Monoclonal antibodies in the detection and
`
`therapy of micrometastatic epithelial cancers
`
`Gert Riethmiiller and Judith P. Johnson
`
`Institute of Immunology, University of Munich, Munich, Germany
`
`The initial promise of monoclonal antibodies as major therapeutic agents
`in human epithelial cancer has not been realized.
`lnaccessibility of cells
`in solid tumors due to factors such as the nature of
`the vascular
`
`endothelia and high pressure in the tumor are primarily responsible
`for the failure of antibody therapy. Although new strategies employing
`recombinant antibodies and immunoglobulins designed to actively engage
`the immune system may prove beneficial, micrometastatic tumor cells (at
`the stage of minimal residual disease) are likely to be the only suitable
`targets for antibody therapy. The diagnostic approaches to identify and
`characterize these cells and their use for prognosis and monitoring
`adjuvant immunotherapy is discussed.
`
`Current Opinion in Immunology 1992, 42647—655
`
`Introduction
`
`The hope that monoclonal antibodies (mAbs), with
`their exquisite specificity and multiple effector func-
`tions, would revolutionize the diagnosis and treatment
`of cancer, has failed to materialize. While mAbs have
`successfully replaced polyclonal antisera for the detec-
`tion of tumor markers in the serum, their therapeutic
`use in patients with solid tumors can only be viewed as
`a disappointment, even with improvements in the ability
`of antibodies to target and destroy tumor cells in model
`systems. Thus, fundamental changes in the strategies of
`using mAbs to treat tumors are required. In this review
`we focus on several such strategies, on the one hand,
`involving new developments in antibody production and
`use, and on the other, focusing on the target of the ther-
`apy itself. As more has been learned about the biology
`of the interaction of tumors with the vascular system and
`the extracellular matrix, it has become clear that much
`of the difficulty with passive antibody therapy is related
`to the accessibility of the tumor cells to these reagents.
`In light of this, micrometastasis, or the stage of minimal
`residual disease, may not be only an important field for
`diagnostic studies but also the best target for antibody
`mediated therapy.
`
`parts of the body can, at present, be diagnosed only in
`retrospect when the true extent of the disease manifests
`itself by an overt clinical relapse. For example, nearly 25%
`of breast cancer patients who do not show signs of dis-
`ease in the regional lymph nodes, do in fact suffer from
`disseminated disease, borne out by the occurrence, often
`many years later, of distant metastases [1]. The concen-
`tration of tumor antigens shed from a few tumor cells is
`too low for detection by the most sensitive immunoassays
`and even the most sophisticated physical diagnostic pro-
`cedures available, including nuclear magnetic resonance
`and photon emission tomography, are not specific and
`sensitive enough to detect individual
`tumor cells dis-
`seminated to distant organs. Thus, in the absence of the
`diagnosis of disseminated disease, the deveIOpment of ef-
`fective antibody-mediated (adjuvant) therapies proceeds
`at a scandalously slow pace. As with all adjuvant thera-
`pies they must first show some efficacy in 5 year trials
`on terminal patients with a heavy tumor burden before
`they can be tested on earlier stages. It is evident that in
`this dilemma, a firm diagnosis of minimal residual disease
`would be of invaluable help to identify patients actually
`in need of adjuvant therapy. Furthermore, the monitor-
`ing of therapeutic effects would enormously benefit the
`development of adjuvant therapies.
`
`Monoclonal antibodies in diagnosis:
`determination of minimal residual disease
`
`Since the early years of the last decade there have
`been several attempts to use mAbs to distinguish in—
`filtrating tumor cells from hematopoietic bone marrow
`cells [2], an approach which was greatly expanded by
`The metasmtic spread of small localized primary tumors
`Neville’s group at the Ludwig Cancer Institute in Lon-
`don [3]. The general feasibility of this method was
`in the absence of clinical signs of the disease in distant
`
`ADCCliantibody—dependent cellemediated cytotoxicity; APAAl’ialkaline phosphatase—anti-alkaline phosphatase;
`CTchytotoxic T lymphocyte; Fv—single chain monoclonal antibody; H—heavy; HLA—human leukocyte antigen; L——light;
`mAb—monoclonal antibody; Mflcemajor histocompatibility complex; PCR—polymerase chain reaction;
`SEA—staphylococcal enterotoxin A; TCR—T-cell receptor; V~variable.
`
`Abbreviations
`
`© Current Biology Ltd ISSN 0952-7915
`
`647
`
`Hospira v. Genentech
`Hospira v. Genentech
`IPR2017-00805
`IPR2017—00805
`Genentech Exhibit 2002
`
`Genentech Exhibit 2002
`
`

`

`Cancer
`
`soon demonstrated using dilferent mAbs in a variety
`of epithelial tumors (Table 1). Because of the gener
`ally low concentration and considerable heterogeneity
`of expression of tumor-associated membrane antigens,
`we chose to use the abundant intracellular cytokeratin
`proteins [4,5] as markers for detecting epithelial cancer
`cells in the bone marrow [6"]. Using an immunocyto-
`chemical alkaline phosphataseanti-alkaline phosphatase
`(APAAP) detection system it is possible to detect epithee
`lial cells at a fequency of 10—5 to 10—6 bone marrow
`cells [7,8",9',10]. Patients without epithelial malignane
`cies essentially lack cytokeratin-positive cells [7,11], while
`between 10 and 40% of these samples exhibit staining
`with antibodies directed against the membrane antigens
`epithelial membrane antigen and human milk fat globe
`ule mucin [6",12,13]. Further evidence for the specificity
`of this method was obtained from double marker stud-
`
`ies using a combination of immunoautoradiography and
`APAAP staining. In the bone marrow from carcinoma pa-
`tients, the expression of the leukocyte common antigen,
`CD45 and cytokeratin 18 were never found on the same
`cell [7].
`
`Although this method appears to have a high level of
`specificity, the low frequency of tumor cells in the bone
`marrow makes it likely that they are not uniformly dis-
`tributed and suggests that a considerable sampling error
`occurs when only one site is sampled [14]. In fact, triple
`site aspiration (left and right iliac crest plus sternum) in-
`creased the percentage of positive stage M0 breast cancer
`patients from 10.7% to 28.2% [15]
`(M denotes distant
`metastases and MO refers to tumors where there is no
`evidence of distant metastases). For routine purposes, a
`two-sided aspiration of the pelvic crest is currently per-
`formed on the operating table, immediately prior to the
`operation.
`
`The judicious use of double staining techniques allows
`one to define additional markers on the individual dis~
`seminated cells. Thus cytokeratinApositive cells in the
`bone marrow of tumor patients were shown to ex-
`press proliferation-associated molecules such as the the
`Ki 67 nuclear antigen and receptors for transferrin and
`epidermal growth factor
`[15]. Similar to cells in the
`metastatic lesions themselves, cytokeratin-positive bone
`marrow cells also often lacked HIA class I expression. In
`one study, 50% of the breast cancer patients with positive
`tumor cells in the bone marrow had only class I nega-
`tive epithelial cells in the bone marrow [8"]. These cells
`not only express proliferationvassociated markers but are
`able to grow in w'tro. Epithelial cells could be expanded
`from bone marrow aspirates by the use of various combi-
`nations of growth factors [16] and the rate of successful
`expansions was substantially increased when the culture
`vessels were precoated with extracellular matrix proteins.
`Using a serum free culture system, Hay e! a].
`[17]
`re-
`cently reported the growth of small-cell lung cancer cells
`in 100% of positive bone marrow samples.
`
`What is the clinical significance of the presence of epi-
`thelial cells in the bone marrow? In a number of studies
`
`on breast carcinoma patients [14,18°°] as well as in colo-
`rectal cancer [19] and neuroblastoma [20], the presence
`of micrometastases in the bone marrow has now been
`shown to be associated with a shorter disease-free in-
`terval. Surprisingly, while bone marrow micrometastatic
`cells were found in 33% of the colorectal tumor pa
`tients (Dukes C stage,
`i.e. a trans-serosal
`tumor with
`regional
`lymph node involvement), most of the man—
`ifest metastases involved the liver,
`indicating that
`the
`presence of tumor cells in the bone marrow is associ~
`ated with the probability of metastases development in
`general, and not necessarily with the manifestation of
`
`
`
`al
`
`1
`
`Table 1. Detection of bone marrow micrometastasis in various types of epithelial cancer.
`
`Origin of tumor
`Breast
`
`Colorectum
`
`Stomach
`Prostate
`
`Small-cell lung
`cancer
`Non-smaII-cell
`lung cancer
`Bladder
`Renal cell
`carcinoma
`
`
`
`
`Cytokeratin
`Cytokeratin
`PSA + Cytokeratin + EMA
`Cytokeratin
`5M1
`LCA1, LCAQ, and LCA3
`Cytokeratin
`
`Cytokeratin
`Cytokeratin
`
`+
`+
`+
`+
`+
`+
`+
`
`+
`+
`
`+
`+
`n.t.
`+
`n.t.
`n.t.
`+
`
`n.t.
`n.t.
`
`[19]
`[6"]
`[72]
`Oberneder“
`l58]
`[59]
`Pantel‘
`
`-
`
`Oberneder‘
`Oberneder‘
`
`*(Unpublished data); n.t., not tested.
`
`Marker antigens
`Epithelial membrane antigen (EMA)
`Cytokeratin
`MBr1
`
`Cytokeratin
`17.1A
`
`Correlation with established
`risk factors
`+
`--
`V
`
`--
`
`Prognostic
`value
`+
`n.t.
`n.t.
`
`n.t.
`
`-[
`
`References
`[14]
`[7]
`[57]
`
`[18"]
`
`
`
`

`

`
`Monoclonal antibodies and epithelial cancer Riethmijller and Johnson
`
`649
`
`skeletal metastasis. Taking all of the published studies
`together, a good correlation exists between the presence
`of epithelial cells in bone marrow and the conventional
`risk factors based mainly on the extent of tumor dissem-
`ination Furthermore, the total tumor burden may be es-
`timated from the number of epithelial cells in the bone
`marrow [18“,20]. The evaluation of tumor cells in the
`bone marrow is, therefore, a potentially powerful diag~
`nostic tool since it allows one to look at a part of the
`tumor burden which the surgeon has left behind. Not
`only could this provide a way to monitor the effective?
`ness of various therapies, but with a further characteri-
`zation of these cells (oncogene, tumor suppressor gene
`expression, etc) it may eventually be possible to more
`accurately predict the metastatic potential of these cells.
`
`Monoclonal antibodies in therapy: new
`strategies for production and use
`
`Although passive antibody therapies have been effective
`against established tumors in many experimental sys
`tems,
`the therapy of solid tumors with mAbs in the
`Clinic has had a history of failure A brief look at the
`literature covering the years of 1984-1991 provides no
`hint of a consistent therapeutic efficacy of mAbs. In a re,
`View of 12 studies comprising 196 patients with a variety
`Of solid tumors, Gisler lists only two complete remissions
`(Table 2) and the number of partial remissions remains
`within the realm of the anecdotal (R Grisler,
`in Com-
`cer and the Immune System, Proceedings of European
`School of Oncology, Venice 1990). The general conclu
`sion that has been drawn from this conspicuous failure
`is that antibodies as naked mouse immunoglobulins are
`
`inefficient, and a number of new strategies are being uni
`dertaken to create new antibodies and to use mAbs to
`
`actively engage the host’s own immune response against
`the autologous tumor.
`
`The development of new reagents that react with tumors
`through recombinant DNA technology
`While emphasis in recent years has been on using genetic
`engineering to ‘humanize’ and ‘Fc customize‘ rodent an-
`tibodies, the future almost certainly lies in the production
`of human antigen-binding molecules from combinatorial
`libraries in Escbericba COZi [21]. In this method, indepen-
`dent cDNA libraries are created from the mRNAs encod
`
`ing variable (V) light (L) and heavy (H) chain antibody re
`gions, using the polymerase chain reaction (PCR). These
`two libraries are then randomly combined and cloned
`into a phage vector which directs the expression of one
`VI. and one VH polypeptide chain. The use of vectors
`which allow the Fab fragments to be expressed as in-
`tact antigen~binding sites on the phage surface, either
`in monomeric or multimeric form, have greatly facili-
`tated screening and selection of desired antibodies [22'].
`In addition, the most recent developments indicate that
`high affinity antibodies of Virtually any specificity can be
`obtained by applying rounds of random mutagenesis and
`selection to the Fabs produced from the cDNA of naive
`IgM+ B lymphocytes, a situation which should obviate
`the need for immune donor cells [23"].
`
`Once the desired antigen-binding moieties have been see
`lectcd, the VL and VH regions can be produced as a sin-
`gle polypeptide (with the regions separated by a flexible
`linker) to generate single chain mAbs or Fv molecules.
`These can be further engineered to contain toxins or
`
`
`
`Table 2. Mouse monoclonal antibodies in clinical trials on solid tumors.
`
`J
`
`Number of
`
`Tumor
`Mouse antibody
`lsotype
`patients
`Effect
`References
`
`
`
`
`
`
`
`Gastrointenstinal
`tumors
`
`CO17—1A
`
`lgGZa
`
`Breast, colon,
`ovarian and
`
`lung cancer
`
`Pancreatic
`adenocarcinoma
`
`Neuroblastoma,
`melanoma
`
`Melanoma
`
`Melanoma
`
`L6
`
`lgGZa
`
`BW494
`CO17-1 A
`
`3F8
`anti-CD2
`
`9.2.27
`
`R24 (anti-GD3)
`
`lgG1
`IgCZa
`
`IgC3
`
`lgCZa
`
`lgG3
`
`20
`22
`8
`20
`
`19
`
`18
`19
`
`17
`
`20
`
`21
`12
`
`20 :l:
`22 d:
`8 —
`20 :l:
`
`1+ +, 182
`
`18 —
`4 +, 15 —
`
`1+ +, 6+, 10—
`
`20 f
`
`4 +, 17
`3+,2::,7—
`
`[60]
`[61]
`[62]
`[63]
`
`[64]
`
`[65]
`[66]
`
`[67]
`
`[68,691
`
`[70]
`[71]
`
`+ +, Complete response; +, partial response;
`
`:l:, minor response; w, no response.
`
`

`

`
`650 Cancer
`
`other effector domains [24"]. Fvs frequently refold into
`a conformation that retains the binding characteristics of
`the divalent mAb and may penetrate tissues much more
`efficiently than intact immunoglobulins. Such characteris-
`tics can be used to predict whether they will be important
`anti-tumor reagents and their preliminary use in mouse
`models confirms this [25].
`
`Use of monoclonal antibodies to engage the
`host’s immune system
`
`tumor-reactive mAbs
`The passive administration of
`is
`increasingly being replaced by immunotherapeutic
`schemes designed to engage the host’s own immune sys»
`tem in the destruction of the autologous tumor. Among
`the most Widely used strategies at present is the attempt
`to exploit the idiotypic network [26“,27' ,28- ]. This ap-
`proach is based on the theory that certain antieidiotypic
`antibodies (the AbZBs) will express the internal image
`of the original immunizing epitope [29]. Treatment with
`such antibodies is, therefore, equivalent to immunizing
`patients with the tumor antigen, or rather with a sin»
`gle epitope of mis antigen.
`In point of fact, patients
`treated with Asz frequently produce Ab3s which re
`act With the original tumor antigen [50,31,32°] and in
`some cases, even appear to develop a cellular reactivity
`against this antigen although the nature of this reactiv-
`ity remains poorly characterized [30,33,34].The clinical
`consequences of active immunization in those instances
`Where the tumor associated antigen, e.g. the 17.1 A colo-
`rectal carcinoma epithelial antigen,
`is also widely ex-
`pressed on normal epithelia are unclear.
`
`Despite the common production of A1355 that react with
`tumor cells in the anti—idiotype treated patients,
`these
`clinical trials have generally not been any more successful
`than those using Abls, antibodies to the original epitope,
`[27' ,28' ]. However, Mittelman et a]. [52'], recently re
`ported a significant increase in survival time in melanoma
`patients producing Ab3s against
`the chondroitin sul-
`phate proteoglycan, a surface antigen of melanoma cells.
`This result could not be accounted for by higher gen
`eral immune reactivity (ie. performance status) of the
`patients. While such discrepant results may reflect,
`in
`part, the fact that immunization with these reagents in,
`duces an immune response against a single epitope that
`may have varying functional and structural Characteristics
`(e.g. density, sensitivity to modulation, functional signif
`icance), studies in a mouse anti-tumor model (reviewed
`in [26"]) indicate that selection of the Ab2, used in such
`trials is of critical importance. In this system, four differ
`ent AbZS, which were very similar in their interaction with
`the Abl and in their ability to induce anti-tumor antigen
`humoral and cellular responses, were examined for their
`ability to vaccinate against tumor growth. Only one AbZ
`was able to induce protective immunity; more disturb,
`ing was the observation that one actually appeared to
`potentiate tumor grovvth [35]. The finding of a linear
`sequence homology between the second hypervariable
`region of the protective Ab2 L chain and the tumor anti-
`
`gen [26"], not only suggests a molecular basis for the
`similarity between the internal image and the tumor anti-
`gen, but means that selection of appropriate AbZS may
`eventually be possible through sequence comparison of
`the antibody variable regions with the sequence of the
`antigen. However, this is not possible for carbohydrate
`antigens that are among the best candidates for tumor
`surrogate antigen vaccines given their effectiveness as tu-
`mor mrget structures [56'], This would be cumbersome
`enough for protein antigens, but it would be even more
`difficult for carbohydrate antigens because of the diffr
`culty of producing them in large quantities; despite this,
`the effectiveness of carbohydrate antigens as tumor target
`structures means that they are among the best candidates
`for surrogate antigen vaccines [56'].
`
`A conceptually simpler approach to engage the host
`immune system is the use of bi-specific antibodies to
`target host elfector cells to the tumor. These reagents
`combine,
`in a single molecule, specificity for a tumor
`associated antigen with specificity for an effector cell
`molecule, most commonly the Tvcell receptor (TCR)—
`associated CDS complex [57,38]. Addition of such cone
`jugates to interleukin-Z or antinDj—activared leukocytes
`prior to their injection reduces the growth of established
`tumors in nude mice more elfectively than the activated
`cells alone [59], a result which was also obtained in a
`small clinical trial [40]. Most cells that are targeted to the
`tumor by this approach are not directed against tumor
`antigens. But bi-specific heteroconjugates can also in-
`crease the killing of autologous tumors by clones of
`tumor-specific tumorinfiltrating lymphocytes and cyto-
`toxic T lymphocytes (CTLs) provided the tumor cells
`express the target antigens [41']. However,
`if the tu
`mors are negative or heterogeneous for targeteantigen
`expression, the presence of heteroconjugates can inhibit
`tumor~specific cytotoxicity, presumably by blocking the
`TCR. Given the universality of tumor cell heterogeneity,
`this could be a serious drawback to the successful use
`
`of such reagents.
`
`d
`
`An approach which might overcome this problem is one
`which takes advantage of the characteristics of bacterial
`toxin superantigens [42]. These molecules bind with
`high affinity to MHC class 11 antigens and once bound,
`demonstrate a specificity for particular families of TCR VB
`chains. Reactivity with the TCR leads to Tcell activation,
`cytokine release and killing of the cell expressing MHC
`class [1 antigen. By coupling a colon carcinoma reactive
`mAb to staphylococcal enterotoxin A (SEA), Dohlstein et
`61]., [43"] have been able to replace class II dependent
`T‘cell activation with a tumor antigen specific Tecell acti
`vation and have achieved killing of carcinoma cell lines by
`CTLs bearing the appropriate VB chains. Since SEA will
`not engage the T cell until it is ‘presented’ by mAb bind-
`ing, activation of the T cells and cytokine release should
`occur only locally, in the immediate environment of the
`tumor cells. Thus, like the use of cytokine transfected tu
`mor cell vaccines [44], which also lead to spatially and
`temporally concordant expression of tumor antigens and
`cytokines, SEAemAb conjugates may also trigger develop.
`ment of the host’s own anti-tumor response.
`
`

`

`
`
`Monoclonal antibodies and epithelial cancer Riethmiiller and Johnson
`
`651
`
`Micrometastatic tumor cells as targets for
`immunotherapy: the way to secondary
`prevention
`
`Even in the face of major improvements in the ability of
`antibodies to bind to and kill tumor cells, the problem
`of the accessibility of the tumor cells to these reagents
`remains unresolved. In fact, recent data on the microcir-
`culation and extracellular matrix of tumors indicate that
`this may in fact be the most important impediment to
`successful antibody treatment of solid tumors [45,46].
`
`The organization of the tumors vascular system is largely
`determined by the histogenic type of tumor (i.e. tissue
`of origin), its differentiation state, proliferation rate and
`location. The non-fenestrated capillaries often found in
`epithelial tumors prevent antibodies from entering the tu-
`mor tissue. Another major factor obviating the transcapil-
`lary transport of macromolecules is the tumor interstitial
`pressure which is generally much higher than the sur-
`rounding normal tissue and which increases with tumor
`size [47]. The pressure gradient from the center to the
`periphery of a tumor leads to an outward convection of
`the interstitial fluid so that extravasated mAb will be trans
`ported to the periphery of the tumor. In the interstitium
`itself, the dense network of glycosaminoglycans presents
`a formidable obstacle for free diffusion of antibody. In
`addition, the antigens shed from the tumor, when immo-
`bilized in this interstitium, may neutralize antibodies and
`lead to precipitation of antigen—antibody complexes. A fie
`nal barrier that must be overcome in differentiated metas-
`tases of adenocarcinomas is the shielding of target anti-
`gens from diifusing antibodies by the intercellular tight
`junctions.
`
`While the clinical trials have only resulted in a very few
`well documented antibody-induced regressions (which
`may more easily be explained by derangements in the
`tumor vasculature or extracellular matrix rather than by
`the inherent characteristics of the antibody), they have
`clearly shown that antibody therapy is remarkably free of
`toxicity and serious side effects. Thus, the stage would
`now seem to be set for applying passive antibody ther-
`apy to its most logical target — minimal residual disease
`(or micrometastasis), a state which applies to 1/3 to 1/2
`of patients with epithelial cancer (the most common type
`of cancer), following (curative) radical surgery of the pri-
`mary lesion.
`
`One of the first hints that micrometastatic cells might be
`particularly suitable targets for passively administered an,
`tibody was provided by Schlimok et a1. [7] who demon
`strated that
`tumor cells in bone marrow of patients
`with colorectal cancer can be labelled with mouse im-
`munoglobulin in 112120. In a later report [8"], the ther-
`apeutic effects of antibody infusions over a period of
`several months up to 2 years were monitored by im-
`munocytochemical analysis of bone marrow. In 12 of
`the 25 Dukes C patients with clinically manifest metas-
`tases, micrometastatic cells were repeatedly identified and
`clinical relapse occurred in nine of these patients.
`
`In order to monitor directly the therapeutic elimina-
`tion of micrometastatic cells, patients with high num-
`
`bers of micrometastatic cells in the bone marrow were
`
`treated with an IgG3 anti-Lewis Y antibody displaying
`high complementadependent cytotoxicity [48']. (This an-
`tibody was selected because IgG?) mouse antibodies are
`particularly effective in the activation of human comple-
`ment and ADCC). While no regressions were observed
`in these patients with extensive metastatic disease, a dra-
`matic decrease in the number of tumor cells in bone
`marrow was Observed, an effect not seen in placebo
`treated patients (G Schlimok, G Riethmiiller, K Pantel et
`61]., abstract 709, ProcAm Soc Clin One 1991, 10212). To
`analyze the efficacy of antibodies against micrometastatic
`cells in colorectal cancer a prospective randomized trial
`was initiated into which only patients with regional dis,
`ease (Dukes C1 and C2; metastases in IAS regional lymph
`nodes and 4 or more, respectively) were recruited. The
`trial comprising a total of 176 patients in a treatment and
`control arm will be closed at the end of 1992 with a me-
`dian observation time of 5 years, A preliminary analysis at
`a median observation time 4.5 years indicates differences
`between treated and control patients in terms of overall
`survival and recurrence rate of distant metastasis (G Rir
`ethmuller, G Schlimok et 61]., unpublished data). Although
`another prospective randomized trial on pancreatic can-
`cer patients treated with an antiecarbohydrate antibody
`failed to show therapeutic effects [49"],
`the state of
`minimal residual disease in these patients can be dis,
`puted as the partial pancreatoeduodenectomy (removal
`of the pancreas and duodenum) employed for tumor re-
`section may not have been complete in many of them.
`
`Conclusions and perspectives
`
`While it is clear that a major consideration for the suc-
`cessful application of antibody therapy in solid tumors
`is the accessibility of the tumor cells, other aspects also
`need to be taken into account. Heterogeneity of tumor
`cells is the major obstacle of any antigen~targeting ther-
`apy. To cope with antigen heterogeneity, which increases
`with tumor progression, a carefully selected combination
`of antibodies directed to independently controlled mem-
`brane antigens should be applied as early as possible,
`i.e. immediately after the removal of the primary tumor.
`Published data demonstrate that selected pairs of anti
`bodies directed against different epitopes of a particular
`antigen exhibit up to a 100—fold synergistic increase in
`cell killing in vitro [50] or in viva [51] or result in
`augmented binding [5264]. As passive antibody ther-
`apy imitates a natural effector mechanism, a well defined
`oligoclonal combination of antibodies, in which each has
`been shown to eliminate micrometastatic cells, should be
`as acceptable for clinical use as current registered poly-
`clonal antithymocyte globulin preparations.
`
`The optimal combination of such antibodies would con-
`sist of human or ‘humanized’ immunoglobulins. Since
`normal epithelia are less accessible to cytotoxic anti-
`bodies (they are located behind a dense basal mem-
`brane which is quasivimpermeable to non-secretory im-
`munoglobulin), an absolute specificity for therapeutic an-
`tibodies appears less critical. Indeed, toxicity to normal
`epithelia has been negligible in the trials reported thus
`far. Although the inactivation of complement by homol-
`
`

`

`652 Cancer
`
`8,
`u
`
`in Vitro Detection with Anti-cytokeratin
`' in Bone Marrow:
`and in Vivo Labelling with Anti-17-1A Monoclonal Antibod-
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`PANTEI. K, SCHUMOK G, KUTI'ER D, SCHALLER G, GENZ T,
`WIEBECKE B, BACKMANN R, FUNKE I, RIETHMULLER G: Frequent
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`This study of HLA class 1 expression reveals an unexpectedly frequent
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`9.
`MouNo A, COLOMBATTI M, BONETrr F, ZARI)INI M, PASINI F,
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`PERINI A, PEIosr G, T'I'IDENTE G, VENERI D, CETI‘O GL: A
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`A methodical paper demonstrating the higher sensitivity of the APAAPV
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`
`10.
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`11.
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`12.
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`OSBORNE MP, WoNG GY, AsINA S, OLD LJ, COTE RJ, ROSEN
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`SCHLIMOK G, SWIERKOT S, HAUSER H, JUNKER A, WARNECKE HH,
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`Drama G, GATTER KC, STEIN H, ERBER WN, PULFORD KAF,
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`TAYLOR-PAPADIMITIROU J, BURCHELL J, Moss F, BEVERLEY P; Mon-
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`SCHLIMOK G, RIETHMULLER G: Detection, Characterization
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`FUNKE I, SCHLIMOK G, BOCK B, SCHWEIBERER B, RlETI-IMULLEK
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`
`ogous restriction factors may contribute to the resistance
`of the tumor cells to lysis, it may be possible to eventually
`inhibit these factors (e.g., C8-binding protein or CD59
`membrane glycoprotein) [55]. As demonstrated by sev-
`eral studies [7] micrometastatic cells can be reproducibly
`identified in individual patients over time, thus offering
`a new way to monitor micrometastasis. Such monitoring
`will be mandatory for the expeditious development of
`an effective passive antibody therapy. Furthermore, new
`techniques such as PCR may replace the more laborious
`immunocytological methods [56].
`
`In conclusion, it would be a major mistake to let anti
`bodies, exquisitely specific and versatile tools, and the
`powerful natural effector mechanisms they can harness
`be misspent on the unsuitable target of advanced solid
`tumors
`
`
`Acknowledgements
`
`The authors are supported by Dr. Mildred Scheel Stiftung, Deutsche
`Krebshilfe, Bonn, Germany. The authors would like to thank Dr.
`Klaus Pantel for helpful discussion.
`
`
`References and recommended reading
`
`Papers of particular interest, published within the annual period of re-
`view, have been highlighted as:
`n
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`u
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`
`7.
`
`SCHLIMOK G, FUNKE I, HOIZMANN B, GO'rI'LIIx‘oER H, SCHMIDT
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