<figure><img src="image_1.png" /></figure>

HAL
open science

# Identification of monoclonal antibodies from naive antibody phage-display libraries for protein detection in formalin-fixed paraffin-embedded tissues

Célestine Mairaville, Morgane Broyon, Margaux Maurel, Myriam Chentouf, Barbara Chiavarina, Andrei Turtoi, Nelly Pirot, Pierre Martineau

<figure><img src="image_2.png" /></figure>

## To cite this version:

Célestine Mairaville, Morgane Broyon, Margaux Maurel, Myriam Chentouf, Barbara Chiavarina, et al. Identification of monoclonal antibodies from naive antibody phage-display libraries for protein detection in formalin-fixed paraffin-embedded tissues. Journal of Immunological Methods, 2024, 532, pp.113730. 10.1016/j.jim.2024.113730. hal-05072900

HAL Id: hal-05072900

https://hal.science/hal-05072900v1

Submitted on 19 May 2025

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_3.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_4.png" /></figure>

# Identification of monoclonal antibodies from naive antibody phage-display libraries for protein detection in formalin-fixed paraffin-embedded tissues

Célestine Mairaville<sup>a</sup>, Morgane Broyon<sup>b</sup>, Margaux Maurel<sup>a</sup>, Myriam Chentouf<sup>a</sup>,
Barbara Chiavarina<sup>a</sup>, Andrei Turtoi<sup>a</sup>, Nelly Pirot<sup>a,b</sup>, Pierre Martineau<sup>a,*</sup>

## ARTICLE INFO

Keywords:

Phage display

Monoclonal antibody

Selection and screening method

Immunohistochemistry

FFPE

## ABSTRACT

Most antibodies used in immunohistochemistry (IHC) have been developed by animal immunization. We wanted to explore naive antibody repertoires displayed on filamentous phages as a source of full-length antibodies for IHC on Formalin-Fixed and Paraffin-Embedded (FFPE) tissues. We used two isogenic mouse fibroblast cell lines that express or not human HER2 to generate positive and negative FFPE pseudo-tissue respectively. Using these pseudo-tissues and previously described approaches based on differential panning, we isolated very efficient antibody clones, but not against HER2. To optimize HER2 targeting and tissue specificity, we first performed 3-4 rounds of in vitro panning using recombinant HER2 extracellular domain (ECD) to enrich the phage library in HER2 binders, followed by one panning round using the two FFPE pseudo-tissues to retain binders for IHC conditions. We then analyzed the bound phages using next-generation sequencing to identify antibody sequences specifically associated with the HER2-positive pseudo-tissue. Using this approach, the top-ranked clone identified by sequencing was specific to the HER2-positive pseudo-tissue and showed a staining pattern similar to that of the antibody used for the clinical diagnosis of HER2-positive breast cancer. However, we could not optimize staining on other tissues, showing that specificity was restricted to the tissue used for selection and screening. Therefore, future optimized protocols must consider tissue diversity early during the selection by panning using a wide collection of tissue types.

## 1. Introduction

Immunohistochemistry (IHC) techniques rely on specific antibodies to detect and localize antigens on tissue sections, using brightfield or fluorescent microscopy. IHC is widely used for research and diagnostic purposes (de Matos et al., 2010; Jaffer and Bleiweiss, 2004; O'Hurley et al., 2014). IHC is a multistep process and many factors can affect its outcome (O'Hurley et al., 2014). One major step is tissue fixation to prevent tissue degradation and putrefaction and to preserve the tissue histomorphology and the subcellular localization of cellular components (Grizzle et al., 2008; Thavarajah et al., 2012). Formalin-Fixation and Paraffin-Embedding (FFPE) is the gold standard for tissue preservation (Dunstan et al., 2011; Matsuda et al., 2010). FFPE tissues are easier and cheaper to store than frozen tissues and their morphology is better preserved (Dunstan et al., 2011; Gaffney et al., 2018). However, formaldehyde can react with different amino acids through the Mannich reaction or the formation of hydroxymethyl adducts or Schiff bases,

ultimately leading to the formation of crosslinks called methylene bridges (Fowler et al., 2008, 2011; Grizzle et al., 2008; Shi et al., 2000; Sompuram et al., 2006; Thavarajah et al., 2012). Antigen retrieval methods can partially reverse these fixation-induced antigen modifications through heating or enzymatic treatment (Ramos-Vara and Miller, 2014; Shi et al., 1991; Yamashita, 2007). Therefore, antigen conformation and accessibility are critical factors that should be taken into account when developing antibodies for IHC.

Most monoclonal antibodies (mAbs) currently used for IHC have been obtained by animal immunization with recombinant proteins, cells or synthetic peptides. Consequently, the conformation of the antigen used to produce antibodies is not directly comparable to that in FFPE tissues. According to Lipman (Lipman et al., 2005), this represents an important source of failure in the development of an efficient mAb "that should be generated to the state of the antigen to which it will eventually need to bind". For many applications, this discrepancy has been solved using display methods, particularly phage display, that allow selecting

<footnote><sup>a</sup> IRCM, Univ. Montpellier, ICM, INSERM, Montpellier, France</footnote>

<footnote><sup>b</sup> BCM, Univ. Montpellier, CNRS, INSERM, Montpellier, France</footnote>

<footnote>* Corresponding author at: IRCM, 208 Rue des Apothicaires, F-34298 Montpellier Cedex 5, France.</footnote>

<footnote>E-mail address: pierre.martineau@inserm.fr (P. Martineau).</footnote>
and screening antibodies under controlled and identical antigen conformation. However, most authors have used it to develop mAbs that are highly specific for native antigens and only few addressed the needs of IHC on FFPE tissues. In a recent review (Mairaville and Martineau, 2021), we identified only eight studies on antibodies developed for IHC using phage display and naive antibody libraries (Gur et al., 2009; Jakobsen et al., 2007; Jarutat et al., 2007; Larsen et al., 2015; Ruan et al., 2006; Su et al., 2018; Sun et al., 2009b; Ten Haaf et al., 2015). None of them tested the resulting antibodies in the IgG format, which is used by most histology laboratories, and limited the validation steps to phages or small recombinant proteins, such as fragment antigen-binding (Fab), single-chain Fragment variable (scFv) or single-domain anti-bodies. However, these recombinant formats are not used in most histology laboratories and are much less effective because secondary reagents have been optimized for the IgG format. Furthermore, reformattin g these binders as full-length IgG is now a routine task, but may affect the binding properties of the selected clones (Chan et al., 2011; Steinwand et al., 2014; Thie et al., 2011).

Traditionally, phage display selection is followed by the screening of 100–1000 clones by ELISA, flow cytometry, or any other medium- or high-throughput method (Schofield et al., 2007). Using IHC for such large screening would be very expensive, time-consuming, and frequently not even possible because of limited tissue availability. However, clone testing should determine whether they can recognize the target protein in the conformation found in FFPE tissues, ideally as early as possible in the screening process. Therefore, it is critical to use the same panning and screening conditions to ensure the final selection of the best clones, but no solution has been proposed yet (Pimenidou et al., 2001).

Here, we describe the development of a selection and screening method for the identification of antibodies for IHC on FFPE tissue sections from a naive phage-displayed antibody library, using on-slide panning, sequence analysis to identify the best candidates and avoid a large-scale screening, and direct validation of the identified clones in a rabbit IgG format. Our objective was to develop a general strategy for antibody discovery without prior knowledge on the targeted antigen. We selected human HER2 as a model system due to its extensive characterization. The wealth of available information on HER2 localization in tissues and cells, established through various methods including IHC, facilitates comprehensive validation of the identified antibodies.

## 2. Materials and methods

### 2.1. Cells lines and tissues

The two murine embryonic fibroblast lines (NIH 3T3-WT and NIH 3T3-HER2) were provided by C. Larbouret. NIH 3T3-HER2 cells are wild type (WT) NIH 3T3 cells stably transduced to express human HER2 (Gaborit et al., 2011). SKOV3 (ovarian cancer) and MDA-MB-468 (breast cancer) cell xenografts were provided by C. Larbouret and HCT116 (colon cancer) cell xenografts by A. Coquelle.

### 2.2. Preparation of cell pellets and pseudo-tissue sections

To prepare pseudo-tissues, 50–100 × 10⁶ fibroblasts were detached with TripLE (Gibco) or scraped with 2 mM EDTA in PBS, both treatments but also regular Trypsin giving identical results with trastuzumab by fluorescence-based flow cytometry. After fixation with 10 mL of 4% buffered formaldehyde at 4 °C for 16 h, formaldehyde was removed. Cell pellets were re-suspended with 750 µL of PBS heated at 65 °C and placed in a hot bath at 65 °C for 5 min, before adding 750 µL of agarose gel (5.5% w/v, dissolved in water and maintained at 65 °C after boiling). After transfer into a mold, cooling at –6 °C and solidification, the resulting gels were impregnated (ASP300 tissue processor from Leica) and embedded in paraffin (HistoCore Arcadia embedding center from Leica). A tissue core of 1 mm of diameter was removed from one of the

NIH 3T3-HER2 cell pellets with a TMA Master tissue microarrayer (3D Histech) and fused to an empty recipient paraffin block.

Sections of 3 µm were obtained with a microtome (MM France). For immunostaining, a positive (NIH 3T3-HER2) and a negative (NIH 3T3-WT) section were placed on each slide. For phage selections, one section of the NIH 3T3-HER2 pseudo-tissue core was placed on a slide with two (selections S1 to S5) or three (selections S6 to S9) sections of the NIH 3T3-WT pseudo-tissue. Slides were dried at 37 °C overnight (O/N). To count cells on slides, additional slides (prepared as for phage selection) were stained with hematoxylin and eosin, scanned using a Hamamatsu NanoZoomer 2.0-HT scanner, and cell number calculated using the QuPath software (https://qupath.github.io) (Bankhead et al., 2017).

### 2.3. Immunohistochemistry and immunofluorescence staining

For panning, IHC and immunofluorescence (IF), an autostainer (Discovery Ultra, Ventana) was used for deparaffinization, antigen retrieval and slide staining [SuperFrost Plus (Thermo Scientific) and Flex (Agilent) slides]. Unless stated otherwise, antigen retrieval was performed by incubation with the Cell Conditioning medium #1 (CC1, Tris-EDTA at pH 8.0–8.5, Ventana), heated at 100 °C for 8 min, followed by incubation with antibodies or phages at room temperature (RT) for 1 h. Antibodies were diluted in Antibody Diluent with Casein (Ventana), with or without horse serum (HS) supplementation. Trastuzumab (Roche) was detected with a rabbit polyclonal anti-human antibody (A0423, Dako), and phages were detected with a rabbit anti-phage polyclonal antibody (B7786, Sigma-Aldrich). The 4B5 rabbit anti-HER2 mAb (Ventana) was used according to the supplier's instructions: 37 °C for 20 min, after antigen retrieval in CC1 medium at 100 °C for 40 min. All rabbit antibodies were revealed with the anti-rabbit OmniMap HRP (rab-MAP) or anti-rabbit HQ (rab-HQ) revaluation system (Ventana) in standard conditions using 3,3'-diaminobenzidine (DAB). The IHC slides were counterstained with hematoxylin. Slides were scanned with a Hamamatsu or MIDI2 scanner (3DHistech).

For IF staining, antigen-antibody reactions were revealed using the rab-MAP system followed by the Tyramide-Cy5 or Tyramide-FAM kit (Roche), and DAPI counterstaining. IF slides were analyzed using a MIDI2 scanner (3DHistech) or a THUNDER Imager (Leica Microsystems; Wetzlar, Germany). Positive and negative cells were determined using the QuPath software (https://qupath.github.io) (Bankhead et al., 2017) and the stardist script (Schmidt et al., 2018).

### 2.4. Phage-display library

The human synthetic HusC1 library in scFv format is based on the sequence of the optimized 13R4 scFv (Martineau et al., 1998) and is displayed on filamentous phages using the pHEN1 vector. Diversity was introduced at positions [Aho numbering (Honegger and Plückthun, 2001)] L38–40, L58, L69, L109, L111, L112, H39–40, H57, H59, H67, H69, and with eight 2–9 residue loops inserted between positions H110–134 of VH-CDR3 (Robin et al., 2014). Diversity was introduced by Kunkel mutagenesis (Kunkel, 1985) using the CDR-deleted 13R4 gene as template and oligonucleotides encoding a mix of five amino acids (Y, S, D, N, G) synthetized using trinucleotide-based chemistry (Caucheteur et al., 2018). The library diversity, as measured by the number of transformants, was 3 × 10¹⁰, and 3 × 10⁹ of fully randomized scFv because of the mutagenesis yield of 70% per CDR, determined by sequencing.

### 2.5. On Slide phage panning

Slides with pseudo-tissue sections were prepared and processed with an autostainer (Discovery Ultra, Ventana). Phages were diluted in Antibody Diluent with Casein (Ventana) supplemented with HS (final concentration 1.4%). After antigen retrieval, 3 × 10¹⁰ to 3 × 10¹²
<figure><img src="image_5.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_7.png" /></figure>

(caption on next page)
<caption>**Fig. 1.** Schematic illustration of the selection and screening process.</caption>

Preparation of the slides with a small FFPE NIH 3T3-HER2 pseudo-tissue section (positive for the target protein) and a large excess of NIH 3T3-WT pseudo-tissue (negative for the target) (A and B1). Scale bars, 250 µm. On-slide panning of the phage library (B3) was carried out directly (S1 to S3) or after pre-selection rounds using recombinant HER2-ECD-Fc or peptides (S4 to S9). The dewaxing and antigen retrieval steps (B2) were performed by an autostainer. Then, phages were manually loaded onto the slides (B3). After simultaneous selection and depletion in the machine (B4), slides were automatically washed. Sections were delineated with a hydrophobic pen and eluted separately (B5). Bacteria were infected with the eluted phages (B6) to produce new, binder-enriched phage stocks (B7) that were used for the subsequent panning rounds (B3) and/or high-throughput sequencing. After sequence analysis, clones were chosen, produced, and tested by IHC as rabbit IgG. C) Schematic illustration of the different protocols tested. Protocols S1 to S3 (section 3.2): The naive phage library was directly panned 2-3 times on tissue sections, phages eluted from the positive tissue were sequenced and ranked by frequencies. Protocols S4 & S5 (section 3.3): The naive phage library was first pre-selected in vitro on the antigen (protein or peptide) using a single panning round, then processed as in protocols S1 to S3. Protocols S6 to S9 (section 3.4): The naive library was enriched in binders by 3-4 rounds of in vitro panning using the antigen (protein or peptide). Bound phages were recovered from all tissue sections (positive and negative), sequenced, and analyzed by clustering and differential analysis. In all protocols, a limited number of clones (typically 1-5) were cloned and purified as rabbit IgGs and tested by IHC. The Figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license (https://creativecommons.org/licenses/by/3.0/).

phages were manually loaded onto the slides (100 µL/slide). The phage solution was automatically covered with mineral oil (Liquid Coverslip, Ventana) to prevent evaporation of the liquid, and nozzles favored homogenization of the phage solution. Phages were incubated at RT for 1 h and washed. Importantly, slides were not allowed to dry because this may affect the phage infection capacity (Sørensen et al., 2010; Sun et al., 2009a). Most of the oil was removed by tapping the slide edge on a paper towel before individual rinsing in 40 mL of fresh PBS twice. The section edges were swiftly dried with paper towel then circled with a hydrophobic barrier pen (Liquid blocker Super pap pen, Daido Sangyo). The HER2-positive section was eluted with 5 µL of freshly prepared elution buffer (50 mM Tris-HCl pH 8, 125 µg/mL trypsin and 1 mM CaCl₂), and one of the HER2-negative sections with 100 µL. After 15 min of incubation at RT in a humid chamber, eluates were collected and 95 µL of PBS was added to the HER2-positive section eluate. Then, 200 µL of exponentially growing TG1 bacteria (OD600 ≈ 0.5) was added to the eluates. After incubation at 37 °C, 150 rpm, for 1 h, serial dilutions of the infected bacteria were prepared and spread on LB plates supplemented with 2% glucose and 100 µg/mL ampicillin (LBGA). The remaining bacteria were centrifuged, re-suspended in 100 µL of LB, and spread on a third LBGA plate. After growth at 37 °C O/N, colonies were counted. Then, colonies were scraped to prepare glycerol and phage stocks.

For phage stocks, bacteria placed in liquid culture were rescued (OD600 ≈ 0.5) with a 20-fold excess of KM13 as helper phage (Kris-
tensen and Winter, 1998). After incubation (37 °C, 150 rpm, for 1 h) and centrifugation (3000 ×g, RT, 10 min), pellets were concentrated twice in 2xTY, supplemented with ampicillin (100 µg/mL) and kanamycin (25 µg/mL). Cultures were incubated at 30 °C, 200 rpm, O/N. After centri-
fugation (11,000 ×g, 4 °C, 10 min), 400 µL of phage-containing super-
natants were added to 100 µL of 20% PEG/2.5 M NaCl (1/5 of the final
volume) and incubated on ice for 1 h before centrifugation (11,000 ×g,
4 °C, 20 min). Supernatants were discarded and after removal of the
remaining traces of PEG/NaCl, pellets were re-suspended in 100 µL of
PBS containing 15% glycerol. The phage concentration was determined
from the UV spectrum using the formula: *phages/mL* = (A269 – A320) ×
6 × 10¹⁶/*(phage base pairs)*.

**2.6. Phage panning using purified antigen**

In vitro pre-selection rounds were performed using 96-well immu-
noplates coated with HER2-extracellular domain (ECD)-Fc (1129-ER-
050, R&D Systems), diluted to 10 µg/mL in PBS, 4 °C O/N (Nunc
MaxiSorp). The library (10¹² cfu) was depleted twice using a human Fc
(Evitria), before the first round. Similarly, in vitro pre-election rounds
were performed using an equimolar mix of three peptides from HER2-
ECD conjugated to magnetic beads: CEVTAEDGT (peptide 1),
PEDECVGEG (peptide 2) and PDEEGAC (peptide 3) (SynBio3, Mont-
pellier). Before the first round of selection, the naive library was
depleted using an irrelevant peptide conjugated to the same magnetic
beads. Captured phages were eluted with 100 µL of freshly prepared
elution buffer (50 mM Tris-HCl pH 8, 125 µg/mL trypsin and 1 mM

CaCl₂). These phages were used to prepare a new phage stock with TG1
bacteria and the KM13 helper phage.

2.7. *Next-generation sequencing (NGS)*

As only 2 × 250 bp were allowed by the Illumina sequencing plat-
form, to keep the VL/VH pairing during NGS, our strategy was improved
throughout the method development. In its final version, phage DNA
from the phage stocks was PCR-amplified with a pair of primers that
hybridize as close as possible to the first variable nucleotide of the H-
CDR1 (bio-TCCTCGAACTTGAGTGGATTCACCTTCAGTAAC) to the last
of the L-CDR3 (CCGAAAACTCGAGTGCT). The forward primer was
biotinylated and contained a restriction site for the type IIS BpuEI
enzyme that cleaves 16/14 nucleotides outside its asymmetric recogni-
tion site. After PCR, amplicons were captured onto streptavidin mag-
netic beads (Pierce, 88,816) and released by BpuEI digestion, allowing
the recovery of cleaved amplicons with a maximum length < 500 bp.
Sequences were analyzed with Python and R scripts.

2.8. *Expression and purification of rabbit IgG*

The genes of the chosen clones were synthesized (Twist Bioscience) and cloned into an expression vector before transient transfection of Human Embryonic Kidney cells cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) with 10% fetal calf serum. After 4-6 h, the culture medium was replaced with serum-free DMEM. Cells were grown at 5% CO₂, 37 °C, for 5 days. The IgG-containing supernatants were purified using protein A agarose beads (Sino Biologicals, ref. 10,600-P07E-RN). The purity of the IgG obtained was controlled by SDS-PAGE.

2.9. Antibody characterization by ELISA

For ELISA on coated proteins, 96-well immunoplates (Nunc MaxiSorp) were coated with 100 µL of protein diluted to 1–10 µg/mL in PBS at 4 °C O/N. Peptides coupled to magnetic beads were used directly. After saturation in PBS-2% milk (RT, 2 h), primary antibodies were incubated at RT for 2 h, washed with PBS/0.1% Tween-20, and detected with secondary antibodies (RT for 2 h). Rabbit IgGs (1–10 µg/mL) were detected with a polyclonal anti-rabbit, HRP conjugate (Santa Cruz Biotechnology, ref. sc-2004), diluted 1/5000 in PBS/2% milk. The anti-Fc-HRP conjugate (Sigma-Aldrich, A0170) and anti-His-HRP conjugate (HIS-1, Sigma-Aldrich, ref. H1029) antibodies were diluted 1/3000 and used as controls. After the last wash, HRP was revealed with 100 µL of TMB (1-Step Ultra TMB-ELISA, Thermo Scientific). After stopping the reaction with 50 µL of 1 M H₂SO₄, absorbance was read at 450 nm.

**3. Results and discussion**

Phage display selection efficacy is limited by the level of unspecific
binding of phage particles. We anticipated that this could be one of the
main limitations of direct tissue selection using a large naive antibody
<figure><img src="image_8.png" /><figcaption>Fig. 2. Tissue staining with trastuzumab.</figcaption></figure>

A) Immunohistochemical staining of sections of NIH 3T3 pseudo-tissues (HER2-positive in a, WT in b), SKOV3 (c, d) and MDA-MB-468 (e) cancer cell xenografts (HER2-positive and -negative, respectively) with trastuzumab at 7 µg/mL (a, b, d, e) or without primary antibody (c) after antigen retrieval in CC1 at 100 °C for 8 min. Antigen-antibody reactions were revealed with a rabbit anti-human antibody (0.57 µg/mL for xenografts or 1.42 µg/mL for pseudo-tissues) and the rab-Map system. B) Immunohistochemical staining of NIH 3T3 pseudo-tissue sections with phages displaying 4D5 (trastuzumab scFv), 13R4 (irrelevant scFv) or nothing (helper phage, KM13), diluted at 10¹¹ phages/mL (RT for 1 h) followed by a rabbit anti-phage antibody and rab-Map system. Scale bars, 20 µm.

library that requires a high number of phages, typically 10¹² phage particles, to fully explore diversity. Therefore, we used an antibody and phage model based on the anti-HER2 mAb trastuzumab to determine the best panning conditions. We used several sources to generate FFPE sections for optimization, panning and antibody characterization: tumors from mice xenografted with human HER2-positive and HER2-negative cancer cell lines, cell pellets made of murine NIH 3T3 fibroblasts transduced or not with the human *ERBB2* gene (encoding HER2) or xenografted on the chorioallantoic membrane of chicken eggs. NIH 3T3-HER2 cells express approximately 1.8 × 10⁵ HER2 molecules at the plasma membrane (Gaborit et al., 2011).

To direct the selection toward the target and avoid the amplification of phages against irrelevant proteins, we used slides that included a small FFPE NIH 3T3-HER2 pseudo-tissue section and two large FFPE NIH 3T3-WT pseudo-tissue sections (Fig. 1A), and an IHC autostainer for panning (Fig. 1B). This approach is similar to the micropipette-assisted microdissection and shadow stick methods developed by Kristensen's group (Lykkemark et al., 2017; Mairaville and Martineau, 2021). The selection specificity was due to the on slide competition between the excess of HER2-negative cells relative to the small number of HER2-positive cells because an irrelevant phage should equally bind to both

cell populations, whereas the anti-HER2 phages should only bind to HER2-positive cells. To minimize the impact of confounding variables like incubation times, temperatures, and dehydration (Eltoum et al., 2001; Scalia et al., 2017) – all of which can influence results during FFPE processing beyond just fixation – we utilized an IHC autostainer to ensure consistent processing conditions.

### 3.1. Optimization of the panning conditions

We first established the antigen retrieval conditions to detect HER2 using humanized trastuzumab (anti-HER2 mAb) in FFPE tissues and cell pellets (i.e. pseudo-tissues). We tested two solutions: the basic CC1 solution and the acidic CC2 solution (citrate buffer at pH 6.0). We incubated FFPE sections with these buffers at 100 or 91 °C for 24 min, followed by trastuzumab at 37 °C for 1 h before revelation with an HRP-conjugated anti-human secondary antibody and staining with DAB. We did not observe any staining with CC2 (data not shown). Then, we tested several conditions: without/with antigen retrieval with CC1 for 4 to 40 min. We obtained the best results using FFPE SKOV3 (HER2-positive) and MDA-MB-468 (HER2-negative) cell xenograft and NIH 3T3 pseudo-tissue sections with antigen retrieval (CC1 for 8 min) followed by
<table><caption>**Table 1**
Selection on tissue sections.</caption><thead><tr><th>Selection</th><th></th><th>S1</th><th>S2</th><th>S3</th><th>S4</th><th>S5</th><th>S6</th><th>S7</th><th>S8</th><th>S9</th></tr></thead><tbody><tr><td rowspan="2">In vitro</td><td>Antigen</td><td>nd</td><td>nd</td><td>nd</td><td>HER2</td><td>pep</td><td>HER2</td><td>HER2</td><td>pep</td><td>pep</td></tr><tr><td>Rounds</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td><td>3</td><td>4</td><td>3</td><td>4</td></tr><tr><td rowspan="2">FFPE</td><td>Functional</td><td>10%</td><td>10%</td><td>10%</td><td>9%</td><td>24%</td><td>84%</td><td>70%</td><td>78%</td><td>55%</td></tr><tr><td>Round</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="3">1</td><td>Ph/mm²</td><td>6</td><td>24</td><td>98</td><td>6000</td><td>759</td><td>727</td><td>413</td><td>680</td><td>730</td></tr><tr><td>Functional</td><td>19%</td><td>10%</td><td>10%</td><td>6000</td><td>12%</td><td>21%</td><td>71%</td><td>61%</td><td>30%</td></tr><tr><td>Ph/mm²</td><td>6</td><td>113</td><td>113</td><td>4%</td><td>117</td><td>9</td><td>69%</td><td>71%</td><td>21%</td></tr><tr><td rowspan="2">2</td><td>Functional</td><td>90%</td><td>30%</td><td>30%</td><td>6000</td><td>759</td><td>727</td><td>413</td><td>680</td><td>730</td></tr><tr><td>Ph/mm²</td><td>3</td><td>192</td><td>95%</td><td>6000</td><td>12%</td><td>21%</td><td>71%</td><td>61%</td><td>30%</td></tr></tbody></table>

Selection, S1 to S9 selection protocols are schematically depicted in Fig. 1C; nd, not done; Ph/mm², number of phages eluted from 1 mm² of FFPE tissue; Functional, percentage of phages encoding a functional scFv; HER2, recombinant HER2-ECD-Fc; pep, mixture of three peptides from HER2-ECD.

incubation with trastuzumab (7 µg/mL) (Fig. 2A).

We then cloned the 4D5 scFv (i.e., the humanized variable domain of trastuzumab) in the phage display vector pHEN1. We used the previously determined antigen retrieval conditions and tested several blocking reagents (bovine serum albumin, HS, milk, soy milk) in several diluents (PBS, Antibody Diluent with Casein from Ventana, Protein-free Blocking buffer from Pierce), with/without Tween-20, using different wash volumes, incubation times and temperatures (4, 20 and 37 °C), different phage concentrations (10¹⁰ to 10¹³ phages/mL), and hyperphages to avoid bald phage particles (Rondot et al., 2001). We obtained the best results with 10¹¹ phages/mL in Antibody Diluent with Casein (Ventana), supplemented with 1.4% HS (Fig. 2B). Irrelevant phages (KM13 helper phage or phages expressing the irrelevant anti-β-galactosidase scFv 13R4) only weakly stained FFPE NIH 3T3-HER2 and -WT pseudo-tissue sections, whereas 4D5-expressing phages gave a strong staining. However, they stained also the NIH 3T3-WT sections, although more weakly than the NIH 3T3-HER2 sections. When we increased the phage concentration to >10¹² phages/mL, all tissues were stained by all phages without specificity. While phage particles are likely the primary contributor to the background noise, 4D5 itself might also play a role since irrelevant or control samples lacking 4D5 (bald control) exhibit a weak or negative signal when tested on NIH3T3-WT pseudo-tissue. First, fusing 4D5 with the g3p moiety might alter its reactivity compared to its free, unconjugated (IgG format) state. Similar observations have been reported for other antibody fragments (Jensen et al., 2002; Larsen et al., 2008). In addition, 4D5 specificity might be affected by the molecular environment as shown with the anti-myc antibody 9E10, whose reactivity is highly dependent on the surrounding molecular context of its epitope (Schüchner et al., 2020).

To conclude, despite assessing many parameters to stain FFPE sections with phages, we did not find the ideal conditions. This could be partly due to the poor ability of 4D5-expressing phages to recognize their target in FFPE samples because this clone strongly recognizes native HER2 at the cell surface. However, the best conditions gave a lower signal with the irrelevant scFv and could be used to select specific phages by panning using tissue sections.

## 3.2. Direct on-slide selection (S1 to S3)

As determined with 4D5-expressing phages, a phage concentration > 10¹¹/mL resulted in high background. This represented only 3 × 10¹⁰ phages/slide, a diversity too low to fully explore the diversity of the HuscI library (>10¹⁰ clones) used in this project. Nevertheless, we tested panning with this low number of phages (S1) but also with 10 (S2) and 100 times (S3) higher concentrations (10¹² and 10¹³ phages/mL). We eluted bound phages by trypsin digestion from the HER2-positive and HER2-negative pseudo-tissue sections, separately (Fig. 1C). After the first round, diversity was strongly reduced and we could elute only a low number of infectious phages (6–6000 phages/mm²; Table 1). For the next rounds, we decreased phage concentration to 10¹² phages/mL. After two rounds of panning, <100 clones were recovered from the HER2-positive section (6–117/mm²), and this number did not increase between round 1 and 2. Therefore, we performed a third round of selection with 10¹³ phages/mL (only for S3), but without any strong increase in the number of eluted phages (Table 1). This low number of eluted phages could be explained by the absence of positive selection or by the limited binding capacity of the FFPE pseudo-tissue sections. As the enriched phage populations contained many binders (see below), the second explanation is more likely. As the positive spot size was 0.78 mm² and contained around 10,400 cells, this means that the highest capture of 192 phages/mm² represented only 1 phage per 70 cells.

When we performed panning with a phage stock that expressed the irrelevant scFv 13R4 (anti-β-galactosidase antibody), the numbers of phages eluted per surface from the HER2-positive and the HER2-negative pseudo-tissue sections were identical and increased with the phage concentration. With 10¹¹ phages/mL, both sections captured
<figure><img src="image_9.png" /><figcaption>Fig. 3. Selection using repeated on-slide panning rounds.</figcaption></figure>

IHC staining of FFPE NIH 3T3-HER2 pseudo-tissue sections with the C1.2, C2.1 and C2.3 clones (rabbit IgG format) from selections S2, S4 and S5, respectively. Detection was performed with the rab-HQ system. 13R4 (anti-β-galactosidase antibody) was used as isotype control. Scale bar, 25 µm (for all images).

~1.5 phages/mm². This number increased to 7 and 21 phages/mm² with 10¹² and 10¹³ phages/mL, respectively. This showed that irrelevant binding was low in the used panning conditions and increased more slowly than the phage concentration. In addition, as the density of the HER2-negative cells was six times lower (2100 versus 13,300 cells/mm² for the HER2-negative and HER2-positive pseudo-tissue section respectively), the constant number of bound phages eluted from both tissues per surface area demonstrated that background was mainly due to the slide and not to the cells.

Conversely, when we did the real panning with the library, the number of phages eluted per surface was higher on the HER2-negative than HER2-positive pseudo-tissue sections despite the lower cell density (Table 1). This was anticipated because the library contains clones that recognize several proteins present in NIH 3T3 cells. However, compared to the irrelevant selection, the number of phages eluted from the HER2-positive section was much higher, and this excess increased linearly with the phage concentration, from 4 times with 10¹¹ phages/mL to 14 times with 10¹² phages/mL and 286 times with 10¹³ phages/mL. Nevertheless, phage binding to FFPE pseudo-tissue sections was low. Indeed, if we consider the cell density, we eluted from the HER2-positive pseudo-tissue section only 1 phage for 2200, 140 and 2 cells with 10¹¹, 10¹² and 10¹³ phages/mL respectively.

To determine whether we selected phages that bind to FFPE HER2-positive pseudo-tissue sections, we analyzed the final phage stocks (round 2 for S1 and S2, and round 3 for S3) by IF using 3 × 10¹⁰ phages/mL and FFPE NIH 3T3-WT and -HER2 pseudo-tissue sections. The three phage stocks specifically stained the HER2-positive cells, much better than 4D5 scFv-expressing phages (Supplementary Fig. 1). This strongly suggested a positive enrichment during panning.

To characterize the clones selected by the positive sections, we sequenced the phage stocks (Illumina sequencing platform) to study the population evolution. Because of the synthetic design of the Husci library, a single 2 × 250 bp paired sequencing experiment was sufficient to cover all the variable positions in the six scFv CDRs. Besides

functional scFv, the Husci library contains non-functional genes with deletions that produce bald phage particles. As the library was constructed using a CDR-deleted template and Kunkel mutagenesis independently of the six CDRs, functional full-length scFv represented only 10% of all clones because the mutagenesis efficacy was ~70% per site (Caucheteur et al., 2018). Therefore, panning success could be estimated by measuring the proportion of functional clones. A successful selection will gradually decrease the defective phage rate, whereas inefficient selection pressure and high background will result in the conservation of these defective phages during panning. The proportion of functional scFv gradually increased during the three selections, leading to 30–95% of functional clones (Table 1). For instance, the selection with the highest initial diversity (S3, 10¹³ phages/mL) gave 4% of functional clones after the first panning round. This rate quickly increased to 89% and 95% after rounds 2 and 3, respectively. This demonstrated that we selected scFv that bind to FFPE HER2-positive pseudo-tissue sections and not random clones due to high background.

For each selection round, we aligned sequences and ranked them in function of the read number. We chose the four sequences found at a frequency ≥ 1% in the final selection rounds for further testing. We synthesized the genes and produced the corresponding clones (C1.1, C1.2, C1.3 and C1.4, Supplementary Table 1) as rabbit IgGs. The resulting antibodies are chimeric, possessing a human Fv region fused to constant domains of rabbit IgGk. Due to the similar size of human and rabbit Fv regions, and because production is done in human cell line HEK293T, these chimeric antibodies are glycosylated and possess a typical molecular weight of 150 kDa. We tested them by IHC (Fig. 3 and Supplementary Fig. 2) with the same cell pellets and conditions used for the selection. The staining obtained with C1.2, C1.3 and C1.4 was so strong that we had to dilute these antibodies to ≤10 ng/mL for IHC. For comparison, according to Ramos-Vara and Miller (Ramos-Vara and Miller, 2014), the recommended working concentrations for primary antibodies are generally between 1 and 5 µg/mL, although they may be decreased by using antigen retrieval techniques and signal amplification. However, these three clones gave a nuclear staining instead of the expected membrane staining. Clone C1.1 stained the membrane/cytoplasm at a more traditional concentration of 1.5 µg/mL (Supplementary Fig. 2). However, all clones similarly stained NIH 3T3-HER2 and NIH 3T3-WT pseudo-tissue sections, showing that they did not specifically recognize human HER2. By western blotting, clones C1.2, C1.3 and C1.4 recognized a nuclear protein with an apparent MW of 30 kDa, present in NIH 3T3-HER2 and -WT histone extracts, and C1.2 also an additional band at 60 kDa (Supplementary Fig. 3 A). The 30 kDa protein could be histone H1 on the basis of its size and nuclear localization. This was confirmed by comparison with an anti-H1 commercial antibody (Supplementary Fig. 3A), and by expressing recombinant His-tagged histone H1 in E. coli. Clones C1.2, C1.3 and C1.4 recognized the seven histone H1 isoforms (western blotting in Supplementary Fig. 3B).

In conclusion, we successfully selected IHC-compatible antibodies from a large naive scFv phage display library by direct panning using FFPE pseudo-tissue sections. Some clones, formatted as rabbit IgGs, were unusually powerful and still gave a strong staining at concentrations <10 ng/mL. However, we could not direct the selection toward a specific protein using on-slide depletion by competition, and we obtained antibodies against histone H1, a ubiquitous protein that is strongly expressed by most cell types (Happel and Doenecke, 2009; Osunsade et al., 2019). We did not quantify the two proteins, but histone H1 is presumably expressed at a much higher level than HER2 in murine fibroblasts and this may explain its preferential selection.

### 3.3. On-slide selection using an in vitro pre-enriched library

As on-slide depletion did not eliminate non-HER2 binders, we first performed a single round of pre-selection against HER2 using recombinant HER2-ECD-Fc (S4) or a peptide mixture (S5) before on-slide panning (Fig. 1C). We anticipated that a single in vitro selection round
<figure><img src="image_10.png" /><figcaption>**Fig. 4.** Selection using in vitro pre-selection and on slide filtering.
Clones expressed as rabbit IgG from selections S7 (C3.4) and S8 (C3.9) were used to stain by IHC FFPE NIH 3T3-HER2 and -WT pseudo-tissue sections (A) and were tested by ELISA for binding to HER2-His or a recombinant human Fc (irrelevant antigen) (B). C) ELISA was used to test the binding of clone C3.9 from selection S8, produced as rabbit IgG and diluted to 10 µg/mL, to the three peptides from HER2-ECD used for the in vitro panning. C3.NS is the most amplified clone during in vitro panning and its sequence was recovered from both HER2-positive and -negative sections during the on slide selection (S6 and S7); 4D5 is trastuzumab, produced as rabbit IgG. Detection was performed with the rab-Map system in (A), and an HRP-coupled anti-rabbit antibody in (B & C). Scale bar, 25 µm (for all images).</figcaption></figure>

would focus the library on HER2-specific clones but would not compromise diversity. For the protein-based selection, we first depleted the library using a recombinant Fc and then did the panning step using recombinant HER2-ECD-Fc. For the selection using the peptide mixture, we depleted the library using an irrelevant peptide conjugated to magnetic beads and then panning using an equimolar mixture of three peptides from HER2-ECD conjugated to the same beads. After these in vitro pre-selections, the library diversity was reduced to ~10⁷ in both cases, allowing us to use only 10¹¹ phages/mL for the on slide panning to reduce background and non-specific binding, as demonstrated with 4D5-expressing phages. The diversity was strongly reduced to <1000 clones/mm² (9–759) after the first and second round using the FFPE pseudo-tissue sections (Table 1). Like during the first selection, it seems that this represents the highest binding capacity of the HER2-positive pseudo-tissue section despite containing >13,000 cells/mm².

As before, we sequenced all the phage stocks by NGS. The percentages of functional clones were similar in the naive library and after two rounds of panning (1 in vitro and 1 using sections) and then they strongly increased to 71–99% after the second round of panning using sections (Table 1). We kept the sequences found at a frequency > 1% in the last round for further testing. Specifically, we recovered two clones from S4 (pre-selection performed using recombinant HER2-ECD-Fc) found at frequencies of 1.2% and 17.1% (C2.1 and C2.2, respectively),

and three clones from S5 (pre-selection performed on peptides) found at frequencies of 41%, 6% and 4.2% (C2.1, C2.3 and C2.4, respectively). One clone (C2.1) was identified in both selections. We cloned and produced the four clones as rabbit IgG (Supplementary Table 1).

By IHC, they all stained the membrane/cytoplasm when used at concentrations between 50 and 500 ng/mL (Fig. 3, Supplementary Fig. 4). However, they similarly stained NIH 3T3-WT and -HER2 cells. As these clones were not HER2-specific, we did not try to identify their target. Again, these clones were very efficient and strongly stained the cells at low concentration, but a single in vitro pre-selection round was not enough to deplete the irrelevant binders from the large diversity of the naive library, as suggested by the still very low frequency of functional clones after this step (9–24%; Table 1).

### 3.4. *In vitro pre-selection with on slide filtering*

Despite one round of in vitro panning using HER2-ECD-Fc or the peptide mixture, we could not avoid the selection of antibodies against other targets. Therefore, we increased the selection strength by four means. First, we increased the number of in vitro rounds from 1 to 3 and 4 to pre-select anti-HER2-enriched libraries. Second, we used a single on slide panning round as a filter for IHC-compatible phages and to avoid the amplification of clones that recognize another protein by
repeated panning. Third, we added a third HER2-negative pseudo-tissue section on the slides and we prepared new cell pellets to obtain sections with a ratio of 1 HER2-positive cell to 235 HER2-negative cells, a 6-fold increase from the 1:41 ratio previously used. Fourth, we also sequenced the pool of clones eluted from the HER2-negative pseudo-tissue section to add a bioinformatic depletion by subtracting these sequences from those of the positive clones (Fig. 1C).

After the in vitro pre-selections, we evaluated the enrichment in HER2/peptide binders by polyclonal and monoclonal phage ELISA. After three and four rounds of pre-selection using recombinant HER2-ECD-Fc, ELISA showed that phage stocks strongly recognized HER2-ECD-Fc with a minimal signal for the recombinant Fc (Supplementary Fig. 5A). When tested individually, after the third round, 65.6% (61/93) of the tested clones gave an ELISA signal at least 5-fold higher than background and 50.5% (47/93) displayed an absorbance >0.5 (Supplementary Fig. 5C). These data demonstrated that the phage pool was strongly enriched in anti-HER2-ECD clones. For the anti-peptide pre-selection, the phage pool recognized strongly the three peptides (Supplementary Fig. 5B), but not recombinant HER2-ECD-Fc by ELISA (Supplementary Fig. 5D). This showed that the selected antibodies recognized peptide conformations that are only minimally present in the native HER2 protein. This was anticipated because anti-peptide antibodies frequently do not recognize the native protein conformation (Chen et al., 2009).

Therefore, we proceeded with on-slide panning using 10<sup>11</sup> phages/mL from the third and the fourth round of the in vitro pre-selections using HER2-ECD-Fc (S6 and S7) or the peptide mixture (S8 and S9). After a single on-slide panning, we recovered separately the phages from the HER2-positive and -negative sections (Table 1). The number of eluted phages (from 343 to1800/mm<sup>2</sup>) was lower compared with the 13,000 cells/mm<sup>2</sup> present in the HER2-positive section. Unlike the previous on-slide selections (S1, S2, S3), comparison of the number of eluted phages per surface did not show a preference for the HER2-negative section. This was expected because the library essentially contained anti-HER2/peptide antibodies. A high percentage (>60%) of recovered phages contained functional scFv without stop codons (Table 1) except those from the anti-peptide selection S9.

After sequencing, we retained the clones present at least at a frequency of 0.5% in the final phage stock and specifically associated with the HER2-positive pseudo-tissue section. This led to the identification of 14 sequences: two and four from S8 and S9 (pre-selection with peptides), and three and five from S6 and S7 (pre-selection with HER2-ECD-Fc), respectively. We produced all these clones as rabbit IgGs as well as the clone most frequently present in the protein-based pre-selection that was similarly present in the HER2-positive and -negative sections (clone C3.NS, Supplementary Table 1). We used clone C3.NS as a control that represented a typical sequence selected by regular phage display.

After purification, we tested all clones by IHC on the same sections (NIH 3T3 cell pellets) used during selection. Among the eight anti-protein clones, two (C3.4 and C3.5) stained the NIH 3T3-HER2 pseudo-tissue section more strongly than the -WT section (Fig. 4A and Supplementary Fig. 6A). The other clones did not (C3.2, C3.3, C3.6 and C3.7) or did stain both sections (C3.1) or gave only a slightly higher signal in the HER2-positive section (C3.8). The signal of the two positive clones was comparable in intensity to that of trastuzumab reformatted as a rabbit IgG (4D5) and used as positive control. However, clones C3.4 and C3.5 mainly stained nuclear and cytoplasmic proteins, respectively, a pattern not compatible with HER2 localization. We also tested clone C3.NS, but did not detect any signal by IHC despite its strong recognition of native HER2 by ELISA (Fig. 4A-B). This underlines the interest of on-slide filtering to focus on IHC-compatible clones. Lastly, we confirmed by ELISA (Fig. 4B and Supplementary Fig. 6B) and cytometry (data not shown) that these clones, except C3.3, C3.6 and C3.8, recognized specifically HER2-ECD-Fc. In conclusion, we successfully selected two anti-HER2-ECD clones (C3.4 and C3.5) that specifically stained our positive sections, were specific for native HER2, but gave a staining pattern not

compatible with an exclusive recognition of HER2 by IHC. It is important to note that previous studies have reported the presence of full-length HER2 in the nucleus (Wang et al., 2004). However, in these studies, the majority of HER2 was consistently found in the membrane and cytoplasm. Additionally, the previously described nuclear staining exhibited a punctate pattern, rather than a uniform distribution throughout the nucleus as observed here. Based on these discrepancies, we believe our antibodies lack specific reactivity with HER2 in tissues. Nevertheless, some antibodies, like C3.4 and C3.5, demonstrate HER2 recognition through a positive ELISA signal against the HER2-ECD and are specific of the HER2-positive pseudo-tissue.

We also tested the six anti-peptide antibodies by IHC on NIH 3T3 cell pellets. Clones C3.9, C3.13 and C3.14 stained only NIH 3T3-HER2 pseudo-tissue sections (Fig. 4A and Supplementary Fig. 6A), whereas the other clones did not give any signal (C3.12) or were non-specific (C3.10 and C3.11). Moreover, C3.9, C3.13 and C3.14 strongly stained sections at low concentration (25–250 ng/mL). However, none of them stained the cell membrane, as expected for a specific anti-HER2 antibody. By ELISA, these three clones recognized mainly peptide 3 (Fig. 4B and data not shown), but not the native protein (Supplementary Fig. 5E). Like for C3.4 and C3.5, C3.9 (protein-based selection), C3.13 and C3.14 are unlikely to recognize specifically HER2 by IHC. However, these five clones (C3.4 and C3.5 and C3.9, C3.13 and C3.14) are as specific as 4D5 for FFPE NIH 3T3-HER2 pseudo-tissue sections, compared with the FFPE NIH 3T3-WT pseudo-tissue sections, with a comparable or stronger intensity but at a 2.3–140-fold lower concentration (1.5 µg/mL – 25 ng/mL vs 3.5 µg/mL).

We also characterized by IHC the strongest specific binder (the anti-peptide C3.9) on cancer cell xenograft tissue sections. None of the observed staining patterns was specific for human HER2. Specifically, C3.9 stained the murine stroma and presented a staining pattern different from trastuzumab (produced as a rabbit IgG) in SKOV3 cell xenografts. It also stained xenografts of HCT116 (Supplementary Fig. 7) and MCF7 cells (data not shown), two cell lines that do not express HER2. Moreover, we could not further improve the staining specificity by optimizing the antigen retrieval conditions, testing different antibody dilutions or incubation conditions.

In the previous experiment we selected the clones from the in vitro pre-selections specifically present in the HER2-positive but not in the HER2-negative section eluate after a single round of panning. However, because of the design of our synthetic library, several highly homologous clones were present in the library, even after four rounds of panning. Due to stochastic selection, a clone may be absent from the HER2-negative section whereas a homolog is present in the HER2-positive section. To eliminate these false positive clones, we introduced two modifications in our pipeline. First, we improved our sequencing method to increase sequencing depth and detect long VH-CDR3 that were previously frequently missed. Second, we added an additional step in our bioinformatic pipeline to consider the co-selection of homologous clones. We ranked clones by frequency and then clustered them around the top 20 clones using a distance of <4 mismatches and an identical VH-CDR3 length. Then, we calculated the frequencies for the whole cluster and we retained the most frequent clones present in a cluster at least 3-fold amplified during panning using sections, and present at a 5-fold higher frequency in the HER2-positive than in the HER2-negative tissue. Only clone C4.3 (peptide-based selection S8) and clone C4.4 (peptide-based selection S9) met these criteria: 5-fold enrichment in the positive versus the negative section, and a 15- and 3-fold amplification during on-slide panning, respectively. In selection S8, C4.3 was not the most frequent cluster. For selections S6 and S7 (pre-selection with HER2-ECD-Fc), these criteria were met by three clones (S6), two of which belonged to the same cluster with three mismatches, and by 16 of the top 20 clones (S7). We retained one clone from each protein-based selection (C4.1 for S6 and C4.2 for S7) because they presented a 25- and 10-fold enrichment in the HER2-positive versus -negative pseudo-tissue sections and a 10- and 35-fold amplification
<figure><img src="image_11.png" /><figcaption>**Fig. 5.** Characterization of an anti-HER2 clone obtained using the optimized protocol.
Clone C4.2 (selection S7), produced as rabbit IgG, was tested (A) by ELISA to investigate its binding to human and mouse HER2-ECD-Fc fusion proteins (huHER2 and moHER2, respectively), and mouse DDR1-ECD-Fc (moDDR1) and human EGFR-ECD-Fc (huEGFR) as irrelevant proteins, and (B) by IHC on FFPE NIH 3T3-HER2 and -WT pseudo-tissue sections followed by the rab-HQ system. Black arrows indicate cells with membrane staining. C) Clones C4.2 and the reference anti-HER2 antibody 4B5 were tested on consecutive FFPE NIH 3T3-HER2 and -WT pseudo-tissue sections using the rab-HQ system and Ty-Cy5 (red). 4B5, an anti-HER2 antibody used in diagnostic tests (Ventana), was detected using the rab-Map system and Ty-FAM (red). Nuclei were counterstained with QD DAPI (blue). White arrows indicate the position of cells that are present in both consecutive sections. Scale bars, 100 and 25 µm in B left and right panels, respectively, and 50 µm in C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
during the last on-slide panning round, respectively. We produced these four clones as rabbit IgGs (Supplementary Table 1). We confirmed by ELISA that C4.1, C4.2 and C4.4 recognized native human and not murine HER2, but C4.3 did not react with HER2 (Fig. 5A and Supplementary Fig. 8A). However, absence of reactivity by ELISA does not mean absence of reactivity by IHC because of the differences in antigen presentation in these two techniques. Indeed, C4.3 and C4.4 recognized specifically NIH 3T3-HER2 cells, but staining was essentially cytoplasmic. C4.1 did not stain NIH 3T3-HER2 and -WT cells, even at 5 µg/mL (Supplementary Fig. 8B). C4.2 showed a better reactivity on the NIH 3T3-HER2 than -WT pseudo-tissue sections when tested at 2.5 µg/mL. Despite a strong cytoplasmic reactivity, this antibody also showed faint membrane staining in some cells (black arrows, Fig. 5B). Changing the demasking conditions (not shown) did not improve the result, indicating that the best reactivity was obtained in the same conditions used for the panning experiment (CC1, 8 min). We further characterized this clone in comparison with the 4B5 anti-HER2 mAb used for the diagnosis of HER2-positive cancers (van der Vegt et al., 2009). We stained two consecutive FFPE NIH 3T3-HER2 and -WT pseudo-tissue sections with C4.2 and 4B5 antibodies by IF. Only 18% (192/1063) of cells were stained in the NIH 3T3-WT pseudo-tissue section (Fig. 5C), possibly because it also recognizes a protein expressed by a subpopulation of this mouse fibroblast cell line. Conversely, 87% (1055/1213) of cells were stained in the NIH 3T3-HER2 pseudo-tissue section and the staining pattern was comparable to that of the reference 4B5 antibody (colocalization indicated by arrows in Fig. 5C). Lastly, we tested the clones on more complex tissues. For this, we grafted NIH 3T3-HER2 cells on the chorioallantoic membrane of chicken eggs. Unlike 4B5 that strongly and specifically stained NIH 3T3-HER2 cells, C4.2 only showed a faint signal and no specificity for the grafted HER2-positive cells (Supplementary Fig. 9).

## 4. Conclusions

Our aim was to develop a method to identify antibodies from a naive antibody phage-display library that recognize a specific protein (human HER2 in our example) for IHC in FFPE tissues. As shown by other authors (review in Mairaville and Martineau, 2021), using a competition between HER2-positive and HER2-negative pseudo-tissue sections, we easily identified phages that recognized specifically the positive tissue demonstrating the efficiency of the subtractive panning approach (Supplementary Fig. 1), as also published by other authors (Mairaville and Martineau, 2021). However, none of the derived mAbs, albeit efficient as IHC reagents, specifically recognized only HER2-positive pseudo-tissue sections, and they equally stained also the HER2-negative pseudo-tissue sections (Fig. 3 and Supplementary Fig. 4). As discussed above (Fig. 2), the fusion of a scFv with the g3p moiety could potentially modify its reactivity compared to its free, unconjugated (scFv, Fab or IgG format) state. This difference in reactivity might explain the variation observed between our phage and IgG experiments (Jensen et al., 2002; Larsen et al., 2008). Therefore, we added additional steps to focus the selection on HER2 (Fig. 1, selections S6 to S9). First, we enriched the library in HER2 binders by in vitro panning using recombinant HER2-ECD-Fc or a mixture of synthetic peptides. Second, we captured IHC-compatible antibody-phages in a single round of panning using FFPE pseudo-tissue sections. We opted for a single round of panning to avoid the amplification of phages that recognize other targets expressed by the used tissues (cell pellets in our example). Third, we performed an experimental depletion by adding a large excess of HER2-negative pseudo-tissue sections during the on-slide panning step and also a bioinformatic depletion by sequencing separately phage outputs from the FFPE HER2-positive and -negative pseudo-tissue sections. In the fourth and last step, we cloned, produced, purified, and tested by IHC between 1 and 5 clones as full-length IgG. Testing a small number of clones was one of the main objectives of our method. Our strategy resulted in a high hit rate: 44% (8/18) of antibodies specifically stained

FFPE HER2-positive pseudo-tissue sections by IHC and IF, and the best one (C4.2) showed membrane but also cytosolic and nuclear staining (Fig. 5). However, when tested on other tissues, this antibody was no longer specific and stained other proteins in addition to the HER2 target recognized in vitro (Fig. 5A).

Our findings highlight the need for further development of the presented methods (Fig. 1) to establish a robust pipeline for antibody selection on FFPE tissues. While the project achieved its primary objective by successfully isolating IgGs from a large naive phage library that specifically stain HER2-positive cells in FFPE sections, the resulting antibodies currently fall short of being suitable reagents for even basic tissue characterization. This is unexpected, considering the proven ability of naive phage libraries to generate highly specific antibodies against native antigens, with several examples of IgGs now approved or under clinical development. A key difference between our approach and traditional panning techniques lies in the very low availability of the targeted antigen. As shown in Table 1, we captured only 100–1000 phages per square mm of positive tissue, even after multiple panning rounds. Translated into HER2 receptors, this translates to a capture efficiency of roughly 1 phage for 13–130 positive cells, i.e. 2.5–25 millions of surface-exposed HER2 molecules. The reasons for this low capture capacity likely stem from a combination of factors: antigen fixation and accessibility within FFPE sections, the large size of phage particles compared to antibodies, and the resulting diffusion limitations within tissues (phages are roughly 100 times larger than IgGs).

Despite these challenges, we firmly believe that selection on tissue sections holds significant promise for future development. Our results support this notion, particularly with the potential benefits of incorporating a library of complex tissues during panning and employing in-depth analysis of the resulting phage pools using NGS. Additionally, implementing techniques like chain shuffling, CDR shuffling, or random mutagenesis could further enhance the specificity of identified clones.

## Funding

CM salary was covered by a grant from Occitanie / Pyrénées-Méditerranée region and Histalim (now Cerba Research). This publication was supported by the ANR grant LabEx MAbImprove ANR-10-LABX-53. MB salary was covered by a REACT-EU grant (Recovery Assistance for Cohesion and the Territories of Europe). Funding agencies were not involved in the conduct of the research and manuscript preparation.

## CRediT authorship contribution statement

**Célestine Mairaville:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Conceptualization.
**Morgane Broyon:** Investigation.
**Margaux Maurel:** Investigation.
**Myriam Chentouf:** Investigation.
**Barbara Chiavarina:** Investigation.
**Andrei Turtoi:** Writing – review & editing.
**Nelly Pirot:** Writing – review & editing, Supervision, Methodology.
**Pierre Martineau:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization.

## Declaration of competing interest

None.

## Data availability

No data was used for the research described in the article.

## Acknowledgments

The authors thank Christel Larbouret and Arnaud Coquelle for providing cell lines and FFPE samples, and Renaud Burrer for
discussions and advice during the project. We acknowledge the imaging facility MRI, member of the France-BioImaging national infrastructure supported by the French National Research Agency (ANR-10-INBS-04 "Investments for the future"). We thank Pascal Verdié from the SynBio3 platform for peptide synthesis (UMR5247 CNRS-ENSCM-Université Montpellier). We thank the histology facility RHEM, supported by SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553, IBiSA and Ligue contre le cancer.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jim.2024.113730.

## References

Bankhead, P., Loughrey, M.B., Fernández, J.A., Dombrowski, Y., McArt, D.G., Dunne, P. D., McQuaid, S., Gray, R.T., Murray, L.J., Coleman, H.G., James, J.A., Salto-Tellez, M., Hamilton, P.W., 2017. QuPath: open source software for digital pathology image analysis. Sci. Rep. 7, 16878. https://doi.org/10.1038/s41598-017-17204-5.

Caucheteur, D., Robin, G., Parez, V., Martineau, P., 2018. Construction of a synthetic antibody gene library for the selection of intrabodies and antibodies. In: Hust, M., Lim, T.S. (Eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology. Springer, New York, NY, pp. 239–253. https://doi.org/10.1007/978-1-4939-7447-4 12.

Chan, C.E.Z., Chan, A.H.Y., Lim, A.P.C., Hanson, B.J., 2011. Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays. J. Immunol. Methods 373, 79–88. https://doi.org/10.1016/j.jim.2011.08.005.

Chen, S.-W.W., Van Regenmortel, M.H.V., Pellequer, J.-L., 2009. Structure-activity relationships in peptide-antibody complexes: implications for epitope prediction and development of synthetic peptide vaccines. Curr. Med. Chem. 16, 953–964.

de Matos, L.L., Trufelli, D.C., de Matos, M.G.L., da Silva Pinhal, M.A., 2010. Immunohistochemistry as an important tool in biomarkers detection and clinical practice. Biomark. Insights 5, 9–20.

Dunstan, R.W., Wharton, K.A., Quigley, C., Lowe, A., 2011. The use of immunohistochemistry for biomarker assessment—can it compete with other technologies? Toxicol. Pathol. 39, 988–1002. https://doi.org/10.1177/ 0192623311491963.

Eltoum, I., Fredenburgh, J., Myers, R.B., Grizzle, W.E., 2001. Introduction to the theory and practice of fixation of tissues. J. Histotechnol. 24, 173–190. https://doi.org/ 10.1179/his.2001.24.3.173.

Fowler, C.B., O'Leary, T.J., Mason, J.T., 2008. Modeling formalin fixation and histological processing with ribonuclease a: effects of ethanol dehydration on reversal of formaldehyde cross-links. Lab. Invest. J. Tech. Methods Pathol. 88, 785–791. https://doi.org/10.1038/labinvest.2008.43.

Fowler, C.B., Evers, D.L., O'Leary, T.J., Mason, J.T., 2011. Antigen retrieval causes protein unfolding: evidence for a linear epitope model of recovered immunoreactivity. J. Histochem. Cytochem. 59, 366–381. https://doi.org/10.1369/ 0022155411400866.

Gaborit, N., Larbourret, C., Vallaghe, J., Peyrusson, F., Bascoul-Mollevi, C., Crapez, E., Azria, D., Chardès, T., Poul, M.-A., Mathis, G., Bazin, H., Pèlegrin, A., 2011. Time-resolved fluorescence resonance energy transfer (TR-FRET) to analyze the disruption of EGFR/HER2 dimers. J. Biol. Chem. 286, 11337–11345. https://doi.org/10.1074/ jbc.M111.223503.

Gaffney, E., Riegman, P., Grizzle, W., Watson, P., 2008. Factors that drive the increasing use of FFPE tissue in basic and translational cancer research. Biotech. Histochem. 93, 373–386. https://doi.org/10.1080/10520295.2018.1446101.

Grizzle, W.E., Fredenburgh, J.L., Myers, R.B., 2008. 4 - fixation of tissues. In: Bancroft, J. D., Gamble, M. (Eds.), Theory and Practice of Histological Techniques, Sixth edition. Churchill Livingstone, Edinburgh, pp. 53–74. https://doi.org/10.1016/B978-0-443- 10279-0.50011-7.

Gur, D., Liu, S., Shukla, A., Pero, S.C., Wicha, M.S., Krag, D.N., 2009. Identification of single chain antibodies to breast cancer stem cells using phage display. Biotechnol. Prog. 25, 1780–1787. https://doi.org/10.1002/btpr.285.

Happel, N., Doenecke, D., 2009. Histone H1 and its isoforms: contribution to chromatin structure and function. Gene 431, 1–12. https://doi.org/10.1016/j. gene.2008.11.003.

Honegger, A., Plückthun, A., 2001. Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool. J. Mol. Biol. 309, 657–670. https://doi.org/10.1006/jmbi.2001.4662.

Jaffer, S., Bleiweiss, I.J., 2004. Beyond hematoxylin and eosin—the role of immunohistochemistry in surgical pathology. Cancer Investig. 22, 445–465. https:// doi.org/10.1081/CNV-200034896.

Jakobsen, C.G., Rasmussen, N., Laenkholm, A.-V., Ditzel, H.J., 2007. Phage display-derived human monoclonal antibodies isolated by binding to the surface of live primary breast cancer cells recognize GRP78. Cancer Res. 67, 9507–9517. https://doi.org/10.1158/0008-5472.CAN-06-4686.

Jarutat, T., Nickels, C., Frisch, C., Stellmacher, F., Hofig, K.P., Knappik, A., Merz, H., 2007. Selection of vimentin-specific antibodies from the HuCAL phage display

library by subtractive panning on formalin-fixed, paraffin-embedded tissue. Biol. Chem. 388, 651–658. https://doi.org/10.1515/BC.2007.070.

Jensen, K.B., Larsen, M., Pedersen, J.S., Christensen, P.A., Alvarez-Vallina, L., Goletz, S., Clark, B.F.C., Kristensen, P., 2002. Functional improvement of antibody fragments using a novel phage coat protein III fusion system. Biochem. Biophys. Res. Commun. 298, 566–573. https://doi.org/10.1016/s0006-291x(02)02484-1.

Kristensen, P., Winter, G., 1998. Proteolytic selection for protein folding using filamentous bacteriophages. Fold. Des. 3, 321–328. https://doi.org/10.1016/S1359- 0278(98)00044-3.

Kunkel, T.A., 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488–492.

Larsen, M., Jensen, K.B., Christensen, P.A., Suarez, E., Paris, D., Sanz, L., Ravn, P., Sauce, D., Saas, P., Goletz, S., Álvarez-Vallina, L., Kristensen, P., 2008. Functionally fused antibodies—a novel adjuvant fusion system. J. Immunol. Methods 339, 220–227. https://doi.org/10.1016/j.jim.2008.09.015.

Larsen, S.A., Meldgaard, T., Lykkemark, S., Mandrup, O.A., Kristensen, P., 2015. Selection of cell-type specific antibodies on tissue-sections using phage display. J. Cell. Mol. Med. 19, 1939–1948. https://doi.org/10.1111/jcmm.12568.

Lipman, N.S., Jackson, L.R., Trudel, L.J., Weis-Garcia, F., 2005. Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J. 46, 258–268. https://doi.org/10.1093/ilar.46.3.258.

Lykkemark, S., Mandrup, O.A., Jensen, M.B., Just, J., Kristensen, P., 2017. A novel excision selection method for isolation of antibodies binding antigens expressed specifically by rare cells in tissue sections. Nucleic Acids Res. 45, e107 https://doi. org/10.1093/nar/gkx207.

Mairaville, C., Martineau, P., 2021. Antibody identification for antigen detection in formalin-fixed paraffin-embedded tissue using phage display and Naïve libraries. Antibodies 10, 4. https://doi.org/10.3390/antib10010004.

Martineau, P., Jones, P., Winter, G., 1998. Expression of an antibody fragment at high levels in the bacterial cytoplasm11Edited by J. Karn. J. Mol. Biol. 280, 117–127. https://doi.org/10.1006/jmbi.1998.1840.

Matsuda, K.M., Chung, Y.-Y., Hewitt, S.M., 2010. Histo-proteomic profiling of formalin- fixed, paraffin-embedded tissue. Expert Rev. Proteomics 7, 227–237. https://doi. org/10.1586/epr.09.106.

O'Hurley, G., Sjöstedt, E., Rahman, A., Li, B., Kampf, C., Pontén, F., Gallagher, W.M., Lindskog, C., 2014. Garbage in, garbage out: a critical evaluation of strategies used for validation of immunohistochemical biomarkers. Mol. Oncol. 8, 783–798. https:// doi.org/10.1016/j.molonc.2014.03.008.

Osunsade, A., Prescott, N.A., Herbert, J.M., Ray, D.M., Jmeian, Y., Lorenz, I.C., David, Y., 2019. A robust method for the purification and characterization of recombinant human histone H1 variants. Biochemistry 58, 171–176. https://doi.org/10.1021/ acs.biochem.8b01060.

Pimenidou, A., Topping, K., Hough, V., Kirkpatrick, N., Monson, J., Greenman, J., 2001. Bacteriophage-derived antibodies in cancer research—diagnosis, imaging, and treatment. Dis. Markers 16, 41–51. https://doi.org/10.1155/2000/979405.

Ramos-Vara, J.A., Miller, M.A., 2014. When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry—the red, brown, and blue technique. Vet. Pathol. 51, 42–87. https://doi.org/10.1177/0300985813505879.

Robin, G., Sato, Y., Desplancq, D., Rochel, N., Weiss, E., Martineau, P., 2014. Restricted diversity of antigen binding residues of antibodies revealed by computational alanine scanning of 227 antibody-antigen complexes. J. Mol. Biol. 426, 3729–3743. https://doi.org/10.1016/j.jmb.2014.08.013.

Rondot, S., Koch, J., Breitling, F., Dübel, S., 2001. A helper phage to improve single- chain antibody presentation in phage display. Nat. Biotechnol. 19, 75–78. https:// doi.org/10.1038/83567.

Ruan, W., Sassoon, A., An, F., Simko, J.P., Liu, B., 2006. Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection * S. Mol. Cell. Proteomics 5, 2364–2373. https:// doi.org/10.1074/mcp.M600246-MCP200.

Scalia, C.R., Boi, G., Bolognesi, M.M., Riva, L., Manzoni, M., DeSmedt, L., Bosisio, F.M., Ronchi, S., Leone, B.E., Cattoretti, G., 2017. Antigen Masking During Fixation and Embedding, Dissected. J. Histochem. Cytochem. Off. J. Histochem. Soc, 65, pp. 5–20. https://doi.org/10.1369/0022155416673995.

Schmidt, U., Weigert, M., Broaddus, C., Myers, G., 2008. Cell Detection with Star-convex Polygons [WWW Document]. arXiv.org. https://doi.org/10.1007/978-3-030-00934- 2.30.

Schofield, D.J., Pope, A.R., Clementel, V., Buckell, J., Chapple, S.D., Clarke, K.F., Conquer, J.S., Crofts, A.M., Crowther, S.R., Dyson, M.R., Flack, G., Griffin, G.J., Hooks, Y., Howat, W.J., Kolb-Kokocinski, A., Kunze, S., Martin, C.D., Maslen, G.L., Mitchell, J.N., O'Sullivan, M., Perera, R.L., Roake, W., Shadbolt, S.P., Vincent, K.J., Warford, A., Wilson, W.E., Xie, J., Young, J.L., McCafferty, J., 2007. Application of phage display to high throughput antibody generation and characterization. Genome Biol. 8, R254. https://doi.org/10.1186/gb-2007-8-11-r254.

Schüchner, S., Behm, C., Mudrak, I., Ogris, E., 2020. The Myc tag monoclonal antibody 9E10 displays highly variable epitope recognition dependent on neighboring sequence context. Sci. Signal. 13 https://doi.org/10.1126/scisignal.aax9730 eaax9730.

Shi, S.R., Key, M.E., Kalra, K.L., 1991. Antigen retrieval in formalin-fixed, paraffin- embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 39, 741–748. https://doi.org/10.1177/39.6.1709656.

Shi, S.-R., Gu, J., Cote, R.J., Taylor, C.R., 2000. Development of the Antigen Retrieval Technique : Philosophical and Theoretical Bases, in: Antigen Retrieval Techniques : Immunohistochemistry and Molecular Morphology. Eaton Publishing, Natick, MA, pp. 17–39.
Sompuram, S.R., Vani, K., Hafer, L.J., Bogen, S.A., 2006. Antibodies immunoreactive with formalin-fixed tissue antigens recognize linear protein epitopes. Am. J. Clin. Pathol. 125, 82-90.

Sørensen, M.D., Agerholm, I.E., Christensen, B., Kølvraa, S., Kristensen, P., 2010. Microselection - affinity selecting antibodies against a single rare cell in a heterogeneous population. J. Cell. Mol. Med. 14, 1953-1961. https://doi.org/10.1111/j.1582-4934.2010.00896.x

Steinwand, M., Droste, P., Frenzel, A., Hust, M., Dübel, S., Schirrmann, T., 2014. The influence of antibody fragment format on phage display based affinity maturation of IgG. mAbs 6, 204-218. https://doi.org/10.4161/mabs.27227.

Su, Y., Bidlingmaier, S., Lee, N.-K., Liu, B., 2018. Combine phage antibody display library selection on patient tissue specimens with laser capture microdissection to identify novel human antibodies targeting clinically relevant tumor antigens. In: Hust, M., Lim, T.S. (Eds.), Phage Display: Methods and Protocols, Methods in Molecular Biology. Springer, New York, NY, pp. 331-347. https://doi.org/10.1007/978-1-4939-7447-4_18

Sun, Y., Shukla, G.S., Kennedy, G.G., Warshaw, D.M., Weaver, D.L., Pero, S.C., Floyd, L., Krag, D.N., 2009a. Biopanning phage-display libraries on small tissue sections captured by laser capture microdissection. J. Biotech Res. 1, 55-63.

Sun, Y., Shukla, G.S., Weaver, D., Pero, S.C., Krag, D.N., 2009b. Phage-display selection on tumor histological specimens with laser capture microdissection. J. Immunol. Methods 347, 46-53. https://doi.org/10.1016/j.jim.2009.06.002.

Ten Haaf, A., Pscherer, S., Fries, K., Barth, S., Gattenlöhner, S., Tur, M.K., 2015. Phage display-based on-slide selection of tumor-specific antibodies on formalin-fixed paraffin-embedded human tissue biopsies. Immunol. Lett. 166, 65-78. https://doi.org/10.1016/j.imlet.2015.05.013.

Thavarajah, R., Mudimbaimannar, V.K., Elizabeth, J., Rao, U.K., Ranganathan, K., 2012. Chemical and physical basics of routine formaldehyde fixation. J. Oral Maxillofac. Pathol. 16, 400. https://doi.org/10.4103/0973-029X.102496.

Thie, H., Toleikis, L., Li, J., von Wasielewski, R., Bastert, G., Schirrmann, T., Esteves, I.T., Behrens, C.K., Fournes, B., Fournier, N., de Romeuf, C., Hust, M., Dübel, S., 2011. Rise and fall of an anti-MUC1 specific antibody. PLoS One 6, e15921. https://doi.org/10.1371/journal.pone.0015921.

van der Vegt, B., de Bock, G.H., Bart, J., Zwartjes, N.G., Wesseling, J., 2009. Validation of the 4B5 rabbit monoclonal antibody in determining Her2/neu status in breast cancer. Mod. Pathol. Off. J. U. S. Can. Acad. Pathol. Inc 22, 879-886. https://doi.org/10.1038/modpathol.2009.37.

Wang, S.-C., Lien, H.-C., Xia, W., Chen, I.-F., Lo, H.-W., Wang, Z., Ali-Seyed, M., Lee, D.-F., Bartholomeusz, G., Ou-Yang, F., Giri, D.K., Hung, M.-C., 2004. Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2. Cancer Cell 6, 251-261. https://doi.org/10.1016/j.ccr.2004.07.012.

Yamashita, S., 2007. Heat-induced antigen retrieval: mechanisms and application to histochemistry. Prog. Histochem. Cytochem. 41, 141-200. https://doi.org/10.1016/j.proghi.2006.09.001.