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. Inaccessibility 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, 4:647—655
`
`Introduction
`
`The hope that monoclonal antibodies (mAbs), with
`their exquisite specificity and multiple effector func‘
`tions, w0uld 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 f0cus 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 development 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 metastatic spread of small localized primary tumors
`Neville’s group at the Ludwig Cancer Institute in Lon-
`in the absence of clinical signs of the disease in distant
`don [3]. The general feasibility of this method was
`
`ADCC—antibody-dependent cell-mediated cytotoxicity; APAAP—alkaline phosphatase—anti-alkaline phosphatase:
`CTLicytotoxic T lymphocyte; Fv—single chain monoclonal antibody; H—heavy; HLA—human leukocyte antigen; L—light;
`mAbgmonoclonal antibody; MHCgmajor histocompatibility complex; PCR—polymerase chain reaction;
`SEA—Staphylococcal enterotoxin A; TCR—T-cell receptor; Vkvariable.
`
`Abbreviations
`
`© Current Biology Ltd ISSN 0952-7915
`
`647
`
`Genentech Exhibit 2025
`
`
`Hospira v. Genentech
`Hospira V. Genentech
`IPR2017-00737
`IPR2017—00737
`Genentech Exhibit 2025
`
`
`
`

`

`Cancer
`
`soon demonstrated using different 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 phosphatas&anti-alkaline phosphatase
`(APAAP) detection system it is possible to detect epithei
`lial cells at a fequency of 10—5 to 10—6 bone marrow
`cells [7,8",9',10]. Patients without epithelial malignan
`cies essentially lack cytokeratinpositive 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 glob-
`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 proliferation associated markers but are
`able to grow in vitro. 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! at [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 [14,180] as well as in cold
`rectal cancer [19] and neuroblastoma [20], the presence
`of micrometastases in the bone marrow has now been
`shown to be associated with a shorter diseasefree 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
`
`
`
`Table 1. Detection of bone marrow micrometastasis in various types of epithelial cancer.
`
`al
`
`1
`
`Origin of tumor
`
`Marker antigens
`
`Correlation with established
`risk factors
`
`Prognostic
`value
`
`References
`
`
`
`
`
`
`
`Breast
`
`Colorectum
`
`Stomach
`Prostate
`
`Small-cell lung
`cancer
`Non-small-cell
`lung cancer
`Bladder
`Renal cell
`carcinoma
`
`Epithelial membrane antigen (EMA)
`Cytokeratin
`MBr‘l
`
`Cytokeratin
`’i7.'lA
`
`Cytokeratin
`Cytokeratin
`PSA + Cytokeratin + EMA
`Cytokeratin
`5M1
`LCA1, LCA2, and LCA3
`Cytokeratin
`
`Cytokeratin
`Cytokeratin
`
`*(Unpublished data); n.t., not tested.
`
`+
`+
`V
`
`--
`
`--
`+
`--
`+
`+
`+
`+
`
`+
`+
`
`+
`n.t.
`n.t.
`
`n.t.
`
`+
`+
`n.t.
`+
`n.t.
`n.t.
`+
`
`n.t.
`nt
`
`[14]
`l7l
`[57]
`
`[18"]
`
`[19]
`[6"]
`[72]
`Oberneder‘
`l58l
`[59]
`Pantel‘
`
`-
`
`Oberneder‘
`Oberneder‘
`
`
`
`

`

`
`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 esmblished 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 Can-
`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 Eschericba coli [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 se
`lected, the VI and VH regions can be produced as a sin-
`gle polypeptide (with the regions separated by a flexible
`linker) to generate single chain mAbs 0r Fv molecules.
`These can be further engineered to contain toxins or
`
`
`
`Table 2. Mouse monoclonal antibodies in clinical trials on solid tumors.
`
`J
`
`+ +, Complete response; +, partial response;
`:l:, minor response; w, no response.
`
`
`_l
`
`Number of
`
`Gastrointenstinal
`tumors
`
`C017—1A
`
`lgCZa
`
`Breast, colon,
`ovarian and
`
`lung cancer
`
`Pancreatic
`adenocarcinoma
`
`Neuroblastoma,
`melanoma
`
`Melanoma
`
`L6
`
`lgGZa
`
`BW494
`CO17-1 A
`
`3F8
`anti-CD2
`
`9.2.27
`
`lgG1
`IgCZa
`
`IgC3
`
`lgCZa
`
`20
`22
`8
`20
`
`19
`
`18
`19
`
`17
`
`20
`
`20 :l:
`22 d:
`8 —
`20 :l:
`
`1+ +, 182
`
`18 —
`4 +, 15 —
`
`1+ +, 6+, 10—
`
`[60]
`[61]
`[62]
`[63]
`
`[64]
`
`[65]
`[66]
`
`[67]
`
`20 —
`
`[68,69]
`
`21
`12
`
`4 +, 17
`3+,2::,7—
`
`[70l
`[71]
`
`
`
`Melanoma
`
`R24 (anti-GD3l
`
`lgGS
`
`Tumor
`Mouse antibody
`lsotype
`patients
`Effect
`References
`
`
`
`
`

`

`

`

`

`

`

`

`

`

`

`

`