near clinical
`trials.
`
`1. Monoclonal Antibody-Based
`Therapies
`
`1.1 Anti-HER2 Monoclonal
`Antibody-Based Therapies
`
`1.1.1 HER2 as a Target for Immunotherapy
`The rodent homologue of HER2, the neu onco-
`gene product, was first identified in neuroblast-
`omas that were generated by in utero treatment of
`rats with ethylnitrosourea.[1] Identification of the
`transforming gene, designated neu because of its
`association with neuroblastoma, revealed it to en-
`code a 185kD membrane-bound glycoprotein closely
`related to the epidermal growth factor receptor
`(EGFR).[2] Subsequent studies demonstrated that this
`original sequence was a mutant allele that differed
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`Biological Therapy of Breast Cancer
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`from the wild type at a single position within the
`transmembrane domain, and which produced con-
`stitutive tyrosine kinase activity.[3] The human
`gene was identified by screening human genomic
`and cDNA libraries for receptors related to EGFR;
`using this strategy, several groups simultaneously
`isolated the human homologue of neu.[4-6] Thisgene
`was designated c-erbB-2 (ErbB2) or HER2 to reflect
`its relation to EGFR (c-erbB-1, HER1), and has
`also been referred to as neu or HER2/neu.
`In contrast to the wild-type rat neu proto-
`oncogene, from which an activated tyrosine kinase
`can arise via a single mutation, analogous muta-
`tions of the HER2 gene have not been observed in
`human cancer.[7] However, the HER2 gene was ob-
`served to be amplified in certain human cancer cell
`lines,[4,5,8] suggesting that overexpression of wild-
`type HER2 might be an alternative oncogenic mech-
`anism. The direct role of HER2 in breast tumour-
`igenesis has since been demonstrated in multiple
`laboratory studies. For example, HER2 functions as
`a classical oncogene that transforms cells in vitro
`and confers tumourigenicity in vivo.[9,10] Transge-
`nic mice overexpressing the mutant or wild-type
`rat neu gene or the mutant or wild-type human
`HER2 gene developed various cancers, including
`mammary cancer.[11-15] Finally, certain MAbs di-
`rected against HER2 or rodent Neu are able to in-
`hibit cancer cell growth in vitro and/or in vivo.
`HER2 amplification was first shown to be clin-
`ically relevant in breast cancer by Slamon and co-
`workers,[16] who observed HER2 amplification in
`approximately 25% of primary breast cancers from
`axillary lymph node–positive patients, which cor-
`relatedwithpoor prognosis. Asubsequent studydem-
`onstrated that HER2 overexpression detected at the
`RNA and protein levels was similarly prognostic
`for poor outcome.[17] It has since been clearly con-
`firmed that HER2 overexpression is associated
`with poor prognosis in node-positive and probably
`also node-negative patients.[18] In addition to its
`prognostic significance, HER2 overexpression has
`also been found to have predictive significance
`with respect to specific therapies. In experimental
`models, HER2 overexpression has induced resis-
`
`tance to tamoxifen therapy.[19,20] In clinical studies
`involving adjuvant chemotherapy of early breast
`cancer, HER2 overexpression correlated with in-
`creased benefit with anthracycline chemother-
`apy,[21-23] and has also been correlated with resis-
`tance to other chemotherapies.[24]
`Among the earliest breast cancer-associated an-
`tigens to be targeted by MAb therapy, HER2 has
`since become a paradigm for immunotherapy of
`solid tumours in general. Indeed, HER2 provides
`an attractive target for MAb-based therapy: it is
`accessible as a cell surface receptor, and when
`overexpressed is present at up to 106 receptors/can-
`cer cell, or 100-fold higher than in normal cells. In
`normal tissues, its expression is detected only in
`certain predominantly epithelial cell types.[25] As
`an oncogene, its continued expression appears to
`remain important throughout malignant progres-
`sion, including metastasis.[26]
`
`1.1.2 Trastuzumab
`As mentioned, certain MAbs against HER2 can
`inhibit cancer cell growth.[27-31] Murine MAb 4D5
`displays cytostatic antiproliferative activity against
`breast cancer cells overexpressing HER2 in both in
`vitro[32] and in vivo models.[33,34] A recombinant
`humanised version of 4D5 (trastuzumab) retains
`these binding and biological activities, but con-
`tains consensus sequences of human IgG1 in place
`of the parental murine MAb sequences.[35] This
`minimises immunogenicity while enhancing the
`potential to recruit human immune effector cells
`via antibody-dependent cellular cytotoxicity, al-
`though this has not been clearly demonstrated in
`patients. The mechanism(s) of the growth inhibi-
`tory activity of trastuzumab are not precisely known.
`Whereas the physiological activator heregulin causes
`activation of the HER2 receptor by heterodimer-
`isation with related receptors EGFR, HER3, and
`HER4, trastuzumab appears to induce altered re-
`ceptor interactions, and possibly generates a differ-
`ent cellular signal.[36]
`Phase I studies of trastuzumab showed that this
`therapy was generally well tolerated, was not asso-
`ciated with significant antibody induction (human
`antihuman antibodies or HAHA), and could be
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`combined with cisplatin chemotherapy. Phase II
`trials showed that trastuzumab has anticancer ac-
`tivity as a single agent[37] as well as in combination
`with cisplatin in patients with advanced and refrac-
`tory metastatic breast cancers that overexpress
`HER2.[38] Based on these early results, 2 expanded
`multicentre studies were initiated to assess the
`efficacy and safety of trastuzumab.[39,40] Results of
`these phase II trials are summarised in table I. In a
`phase II clinical trial of 222 stage IV patients,
`trastuzumab was associated with an objective re-
`sponse rate of 14% (table I).
`In these studies, the likelihood of responding to
`trastuzumab appeared to be higher in patients who
`had not previously received chemotherapy for met-
`astatic breast cancer. Similar tumour response rates
`were seen in patients with visceral metastases and
`in patients who had previously undergone high
`dose chemotherapy with stem cell or bone marrow
`transplant. In the study that included patients pre-
`viously untreated for metastatic disease, no differ-
`ence in response rates was seen between the 2
`trastuzumab doses. Toxicities included an infusion
`reaction consisting of self-limited fevers and chills,
`mostly after the first infusion only, mild upper air-
`way congestion, and diarrhoea. Cardiac dysfunc-
`tion, defined as symptoms of congestive cardiomy-
`opathy or subclinical declines in cardiac ejection
`fraction, was seen in 3 to 5% of patients. The mech-
`anisms of cardiotoxicity remain unclear, since my-
`ocytes do not express HER2 at appreciable levels,
`although the HER2 pathway may be important in
`prenatal cardiac development and possibly in myo-
`cardial remodelling.
`
`A phase III study was performed to directly
`compare chemotherapy plus trastuzumab versus
`chemotherapy alone in patients with HER2-over-
`expressing breast cancers as first-line therapy for
`metastatic (stage IV) breast cancer.[41] Chemother-
`apy plus trastuzumab was associated with a median
`time to progression of 7.2 months, versus 4.5
`months for chemotherapy alone (p < 0.001), and a
`response rate of 45% versus 29% for chemotherapy
`alone (p = 0.001) [table II]. Chemotherapy was
`given for 6 cycles or longer (at the investigator’s
`discretion) in conjunction with weekly trastuzu-
`mab. Trastuzumab therapy was given until disease
`progression, which was the primary end-point of the
`study.
`Patients who had previously received an anthra-
`cycline-based chemotherapy regimen in the adjuvant
`setting received paclitaxel 175 mg/m2; the others re-
`ceived doxorubicin 60 mg/m2 (or epirubicin 75 mg/
`m2) plus cyclophosphamide 600 mg/m2 (AC), with
`all chemotherapy given every 3 weeks. Improve-
`ments in time to disease progression, response rate
`and 1-year survival were seen with the addition of
`trastuzumab to chemotherapy, particularly in the
`paclitaxel stratum (table II). Updated information
`continues to demonstrate an improvement in sur-
`vival due to trastuzumab therapy, with a median
`survival of 20.3 months with chemotherapy alone
`versus 25.4 months with chemotherapy plus
`trastuzumab (p = 0.025).[42] This result is particu-
`larly noteworthy, since not all patients responded,
`and also since 65% of patients progressing on che-
`motherapy alone crossed over to receive tras-
`tuzumab following progression, as allowed by pro-
`tocol.
`
`Table I. Phase II studies of trastuzumab alone or with chemotherapy
`Therapy
`No. of
`Prior chemotherapy for
`Response rate
`patients
`advanced disease
`(%)
`46
`Any
`12
`39
`1 or 2 prior regimens
`24
`
`Median response
`duration (mo)
`6.6
`5.3
`
`Median time to disease
`progression (mo)
`5.1
`Not reported
`
`Trastuzumab
`Trastuzumab plus
`cisplatin
`Trastuzumaba
`3.0
`9.1
`15
`1 or 2 prior regimens
`222
`Trastuzumabb
`3.5
`9 (estim)
`26
`None
`113
`a Trastuzumab was given as a loading dose of 4 mg/kg followed by 2 mg/kg intravenously every week.
`b Patients were randomised to 4 mg/kg followed by 2 mg/kg intravenously every week vs 8 mg/kg followed by 4 mg/kg every week.
`
`Reference
`
`37
`38
`
`39
`40
`
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`Biological Therapy of Breast Cancer
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`225
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`Table II. Phase III randomised trial of chemotherapy versus chemotherapy plus trastuzumab[41]
`Treatment
`No. of
`Median time to disease
`1-year survival
`patients
`progression (mo) [p value]
`(%) [p value]
`234
`4.5 [<0.001]
`68 [0.01]
`Chemotherapy
`Chemotherapy + trastuzumab
`235
`7.2
`79
`138
`5.7 [0.001]
`73 [0.04]
`AC
`AC + trastuzumab
`143
`7.6
`83
`96
`2.5 [0.0001]
`61 [0.08]
`Paclitaxel
`Paclitaxel + trastuzumab
`92
`6.7
`73
`AC = anthracycline (doxorubicin or epirubicin) plus cyclophosphamide.
`
`Response rate
`(%) [p value]
`29 [0.001]
`45
`38 [0.1]
`50
`15 [0.001]
`38
`
`Median response
`duration (mo) [p value]
`5.8 [0.0001]
`8.3
`6.4 [0.0025]
`8.4
`4.3 [0.0001]
`8.3
`
`Toxicities in the phase III trial attributable to tras-
`tuzumab were generally similar to those seen in the
`single agent studies. Cardiotoxicity was higher in
`the trastuzumab group, especially in the substra-
`tum of patients receiving AC chemotherapy.[43]
`FDA-approved indications for
`the use of
`trastuzumab currently include treatment of patients
`with advanced metastatic breast cancer whose tu-
`mours overexpress HER2. For those who have not
`received chemotherapy for advanced disease, tras-
`tuzumab is indicated in combination with pacli-
`taxel. For previously treated patients, trastuzumab
`alone is indicated. Patients should receive a load-
`ing dose of 4 mg/kg intravenously over 90 minutes,
`and premedication with paracetamol (acetamino-
`phen) and diphenhydramine may lessen the poten-
`tial for infusion reactions. Subsequent doses of 2
`mg/kg are given weekly; the drug can be adminis-
`tered over 30 minutes if there are no infusion-re-
`lated symptoms. Baseline evaluation of cardiac
`function and extreme caution in patients with car-
`diac problems are recommended.
`The FDA has recently approved an immunohis-
`tochemical kit to determine candidates for trastu-
`zumab therapy (HercepTest™ 1). The performance
`of this kit is somewhat concordant with the method
`used in the trastuzumab clinical trials, but there is
`more discordance in the intermediate (1-2+) ex-
`pression level. A gene-based assay for HER2 amp-
`lification, fluorescence in situ hybridisation (FISH),
`has also been approved to stratify risk and aid in
`the choice of adjuvant chemotherapy for patients
`
`1 Use of a trade names is for product identification purposes
`only, and does not imply endorsement.
`
`with early stage breast cancer. Since the likelihood
`of response may depend on the actual level of over-
`expression, further studies will be required to clarify
`the optimal method and cutoffs for choosing patients
`most likely to benefit from trastuzumab. As with
`other biological therapies, many mechanisms for
`resistance to therapy are likely to exist or develop
`with time. Furthermore, many HER2-overexpress-
`ing tumours may use alternative oncogenic path-
`ways that are not modulated by trastuzumab.
`Trials are ongoing or planned to study the effi-
`cacy of trastuzumab with other chemotherapeutic
`agents, such as docetaxel, carboplatin, vinorelbine,
`gemcitabine and capecitabine. Preliminary results
`from a phase II trial using weekly paclitaxel at 90
`mg/m2 with weekly trastuzumab showed a re-
`sponse rate of 62% in patients with HER2-over-
`expressing tumours, compared with 44% in pa-
`tients with non-overexpressing tumours who were
`also enrolled.[44] Likewise, early results of a study
`using vinorelbine plus trastuzumab as first-line
`therapy for metastatic breast cancer show a re-
`sponse rate of 71%.[45] Combinations with hor-
`monal therapies will also be studied; as mentioned,
`HER2-overexpression may mediate resistance to
`tamoxifen, and this potentially may be reversible
`with trastuzumab. The use of trastuzumab as part
`of neoadjuvant therapy for locally advanced breast
`cancer and adjuvant therapy for early stage breast
`cancer will be tested in large cooperative group trials.
`
`1.1.3 Anti-HER2 Bispecific Antibodies
`Bispecific antibodies (BsAbs) are hybrid con-
`structs in which two MAb fragments recognising
`distinct antigenic targets are linked together. This
`
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`linkage can be achieved by chemical cross-linking
`of MAb fragments, cell fusion of hybridomas to pro-
`duce quadromas, or recombinant DNA techniques
`using cloned antibody genes. The rationale behind
`most BsAbs has been to combine a MAb fragment
`directed against a tumour-associated antigen with
`a MAb fragment directed against an antigen pres-
`ent on immune effector cells; the resulting con-
`struct can potentially mediate enhanced immune-
`mediated killing of target cells (‘directed killing’).
`MDX-210 is a bispecific MAb that recognises
`both HER2 and CD64, the high affinity type I Fc
`(FcγRI) expressed by mononuclear
`receptor
`phagocytes and activated neutrophils. In a phase I
`clinical study in advanced breast and ovarian can-
`cer, MDX-210 was observed to produce immuno-
`logical effects including transient monocytopenia
`and cytokine release.[46] One out of 10 assessable
`patients achieved a partial response. Another bi-
`specific MAb, 2B1, recognises both HER2 and
`CD16,
`the low affinity type III Fc receptor
`(FcγRIII) expressed by mononuclear phagocytes
`and natural killer cells.[47] 2B1 was evaluated in a
`phase I clinical trial and shown to induce cytokine
`release; several minor antitumour responses were
`observed.[48]
`
`1.1.4 Anti-HER2 Immunotoxins
`Immunotoxins consist of a MAb or MAb frag-
`ment conjugated or fused to a toxin molecule, and
`provide a strategy to greatly increase the potency
`of MAbs against targeted cancer cells. Erb-38 is a
`recombinant
`immunotoxin consisting of anti-
`HER2 Fv sequences fused to sequences derived
`from Pseudomonas exotoxin A (PE). In a phase I
`clinical trial, erb-38 was administered to 6 patients,
`and all 6 experienced significant hepatotoxicity
`with elevated transaminase levels.[49] Hepatotoxic-
`ity was unexpected in view of the very low levels
`of HER2 expression in hepatocytes as well as the
`low doses of erb-38 administered. This result indi-
`cates that some immunotoxin strategies can be too
`potent, since even normal tissues with very low levels
`of antigen expression (such as HER expression in
`hepatocytes) can still be targeted by these other-
`wise nonspecific and exquisitely active cytotoxins.
`
`1.1.5 Anti-HER2 Immunoliposomes
`Anti-HER2 immunoliposomes (ILs) combine
`the tumour-targeting properties of anti-HER2 MAb
`fragments with the pharmacokinetic and drug de-
`livery advantages of long circulating liposomes.[50]
`ILs displayed long circulation after single or multi-
`ple doses in normal adult rats that was identical to
`that of sterically stabilised (‘stealth’) liposomes.[51]
`Unlike nontargeted liposomes, anti-HER2 ILs
`bound avidly to HER2-overexpressing target cells
`in vitro.[52,53] Importantly, binding was accompa-
`nied by efficient internalisation via receptor-medi-
`ated endocytosis, resulting in intracellular drug de-
`livery.[52,53] Furthermore, in HER2-overexpressing
`tumour xenograft models, intravenous treatment
`with gold-labelled ILs produced markedly differ-
`ent intratumoural distribution and mechanism of
`delivery from that produced by nontargeted lipo-
`somes: while liposomes had accumulated extracel-
`lularly or within macrophages, ILs had penetrated
`extensively throughout tumour tissue and had
`internalised into tumour cell cytoplasm.[54]
`This novel mechanism may account for the sig-
`nificantly enhanced efficacy of anti-HER2 ILs
`against HER2-overexpressing tumours. In 4 differ-
`ent HER2-overexpressing tumour xenograft mod-
`els, doxorubicin-loaded anti-HER2 ILs (ILs-dox)
`produced growth inhibition, regressions and cures.
`Anti-HER2 ILs-dox was significantly superior to
`all other treatments tested, including free doxorub-
`icin, liposomal doxorubicin and anti-HER2 MAb
`(trastuzumab). Anti-HER2 ILs-dox was also sig-
`nificantly superior to combination therapies in-
`cluding free doxorubicin plus free trastuzumab and
`commercial liposomal doxorubicin plus free tras-
`tuzumab. Based on these studies, anti-HER2 ILs-
`dox is undergoing production scale-up in prepara-
`tion for phase I clinical testing.
`The immunoliposome approach offers a number
`of theoretical advantages over other MAb-based
`strategies against HER2. For example, anti-HER2
`delivery of doxorubicin may circumvent the pro-
`hibitive cardiotoxicity associated with combined
`trastuzumab plus doxorubicin treatment. Anti-
`HER2 ILs can be constructed using single-chain Fv
`
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`fragments (scFv) that lack antiproliferative activ-
`ity, are incapable of antibody-dependent cellular
`cytotoxicity, and require threshold levels of HER2
`expression for delivery.[51] Such ILs can provide
`targeted drug delivery without the antiproliferative
`or immunological activity associated with steady-
`state exposure to trastuzumab, and are thus likely
`to avoid the attendant cardiotoxicity. In compari-
`son with immunotoxin strategies, ILs provide an
`additional level of specificity through the use of
`chemotherapeutic agents with their own intrinsic
`therapeutic index. Hence, normal cells and tissues
`with low HER2 expression are likely to receive no
`greater (and potentially much less) exposure than
`occurs with free drug. Finally, unlike many im-
`munotoxin or other immunoconjugate constructs,
`ILs appear to be completely nonimmunogenic and
`capable of long circulation as stable constructs
`even with repeat administrations.
`
`1.2 Monoclonal Antibody-Based Therapies
`Against Other Antigens
`
`A number of MAbs directed against other breast
`cancer-associated antigens have been developed.
`EGFR is the prototypic member of the receptor
`tyrosine kinase family that also includes the pre-
`viously discussed HER2, as well as HER3 (ErbB3)
`and HER4 (ErbB4). EGFR is overexpressed in a
`number of cancers, including breast cancer.[55,56]
`Overexpression of EGFR occurs in 10 to 50% of
`breast cancers, although gene amplification is in-
`frequent. A MAb directed against EGFR, MAb
`225, competitively inhibits endogenous ligands
`such as epidermal growth factor and transforming
`growth factor-α from binding and activating
`EGFR.[57,58] Preclinical studies demonstrated that
`MAb 225 could inhibit the growth of cancer cells
`in vitro and human tumour xenografts in nude
`mice.[58] MAb 225 has also been shown to enhance
`the antitumour activity of radiation, as well as che-
`motherapeutic agents such as doxorubicin[59] and
`cisplatin.[60] C225, a chimeric version of MAb 225,
`retains the ability to block EGFR-mediated signal-
`ling and to inhibit cancer cell growth.[61,62] In
`
`phase I clinical trials, C225 displayed activity
`against several types of solid tumours.[63]
`Antibody-based therapies against other tumour-
`associated antigens have been developed. While
`oncogene products such as HER2 and EGFR are
`attractive targets because of their central role in the
`pathogenesis of many cancers, cell surface compo-
`nents that are not themselves oncogenic but are
`frequently associated with cancer cells have been
`identified and pursued as antigenic targets. For ex-
`ample, the MAb L6 recognises an integral mem-
`brane glycoprotein antigen (‘L6 antigen’) that is
`expressed in many epithelial tumour types.[64] Mu-
`rine MAb L6 was evaluated in phase I clinical tri-
`als, which showed a high rate of human antimouse
`antibody (HAMA) formation.[65,66] Achimeric ver-
`sion of L6 was developed, and showed somewhat
`reduced immunogenicity.[67] To increase potency,
`immunoconjugate versions of L6 including con-
`structs for radioimmunotherapy have also been de-
`veloped.[68]
`Another antibody-based strategy involves tar-
`geting the Lewis-Y antigen, a cell surface glyco-
`protein present in many cancers, including breast,
`lung, colorectal and ovarian cancers. Certain nor-
`mal tissues, particularly gastrointestinal sites, also
`express Lewis-Y. An immunoconjugate, BR96-
`dox (BMS 182248), was developed for antibody-
`directed drug delivery in Lewis-Y-expressing can-
`cers.[69] This novel agent consists of a chimeric
`anti-Lewis-Y MAb (BR96) conjugated via a dis-
`ulphide or thioether bond to acid-labile hydrazone
`linkers, which in turn are conjugated to doxoru-
`bicin. In this way, these immunoconjugates were
`designed for internalisation in Lewis-Y-expressing
`cells, followed by doxorubicin release intracellu-
`larly.
`In preclinical studies, BR96-dox demonstrated
`considerable efficacy against a variety of tumour
`xenografts, and was markedly superior to free
`doxorubicin alone.[69,70] A phase I trial of BR96-
`dox in 66 patients, with mainly metastatic breast or
`colon cancer, indicated severe gastrointestinal tox-
`icities at higher doses, including nausea, vomiting,
`gastritis and haematemesis.[71] The optimal dosage
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`Park et al.
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`of BR96-dox was determined to be 700 mg/m2 in-
`travenous infusion over 24 hours every 3 weeks,
`which corresponded to only 19 mg/m2 of doxoru-
`bicin. Interestingly, 1 patient received unconju-
`gated BR96 without doxorubicin and also experi-
`enced bloody diarrhoea and haematemesis. This
`suggests that binding of MAb BR96 to Lewis-Y-
`expressing gastrointestinal mucosa is sufficient to
`induce cytotoxicity, and that the observed gastro-
`intestinal toxicities may have been unrelated to
`doxorubicin delivery. Additional issues were the
`observation of immune responses to BR96-dox in
`37% of patients, which may have been due to the
`immunogenicity of the linker; and the progressive
`loss of doxorubicin molecules from the conjugate
`during circulation.
`BR96-dox was further evaluated in a random-
`ised phase II trial in patients with metastatic breast
`cancer,[72] using the optimal regimen previously
`defined. BR96-dox was associated with 1 partial
`response in 14 patients (7%), while doxorubicin
`alone produced 1 complete and 3 partial responses
`in 9 patients (44%). Gastrointestinal toxicities
`were again prominently observed with BR96-dox.
`The authors concluded that while tumour-targeted
`drug delivery remains promising, the MAb (BR96)
`and/or the antigen target (Lewis-Y) used were in-
`sufficiently specific for this strategy.
`
`2. Active Specific
`Immunotherapy/Vaccines
`
`2.1 Breast Cancer Vaccines:
`General Considerations
`
`Attempts to develop vaccines for cancer treat-
`ment, despite many decades of experimental work,
`have yet to yield clinically useful agents. However,
`a tremendous increase in interest has been gener-
`ated by recent advances in the areas of tumour biol-
`ogy, immunology and molecular therapeutics, which
`have led to more sophisticated and promising vac-
`cine strategies than previously available. In malig-
`nant lymphoma, for example, studies have shown
`that vaccination using idiotype antigens can protect
`animals against tumour transfer and can induce
`
`cure of established lymphomas.[73-79] In clinical tri-
`als, idiotype vaccines have been associated with
`clear evidence of clinical benefit in some lym-
`phoma