A PDGFRB- and CD40-targeting bispecific AffiMab induces stroma-targeted immune cell activation

ABSTRACT

CD40 agonism by systemic administration of CD40 monoclonal antibodies has been explored in clinical trials for immunotherapy of cancer, uncovering enormous potential, but also dosing challenges in terms of systemic toxicity. CD40-dependent activation of antigen presenting cells is dependent on crosslinking of the CD40 receptor. Here we exploited this requisite by coupling crosslinking to cancer-receptor density by dual-targeting of CD40 and platelet-derived growth factor receptor beta (PDGFRB), which is highly expressed in the stroma of various types of tumors. A novel PDGFRBxCD40 Fc-silenced bispecific AffiMab was developed to this end to test whether it is possible to activate CD40 in a PDGFRB-targeted manner. A PDGFRB-binding Affibody molecule was fused to each heavy chain of an Fc-silenced CD40 agonistic monoclonal antibody to obtain a bispecific “AffiMab”. Binding of the AffiMab to both PDGFRB and CD40 was confirmed by surface plasmon resonance, bio-layer interferometry and flow cytometry, through analysis of cells expressing respective target. In a reporter assay, the AffiMab displayed increased CD40 potency in the presence of PDGFRB-conjugated beads, in a manner dependent on PDGFRB amount/bead. To test the concept in immunologically relevant systems with physiological levels of CD40 expression, the AffiMab was tested in human monocyte-derived dendritic cells (moDCs) and B cells. Expression of activation markers was increased in moDCs specifically in the presence of PDGFRB-conjugated beads upon AffiMab treatment, while the Fc-silenced CD40 mAb did not stimulate CD40 activation. As expected, the AffiMab did not activate moDCs in the presence of unconjugated beads. Finally, in a co-culture experiment, the AffiMab activated moDCs and B cells in the presence of PDGFRB-expressing cells, but not in co-cultures with PDGFRB-negative cells. Collectively, these results suggest the possibility to activate CD40 in a PDGFRB-targeted manner in vitro. This encourages further investigation and the development of such an approach for the treatment of solid cancers.

KEYWORDS:

• Cancer
• CD40
• immuno-oncology
• microenvironment
• PDGFRB

Introduction

Immunotherapy has recently emerged as a promising approach for the treatment of several types of cancer. One emerging target for immunotherapy is CD40, a member of the TNF (tumor necrosis factor) receptor superfamily characteristically expressed by antigen-presenting cells (APC) such as dendritic cells (DCs), B cells and macrophages.¹ Cells of the tumor microenvironment such as cancer associated fibroblasts (CAFs) favor immune escape and an immune suppressive milieu in cancer.² Activation of CD40 on APC has the potential to revert immune suppression by activating both innate and adaptive immune response against cancer.³ CD40-expressing cells are a major component of tumor-infiltrating leukocytes.⁴ The activation of CD40 is governed by the buildup of receptor superclusters to elicit downstream signaling, wherein the receptor’s natural agonistic ligand confers signaling in its trimeric form. An important characteristic of CD40 agonism is that receptor clustering on the membrane of CD40 expressing cells is the key to activation of the downstream signaling.³

DCs respond to CD40 stimulation with upregulation of co-stimulatory molecules including CD80 and CD86, subsequentially becoming more prone to antigen presentation and generation of an antigen-specific T cell response.⁵ Furthermore, B cells respond to CD40 agonism by upregulating co-stimulatory molecules and are able to mediate priming of T cells.⁶ Upon CD40 stimulation, macrophages release immune cell attractive factors such as C-C motif chemokine ligand 5 and interferon-gamma and tumor-associated macrophages (TAMs) have been shown to acquire an anti-tumor phenotype.^7,8

Dose-limiting toxicities of clinically evaluated CD40 agonistic mAbs together with positive signs of efficacy have elicited scientific interest in the development of strategies for local, tumor-targeted activation of CD40.⁹ Substantial efforts have been invested in the understanding and optimization of agonistic anti-CD40 antibodies with regard to their mode-of-action in relation to selection of IgG subtype, epitope, FcγR engagement, mAb hinge engineering, and valency to achieve optimal receptor super-clustering and downstream signaling.^9,10 The high affinity agonistic anti-CD40 antibody APX005M was developed based on an IgG1 scaffold conferring CD40 agonistic signaling via FcγR mediated clustering.¹¹ It has been shown that local administration of a CD40 agonist at the tumor site results in superior antitumor effects compared to systemic administration and improved toxicity profile.^12–14 Delivery of CD40 agonistic molecules at the tumor site via a stromal target is a promising strategy to reproduce and enhance such effects.⁹ Bispecific molecules have been developed with the ability to cluster and activate CD40 by tumor associated antigen or stromal target.¹⁵ A stromal target explored in such an approach is fibroblast activation protein (FAP),^16,17 with FAPxCD40 bispecific molecules showing positive signs of efficacy in vivo and currently being evaluated in clinical trials (e.g., DARPin® MP0317 in NCT05098405; CrossMab RO7300490 in NCT04857138).

Platelet-derived growth factor receptor beta (PDGFRB) is expressed in the stroma of several types of cancer. Higher expression has been associated with poorer prognosis in various cancer types.^18–20 The signaling axis involving PDGFRs and their ligands has been shown to be essential for the contribution of CAFs to several hallmarks of cancer, including proliferation, migration, and response to treatment.¹⁹ Recently, a set of studies have corroborated the importance of PDGFRB as a stromal marker by associating the presence of PDGFRB⁺ cells in the tumor stroma and subsets of PDGFRB⁺ CAFs with modulation of radiosensitivity of cancer cells,²¹ survival,²² disease recurrence,²³ and immune features.²⁴ Hence, PDGFRB displays properties making it an attractive tumor stroma target to explore.

Affibody molecules are small (6.5 kDa) and very stable affinity proteins based on a three-helix bundle domain. Through combinatorial protein engineering, Affibody molecules can be selected with the ability to bind any given target.²⁵ Their small size, lack of disulfide bonds and robustness of the molecules make them ideal tools for tumor targeting²⁶ and as fusion partners with monoclonal antibodies (mAbs) to create multispecific antibody-based affinity proteins, denoted AffiMabs.²⁷ Clinical utility of Affibody molecules has been demonstrated both for tumor targeting in patients with metastatic breast cancer,²⁸ as well as for chronic treatment of inflammatory disorders with data of well-tolerated and efficacious treatment of patients with plaque psoriasis up to three years.²⁹ An Affibody molecule is available for binding to PDGFRB, named Z_{PDGFRb_3,}³⁰ and has been shown in imaging studies to target PDGFRB-expressing remodeling tumor stroma in syngeneic tumors and in xenografts of glioma (U87) in vivo.^30,31

Here, an agonistic CD40 antibody governed by FcγR cross-linking was redesigned with a silenced Fc region into an AffiMab, a bispecific fusion protein by linkage of a PDGFRB-binding Affibody molecule to the antibody structure. Through in vitro testing, we provide a set of observations supporting the potential of the AffiMab functioning as a bispecific immune-tumor cell bridging engager, conferring conditional CD40 agonistic signaling in a PDGFRB-dependent manner. The bispecific molecule and its mode-of-action demonstrated in vitro presents itself as an interesting candidate to achieve local CD40 activation that could be evaluated for in vivo activity in solid tumors.

Results

The Z_{PDGFRb_3} Affibody molecule binds PDGFRB without inducing receptor activation or internalization

Prior to generating a PDGFRBxCD40 hybrid construct, the PDGFRB binding Affibody molecule (Z_{PDGFRb_3}) was characterized with regard to its effects on PDGFRB. MG−63 cells express PDGFRB as evaluated by flow cytometry (Figure 1a). Compared to another Z molecule binding an irrelevant target, Z_{PDGFRb_3} binds the PDGFRB-expressing cell line MG−63. (Figure 1b)

Figure 1. Z_{PDGFRb_3} binds PDGFRB without inducing receptor activation or internalization. (a) MG − 63 cell lines are positively stained by an anti-PDGFRB antibody via a secondary FITC (fluorescein isothiocyanate)-conjugated secondary antibody, compared to FITC-conjugated secondary antibody alone. (b) Z_{PDGFRb_3} binds MG − 63 cells when compared to an Affibody molecule binding an irrelevant target. (c) BUD − 8 cell line is stimulated with PDGF-BB (PDGFRB natural ligand) or Z_{PDGFRb_3} and cell proliferation is assessed after 48 hours. Z_{PDGFRb_3} does not stimulate cell proliferation when compared to the natural ligand. (d) BJhTERT cells are treated with either Z_{PDGFRb_3} or PDGF-BB, at 37°C or on ice to assess PDGFRB activation and internalization-dependent degradation by western blot. Receptor phosphorylation (P-Tyr) is observed in the presence of PDGF-BB but not with Z_{PDGFRb_3} (performed on ice to prevent receptor internalization). Lower levels of PDGFRB due to receptor internalization-dependent degradation are observed in presence of PDGF-BB but not with Z_{PDGFRb_3}.

Alt Text: Z_{PDGFRb_3} binds PDGFRB on cells and does not induce cell proliferation or receptor internalization or activation.

To rule out any unwanted effects of Z_{PDGFRb_3} binding to PDGFRB, proliferation, and receptor activation were analyzed in PDGFRB-expressing cells. BUD−8 cells were treated either with a dilution series of Z_{PDGFRb_3} or with PDGF-BB (PDGF, two B subunits, one natural ligand to PDGFRB). Proliferation of PDGFRB⁺ BUD−8 cells (Supplementary Figure S1) was assessed with the CCK−8 viability test, showing that Z_{PDGFRb_3} did not stimulate BUD−8 proliferation (Figure 1c).

BJhTERT cells were seeded in 6-well plates and treated with either Z_{PDGFRb_3} or with PDGF-BB, for 90 min at either 37°C or on ice, the latter to prevent receptor internalization. Protein extracts were prepared and analyzed by western blot to assess PDGFRB levels and phosphorylation status, using B-actin as loading control. Compared to the natural ligand, which induces both PDGFRB internalization-induced degradation and phosphorylation, Z_{PDGFRb_3} does not stimulate PDGFRB activation or internalization-induced degradation (Figure 1d).

Taken together, these data support that Z_{PDGFRb_3} binds PDGFRB expressing cells without stimulating either cell proliferation or receptor internalization or activation. These properties are optimal for a molecule meant to only anchor to PDGFRB on cells such as CAFs without activating them.

The bispecific PDGFRBxCD40 AffiMab binds both PDGFRB and CD40

A bispecific PDGFRBxCD40 AffiMab was generated by linking a PDGFRB-binding Affibody molecule to a CD40 agonistic, non-FcγR-binding³² mAb (CD40 mAb-LALAPG) as schematically shown in Figure 2a. The anti-CD40 agonistic antibody relies on FcγR mediated clustering of CD40 to elicit downstream signaling.¹¹ By introduction of non-FcγR-binding mutations (LALAPG) to the AffiMab design, we postulated a mode-of-action governed by a PDGFRB-dependent clustering for CD40 activation. The binding properties of the bispecific construct along with the CD40 mAb and CD40-LALAPG mAb as controls, against CD40 and PDGFRB were assessed by surface plasmon resonance (SPR), bio-layer interferometry (BLI), and flow cytometry.

Figure 2. The bispecific PDGFRBxCD40 AffiMab binds both PDGFRB and CD40. (a) the AffiMab is designed from a CD40 agonistic, LALAPG-mutated antibody (unable of binding the FcγR). One Z_{PDGFRb_3} is linked to the C-terminus of each heavy chain. (b) the binding properties of the AffiMab are tested against CD40 and PDGFRB by SPR. (c) Simultaneous binding of the AffiMab toward PDGFRB and CD40 confirmed by BLI. (d) All the three molecules in (a) bind the CD40 expressing cell line HEK (human embryonic kidney)-Blue CD40L while only the AffiMab binds PDGFRB expressing cells MG − 63, assessed by flow cytometry.

Alt Text: The AffiMab, an Fc-silenced CD40 monoclonal antibody bound to a PDGFRB binding Affibody molecule, has been characterized for its binding to PDGFRB and CD40.

The ability of the AffiMab to bind to both targets was primarily evaluated by SPR and BLI. Binding to each target in a concentration-dependent manner by the AffiMab was demonstrated by SPR. (Figure 2b). As expected, no binding was observed by the monospecific counterparts toward the non-intended target. Furthermore, simultaneous binding toward both antigens by the AffiMab was confirmed by BLI analysis (Figure 2c).

Binding to HEK-Blue CD40L cells was analyzed for the CD40 mAb, the CD40 mAb-LALAPG and the AffiMab by flow cytometry, concluding that CD40 binding capability was retained by the three molecules (Figure 2d). As expected, binding to PDGFRB on MG−63 and NIH−3T3 cells was instead only a property of the AffiMab, as tested by flow cytometry (Figure 2d, Supplementary Figure S2).

Collectively, the data show that the AffiMab is capable of engaging both PDGFRB and CD40 simultaneously, indicating a potential for cell–cell bridging capability, as well as functioning as an immune-cell engaging bispecific molecule.

The AffiMab activates CD40 in a PDGFRB-targeted manner in a reporter gene cell assay

HEK-Blue CD40L cells were used to test the ability of the AffiMab to activate CD40 in a PDGFRB-dependent manner. This reporter gene cell line overexpresses CD40 and responds to CD40 stimulation with the release of secreted embryonic alkaline phosphatase in the supernatant, the levels of which can be assessed through a colorimetric assay.

To be able to distinguish between PDGFRB-independent and -dependent CD40 agonism, we cultured the HEK-Blue CD40L cells together with either unconjugated beads or PDGFRB-conjugated beads (Figure 3a). Upon treatment with the AffiMab, a potency shift could be observed when PDGFRB-conjugated beads were provided, supporting the hypothesis that the AffiMab is crosslinked in the presence of PDGFRB, resulting in enhanced clustering of CD40 on HEK-Blue CD40L cells, and subsequently leading to increased CD40 activation (Figure 3b).

Figure 3. PDGFRB-targeted CD40 activation in HEK-Blue CD40L reporter gene cell assay. (a) Schematic illustration of the assay using HEK-Blue CD40L cells as reporter gene cell assay with either PDGFRB-conjugated, PDGFRB-conjugated beads in different ratios with Chicken IgY and unconjugated beads or mono and co-cultures in presence of the AffiMab. (b) CD40 activation occurs at lower AffiMab concentrations in presence of PDGFRB conjugated beads compared to unconjugated beads. (c) PDGFRB-targeted CD40 activation is dependent on PDGFRB availability/density on the beads. Beads were used with PDGFRB at a ratio of 1/4, 2/4, or 3/4 of the total amount of protein bound to the beads. An anti-chicken IgY biotinylated antibody was used to occupy the beads together with PDGFRB. Beads carrying only PDGFRB or anti-chicken IgY antibody were also included. The CD40 activation signal intensity dependent on PDGFRB availability on the beads. Mean and standard deviation of one representative experiment of at least two independent replicates are displayed on (B-C). (d) MG − 63 Co-culture (f). NIH − 3T3 Co-culture. Mean and standard deviation of two independent replicates shown (D-E).

Alt Text: The AffiMab activates CD40 in a reporter gene assay with increased potency in the presence of either PDGFRB bound to beads or provided by human cells or murine PDGFRB provided by murine cells.

To better study the dependency of this phenomenon on PDGFRB density, beads were generated with different amounts of PDGFRB and anti-chicken IgY (chosen as an irrelevant molecule). The following ratios (PDGFRB to total conjugated protein amount) were chosen for the experiment: 3:4, 2:4, and 1:4. Beads with only PDGFRB and anti-chicken IgY were also evaluated (Figure 3c). This experiment showed a gradually lower CD40 potency shift upon AffiMab treatment of HEK-Blue CD40L with lower ratios of PDGFRB on the beads, supporting PDGFRB-dependent AffiMab crosslinking as the mediator of enhanced CD40 activation in the reporter cell assay. To further confirm a PDGFRB-dependent activation of CD40 by the AffiMab, HEK-Blue cells were employed in a co-culture set-up with either the human PDGFRB⁺ MG−63 fibroblast cell line or murine PDGFRB⁺ NIH−3T3 fibroblast cell line (Figure 3a). As postulated, the AffiMab conferred a stronger CD40 activation in the presence of fibroblast expressing PDGFRB, resulting in a 36% increase and 2.1-fold increase in recorded signal, respectively, in the reporter assay for the AffiMab at the highest concentration used for the co-culture set-up compared to treated HEK-Blue monocultures (Figure 3d, e).

The AffiMab activates CD40 in a PDGFRB-targeted manner in donor-derived immune cells

To assess the translational potential of our observations, the PDGFRB-dependent CD40 activation was evaluated in a more physiologically relevant system taking advantage of in vitro assays implementing donor-derived blood cells. Specifically, monocyte-derived dendritic cells (moDCs) and B cells were investigated for this purpose.

MoDCs were generated by isolating monocytes from blood donors and differentiating them into dendritic cells. They were subsequently cultured in the presence of either unconjugated beads or PDGFRB-conjugated beads, similarly to the reporter gene cell assay, and treated with the AffiMab or the CD40 mAb-LALAPG. CD40 activation was assessed by flow cytometry, evaluating upregulation of activation marker CD86. The AffiMab specifically stimulated CD40 activation in the presence of PDGFRB-conjugated beads and not in the presence of unconjugated beads, while, as expected, the CD40 mAb-LALAPG did not stimulate CD40 activation in the presence of PDGFRB (Figure 4a).

Figure 4. AffiMab mediated, PDGFRB-dependent CD40 activation in moDcs. (a) in a similar fashion as in Figure 2a, the AffiMab is evaluated on moDcs in presence of PDGFRB-conjugated or unconjugated beads. The CD40 mAb-LALAPG is also tested in the presence of PDGFRB-conjugated beads. The same experiment is repeated using BJhTERT cells as source of PDGFRB, performing a mono and co-culture. (b) Only the AffiMab activates moDcs in bead-based set-up specifically in presence of PDGFRB, as measured by upregulation of CD86 in flow cytometry. (c) CD86 is upregulated on moDcs following AffiMab treatment only in presence of PDGFRB expressing cells. Average mean fluorescence intensity (MFI) of one representative experiment of at least two independent replicates is displayed on (B-C).

Alt Text: The AffiMab activates CD40 in a PDGFRB-dependent fashion in monocyte-derived dendritic cells, while the Fc-silenced CD40 monoclonal antibody does not activate monocyte-derived dendritic cells, as evaluated by CD86 upregulation.

To assess whether this PDGFRB-targeted CD40 activation can be observed also in the presence of PDGFRB-expressing cells, additional co-culture experiments were performed. MoDCs were cultured in mono-culture or co-cultured with the PDGFRB expressing cell-line BJhTERT and CD40 activation was subsequentially monitored following AffiMab treatment (Figure 4b). Notably, a clearly stronger AffiMab-induced CD40 activation was observed in the moDC+BJhTERT co-culture than in the moDC monoculture (Figure 4c).

With the aim of reproducing the observations in an additional donor-derived immune cell type, B cells were isolated from human blood, co-cultured with the PDGFRB-negative A431 or the PDGFRB-positive BJhTERT (Figure 5a) and treated with the AffiMab (Figure 5b). Upregulation of CD86 was observed at low AffiMab concentrations in the presence of PDGFRB-positive BJhTERT cells, while only the highest tested concentration achieved an activation in the co-culture with the PDGFRB-negative A431 cells (Figure 5c).

Figure 5. AffiMab mediated, PDGFRB-dependent CD40 activation in B cells. (a) B cells are co-cultured with either PDGFRB⁺ BJhTERT or PDGFRB⁻ A431 cells in presence of the AffiMab. (b) PDGFRB expression is evaluated on BJhTERT and A431 cells by flow cytometry. The former is confirmed PDGFRB⁺ as also shown by western blot data in Figure 1D. A431 cells are identified as PDGFRB⁻. Unstained cells are included as negative control. (c) CD86 fraction of CD20⁺ B cells is higher at lower AffiMab concentrations when PDGFRB is provided by cells. One representative experiment of at least two independent replicates is plotted with CD86+ cells (on CD20+ B cells) calculated on at least 1 × 10⁴ collected cell events.

Alt Text: The AffiMab activates CD40 in a PDGFRB-mediated fashion in B cells as evaluated by CD86 upregulation.

Taken together, these results suggest that CD40 can be activated in donor-derived immune cells by the AffiMab in a manner dependent on the presence of PDGFRB-expressing cells.

PDGFRB is expressed in the stroma of clinical tumor specimens

To explore the translational potential of our in vitro data, PDGFRB expression was analyzed by immunohistochemistry on a tissue microarray (TMA) containing a set of cores from multiple cases of a variety of tumor types, namely estrogen receptor positive (ER⁺), human epidermal growth factor receptor 2 positive (HER2⁺), and triple negative breast cancer (TNBC); colorectal cancer (CRC); non-small cell lung cancer (NSCLC); and prostate adenocarcinoma (ADC). PDGFRB staining showed a variety of stromal patterns across different cores (Figure 6a). Consistently with existing literature data, positive PDGFRB staining was observed in most of the analyzed cases (Figure 6b). These observations corroborate the hypothesis that PDGFRB is an interesting stromal target for CD40 directed immuno-oncology.

Figure 6. PDGFRB immunohistochemical staining of clinical tumor specimens. Staining settings: PDGFRB-DAB (brown) and Hematoxilin (blue). (a) Representative tumor cores of a tumor TMA, showing different patterns of PDGFRB in the tumor stroma. (b) Quantification of PDGFRB fraction (positive staining area as percentage tumor core area) showing overall representation of PDGFRB staining across different tumor types.

Alt Text: PDGFRB is expressed in the stroma of clinical tumor specimens as assessed by immunohistochemical staining.

Discussion

CD40 agonism is a promising approach for immunotherapy of cancer despite challenges related to dose-limiting toxicities mostly due to systemic activation of CD40.^33,34 Activation of CD40 is dependent on receptor clustering on the membrane of cells such as APC. Herein, we present in vitro support that CD40 clustering can be achieved via PDGFRB-dependent crosslinking of a PDGFRBxCD40 bispecific AffiMab, composed of a CD40 agonistic, FcγR-silenced antibody and two PDGFRB binding Affibody molecules. With PDGFRB being a tumor stroma marker, such an approach could allow conditional, tumor restricted CD40 activation.

The Z_{PDGFRb_3} was shown to bind PDGFRB-expressing cells without stimulating their proliferation and without mediating receptor internalization or activation. Such properties are optimal, given the fact that PDGFRB serves as anchor for the AffiMab and should not be internalized to provide the expected mechanism-of-action. The absence of growth stimulation and PDGFRB activation is also optimal, considering that PDGFRB⁺ cells such as CAFs in the tumor microenvironment are mostly identified as pro-tumoral, and PDGFRB signaling has also been described as mediator of tumor supportive effects.¹⁹

The AffiMab activates CD40 on the HEK-Blue CD40L reporter gene cell assay, with increased potency in the presence of PDGFRB. The presence of unspecific activation is not surprising given the fact that the cell lines used in this assay express the receptor significantly higher than physiological levels. In addition, the agonistic CD40 mAb itself is a potent and efficacious CD40 activator. The presence of a potency shift suggests, however, the possibility to envision a favorable therapeutic window, which in the clinic should be assessed by a careful dose escalation study. The PDGFRB-mediated shift in CD40 activation potency of HEK-Blue CD40L cells was additionally observed in co-cultures performed with human and murine PDGFRB positive cell lines.

In the immunologically relevant assays on donor-derived cells, CD40 activation appears more strictly dependent on AffiMab crosslinking via PDGFRB, suggesting that the reporter gene assay, albeit useful for activities such as CD40 agonist screening, may be hyper-responsive to CD40 stimulation. The ability to activate CD40-expressing APC in the tumor microenvironment holds potential to revert the tumor-suppressive environment of cancer and stimulate innate and adaptive immunity against the tumor.³⁵ The in vitro moDC and B cell co-culture experiments demonstrate the ability of the AffiMab to mediate CD40 activation at low concentrations compared to the parental mAb in co-cultures with PDGFRB-positive cells. We note a difference in potency values of the AffiMab across the assays. This may be due to the diverse assay conditions, distinct levels of CD40 expression on analyzed cell lines and PDGFRB density/amount between beads and cells.

The ability to activate CD40-expressing APC in the tumor microenvironment holds potential to revert the tumor-suppressive environment of cancer and stimulate innate and adaptive immunity against the tumor.³⁵ Conditional, stroma-targeted CD40 activation has been previously demonstrated in vitro and in vivo in an approach evaluating FAP as a target.^16,17 The differential expression of PDGFRB and FAP across various tumor types has not as yet been systematically reviewed. Nevertheless, given the existence of literature supporting high PDGFRB expression in the stroma of a broad spectrum of tumor types, and recent literature suggesting the presence of CAF subsets with mutually exclusive expression of FAP or PDGFRB,²⁴ we believe that PDGFRB qualifies as an alternative stroma target for such an approach.^36–43

Interestingly, Tao et al.^44,45 have linked Z_{PDGFRb_3} to TNF-related apoptosis-inducing ligand (TRAIL) in order to deliver TRAIL to PDGFRB-expressing pericytes at the tumor site. The antitumor effect of TRAIL in mouse xenografts was enhanced with such an approach, highlighting the capability of Z_{PDGFRb_3} to engage its target in vivo.

Bispecific molecules targeting CD40 in a tumor or stroma localized manner are currently being evaluated in the clinic. Two FAP-targeting CD40-engagers^16,17 and one mesothelin-targeting CD40-engager⁴⁶ are currently being evaluated in Phase 1 clinical trials. Positive results from more advanced phases of clinical trials involving the aforementioned molecules will empower the strategy of targeting a stromal marker while activating CD40 using bispecific molecules.

PDGFRB is expressed at prominent levels in the tumor stroma of clinical specimens across diverse types of cancer, as we concluded from immunohistochemical staining in accordance with available literature.^36–43 This is a promising observation that suggests that the target would be available for a bispecific PDGFRBxCD40 AffiMab to anchor the tumor and stimulate the immune response. It also highlights the fact that such an approach could be potentially applicable across different cancer types.

In conclusion, our study provides in vitro evidence that CD40 activation can be achieved in immune cells by PDGFRB-mediated crosslinking of a bispecific PDGFRBxCD40 AffiMab. PDGFRB is highly expressed in the stroma of several types of tumors and may be used as a target to direct CD40 agonism at the tumor site. In vivo studies would be needed to support the translational potential of this hypothesis. Such a targeted approach could result in enhanced therapeutic window of CD40 agonism.

Materials and methods

AffiMab generation

The AffiMab was designed by linking PDGFRB-binding Affibody molecules^30,31 in the C-termini of the heavy chain of an Fc-silenced analog of the CD40 agonistic mAb APX005,^11,32 resulting in a symmetrical bispecific format reported earlier.⁴⁷ The amino acid sequence for APX005M was retrieved for research purposes using publicly available information.^11,48 The linked PDGFRB Affibody molecule displays binding to murine and human PDGFRB. The AffiMab was produced in Chinese hamster ovary cells and appropriate quality control was carried out, including purity and monomericity (>99%) and endotoxin (<1 EU/mg) test.

Surface plasmon resonance

Affinity determinations of the antibody and AffiMab toward human CD40 (ref. AF632, R&D systems) and human PDGFRB (ref. 10676-PR−100, R&D systems) were made using a BIAcore 8K instrument (Cytiva). The antigens were injected at a concentration of 20 ug/mL and immobilized on a CM5 chip via amine coupling according to manufacturer’s instruction (ref. 29104988, Cytiva). The analytes were injected in a twofold dilution series of six dilutions starting at 100 nM in phosphate-buffered saline (PBS)-Tween 20 (0.05%).

Biolayer interferometry

Dual binding toward human CD40 (ref. AF632, R&D systems) and biotinylated PDGFRB (ref. 10514-H08H-B, Sino Biological) by the AffiMab was evaluated in an Octet RED86 system (Sartorius). Biotinylated PDGFRB at a concentration of 50 nM diluted in PBS was immobilized on streptavidin-coated octet probes (Sartorius). Subsequently, probes were lowered in wells with 100 nM AffiMab or mAb diluted in PBS and final dip-and-read in wells filled with 50 nM CD40 diluted in PBS.

Cell cultures

The supplier’s instructions were followed regarding cell-line handling, unless otherwise specified. The MG−63 (ref. CRL−1427), BUD−8 (ref. CRL−1554) and NIH−3T3 (ref. CRL−1658) cell lines were bought from ATCC. HEK-Blue CD40L cells were bought from Invivogen (ref. hkb-cd40). The A431 cell line was bought from ECACC (ref. 85090402) and was cultured according to ATCC’s recommendations for the same cell line. BJhTERT cells were a kind gift from Arne Östman’s lab. BJhTERT and A431 cell identity has been confirmed through cell-line authentication using the FTA Sample Collection Kit for Human Cell Authentication Service (ref. 135-XV−10, ATCC) and following the manufacturer’s recommendations. B cells and moDCs were cultivated in Roswell Park Memorial Institute (RPMI) medium (see Immunological cell assays in Materials and Methods). Reagents used for cell culturing included Dulbecco’s Modified Eagles Medium + Glutamax (ref. 31966047, Gibco), Eagle’s Minimum Essential Medium (ref. 30–2003, ATCC), RPMI medium (ref. R8758, Sigma Aldrich), Dulbecco’s PBS 1× without calcium or magnesium (21–031-CV, Corning), fetal bovine serum (FBS), qualified, heat inactivated (ref. 10500–064, Gibco) and TrypLE Express Enzyme (1×) phenol red (ref. 12605010, Gibco). Cell culturing incubation conditions consisted of 37°C, 5% CO₂ and 95% relative humidity unless otherwise specified.

Reporter gene cell assay (HEK-Blue CD40L)

Dynabeads^TM M−280 Streptavidin (ref. 11205D, Invitrogen) were used in experiments where PDGFRB was provided by beads. They were conjugated with recombinant human biotinylated PDGFRB (ref. 10514-H08H-B, Sino Biological) using manufacturer’s protocol and indication about molar quantity of conjugated peptide.

HEK-Blue CD40L cells were seeded in 1:2 ratio with unconjugated or PDGFRB-conjugated beads and treated with a dilution series of the AffiMab. After 24 h, supernatants were collected and incubated for 2 h with QuantiBlue solution 1× (ref. rep-qbs2, Invivogen) according to the manufacturer’s protocol. Absorbance at 620 nm was read using Molecular Devices SpectraMax iD5 and data were analyzed in GraphPad.

For the co-cultures, MG−63 or NIH−3T3 cells were seeded at a 1:2 ratio with HEK-Blue CD40L cells (5 × 10⁴ to 1 × 10⁵ cells). Cells were treated with a threefold serial dilution of antibody or AffiMab comprising six concentrations starting at 30 pM. Subsequently, 24 h post treatment, supernatants were collected and analyzed in the same manner as described above.

Proliferation assay

The CCK−8 assay (ref. 96922, Sigma-Aldrich) was used to assess cell proliferation. Briefly, 1 × 10⁴ BUD−8 cells/well were added in a 96-well plate. After 48 h, a serial dilution of the AffiMab was added to the wells. PDGF-BB (ref. 220-BB, R&D Systems) was used as positive control. After further 48 h, 10 µL of CCK−8 were added to each well of the plate, which was subsequently incubated for 4 h before reading absorbance at 450 nm. Data were analyzed in GraphPad.

Immunological cell assays

Buffy coats were ordered at Karolinska University Hospital. For moDC generation, monocytes were isolated using RosetteSep^TM Human Monocyte Enrichment Cocktail (ref. 15028, StemCell Technologies) followed by stratification on Lymphoprep^TM (ref. 07801, StemCell Technologies). Monocytes were in vitro differentiated to moDCs for 6 days in the presence of granulocyte-macrophage colony stimulating factor (ref. 300–03, PeproTech) at 250 ng/mL and interleukin−4 (ref. 204-IL−010, R&D Systems), with cytokine medium renewal after 3 days. In wells of a 96-well plate, 2 × 10⁵ moDCs were seeded in a 1:2 ratio with either empty or PDGFRB-conjugated beads (see Reporter gene assay in Materials and Methods for details about the beads). In the co-culture experiment, 5 × 10⁴ partner cells were seeded overnight and 2 × 10⁵ moDCs were added on the following day, together with a dilution series of the AffiMab. After 72 h of incubation, moDCs were subjected to staining (see Flow cytometry section) using a dead cell marker and CD1a to gate live moDCs and CD86 and CD83 as activation markers.

B cells were isolated by negative selection using the B Cell Isolation Kit II, human (ref. 130-091-151, Miltenyi) according to manufacturer’s recommendations starting from peripheral blood mononuclear cells isolated by density gradient on Ficoll-Paque PLUS (ref. 17144002, Cytiva). In the beads experiment, B cells were seeded in a 1:2 ratio with beads. In the co-culture experiment, 5 × 10⁴ partner cells were seeded overnight and 1 × 10⁶ B cells were added on the following day, together with a dilution series of the AffiMab. After 24 h of incubation, cells were subjected to staining using a dead cell marker and CD20 to gate live B cells and CD86, CD54 and CD69 as activation markers.

Flow cytometry

Cells from co-culture systems (or adherent cells in general) were detached through a short incubation with TrypLE or Trypsin-EDTA (Sigma-Aldrich, ref. T3924) prior to washing in flow buffer, consisting of PBS with 1% FBS. Suspension cells were directly spun down and washed in flow buffer. Flow buffer was used for further washes and antibody dilution steps, except the washes prior and after adding the viability marker, where PBS only was used. Antibodies with their working dilution used in this study are listed in Supplementary Table S1.

Cells were resuspended in FcR-blocking reagent (Purified NA/LE Human BD Fc Block^TM, ref. 564765, BD Biosciences) And incubated for 10 min at room temperature (RT). Subsequently, primary antibodies were added, and incubation was performed at 4°C in the dark for 30 min. In the case of non-directly fluorophore-conjugated primary antibody, the cells were washed two times and resuspended in the secondary antibody and incubated at 4°C. Two further washes were performed, and the viability marker was added. Two more washes were performed, and the samples were resuspended in flow buffer and acquired using BD FACSCanto II or Agilent Novocyte Advanteon. Data analysis was performed in FlowJo or NovoExpress, and further data plotting was performed in Prism GraphPad. Gating was performed on live cells. Subsequently, the markers CD20 and CD1a were used for B cells and moDCs, respectively.

Immunoblotting

Samples were prepared seeding BJhTERT 6-well plates and letting them grow overnight. Desired stimuli were added to each well as described in Results, across two conditions: on ice or at 37°C. Protein lysates were collected and analyzed following the previously described protocol from Östman’s lab.^23,49

Immunohistochemistry

The multi-tumor TMA was purchased from Tristar (ref. 69573165–2995). The staining protocol was obtained from the Östman’s lab as described in the literature.⁵⁰ Briefly, the TMA slide was baked at 56°C for 3 h, deparaffinized and hydrated. Antigen retrieval was performed at 110°C for 5 min, with Dako Target Retrieval Solution, pH 9 (10×), Dako (ref. S2367), which was later exchanged to distilled water. After a first wash in PBS-T (PBS with 0,05% Tween−20, used throughout the protocol for the washes), endogenous peroxidase activity was quenched with 3% hydrogen peroxide. Animal-free blocker 5× (ref. SP−5030, Vector Laboratories) was used as blocking solution, and PDGFRB (28E1) Rabbit mAb (ref. #3169S, Cell signaling) was added to the slides and incubated overnight at 4°C. The slide was then washed and incubated with EnVision+ System horseradish peroxidase-labeled Polymer Anti-Rabbit (ref. K4002, Dako) for 1 h at RT, washed again and staining was disclosed with DAB (3,3’-diaminobenzidine) Substrate kit, peroxidase (ref. SK−4100, Vector Laboratories). The slide was then counterstained with hematoxylin, dehydrated and cleared in xylene, and mounted in xylene containing mounting medium. The slide was digitalized at the Karolinska Institutet HistoCore facility using the VS200 Research Slide Scanner (Olympus). Images were analyzed using QuPath v0.3.2.

Abbreviation list

ADC	=	Prostate adenocarcinoma
APC	=	Antigen-presenting cell
BLI	=	Bio-layer interferometry
CRC	=	Colorectal cancer
DAB	=	3,3’-diaminobenzidine
ER	=	Estrogen receptor
FAP	=	Fibroblast activation protein
FBS	=	Fetal bovine serum
FITC	=	Fluorescein isothiocyanate
HEK	=	Human embryonic kidney
HER2	=	Human epidermal growth factor receptor 2
mAb	=	Monoclonal antibody
MFI	=	Mean fluorescence intensity
NSCLC	=	Non-small cell lung cancer
PBS	=	Phosphate buffer saline
PDGF-BB	=	Platelet-derived growth factor, two B subunits
PDGFRB	=	Platelet-derived growth factor receptor beta
RPMI	=	Roswell Park Memorial Institute
RT	=	Room temperature
SPR	=	Surface plasmon resonance
TMA	=	Tissue microarray
TNBC	=	Triple negative breast cancer
TNF	=	Tumor necrosis factor
TRAIL	=	Tumor necrosis factor-related apoptosis-inducing ligand

Data availability statement and data analysis

Data analysis information are provided in the corresponding Materials and Method sections. Details about specific experiments are provided in figure legends. The data that support the findings of this study are available to any researcher wishing to use them for non-commercial purposes from the corresponding author, Frejd FY, upon reasonable request.

Disclosure statement

A. Mega, E. Ryer, A. Sköld, A. Sandegren, E. Backström Rydin and F.Y. Frejd are employed at Affibody AB. A. Mebrahtu, G. Aniander, J. Rockberg and A. Östman have no conflicts of interest that are directly relevant to the content of this article.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2223750

Additional information

Funding

The work was funded by Swedish innovation agency Vinnova through GeneNova and CellNova.