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Comparative immunohistochemistry of L19 and F16 in non-small cell lung cancerand mesothelioma: Two human antibodies investigated in clinical trials inpatients with cancer
Marta Pedretti a, Alex Soltermannb, Stephan Arnic, Walter Wederc, Dario Neria,∗, Sven Hillingerc,∗∗
a Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerlandb Institute of Surgical Pathology, University Hospital Zurich, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerlandc Department of Thoracic Surgery, University Hospital Zurich, Rämistrasse 100, CH-8091 Zurich, Switzerland
a r t i c l e i n f o
Article history:Received 9 April 2008Received in revised form 18 June 2008Accepted 25 July 2008
Keywords:Therapeutic antibodies
a b s t r a c t
The antibody-mediated targeted delivery of therapeutics to tumor sites is an attractive avenue for com-bating cancer while sparing normal tissues. Indeed, five derivatives of the human monoclonal antibodiesL19 and F16, specific to splice isoforms of fibronectin and tenascin-C, are currently being investigated inclinical trials in patients with malignancies. Until now, a comparative immunohistochemical analysis ofthese antibodies, which recognize components of the modified extracellular matrix, was missing. Here,we report that the majority of NSCLC and mesothelioma specimens are stained with both antibodies inthe stroma, while non-tumoral lung and mesothelium samples rarely exhibit reactivity with either L19 or
Extra-domain B of fibronectinExtra-domain A1 of tenascin-CNon-small cell lung cancerMT
F16. In our analysis, the anti-tenascin F16 antibody was found to generally exhibit a stronger staining ofdesmoplastic stroma surrounding tumor. This superior performance was found to be particularly striking
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. Introduction
The antibody-based delivery of therapeutic agents to tumorite is an attractive area of modern anti-cancer research, whichromises to concentrate bioactive agents onto neoplastic lesionshile sparing normal tissues [1–3]. In particular, the antibody-
ased targeting of the tumor neo-vasculature and/or the tumortroma has attracted considerable interest in the recent past,ecause of the pathological relevance of angiogenesis and tissueemodeling in cancer, and because of the attractiveness of tacklingifferent types of malignancy using the same antibody [4–6].
The alternatively spliced EDB domain of fibronectin [7–9] and A1omain of tenascin-C [10] represent two of the best-characterized
arkers of angiogenesis [4–5], which have been reported to be
xpressed around the neo-vasculature and in the stroma of virtu-lly all types of aggressive solid tumors. In collaboration with theroup of Luciano Zardi, our group has developed and extensively
∗ Corresponding author at: Institute of Pharmaceutical Sciences, Department ofhemistry and Applied Biosciences, Swiss Federal Institute of Technology Zurich,TH Zurich, Wolfgang-Pauli-Strasse 10, ETH Hoenggerberg, HCI G396, CH-8093urich, Switzerland. Tel.: +41 44 6337401; fax: +41 44 6331358.∗∗ Corresponding author. Tel.: +41 44 2554101; fax: +41 44 2558805.
haracterized the high-affinity human monoclonal antibodies L19nd F16, which recognize the EDB domain of fibronectin and the1 domain of tenascin-C, respectively [11,12]. These antibodiesave been conjugated to drugs, photosensitizers, cytokines, pro-oagulant factors, enzymes, fluorophores, microbubbles, liposomesnd radionuclides, and evaluated for their performance in animalodels of cancer [12–22]. As a result, three derivatives of the L19
ntibody and two derivatives of the F16 antibody are currentlyeing tested in clinical trials for the therapy of cancer.
A direct comparative analysis of the immunohistochemicaltaining of NSCLC and mesothelioma using the L19 and F16 anti-odies has not been reported so far. Such studies are complicated byhe fact that the two antibodies do not work well in formalin-fixed,araffin-embedded specimens, but rather require the analysis ofreshly frozen samples [8,23]. In this article, we have performed
comparative analysis of 103 freshly frozen specimens obtainedfter thoracic surgery, including 52 tumors and 51 matched normalamples.
. Materials and methods
.1. Subjects characteristics
The study group comprised 54 patients with primary lung car-inomas or mesotheliomas. Tissue specimens were taken during
urgical resection and immediately processed according to theuidelines of the Swiss Society of Pathology for frozen sections.issue pieces were snap-frozen in OCT and stored at −80 ◦C inhe Thoracic Surgery cryopreserved tumor tissue bank of Univer-itätSpital Zürich. The specimens consisted of 52 tumors and 51djacent portions of normal lung or normal mesothelium. Dataollected included age, gender, histopathologic diagnosis, grade ofumor differentiation (for NSCLC only) and TNM stage. Pathologi-al TNM staging and grading was performed according to the WHOlassification of malignant tumors [24,25].
.2. Immunohistochemistry
The F16 antibody, specific to the extra-domain A1 of tenascin-, and the L19 antibody, specific to the EDB domain of fibronectin,ave been described before [11,12]. Both antibodies were obtained
rom the ETH2 phage display libraries [11], underwent affinityaturation [11,12], had dissociation constants in the low nanomo-
ar range and exhibited kinetic dissociation constants koff towardshe respective cognate antigens <10−2 s−1 in real-time interactionnalysis experiments performed on a BIAcore 3000 instrumentGE Healthcare, Otelfingen) [11,12]. For immunohistochemical pro-edures, the two antibodies carrying a C-terminal myc tag formmunodetection with the 9E10 antibody [26] were used in scFvormat in identical conditions (final concentration of 7 �g/ml foroth antibodies). Aliquots of antibodies were prepared from aingle batch, stored frozen and were used only once, thus con-ributing to excellent reproducibility of immunohistochemicalesults.
The freshly frozen tissues were stored at −80 ◦C. Sections of0 �m thickness were treated with ice-cold acetone, rehydratedn TBS (50 mMTris, 100 mM NaCl, 0.001% Aprotinin, pH 7.4) andlocked with TBS 20% fetal calf serum. The scFv(F16)-myc andcFv(L19)-myc were added onto the sections, together with theecondary antibody monoclonal mouse anti-myc 9E10 (final con-entration of 1.7 �g/ml) in a TBS 3% bovine serum albumin (BSA)olution. Bound antibodies were detected with polyclonal rabbitnti-mouse immunoglobulins (Dako Cytomation, final concentra-ion of 125 �g/ml) in a TBS 3% BSA solution, followed by monoclonal
ouse anti-alkaline phosphatase immunocomplex with alkalinehosphatase (APAAP, Dako Cytomation, final concentration of.92 �g/ml) in a TBS 3% BSA + 2 mM MgCl2 solution. Fast RedTablets Set, Sigma) was used as phosphatase substrate, and sec-ions were counterstained with Gill’s hematoxilin No. 2 (Sigma).or every immunohistochemical experiment, in addition to stain-ng with the L19 and F16 antibody, a negative control experimentas performed by omitting the primary antibodies. Additionally,control tumor section which strongly stained with the F16 anti-ody was always included in the analysis series as positive controlSupplementary Figure 1).
.3. Quantification of immunohistochemical results, scoring andtatistical analysis
An optic microscope (Zeiss) at 5×, 10× and 20× magnificationsas used to evaluate the expression of the A1 domain of tenascin C
nd the EDB domain of fibronectin, as revealed by the staining usinghe F16 and L19 antibodies, respectively. The staining intensity wascored using four possible levels: no staining, weak, strong and verytrong (0, 1, 2, 3). A reference “training” table with three examples
f each level of staining was created before immunohistochemicalesults, following the recommendation of a certified pathologistA.S.). Using the training table as reference, sections stained with19 and F16 were scored blindly by three independent investiga-ors (Supplementary Figure 2). The results were then compiled as
sdbwl
Grade 3 18
a Values are expressed as number of subjects.b Available for NSCLC only.
verage of the three independent scores, indicating the standardeviation for each analysis. The grouping of scores in two stain-
ng categories (0–1 and 2–3) facilitated the concordance amongndividual assessments.
Significance levels for pairwise comparisons between L19 and16 scores were calculated using Student’s t-test.
. Results
The hundred and three specimens, derived from the Divisionf Thoracic Surgery of UniversitätSpital Zürich, were collectedmmediately after surgery and snap-frozen in OCT for optimalryopreservation. For each patient, a tumor sample and the cor-esponding normal tissue from the same lobe were collected toe available for comparative analysis and correlation with clinicalutcome data.
Adenocarcinoma was the most common histological type53%) of lung cancer, followed by squamous cell carcinoma41%) and small cell carcinoma (6%). Furthermore, there were 20
esothelioma specimens. The male/female ratio was 4:1. Clinico-athological data are summarized in Table 1.
Both F16 and L19 displayed no detectable staining in the major-ty of normal lung and mesothelium specimens, with only 3%nd the 2% of normal tissues exhibiting a strong reaction in thearenchyma, respectively (Fig. 1a and b). Interestingly, F16 gave
n general a stronger staining pattern for tumors compared to19 (strong and very strong staining 79% ± 2.4 vs. 34% ± 2.4, withconfidence level >99.9%), even though the two reagents were
sed at identical concentrations and have a comparable affin-ty to the cognate antigen. 78% of tumors displayed an intensetaining pattern with F16 (levels 2–3), whereas only 34% of themhowed a strong staining (levels 2–3) with L19 (Fig. 1a and). Importantly, no substantial difference in staining intensityas observed for the major tumor types (Fig. 1c and d). When
onsidering non-small cell lung carcinomas, we observed thathe majority of specimens were strongly stained both with F16nd L19 (Fig. 1c–h). Interestingly, L19 preferentially displayed atrong staining towards grade 3 tumors (strong and very strongtaining 80% ± 0 vs. 25% ± 0, with a confidence level >99.9%),hile F16 was able to intensely stain also grade 1–2 lesions
Fig. 1e–h).Fig. 2 shows a representative collection of the immunohisto-
hemical findings observed with the two antibodies. Panels a andshow normal lung sections from the same specimen, which were
cored negative with both F16 and L19. By contrast, panels c andexhibit normal lung sections which gave a weak staining with
oth antibodies. Panels e–j show pairs of tumor sections stainedith comparable intensity by the two antibodies. Panels k andindicate one of the two cases in which the L19 staining was
30 M. Pedretti et al. / Lung Cancer 64 (2009) 28–33
F 6 anda gh grat the dif2 ers we
siotvl
t
ig. 1. Analysis of the immunohistochemical staining results obtained with the F1nd b), results for different tumor types (c and d), a comparison of low grade vs. hiowards low grade and high grade NSCLC (g and h). The standard deviation refers to. Standard deviation bars are not visible, whenever the assessments of the examin
tronger compared to the F16. By contrast, for the majority of spec-
mens (e.g., panels m–t), F16 gave a substantially stronger staining,ften with a diffuse stromal reaction. The patterns of staining inumors with the two antibodies ranged between predominantlyascular and predominantly stromal. The behavior did not corre-ate with histological tumor types, but was generally similar for the
cibam
L19 antibodies. The figure presents a comparison of normal tissues vs. tumors (ade NSCLC (e and f), as well as a comparison of the reactivity of the two antibodiesferences in scores obtained from independent assessments, as described in Sectionre completely concordant.
wo antibodies within a given tumor specimen. Individual tumor
ells did not exhibit preferential staining with the two antibod-es, even though it is known that tumor cells contribute to theiosynthesis of oncofetal fibronectin and tenascin-C isoforms, inddition to endothelial cells and fibroblasts of the tumor environ-ent [4,5,27,28].
M. Pedretti et al. / Lung Cancer 64 (2009) 28–33 31
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ig. 2. Immunohistochemical findings obtained with the F16 and L19 antibodies in pntibody are indicated. Scale bars indicate 100 �m.
. Discussion
Oncofetal splice isoforms of fibronectin and tenascin-C have
ong been recognized as good-quality tumor associated antigens4,28–31], which are suitable for the antibody-based delivery ofherapeutic agents, such as radionuclides [32–38] or cytokines4,5,39]. In the adult, the EDB domain of fibronectin (recognizedy the L19 antibody) and the A1 domain of tenascin-C are vir-
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f sections derived from the same surgical specimen. For each panel, tissue type and
ually undetectable in normal tissues (exception made for thendometrium in the proliferative phase), but are strongly expressedn conditions of tissue remodeling, often with a prominent vascular
attern of staining. The alternative splicing process appears to beegulated by small changes towards basic intracellular pH values40], a situation which is often encountered during fetal develop-
ent and in neoplastic cells, in this case as a consequence of activeetabolism and Na+/H+ antiporter function.
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Neither the EDB domain of fibronectin nor the A1 domain ofenascin-C appear to have an obvious biological function. Indeed, inpite of the fact that the EDB domain is the most conserved domainn the fibronectin molecule (being identical between mouse and
an), two groups have reported that the targeted deletion of theDB exon in mouse yields animals which develop normally, healone fracture normally and which can develop tumors [41–43].imilarly, the deletion of the tenascin-C gene in the mouse isot lethal [44]. As a consequence of these findings, it is unlikelyhat anti-EDB and anti-EDA antibodies may display a therapeu-ic function on their own (and indeed, in our hands, neither L19or F16 display a therapeutic action in the mouse unless thewo antibodies are conjugated to a bioactive agent). By contrast,he restricted expression pattern of the antigens in normal tis-ues and the strong expression in many tumor types make L19nd F16 ideal candidates for antibody-based pharmacodeliverypplications. To our knowledge, only few immunohistochemicaleports on the expression of EDB domain of fibronectin and A1omain of tenascin-C in lung cancer and in mesothelioma haveeen published so far [45–56]. The vast majority of the normal
ung specimens gave only a faint staining or no detectable stain-ng (levels 0–1). For the few samples that exhibited some staining
ith the L19 and/or the F16 antibody, one should consider thathe presence of the antigen may be tumor-derives, as the nor-
al lung portion was adjacent to the tumor and was removedurgically together with the lesion. In animals, immunohisto-hemical analysis of normal lung reveals no detectable stainingsing antibodies specific to oncofetal fibronectin or tenascin-C12,57–59]. This is consistent with the observation of no preferen-ial uptake of L19 or F16 in normal lung in biodistribution studies12,15,27,35,38,60].
Three derivatives of the L19 antibody (L19-131I, L19-IL2 and19-TNF) are currently being studied in 7 Phase I and Phase IIlinical trials in patients with cancer (alone or in combinationith chemotherapy). Similarly, the radiolabeled antibody F16-131Ias recently entered clinical trials for the radioimmunotherapyf both solid tumors and hematological malignancies, while themmunocytokine F16-IL2 (a fusion proteins between the F16 anti-ody in scFv format and the human pro-inflammatory cytokine
nterleukin-2) has recently entered Phase Ib clinical trials in com-ination with doxorubicin for the treatment of breast and ovarianancer, and in combination with paclitaxel for the treatmentf breast and lung cancer. The clinical trials with L19-131I and16-131I feature the use of the recombinant antibodies in SIPormat which displays more favorable pharmacokinetic proper-ies compared to the conventional IgG format [15,35,38]. Patientsith any type of malignancy (including patients with lung can-
er and mesothelioma) are in principle eligible for these studies,rovided that they are in sufficiently good health conditions.ollowing the administration of a first diagnostic dose of radio-abeled antibody, only patients which display a sufficiently largeumor radiation dose compared to the bone marrow becomeligible for a radioimmunotherapeutic treatment in the 150 mCi-00 mCi dose range. The observation, reported in this paper,hat F16 generally stains thoracic tumors more strongly than L19,ill allow to resolve a fundamental open question in the field
f antibody-based tumor targeting, i.e. whether targeting perfor-ance correlates with absolute antigen expression at the tumor
ite.
onflict of interest statement
Dario Neri is shareholder of Philogen (www.philogen.com), aompany that has licensed the F16 and L19 antibodies from theTH Zurich.
[
[
cer 64 (2009) 28–33
cknowledgments
We have received financial support from the Swiss National Sci-nce Foundation, the Gebert-Rüf Foundation, the Schweizerischerebsliga, the ETH Zurich and the European Union projects STROMAnd IMMUNOPDT.
We also thank Dr. Christoph Schliemann for the contribution inhe scoring of immunohistochemical tissue sections.
ppendix A. Supplementary data
Supplementary data associated with this article can be found,n the online version, at doi:10.1016/j.lungcan.2008.07.013.
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