# A multispecific checkpoint inhibitor Nanofitin with a fast tumor accumulation property and anti-tumor activity in immune competent mice

Perrine Jacquot, Javier Muñoz-Garcia, Antoine Léger, Antoine Babuty,

Manon Taupin, Laurie Fradet, Fabio Dupont, Marie-Françoise Heymann,

Mathieu Cinier, Dominique Heymann

► To cite this version:

Perrine Jacquot, Javier Muñoz-Garcia, Antoine Léger, Antoine Babuty, Manon Taupin, et al. A multispecific checkpoint inhibitor Nanofitin with a fast tumor accumulation property and anti-tumor activity in immune competent mice. Biomolecules, 2025, 15 (4), pp.471. 10.3390/biom15040471. inserm-05005361

HAL Id: inserm-05005361

https://inserm.hal.science/inserm-05005361v1

Submitted on 25 Mar 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished 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_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# A multispecific checkpoint inhibitor Nanofitin with a fast tumor accumulation property and anti-tumor activity in immune competent mice

Perrine Jacquot<sup>1,2,3*</sup>, Javier Muñoz-Garcia<sup>2,3</sup>, Antoine Léger<sup>1</sup>, Antoine Babuty<sup>2,3,4</sup>, Manon Taupin<sup>5</sup>, Laurie Fradet<sup>2,3</sup>, Fabio Dupont<sup>1</sup>, Marie-Françoise Heymann<sup>2,3,5</sup>, Mathieu Cinier<sup>1</sup>, Dominique Heymann<sup>2,3,6</sup>

**Abstract:** Immune checkpoint inhibitors have revolutionized cancer treatment, but remain limited by on-target/off-tumor effects that narrow their therapeutic window. Although PD-L1 is mainly expressed by tumor cells, these effects could reduce bloodstream availability and tumor accumulation of PD-L1 inhibitors. Enhancing tumor specificity through bispecific proteins targeting two tumor-associated antigens offers a promising strategy. This study evaluated a bispecific Nanofitin, B10-B11, targeting PD-L1 and EGFR. *In vitro*, B10-B11 efficiently bound to human A431 and murine CT26 cell lines, validating these models for *in vivo* studies. Nanofitins accumulation in tumors and their anti-tumor efficacy were assessed respectively in an A431 xenograft and a CT26 immunocompetent mouse models. In both experiments, B10-B11 was compared with its albumin binding fused counterpart (B10-B11-ABNF). The study showed that the dual-targeting approach with the bispecific Nanofitin enhanced *in vitro* PD-L1 neutralization compared to the monomeric form and led to *in vivo* anti-tumor activity evidenced by reduced tumor growth and increased CD3+ T cells and F4/80+ macrophages in tumors. Nanofitin efficacy correlated with its tumor accumulation at 7 hours post-injection, which was higher for the B10-B11-ABNF. This study highlights the potential of bispecific Nanofitins, particularly with albumin binding to enable rapid and uniform tumor accumulation of effective PD-L1 immunotherapy.

**Keywords:** bispecific Nanofitin; programmed cell death ligand 1 (PD-L1); epidermal growth factor receptor (EGFR); on-target/off-tumor effect; tumoral accumulation.

## 1. Introduction

Clinical successes and Food and Drug Administration (FDA) approvals of Immune Checkpoint Inhibitors (ICIs) Ipilimumab (Yervoy®, 2010), Pembrolizumab (Keytruda®, 2016), and Atezolizumab (Tecentriq®, 2016) respectively targeting Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4), Programmed cell Death protein 1 (PD-1), and Programmed Death-Ligand 1 (PD-L1), have highlighted the relevance of addressing tumor evasion mechanisms in the management of cancer. While these therapies have fundamentally changed the paradigm of cancer treatment, their therapeutic window is restricted by on-target/off-tumor effects. These limitations hinder the ability to tailor treatment protocols to overcome primary or acquired resistances responsible for variable efficacies across patients, tumor entities and over time [1-3]. The mechanism of action of ICIs involves reactivating the immune system, that has become tolerant to tumors, which can result in Immune-Related Adverse

<footnote n="1">¹ Affilogic SAS, 24 rue de la Rainière, 44300 Nantes, France</footnote>

<footnote n="2">² Nantes Université, CNRS, UMR6286, US2B, Nantes, France</footnote>

<footnote n="3">³ Institut de Cancérologie de l'Ouest, Tumor Heterogeneity and Precision Medicine Lab, Saint-Herblain, France</footnote>

<footnote n="4">⁴ Nantes University Hospital, Nantes, France</footnote>

<footnote n="5">⁵ Institut de Cancérologie de l'Ouest, Research Pathology Platform, Saint-Herblain, France</footnote>

<footnote n="6">⁶ The University of Sheffield, School of Medicine and Population Health, Sheffield, UK</footnote>

<footnote>* Correspondence: ferrine@affilogic.com</footnote>
Events (irAEs) due to uncontrolled off-tumor immune activation [4]. Anti-CTLA-4 therapy is associated with the lowest overall response rate and the highest incidence of overall and organ-specific irAEs of any grade, including grade 3 or above. This is explained by the early and systemic T-cell activation resulting from CTLA-4 neutralization whereas the PD-1/PD-L1 pathway modulates a more restricted T-cell activation within the tumor microenvironment [5].

Nonetheless, PD-1 and PD-L1 blockers are not exempt from on-target/off-tumor effects, albeit with distinct repercussions. PD-1 neutralizing antibodies provide a general inhibition of the receptor by blocking its interaction with both Programmed Death-Ligand 1 (PD-L1) and Programmed Cell Death Ligand 2 (PD-L2), hence exposing to risks of pneumonitis [6]. Although PD-L1 neutralizing antibodies are more selective for the PD-1/PD-L1 axis, their systemic exposure is limited by the expression of the target protein on various non-tumoral cell types [7]. This widespread expression creates a target sink, reducing the clinical effectiveness of these antibodies. While systemic exposure can be improved by increasing the dose and saturating the non-tumoral tissues, this does not necessarily optimize the therapeutic window from a safety perspective [8]. Reducing the on-target/off-tumor effect of PD-L1 inhibitors by improving their selectivity for the tumor microenvironment could enhance their accumulation in tumors while managing safety, thus optimizing the therapeutic window.

Such selectivity improvements can be achieved by conditioning cell engagement to the
recruitment of two receptors overexpressed in the context of malignant cells. Consequently, bispecific
agents that conditionally inhibit PD-L1 only when both PD-L1 and another receptor are overexpressed
on tumor cells can mitigate on-target/off-tumor effects. This approach has been described with a
bispecific antibody targeting PD-L1 and Epithelial Growth Factor Receptor (EGFR) [9] and more
recently by our group with a bispecific construction of the small Nanofitin alternative scaffold
targeting the same two receptors [10]. In these cases, the selectivity of PD-L1 immune checkpoint
blockade activity for tumor cells relies on monovalent and low-affinity interactions for each receptor,
which, in the context of the co-engagement of both receptors, leads to a high-affinity interaction and
subsequent immune checkpoint blockade via the so-called avidity effect.

The tumor microenvironment is characterized by elevated interstitial pressure. In this context, the distribution of macromolecules is limited by their diffusion coefficient, which decreases as their size increases [11]. Considering the high molecular weight of antibodies, they exhibit a slow and heterogeneous diffusion in tissues, which is compensated by their long plasma residence time [12]. Conversely, small alternative scaffolds like Nanofitins (7 kDa) display a high diffusion coefficient, enabling fast and efficient accumulation within the tumor tissue, despite a rapid clearance from the bloodstream. This was particularly highlighted in the context of a monomeric anti-EGFR Nanofitin showing a strong and homogeneous distribution within the tumor, with a labeling of nearly all the cancer cells as fast as 90 min post-injection. Its conjugation with a cytotoxic payload provided a potent drug conjugate enabling complete inhibition of the tumor growth in a xenograft mouse tumor model [13].

In this study, we further characterized the anti-PD-L1 x EGFR bispecific Nanofitin, previously
described to exhibit *in vitro* higher tumor cells selectivity and superior anti-tumor cells activity as
compared to the monomeric anti-PD-L1 Nanofitin alone [10]. We evaluated its *in vivo* tumor
accumulation capacity in the same tumor xenograft model as previously described for the monomeric
anti-EGFR Nanofitin [13]. We also took advantage of the cross-reactivity of the anti-PD-L1 and anti-
EGFR Nanofitin toward the murine targets to demonstrate the *in vivo* anti-tumor activity of the
bispecific molecule in a syngeneic immunocompetent model.

**2. Materials and Methods**

2.1. *Plasmid Construct, Expression and Purification of Proteins*
Monomeric B10, B11 Nanofitins and Albumin Binding Nanofitin (ABNF) respectively specific of EGFR, PD-L1 and Human Serum Albumin (HSA) were used to develop multispecific Nanofitins. DNA constructs were obtained by gene synthesis (Genscript) with codon optimization for E. coli expression and cloned in the pET21a vector between the NdeI and HindIII restriction sites. All final constructs were fused at the N-terminal and C-terminal with the respective hexahistidine and hemagglutinin tags. The BL21 Glod (DE3) strains were transformed by heat shock and incubated overnight at 37 °C in 10 mL of 2YT, 1% glucose, 5 ng/mL Tetracycline and 100 ng/mL Ampicillin (growing medium) under gentle agitation (200 RPM, Innova® S44i, Eppendorf). Then, the overnight pre-culture was diluted in 200 mL of growing medium and incubated at 37 °C under gentle agitation (200 RPM, Innova® S44i, Eppendorf) with the regular measurement of the OD at 600 nm. When the exponential phase was reached, the protein expression was induced by adding IPTG at the final concentration of 0.5 mM (Merck, #16758-10G) for 4 h at 30 °C. After their extraction using BugBuster® 1X (Merck, #70921) for 15 min, the proteins were purified by IMAC with the NGC Quest 10 Plus System (HisTrapHP, Dutscher, #17-5248-02) and following by size exclusion chromatography using a HiLoad 26/600 Superdex 75 pg. All Nanofitins were formulated in PBS1X 0.1M L-Arginine, 40 mg/mL Trehalose and 0.01% Tween and filtered (Acrodisc® membrane mustang® E, #514-4235) to remove endotoxins below 0.5 EU/mg.

## 2.2. Biolayer Interferometry Analyses

Nanofitin affinity and targeting capacity were determined by biolayer interferometry (BLI) on an Octet Red instrument (Fortebio). All biolayer interferometry analyses were performed in 96 multi-well plates (Dutsher) at 30 °C, 1000 RPM. The binding kinetic parameters of Nanofitins were defined by loading of 10 µg/mL mouse EGFR Fc Chimera Protein (R&D, #1280-ER-050) and 5 µg/mL recombinant mouse PDL1/B7-H1 Fc chimera protein (R&D, #1019-B7-100) at 2 nm on protein A biosensors (Sartorius). All measure steps were performed in TBS1X containing 0.002% Tween 20 and 0.01% BSA. Between each measurement, biosensors were regenerated using three cycles of alternating washes for 10 s in Glycine 10 mM, pH 2 and TBS1X. The biosensor unexposed to Nanofitin was used as a background reference. Sensorgrams were obtained after a reference subtraction a background correction, a smoothing with the Savitzky-Golay algorithm and a fitting with a 1:1 model using the Octet Data Analysis software 7.1.

## 2.3. Cell Culture

All cell lines were obtained from American Type Culture Collection (ATCC) and were cultured in a 37 °C incubator with 5% CO2 saturated in humidity. The human A431 cell line (#CRL-1555, epidermoid carcinoma) was cultured in DMEM with 5% FBS and 1% L-glutamine and the murine CT26 cell line (#CRL-2638, colorectal carcinoma) was cultured in RPMI 1640 with 5% FBS and 1% L-glutamine. All cell lines were tested negative for mycoplasma before use.

## 2.4. Cell Surface Binding by Flow Cytometry

Proliferating cells were washed with PBS and detached by 0.2% EDTA. After 5 min centrifugation at 450 x g, the cell pellets were washed twice with cold PBS and then resuspended at 2 x 10⁶ cells/mL. Cells were distributed in 96 multi-well plates and incubated for 15 min in PBS1X + 1% BSA. The expression levels of EGFR and PD-L1 on A431 cells were studied by respectively incubating cells with 100 µL of PE anti-human EGFR antibody (Biolegend, clone AY13, 1/20) and recombinant Alexa Fluor 488 anti-PD-L1 antibody (Abcam, clone 28-8, 1/50). To evaluate the EGFR and PD-L1 level expression on A431 cells, the respective control antibodies were used: PE mouse IgG1 kappa isotype control antibody (Biolegend, clone MOPC-21, 1/20) and recombinant Alexa Fluor 488 Rabbit IgG (Abcam, clone EPR25A, 1/500). The expression level of EGFR on CT26 cells was studied by incubating permeabilized cells with 100 µL of EGFR polyclonal antibody (Thermo Fisher Scientific, #BS-0165R,
1/100) or 100 µL of the Rabbit IgG isotype control (Thermo Fisher Scientific, #02-6102, 1/100) followed by adding the secondary antibody anti-rabbit IgG (H+L), F(ab')2 fragment Alexa Fluor® 488 conjugate (Cell Signaling Technology, #4412, 1/100). The expression level of PD-L1 on CT26 cells was studied by incubating cells with 100 µL of Purified anti-mouse CD274 (B7-H1, PD-L1) antibody (Biolegend, clone 10F.9G2, 1/50) or 100 µL of the Purified Rat IgG2b, κ isotype control antibody (Biolegend, clone RTK4530, 1/50) followed by adding the secondary antibody Alexa Fluor® 488 anti-rat IgG2b Antibody (Biolegend, clone MRG2B-85, 1/200). The cell binding capacity of Nanofitins was analyzed by incubating A431 cells or CT26 previously serum starved cells with 100 µL of Nanofitin at 10 µM followed by the addition of DyLight650 anti-HA tag antibody (Abcam, clone 16B12, 1/200). In order to evaluate the Nanofitins binding capacity to cells, the control condition was performed by adding the DyLight650 anti-HA tag antibody on cells without previous incubation of Nanofitin. The FL-1 (Alexa Fluor 488), FL-2 (PE) and FL-4 (DyLight 650) fluorescence on cells were analyzed using flow cytometry (BD AccuriTM C6 Plus System). For all analyses, 10,000 events were recorded.

## 2.5. Agilent X-Celligence Real-Time Cell Analysis

1 x 10⁴ A431 cells were seeded in 96 multi-well electronic plates (Agilent Technologies). After 24 h, treatment of A431 cells was initiated by removing the media and then adding fresh media, with or without Nanofitins. After 1 h, another fresh media containing or not a mix of Jurkat cells with an anti-Epithelial Cell Adhesion Molecule (EpCAM) and anti-Cluster of Differentiation 3 (CD3) Bi-specific T-cell Engager (BiTE) antibody (produced by Sinobiological) was added. In this experiment, the A431 cells were co-cultured with Jurkat cells at a final Effector/Target (E:T) ratio of 10:1. Nanofitin treatments were performed at a final concentration of 10 µM. The anti-EpCAM x CD3 BiTE was used at a final suboptimal concentration (75 ng/mL) in order to develop the background activity of Jurkat cells as previously described by Koopmans et al. [9]. The real-time kinetic proliferation of tumor cells was analyzed for 100 h with the Real-Time Cell Analysis (RTCA) system (Agilent).

## 2.6. Animal Experiments

All protocols applied in the present study were first validated by the French ethical committee of the "Pays de la Loire" (CEEA-PdL-06) and authorized by the French ministry of agriculture and fisheries (authorization APAFIS # 2168-2019080709313558 v6).

Evaluation of intra-tumoral diffusion of Nanofitins: 8-week-old female BALB/c nude mice (Janvier Lab, France) were anesthetized by inhalation of an isoflurane/air mixture (2%, 1 L/min) before subcutaneous inoculation of 1 x 10⁶ A431 cells in PBS into the left flank. Tumors volumes were measured every two days with a caliper according to the following formula: V= 0.5 x L x (l)², in which L and l are, respectively, the largest and the smallest perpendicular diameters. At day 10 after tumor injection, homogeneous tumor sizes were obtained and mice were randomly split into three groups of three. A single dose of B10 (66 µg), B10-B11 (131 µg) and B10-B11-ABNF (197 µg) Nanofitins were injected intravenously to achieve the same molar dose. Injected Nanofitins quantities were determined to distribute an equivalent quantity of anti-PD-L1 Nanofitin around 60 µg as previously described [14]. Mice were sacrificed 90 min or 7h post-injection to harvest the tumor tissues. After tumor resection and standard formalin-fixed and paraffin-embedded tissue processing, serial tumor sections were prepared for histopathological assessment.

Evaluation of Nanofitins effect on the tumor growth: At day 0, 8-week-old female BALB/cJRj mice (Janvier Lab, France) were inoculated with 0.5 x 10⁶ CT26 cells in PBS by a subcutaneous injection into the left flank. Tumor volumes were monitored as described above. At day 4, mice were randomly split into three groups of six and Nanofitins treatments were started. The control group received intraperitoneal injection of vehicle five times a week. The B10-B11 and B10-B11-ABNF Nanofitin groups received intraperitoneal injection of 20 mg/kg Nanofitins five and three times a week respectively for three weeks. The mice were sacrificed by cervical dislocation for ethical reasons when
the first critical point was detectable (e.g. high tumor size, pain, tissue necrosis). The study was stopped after the sacrifice of all mice of control group.

## 2.7. Immunohistochemistry analyses

All immunohistochemistry (IHC) experiments were performed using BOND Polymer Refine Detection system (Leica Biosystems). 3 µm-thick sections were deparaffinized and rehydrated. Heat-induced epitope retrieval was performed using BOND Epitope Retrieval Solution 2 at pH 9 for 20 min at 95 °C (Leica Biosystems). The revelation was done by adding a solution of an anti-rabbit polymer coupled to HRP for 8 min and then by the addition a DAB solution. The washes were carried out in cycles of 3 in Wash Solution (Leica Biosystem). Nuclei were counterstained by adding hematoxylin for 5 min. Images were acquired using a Nanozoomer digital slide scanner (Hammamatsu, Japan).

Evaluation of intra-tumoral diffusion of Nanofitins: Nanofitins were HA-tagged for tumoral detection by IHC. Following the blockade of endogenous peroxidase, tissue sections were incubated with the HA-tag rabbit mAb (Cell signaling, clone C29F4, 1/300) for 1 h.

Evaluation of Nanofitins effect on the immune cell recruitment: Following the blockade of endogenous peroxidase, tissue sections were incubated with the anti-CD3 epsilon antibody (Abcam, ab16669, 1/150) or the F4.80 (D2S9R) XP® Rabbit mAb (Cell signaling, 70076, 1/100) for 15 min. CD3 and F4.80 staining percentages were estimated using ImageJ.

# 3. Results

## 3.1. *The bispecific B10-B11 Nanofitin binds to A431 cells and induces a cytotoxic effect*

In the present study, we compared the cell labelling efficiency of the bispecific B10-B11 Nanofitin with its two monomeric counterparts on A431 cells using flow cytometry. Flow cytometry analyses revealed a stronger capacity of B10 to label A431 cells than B11 (Figure 1A, Table S1). This observation was correlated with the level of EGFR and PD-L1 expressions on these cells (Figure 1B). Interestingly, the bispecific B10-B11 Nanofitin demonstrated a lower cell labelling intensity than B10 but a slightly higher labelling intensity than B11 (Figure 1A, Table S1). The cell cytotoxic assay demonstrated that both B11 and bispecific B10-B11 Nanofitins induced cytotoxic effects on A431 cells cultured in the presence of Jurkat cells, with a more pronounced impact for bispecific B10-B11 (Figure 1C). In contrast, as we previously observed, the B10 monomeric Nanofitin was ineffective for inhibiting EGFR signaling in A431 cell line [10]. Of note, bispecific B10-B11 Nanofitin also showed a slight cytotoxicity in absence of Jurkat cells through a mechanism that remains to be identified (Figure 1D).
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Cell labelling efficiency of the bispecific B10-B11 Nanofitin on the A431 and its cytotoxic effect on tumor cell line. (A) Cell labelling efficiency evaluation by flow cytometry of B10, B11 and bispecific B10-B11 Nanofitins on A431 cell line. Dotted lines: alignment to isotype control. (B) Expression level of Programmed Death-Ligand 1 (PD-L1) (left) and Epithelial Growth Factor Receptor (EGFR) (right) on A431 cell line. In grey: isotype control; In black: anti-PD-L1 or anti-EGFR antibody. (C) The real-time proliferation of A431 cells co-cultured with a mix of Jurkat cells and anti-EpCAM x CD3 BiTE. In black: A431 cells co-cultured with a mix of Jurkat cells and anti-EpCAM x CD3 BiTE without Nanofitin treatment; In red: A431 cells co-cultured with a mix of Jurkat cells and anti-EpCAM x CD3 BiTE and treated by the B11 Nanofitin (10µM); In purple: A431 cells co-cultured with a mix of Jurkat cells and anti-EpCAM x CD3 BiTE and treated by the B10-B11 Nanofitin (10µM). (D) The real-time proliferation of A431 cells. In black: A431 cells alone; In red: A431 cells treated by the B11 Nanofitin (10 µM); In purple: A431 cells treated by the B10-B11 Nanofitin (10 µM).</figcaption></figure>

## 3.2. Tumor accumulation of the bispecific B10-B11 Nanofitin in an A431 xenograft model

To rely on a previous report of tumor accumulation potential of the anti-EGFR Nanofitin B10 [13], tumor accumulation of the bispecific B10-B11 Nanofitin was monitored similarly in this study using the A431 tumor xenograft model, and compared either to the monomeric B10 Nanofitin at 90 min post-injection, or to the bispecific Nanofitin fused to an Albumin Binding Nanofitin (ABNF) at 7 h post-injection. Localization of the different Nanofitin constructs within the tumor was revealed by immunohistochemistry on tumor tissues (Figure 2, Figure S1).

The B10 Nanofitin showed both a fast tumor accumulation and diffusion, as revealed by the positive immunostaining of A431 cells membrane on nearly all the tumor tissue at 90 min post-injection. The bispecific B10-B11 Nanofitin showed a significantly lower tumor accumulation and diffusion at the same time point, as shown by a partial staining of the tumor. At 7 h post-injection, the staining of the tumor by the bispecific B10-B11 Nanofitin remained only barely visible, suggesting that what had accumulated at 90 min post-injection had been cleared by 7 h post-injection. A counterpart of this bispecific B10-B11 Nanofitin obtained by its fusion to an ABNF [15] (B10-B11-ABNF) displayed a strong accumulation and complete diffusion within the tumor tissue at 7h post-injection.
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Intratumoral infiltration of Nanofitins after systemic administration on a A431 cell xenograft model. Intratumoral infiltration of B10 and bispecific B10-B11 at 90 min and of bispecific B10-B11 and the bispecific Nanofitin fused to an Albumin Binding Nanofitin (ABNF) at 7 h post-intravenous injection. Nanofitins staining are represented on the whole tumor (bar scale: 2.5 mm) and on a zoom of selected regions (bar scale: 100 µm). n=3 mice per group. All tumors were analyzed and showed similar results. Representative images are presented.</figcaption></figure>

### 3.3. *The bispecific B10-B11 Nanofitin is cross-reactive with mouse EGFR and PD-L1 and binds to CT26 cells*

The isograft model based on inoculation of murine CT26 cells in immunocompetent mice was used to monitor an immunologic-based anti-tumor activity of the Nanofitins. Expression of EGFR and PD-L1 on the murine CT26 cell line was demonstrated by flow cytometry (Figure 3A). Affinity of the bispecific B10-B11 Nanofitin for murine EGFR and PD-L1 was confirmed and measured by interferometry respectively at 1740 nM and 387.3 nM (Figure 3B). The respective affinity of the bispecific B10-B11 Nanofitin for each of the target receptors enable its engagement on the CT26 cells at higher level compared to its monomeric counterparts (Figure 3C, Table S1).

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Mouse cross reactivity validation of the bispecific B10-B11 Nanofitin. (A) Expression level of PD-L1 (left) and EGFR (right) on CT26 cell line. In grey: isotype control; In black: anti-PD-L1 or EGFR antibody. (B) Biolayer interferometry sensorgrams and binding kinetic parameters of bispecific B10-B11 Nanofitin on mouse EGFR and PD-L1. Fittings (1:1 model) are represented as solid gray lines. (C) CT26 cell labelling efficiency evaluation by flow cytometry of monomeric B10 and B11 and bispecific B10-B11 Nanofitins. Dotted lines: alignment to isotype control.</figcaption></figure>

### 3.4. *The bispecific B10-B11 Nanofitin reduces tumor proliferation in an immunocompetent murine model*

Immunocompetent mice bearing CT26 tumors received intraperitoneal injections starting 4 days after tumor cell inoculation when tumors were palpable for each animal. Treatments involved either daily administration of the bispecific B10-B11 Nanofitin or administration every two days of the B10-
B11-ABNF Nanofitin (Figure 4A). A control group was performed with a daily injection of the vehicle only (PBS). Mouse weight was followed throughout the experiment. Except for one mouse of the control group, along the study, no significant weight loss was observed for all the mice in the three treatment groups, suggesting that in all cases treatments were well tolerated (Figure S2). Four mice treated with the bispecific B10-B11 Nanofitin demonstrated a partial response to the treatment. For this group, a slight reduction of the tumor growth (35%) as compared to the control group was observed though that did not reach statistical significance. However, despite a less frequent administration, the treatment using the B10-B11-ABNF Nanofitin produced a much more pronounced effect. All treated mice initially showed a reduction of tumor growth, and complete inhibition of tumor growth was observed in four of them. For this group, the final tumor volume has been reduced by around 92% compared to the control group (Figure 4B).

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Therapeutic efficacy evaluation of the bispecific Nanofitin on an immunocompetent CT26 cell isograft model. (A) Study details with timeline and injection frequencies. CT26 cells (0,5 x 10⁶) were injected (s.c.) on day 0. Vehicle and B10-B11 were injected five times a week (I.P. and 20 mg/kg). B10-B11-ABNF was injected three times a week (I.P. and 20 mg/kg). Mice were sacrificed on day 20. (B) Tumor progression evaluation for the 3 groups of treatment. Significance was assessed by two-way ANOVA using 6 Prism software (GraphPad). **** P < 0.0001. Each group consisted of 6 mice (n=6).</figcaption></figure>

### 3.5. The bispecific B10-B11 Nanofitin promotes immune cell recruiting

The immune infiltration of CT26 tumors were analyzed by immunohistochemistry. At end point tumor tissues were infiltrated by T cells and macrophages in treated or non-treated groups (Figure 5, Figure S3). The semi quantitative evaluation of these infiltrates showed a significant increase in the mean percentages of CD3+ T cells (Figure 5A,C) for the B10-B11-ABNF group and of F4.80+ macrophages (Figure 5B,C) for both treated groups compared to non-treated group. This observation demonstrated the ability of the bispecific B10-B11 Nanofitin to recruit immune cells inside the tumor masses. Similarly, significant increases in the mean staining percentages of CD3+ T cells and F4.80+ macrophages, with respective factors 40 and 4, revealed the superiority of the B10-B11-ABNF Nanofitin to recruit immune cells.
<figure><img src="image_6.png" /></figure>

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

<figure><img src="image_8.png" /><figcaption>**Figure 5.** Immune cell recruitment into CT26 tumors after mice treatments. Mean staining percentages of (A) CD3 positive cells and (B) F4.80 positive cells in CT26 tumors of each treated group (control, B10-B11 and B10-B11-ABNF). Significance was assessed by one-way ANOVA using 6 Prism software (GraphPad). **** P < 0.0001. Each group consisted of 90 Region of Interest (ROI) measured from 3 samples per group. (C) CD3 and F4.80 staining of CT26 tumors for one mouse in each treated group (control, B10-B11 and B10-B11-ABNF).</figcaption></figure>

**4. Discussion**

Over the past few years, cancer immunotherapies have made significant progress. However, challenges persist, particularly concerning the enhancement of their therapeutic window. The high molecular weight of anti-PD-L1 antibodies, combined with their on-target/off-tumor uptake impairs their accumulation in tumors, potentially compromising their effectiveness. Compounds with lower molecular weights and more tumor-specific PD-L1 targeting could address these limitations, enhancing tumor accumulation and potentially improving the therapeutic window of PD-L1 immune checkpoint inhibitors.

4.1. *Bispecific B10-B11 Nanofitin simultaneously engages EGFR and PD-L1 on human A431 and murine CT26 cell lines*

Previously, we reported the development of a bispecific Nanofitin, engineered by fusing the anti-EGFR B10 [16] and anti-PD-L1 B11 [17] Nanofitins. This bispecific B10-B11 Nanofitin demonstrated its ability to inhibit PD-L1 in an EGFR dependent manner. Our prior study revealed that its affinities for human EGFR (892 nM) and PD-L1 (229 nM) prevented its monovalent binding to non-target cells that only expresses PD-L1, even at a high level. Conversely, compared to its monovalent counterparts, the bispecific B10-B11 Nanofitin exhibited enhanced targeting on cells with high levels of both EGFR and PD-L1. This dual targeting enabled a selective anti-tumor effect in vitro on MNNG-HOS tumor cells through the PD-L1 immune checkpoint neutralization activity of B11, whereas B10 was previously demonstrated unable to inhibit EGFR signaling [10]. In this study, we evaluated the ability of the bispecific B10-B11 Nanofitin to simultaneously bind EGFR and PD-L1 on A431 and CT26 cell lines, thereby validating these models for subsequent in vivo experiments.

First, we documented the co-expression of both EGFR and PD-L1 on A431 cells. Flow cytometry confirmed the binding of the bispecific B10-B11 Nanofitin to A431 cells. Interestingly, the cell labeling
efficiency of B10-B11 was not superior to the monomeric B10 Nanofitin but matched that of the monomeric B11 Nanofitin. This finding suggests that, unlike the Nanofitin B10, which can target cells through monovalent binding to EGFR, the affinities of the bispecific Nanofitin for both human EGFR and PD-L1 prevent its monovalent binding. Consequently, its binding to these cells is limited to the co-engagement of both receptors, thus aligning its labeling efficiency with that of the less expressed receptor, i.e., PD-L1. The superior *in vitro* ICI activity of the bispecific B10-B11 observed in a T cell killing assay, compared to the monomeric anti-PD-L1 B11 Nanofitin, likely resulted from the avidity effect due to EGFR co-engagement (Figure 1). As described in a previous study [10], optimizing the spacing between the two Nanofitins for a better fit with these unbalanced densities of EGFR and PD-L1 would enhance simultaneous targeting on this A431 cell line. A tumor xenograft model based on A431 cells was later used to evaluate the accumulation in tumors of different Nanofitin constructs.

Second, the cross-reactivity of the B10 Nanofitin on murine EGFR and the B11 Nanofitin on murine PD-L1 were confirmed with their respective affinities of 90 nM and 53.81 nM (Figure S1) and their ability to targets CT26 cells (Figure 3, Table S1). CT26 cells have been previously characterized by low PD-L1 expression on the cell surface (62 ± 5 receptors/cell) [18], and yet to be sensitive to therapies based on immune checkpoint inhibition. The weak ability of the monomeric B11 Nanofitin to target CT26 cells highlights the need of enhancing its PD-L1 neutralizing activity through dual targeting of EGFR and PD-L1. The affinities of the bispecific Nanofitin B10-B11 for murine EGFR (1740 nM) and murine PD-L1 (387.3 nM) tend to suggest that, as previously observed in cell lines expressing human antigens [10], these affinities would prevent monovalent targeting in favor of cross-arm binding. The bispecific B10-B11 Nanofitin's superior targeting ability for CT26 cells compared to its monovalent counterparts suggest its simultaneous binding to both EGFR and PD-L1 on this murine cell line as well (Figure 3, Table S1). *In vivo* implantation of CT26 cells enabled the creation of an immunocompetent isograft tumor model, which was used to evaluate the therapeutic efficacy of the bispecific B10-B11 Nanofitin.

## 4.2. Nanofitin accumulation in tumor is driven by its size

Previously, the monomeric anti-EGFR Nanofitin B10 has demonstrated high accumulation and broad diffusion in tumors in an immunodeficient murine A431 tumor xenograft model, effectively targeting nearly all tumor cells as early as 90 min post-injection [13]. Here, we used the same model to evaluate the *in vivo* tumor accumulation of the bispecific B10-B11 Nanofitin by IHC in a side-by-side study with the monomeric B10 as a benchmark for fast and efficient tumor accumulation. At 90 min post-injection, the B10 monomeric Nanofitin (~7,600 Da) was found throughout all regions of the tumor, while the bispecific B10-B11 Nanofitin (~16,140 Da) displayed only partial diffusion within the tumor mass (Figure 2). This result is consistent with other studies comparing tumor accumulation of monomeric versus dimeric constructs of other alternative scaffold technologies, where the dimeric format consistently exhibited lower tumor accumulation. Zahnd et al. described lower tumor accumulation of the radiolabeled anti-Human Epidermal Growth Factor Receptor-2 (HER2) DARPin G3 in its dimeric format compared to its monomeric format in an HER2-positive SKOV3 tumor xenograft mouse model [19]. Additionally, Debie et al. compared the efficiency of tumor infiltration of the fluorescently labeled monomeric anti-HER2 Nanobody 2Rb17c to a dimeric construct made by fusion with an irrelevant Nanobody or with itself. In their study, the monomeric Nanobody displayed faster and more homogeneous infiltration than its dimeric counterparts, with better homogeneity observed in the monospecific dimeric construct compared to the bivalent anti-HER2 Nanobody [20]. These observations, including ours, can be explained by a correlation between compound size and its permeability to the vasculature and diffusivity within the tumor, with a greater contribution from smaller sizes. Interestingly, we observed a weak tumor accumulation in the case of the bispecific B10-B11 Nanofitin (~16,140 Da) at 90 min post-injection, while compound of similar molecular mass like DARPin (~16,900 Da) are displaying efficient tumor infiltration capacity at the same time point. This
can be explained by the fact that the bispecific B10-B11 Nanofitin is not expected to be a globular protein and, therefore does not follow the correlation observed between molecular weight and size for globular proteins. The hydrodynamic radius of the bispecific Nanofitins (~2.39 nm) is greater than that of a monomeric DARPin (~1.944 nm), and approximating that of a Single-Chain Variable Fragment (ScFv) (~2.346 nm) (Table 1). The comparison with the ScFv here is intentional. *In silico* analysis has shown that the accumulation potential of a protein ligand as a function of its molecular size exhibits a U-shaped profile. This means there is a "Death Valley" where molecules are still too small to avoid first-pass systemic clearance and too large for efficient permeability and diffusivity, which happens to be around the size of an ScFv [22].

<table><caption>**Table 1.** Molecular Weight (kDa) versus Size of scaffold and antibody-derived proteins. Molecular Weight was estimated using Expasy ProtParam. Stokes Radius in Ångströms was estimated using HullRad and converted to Nanometer applying the formula $1Å=0.1nm$. * Dimeric Nanofitin was generated by combining two structures of Sac7d (1AZP) with a 15-mer peptide linker, modeled using Modeller software [21].</caption><thead><tr><td>Protein</td><td>PBD</td><td>Molecular Weight (kDa)</td><td>Stokes Radius (Å)</td><td>Rh (nm)</td></tr></thead><tbody><tr><td>Nanofitin</td><td>1AZP</td><td>7.60</td><td>15.81</td><td>1.581</td></tr><tr><td>Dimeric Nanofitin</td><td>*</td><td>16.14</td><td>23.90</td><td>2.390</td></tr><tr><td>DARPin</td><td>5KNH</td><td>18.21</td><td>19.44</td><td>1.944</td></tr><tr><td>scFv</td><td>8DGR</td><td>29.59</td><td>23.46</td><td>2.346</td></tr></tbody></table>

### 4.3. Bispecific B10-B11 Nanofitin demonstrates a potent anti-PD-L1 activity in an immunocompetent mice model

Efficiency of the bispecific Nanofitin was evaluated in presence and absence of a third Nanofitin module targeting the Human Serum Albumin (HSA) and named ABNF. Due to its plasma half-life of 10 to 15 min in mice driven by rapid clearance correlated with its molecular weight being below the kidney's glomerular filtration cutoff, the bispecific B10-B11 Nanofitin was administered daily. The ABNF was shown to bind to serum albumin at its domain II, thereby hijacking the natural recycling mechanism of albumin to extend the plasma half-life of itself and its cargo molecule up to 20 h in mice [15]. Consequently, the B10-B11-ABNF Nanofitin was administered every other day, accounting for its longer residence in the bloodstream by the ABNF contribution.

While both constructs showed inhibition of tumor growth, only the B10-B11-ABNF Nanofitin provided significant results, including shrinkage of the tumor by the end of the study (day 20) (Figure 4). The potency of the two constructs was directly correlated to the increased level of immune cell infiltrates, especially CD3+ T cells and F4.80+ tumor infiltrated macrophages quantified by IHC, compared to the vehicle control (Figure 5). The increased frequency of Tumor-Associated Macrophages (TAM) is associated with enhanced antigen-specific T cell activation in CT26 syngeneic tumor model but in a PD-L1/PD-1 interaction dependent manner [23]. Indeed, Gordon et al. demonstrated a reduced degree of phagocytosis by PD1+ TAM compared to PD1- TAM [24]. Therefore, we can hypothesize that the PD-L1 blockade by the bispecific Nanofitin could release the tumor-driven hindered phagocytic activity of the TAM and thus promote antigen-specific T cell activation but has to be elucidated by further preclinical experiments. It would be interesting to evaluate in a subsequent study whether this can lead to an anti-tumor systemic immunity by rechallenging the treated mice with a second tumor cells implantation on the other flank. Furthermore, we observed a correlation between the efficacy of these two compounds in the CT26 syngeneic model and their respective tumoral accumulation at 7 h post-injection in the A431 tumor xenograft model (Figure 2). It is assumed that the low accumulation of the bispecific B10-B11 Nanofitin resulted from an imbalance between systemic exposure and speed of accumulation, which was compensated by the incorporation of ABNF. These results suggest that the bispecific B10-B11
Nanofitin displays anti-tumor activity and that a minimal exposure time is necessary to trigger a significant anti-tumoral effect, which requires the addition of the ABNF.

### 4.4. Taking advantages of small and bispecific PD-L1 immune checkpoint inhibitor

As discussed before, anti-PD-L1 antibody-based therapies size and on-target/off-tumor effects may restrict their delivery to tumors and limit their efficacies. A previous biodistribution study of the anti-EGFR Nanofitin demonstrated significant tumor specificity in vivo, with high accumulation in EGFR-expressing tumors and low accumulation in non-EGFR-expressing tumors or healthy organs (heart, lungs, skin) 2.5 hours post-injection [16]. In addition of amplifying specificity to EGFR positive tumors, dual targeting of both EGFR and PD-L1 by a bispecific Nanofitin could offer a potential solution to on-target/off-tumor effects linked to PD-L1 monospecific targeting.

Moreover, smaller compounds penetrate tumors faster, which in this case is advantageous for the ABNF-bispecific Nanofitin compared to monoclonal antibodies. This assumption is well aligned with the findings of Tijink et al., who demonstrated identical tumor accumulation between Cetuximab and a Nanobody trimer involving two binding sites for EGFR and one binding site for albumin, but at different timelines as the Nanobody trimer showed a faster and deeper tumor accumulation than the antibody [25]. Similarly, the level of tumor accumulation observed as soon as 7 h post-injection for the ABNF-bispecific Nanofitin reflects that of reported with monoclonal antibodies at longer time point of at least 24 h post-injection [12].

In the present study, no weight loss or abnormal behavior was observed in mice treated with the Nanofitin, suggesting good tolerability of the compound. Furthermore, no histological changes were detected in the heart, liver, kidney, or spleen (data not shown). Consequently, PD-L1 immune checkpoint inhibition with the ABNF-bispecific Nanofitin, due to its small size and high tumor specificity, constitutes an effective strategy to bypass both target sink and on-target/off-tumor effects, thereby enhancing the therapeutic window of this therapy.

## 5. Conclusions

Dual targeting of two overexpressed receptors on tumor cells is a promising strategy to circumvent the on-target/off-tumor effects that limit the effectiveness of PD-L1 inhibitors. Our *in vitro* and *in vivo* experiments demonstrate that the bispecific B10-B11 Nanofitin efficiently engages and achieves immune checkpoint inhibition specifically through the co-engagement of two tumor associated antigens, EGFR and PD-L1. When fused to an albumin binding Nanofitin, this bispecific construct generated a potent anti-tumor effect, fully inhibiting the tumor growth of a CT26 syngeneic immunocompetent tumor model. The anti-tumor potency of the molecule was well correlated with the recruitment of CD3+ T cells and F4.80+ tumor infiltrated macrophages, as well as its rapid and homogeneous distribution within the tumor, observed as early as 7 h post-injection. Given its reported potency and selectivity, we believe that this approach has the potential to expand the therapeutic window of currently approved anti-PD-L1-based therapies.

**Supplementary Materials:** The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: A431 tumor staining by only the anti-rabbit polymer coupled to HRP. The mice were sacrificed 90 min after intravenous injection of the B10 Nanofitin. Here is represented the staining for one mouse of the group; Figure S2: Mice weight monitoring of (A) Control group, (B) B10-B11 group and (C) B10-B11-ABNF group. Measurements were taken every two days; Figure S3: CT26 tumors staining by only the anti-rabbit polymer coupled to HRP. Data of one mouse per treated group are shown. (A) Control group, (B) B10-B11 group, (C) B10-B11-ABNF group; Table S1: Mean Fluorescence Intensity values of Nanofitins binding to A431 and CT26 cell lines. Comparison of mean fluorescence intensity of B10, B11, and B10-B11Nanofitins. A DyLight650 anti-HA tag antibody was used to detect Nanofitins and as an isotype control by incubating on cells without Nanofitins. FL-4
(DyLight 650) fluorescence on cells were analyzed using flow cytometry (BD AccuriTM C6 Plus System). For each acquisition, fluorescence measurements were conducted on a total of 10,000 recorded events.

**Author Contributions:** Conceptualization, M.C. and D.H.; methodology, P.J.; validation, M.C., D.H.; formal analysis, P.J.; investigation, P.J., J.M.-G., A.L., M.T., L.F. and F.D.; resources, D.H., A.L., M.T. and M.-F.H.; data curation, P.J.; writing—original draft preparation, P.J.; writing—review and editing, M.C. and D.H.; visualization, P.J.; supervision, J.M.-G., A.B., M.T., M.-F.H., D.H.; project administration, M.C., P.J., D.H.; funding acquisition, M.C., D.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was partially sponsored by a grant from the European Regional Development Fund (ERDF/FEDER) via the Region Pays de Loire (PL0015680) and by the Agence Nationale de la Recherche et de la Technologie (ANRT) via a Convention Industrielle de Formation par la Recherche (CIFRE n°20190120).

**Institutional Review Board Statement:** The animal study protocol was validated by the French ethical committee of the "Pays de la Loire" (CEEA-PdL-06) and authorized by the French ministry of agriculture and fisheries (authorization APAFIS # 2168-2019080709313558 v6).

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We are grateful to Sébastien Viollet, Simon Huet, Caroline Roze, Julien Lenoir, Elena Alvarez Sanchez, Maurine Fleury, Rémi Gaillard, Justine Picot, Elise Enouf, Anaelle Perrocheau, Chloé Savignard, Jessy Pedrault, Marine Tanou and Léo Candela from Affilogic for their involvement and technical support. We acknowledge Emilie Ollivier, Camille Jubelin, Nolwenn Dubois, Axelle Renodon-Cornière and Denis Cochonneau, from Institut de Cancérologie de l'Ouest for helpful discussions, suggestions and implications in this project. We thank Atlantic Bone Screen for animal housing, formation and supervision of animal experiments.

**Conflicts of Interest:** The authors declare no conflicts of interest. P.J., A.L., F.D. and M.C. hold employment in Affilogic. The Nanofitin technology described in this study, commercialized by Affilogic, uses the patent application owned by Institut Pasteur and Centre National de la Recherche Scientifique (CNRS): "OB-fold used as scaffold for engineering new specific binders"; PCT/IB2007/004388. Affilogic SAS, Nantes, France, provided support for the study and participated in study design, conducted the study, and provided data collection, management and interpretation. This does not alter the authors' adherence to all the journal policies on sharing data and materials. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

## Abbreviations

The following abbreviations are used in this manuscript:

<table><tr><td>FDA</td><td>Food and Drug Administration</td></tr><tr><td>ICI</td><td>Immune Checkpoint Inhibitors</td></tr><tr><td>CTLA-4</td><td>Cytotoxic T-Lymphocyte-Associated protein 4</td></tr><tr><td>PD-1</td><td>Programmed cell Death protein 1</td></tr><tr><td>PD-L1</td><td>Programmed Death-Ligand 1</td></tr><tr><td>irAE</td><td>Immune-Related Adverse Events</td></tr><tr><td>PD-L2</td><td>Programmed Cell Death Ligand 2</td></tr><tr><td>EGFR</td><td>Epithelial Growth Factor Receptor</td></tr><tr><td>ABNF</td><td>Albumin Binding Nanofitin</td></tr><tr><td>HSA</td><td>Human Serum Albumin</td></tr><tr><td>EpCAM</td><td>Epithelial Cell Adhesion Molecule</td></tr><tr><td>CD3</td><td>Cluster of Differentiation 3</td></tr><tr><td>BiTE</td><td>Bi-specific T-cell Engager</td></tr><tr><td>RTCA</td><td>Real-Time Cell Analysis</td></tr><tr><td>IHC</td><td>Immunohistochemistry</td></tr><tr><td>scFv</td><td>Single-Chain Variable Fragment</td></tr></table>
References

1. Zhao B, Zhao H, Zhao J. Efficacy of PD-1/PD-L1 blockade monotherapy in clinical trials. Ther Adv Med Oncol. 2020 Jan;12:175883592093761.

2. Hiltbrunner S, Cords L, Kasser S, Freiberger SN, Kreutzer S, Toussaint NC, et al. Acquired resistance to anti-PD1 therapy in patients with NSCLC associates with immunosuppressive T cell phenotype. Nat Commun. 2023 Aug 24;14(1):5154.

3. Kawashima S, Togashi Y. Resistance to immune checkpoint inhibitors and the tumor microenvironment. Experimental Dermatology. 2023 Mar;32(3):240-9.

4. Yin Q, Wu L, Han L, Zheng X, Tong R, Li L, et al. Immune-related adverse events of immune checkpoint inhibitors: a review. Front Immunol. 2023 May 25;14:1167975.

5. El Osta B, Hu F, Sadek R, Chintalapally R, Tang SC. Not all immune-checkpoint inhibitors are created equal: Meta-analysis and systematic review of immune-related adverse events in cancer trials. Critical Reviews in Oncology/Hematology. 2017 Nov;119:1-12.

6. Khunger M, Rakshit S, Pasupuleti V, Hernandez AV, Mazzone P, Stevenson J, et al. Incidence of Pneumonitis With Use of Programmed Death 1 and Programmed Death-Ligand 1 Inhibitors in Non-Small Cell Lung Cancer. Chest. 2017 Aug;152(2):271-81.

7. Shklovskaya E, Rizos H. Spatial and Temporal Changes in PD-L1 Expression in Cancer: The Role of Genetic Drivers, Tumor Microenvironment and Resistance to Therapy. IJMS. 2020 Sep 27;21(19):7139.

8. Kurino T, Matsuda R, Terui A, Suzuki H, Kokubo T, Uehara T, et al. Poor outcome with anti-programmed death-ligand 1 (PD-L1) antibody due to poor pharmacokinetic properties in PD-1/PD-L1 blockade-sensitive mouse models. J Immunother Cancer. 2020 Feb;8(1):e000400.

9. Koopmans I, Hendriks D, Samplonius DF, Van Ginkel RJ, Heskamp S, Wierstra PJ, et al. A novel bispecific antibody for EGFR-directed blockade of the PD-1/PD-L1 immune checkpoint. OncoImmunology. 2018 Apr 24;e1466016.

10. Jacquot P, Muñoz-Garcia J, Fleury M, Cochonneau D, Gaussin R, Enouf E, et al. Engineering of a Bispecific Nanofitin with Immune Checkpoint Inhibitory Activity Conditioned by the Cross-Arm Binding to EGFR and PDL1. Biomolecules. 2023 Mar 31;13(4):636.

11. Dewhirst MW, Secomb TW. Transport of drugs from blood vessels to tumour tissue. Nat Rev Cancer. 2017 Dec;17(12):738-50.

12. Lee CM, Tannock IF. The distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab within solid tumors. BMC Cancer. 2010 Dec;10(1):255.

13. Huet S, Zeisser Labouebe M, Castro R, Jacquot P, Pedrault J, Viollet S, et al. Targeted Nanofitin-drug Conjugates Achieve Efficient Tumor Delivery and Therapeutic Effect in an EGFRpos Mouse Xenograft Model. Molecular Cancer Therapeutics. 2023 Nov 1;22(11):1343-51.

14. Nedrow JR, Josefsson A, Park S, Ranka S, Roy S, Sgouros G. Imaging of Programmed Cell Death Ligand 1: Impact of Protein Concentration on Distribution of Anti-PD-L1 SPECT Agents in an Immunocompetent Murine Model of Melanoma. J Nucl Med. 2017 Oct;58(10):1560-6.

15. Michot N, Guyochin A, Cinier M, Savignard C, Kitten O, Pascual MH, et al. Albumin binding Nanofitins, a new scaffold to extend half-life of biologics – a case study with exenatide peptide. Peptides. 2022 Jun;152:170760.

16. Goux M, Becker G, Gorré H, Dammicco S, Desselle A, Egrise D, et al. Nanofitin as a New Molecular-Imaging Agent for the Diagnosis of Epidermal Growth Factor Receptor Over-Expressing Tumors. Bioconjugate Chem. 2017 Sep 20;28(9):2361-71.

17. Marcion G, Hermetet F, Neiers F, Uyanik B, Dondaine L, Dias AMM, et al. Nanofitins targeting heat shock protein 110: An innovative immunotherapeutic modality in cancer. Intl Journal of Cancer. 2021 Jun 15;148(12):3019-31.
18. Heskamp S, Wierstra PJ, Molkenboer-Kuenen JDM, Sandker GW, Thordardottir S, Cany J, et al. PD-L1 microSPECT/CT Imaging for Longitudinal Monitoring of PD-L1 Expression in Syngeneic and Humanized Mouse Models for Cancer. Cancer Immunology Research. 2019 Jan 1;7(1):150-61.

19. Zahnd C, Kawe M, Stumpp MT, De Pasquale C, Tamaskovic R, Nagy-Davidescu G, et al. Efficient Tumor Targeting with High-Affinity Designed Ankyrin Repeat Proteins: Effects of Affinity and Molecular Size. Cancer Research. 2010 Feb 15;70(4):1595-605.

20. Debie P, Lafont C, Defrise M, Hansen I, Van Willigen DM, Van Leeuwen FWB, et al. Size and affinity kinetics of nanobodies influence targeting and penetration of solid tumours. Journal of Controlled Release. 2020 Jan;317:34-42.

21. Fiser A, Do RKG, Šali A. Modeling of loops in protein structures. Protein Science. 2000 Jan;9(9):1753-73.

22. Wittrup KD, Thurber GM, Schmidt MM, Rhoden JJ. Practical Theoretic Guidance for the Design of Tumor-Targeting Agents. In: Methods in Enzymology [Internet]. Elsevier; 2012 [cited 2024 Aug 6]. p. 255-68. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780123969620000100

23. Modak M, Mattes AK, Reiss D, Skronska-Wasek W, Langlois R, Sabarth N, et al. CD206+ tumor-associated macrophages cross-present tumor antigen and drive antitumor immunity. JCI Insight. 2022 Jun 8;7(11):e155022.

24. Gordon SR, Maute RL, Dulken BW, Hutter G, George BM, McCracken MN, et al. PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity. Nature. 2017 May;545(7655):495-9.

25. Tijink BM, Laeremans T, Budde M, Walsum MS van, Dreier T, De Haard HJ, et al. Improved tumor targeting of anti-epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology. Molecular Cancer Therapeutics. 2008 Aug 1;7(8):2288-97.