A Bispecific Antibody Targeting the Αv and Α5β1 Integrins Induces Integrin Degradation in Prostate Cancer Cells and Is Superior to Monospecific Antibodies

Author Manuscript Published OnlineFirst on October 21, 2019; DOI: 10.1158/1541-7786.MCR-19-0442 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A bispecific antibody targeting the αv and α5β1 integrins induces integrin degradation in prostate cancer cells and is superior to monospecific antibodies

Raghav Joshi1, Wenying Ren1, Paul Mathew1

Tufts Medical Center, Boston, MA

Corresponding author:

Paul Mathew MD

Division of Hematology-Oncology

Tufts Medical Center,

800 Washington Street, Box 245

Boston, MA 02111

Email: [email protected]

Tel: 617-636-8483

Fax: 617-636-8538

Running Title: Bispecific antibody targeting of αv and α5β1 integrins

Keywords: bispecific antibody, integrin alpha v, integrin alpha 5 beta 1, prostate cancer, bone metastases, epithelial-stromal interactions

Conflict of Interest: A patent pertinent to the strategy and composition of the bispecific antibody has been filed. No other conflicts of interest are identified.

Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 21, 2019; DOI: 10.1158/1541-7786.MCR-19-0442 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Fibronectin-binding integrins α5β1 and αv collaborate in prostate cancer -bone stromal interactions relevant to the colonization of the bone-marrow microenvironment. Combinatorial inactivation of these integrins on prostate cancer cells was assessed. Monospecific antibodies to α5β1and αv integrins alone (MAbs) and in combination (cMAbs), and a bispecific antibody that simultaneously targets α5β1and αv integrins (BsAbα5β1/αv) were compared in assays of chemotaxis, clonogenic survival and induction of endothelial migration. Cellular expression of integrins, their transcription, translation and degradation fate was compared. The BsAbα5β1/αv was superior to MAbs and cMAbs in abrogating adhesion, migration, clonogenic survival and induction of endothelial migration responses by prostate cancer cells. Integrin upregulation observed with MAbs or cMAbs was abrogated with the BsAbα5β1/αv. Loss of integrin expression was uniquely induced by the BsAbα5β1/αv and blocked by lysosomal inhibition. Implications: A novel and effective combinatorial strategy to target α5β1and αv integrins is defined for translational studies.

Introduction

The preferential colonization of the bone marrow microenvironment by disseminated prostate cancer cells underpins its lethal metastatic phenotype. Deconvolution of the molecular mechanisms that mediate this behavior can define novel therapeutic strategies to improve mortality and morbidity from the disease. Bone-marrow derived mesenchymal stromal cells have been implicated as architects of both the hematopoietic1 and bone metastatic niche.2 Earlier studies indicated that human bone- marrow derived mesenchymal stromal cells (hBM-MSCs) induce a strong chemotactic and adhesive response in prostate cancer cells. The relevant bioactive fraction of the hBM-MSC secretome was purified and proteolytic fragments of fibronectin (FNFr) signaling via the α5β1 integrin in prostate cancer cells identified as the basis of the chemotactic response.3 Genetic inactivation of the α5 integrin induced programmed cell death in prostate cancer cells indicating a role in adhesion-mediated survival.4 Accordingly, the FNFr-α5β1 integrin interaction was proposed as a seed-and-soil mechanism of bone colonization by prostate cancer.3

We hypothesized that the αv integrin, an alternative fibronectin-binding integrin 5 cooperates with the α5β1 integrin in mediating the metastatic niche interactions of prostate cancer cells. By comparing the impact of monospecific neutralization of α5β1 and αv integrins alone (MAbs) and in combination (cMAb) to a first-in-class integrin- targeting bispecific antibody, BsAbα5β1/αv, we find that the BsAbα5β1/αv optimally neutralizes tumor-stromal interactions with a novel mechanism of action. Methods

Prostate cancer, stromal and endothelial cell lines and culture:

Prostate cancer lines PC-3, DU-145, LNCaP, VCaP and C4-2B and human umbilical vein endothelial cells (HUVEC) were obtained from American Type Culture Collection (ATCC) (Manassas, VA) and the Characterized Cell Line Core Facility (MD Anderson Cancer Center, TX). Plastic adherent hBM-MSCs were generated from human bone marrow aspirates (Lonza, MD) as previously described. Cell lines represent genomically and phenotypically diverse prostate cancer cell lines: integrin α5/αv co- expressing lines include bone-derived PTEN-null Androgen Receptor (AR)-negative PC- 3 cells, bone-derived PTEN-null AR-positive hormone-resistant C4-2B cells, lymph-node derived PTEN-null AR-positive hormone sensitive LnCAP cells, and PTEN- wild type AR-negative DU-145 cells. TMPRSS2-ERG-positive, PTEN-wild type AR-positive VCAP cells lack membrane α5 expression.

Generation and validation of a bispecific antibody to integrins α5β1 and αv

A genetic construct was designed to express a bispecific antibody that targets αv6 and α5β1 integrins.7 The construct was expressed in 293T cells, and the resulting supernatant was purified by protein A chromatography (Creative Biolabs, NY). A similar approach was used to generate α5β1 and αv IgG control antibodies with the same antigen-binding sequences as BsAbα5β1/αv. Further description of structure, purity and binding of BsAbα5β1/αv is provided in supplementary figure 1.

Generation of Conditioned Media (CM)

hBM-MSC CM was generated as previously described. 3 Briefly, confluent hBM-MSCs were cultured in serum-free media with CM harvested after 24h and gently centrifuged before being stored at 4°C.

Cell Migration and Adhesion assays

Prostate cancer adhesion and migration assays were performed as described previously.3 Briefly, migration of cancer cells to hBM-MSC CM in a Boyden chamber was resolved after 24h with calcein AM or crystal violet staining with average counts of 5 representative fields reported. For endothelial migration, 50,000 prostate cancer cells were layered onto confluent hBM-MSC in the lower chamber. After 24h of co-culture, migration of 10,000 HUVEC was assessed as above. Adhesion of cancer cells to hBM- MSC CM coated wells was assessed after 1h (PC-3, DU-145) or 6h (C4-2b) using MTS reagent (Promega Madison WI) and was reported directly as optical density or RFU (relative fluorescent units).

Cell Clonogenic Potential Assay

Cancer cells (20,000) were treated and then plated in a 24-well plate in full culture media. 48 hours later, cells were trypsinized and reseeded in a series of limiting dilutions (1000, 500, 250, 100 cells per well) in a 24-well format. After 14 days, cells were fixed and stained with crystal violet and colonies of greater than 50 cells were counted. Plating efficiencies are reported as a ratio to the number of inoculated cells in respective wells. Only one limiting dilution was used for calculations for each experiment.

Quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) RNA extractions, cDNA syntheses and qRT-PCR reactions were performed as previously described.4 Primer sequences are provided in supplementary methods.

Flow Cytometry

Cells were labeled with conjugated primary antibodies or with unconjugated primary antibodies followed by incubation with fluorescently-labeled secondary antibodies. All incubations were for 1h at room temperature. For intracellular detection, cells were stained in the presence of 0.2% saponin. Median fluorescence intensity of stained populations was detected using a CyAn™ ADP Analyzer (Beckman Coulter).

Western Blot

Western blot was conducted as previously described.4 See supplementary methods for a list of antibodies used.

Results Combinatorial integrin α5 and αv blockade is superior to blockade of either integrin in inhibiting prostate cancer cell chemotaxis and induced endothelial migration

A strong chemotactic and adhesive response of prostate cancer cells to hBM-MSC conditioned media (CM ) was previously identified.3 Further, co-culture of prostate cancer cells with hBM-MSCs strongly induces the migration of human endothelial cells. Using these in vitro tumor-niche interaction models, we tested the hypothesis that combinatorial blockade of integrins α5 and αv would be superior to single integrin blockade alone across a panel of genomically diverse, integrin α5/αv co-expressing prostate cancer cells. As hypothesized, combined α5 and av blockade with dual monospecific antibodies was superior to individual single agents in inhibiting prostate cancer cell migration, adhesion and induced endothelial migration in each of these cell lines (Fig. 1A).

Generation of a bispecific antibody to integrins α5β1 and αv

Based on the hypothesis that a bispecific antibody targeting these two integrins would be superior to a combination of monospecific antibodies, we designed and generated a bispecific antibody. The molecule is composed of an IgG that targets the αv integrin 6 and an scFv that targets the α5β1 integrin 7 (Suppl Fig. 1A). By including a pan-αv integrin targeting sequence, the problem of diverse αv heterodimers with overlapping functions was skirted whereas α5β1 is the obligate heterodimer of α5 integrin. BsAbα5β1/αv purity was demonstrated by gel electrophoresis (Suppl Fig. 1B). BsAbα5β1/αv binding to target integrin receptors was correlated with commercially available α5 and αv antibodies across a panel of variably expressing integrin α5 and αv cell lines (Suppl Fig. 1C). High affinity, specific binding of BsAbα5β1/αv to target integrins was demonstrated using the Biacore platform (Suppl Fig. 2).

BsAbα5β1/αv is superior to monospecific combination in inhibiting prostate cancer cell chemotaxis and induced endothelial migration

We hypothesized that BsAbα5β1/αv would be superior to cMAbs in assays of tumor- hBM-MSC interaction. Bsα5/αvAb was superior to the combination of sequence- matched monospecific IgG controls (MAbα5β1 or MAbαv) in inhibiting prostate cancer cell migration, adhesion and induced endothelial migration (Fig. 1B). An scFv control was not considered to be an optimal control as its half-life in vivo is expected to be low given its low molecular-weight and susceptibility to rapid renal clearance.

BsAbα5β1/αv treatment uniquely induces loss of integrin expression and/or blocks integrin upregulation in prostate cancer cells

In assessment of the pharmacodynamic effects of the BsAbα5β1/αv, a marked loss of total cellular integrin αv and α5 in PC-3 cells at 48h following treatment was observed (Fig. 2A). This reduction in expression was not observed with MAbs or cMAbs. In DU- 145, C4-2B and VCAP cells, treatment with MAbs and/or cMAbs, variably resulted in upregulation of either or both integrins. However, in each case this upregulation was either abrogated or markedly inhibited by BsAbα5β1/αv treatment. (Fig. 2A). By contrast, preferential impact of the BsAbα5β1/αv on focal adhesion kinase, Akt or Erk signaling was inconsistent across cell lines (Suppl Fig. 3). MMP-14, a master regulator of matrix degradation, MMP-2 and TGF-beta activation, was strongly downregulated with cMAbs and the BsAbα5β1/αv but not MAbs (Suppl Fig 4).

Loss of integrin expression induced by BsAbα5β1/αv treatment is correlated with inhibition of prostate cancer chemotaxis and clonogenic survival

We hypothesized that the persistent reduction of integrin expression observed with BsAbα5β1/αv treatment at the 48 hour timepoint would correlate with a differential functional impact on chemotaxis and clonogenic survival of prostate cancer cells compared to MAbs or cMAbs. Accordingly, in PC-3 and DU-145 cells recovered 48h after antibody exposure, chemotaxis was most significantly impaired in cells treated with the BsAbα5β1/αv (Fig. 2B, Suppl Fig. 5) compared to MAbs and cMAbs. Similarly, clonogenic survival of both PC-3 and DU-145 cells was maximally impaired by BsAbα5β1/αv compared to cMAbs (Fig. 2B). In androgen receptor positive lines C4-2B, VCaP and LNCaP, αv-directed treatments universally resulted in a marked loss in replating and migratory capacity, prolonged proliferative arrest and reduced numbers of filopodia-like projections (Fig. 2B, Fig. 2C).

Regulation of integrin expression dynamics by BsAbα5β1/αv is post-translational and related to altered trafficking and lysosomal degradation of target integrins

Using PC-3 prostate cancer cells as a model line, we investigated the mechanism by which the BsAbα5β1/αv induced a marked decrease in cellular integrin expression. We initially hypothesized that these dynamic changes might be explained by rapid endocytosis and degradation of antibody-bound receptor as seen previously with other antibodies. However, BsAbα5β1/αv treatment resulted in slow progressive loss of αv membrane expression observed over 24-48 hours post-treatment (Fig. 3A). This loss of membrane expression correlated with diminished total cellular expression of αv. (Data not shown).

To assess the fate of these integrins more closely, we assessed whether integrin synthesis or degradation were specifically impacted by BsAbα5β1/αv treatment. Quantitative reverse transcription polymerase chain reaction studies showed no significant change in ITGA5 or ITGAV transcript expression following either MAbs, cMAbs or BsAbα5β1/αv treatment (Fig. 3B). Treatment with cycloheximide, an inhibitor of protein translation, failed to abrogate the differential impact of BsAbα5β1/αv on target expression (Fig. 3C), indicating the regulation was post-translational. Because the major post-translational regulator of integrin expression is transition from recycling pathways to lysosomal/endosomal degradation,8 we hypothesized that BsAbα5β1/αv induced preferential lysosomal degradation of bound target receptors. Treatment with chloroquine, a disruptor of lysosomal integrity and function, blocked the downregulation of integrin expression observed with BsAbα5β1/αv therapy (Fig. 3D, Suppl Fig 6).

Discussion

Our studies have implicated fibronectin-binding α5 and αv integrins in the key interactions of prostate cancer cells with hBM-MSCs, the putative metastatic niche- regulating cell in the bone microenvironment. We show that combined antibody blockade of these integrins is superior to single integrin blockade and that a bispecific antibody strategy optimally abrogates chemotaxis, clonogenic tumor survival and tumor- induced endothelial migration in prostate cancer cells. A distinct mechanism of action for the BsAbα5β1/αv is demonstrable. Membrane-depletion, blocked upregulation and induced lysosomal degradation of target integrins by the BsAbα5β1/αv contrasts strongly with upregulated integrin expression following single or combined monospecific antibody therapy. The data presented in this report provide foundational data to advance in vivo and translational studies of the novel BsAbα5β1/αv strategy.

From a historical perspective in targeting integrins in prostate cancer, the α5β1 monoclonal antibody volociximab was abandoned in the absence of discernible clinical efficacy in a limited set of solid tumor studies.9 Only a handful of patients with prostate

cancer were included, and the efficacy data are insufficient to draw conclusions. Synthetic peptide approaches to block the α5β1 integrin10 did not advance in clinical trials and it is unclear if peptide-based competitive binding strategies are capable of interdicting both ligand-dependent and ligand-independent integrin functions both of which are implicated in tumor survival and dissemination.11 From other lines of investigation, the αv integrin has been shown to be expressed in primary tumors, mediate bone-homing in experimental models, mediate interaction with endothelial cells and bone-matrix components to drive tumor progression and endow prostate cancer cells with stem-repopulating capacity.12 Integrin αv was targeted with the cyclic peptide cilengitide but without significant clinical impact.13 Contrasted to α5β1, the αv integrin has multiple heterodimeric forms implicated in the progression of prostate cancer including αvβ1 and αvβ614 15. The restricted αvβ3 and αvβ5 heterodimeric targeting by celingitide, it’s short half-life administered by intermittent intravenous infusion and rapid adaptive resistance by membrane recycling of α5β116 likely contribute to treatment failure. A pan-αv monoclonal antibody, abituzumab, demonstrated intriguing biological activity by delaying progression of bone metastases in men with metastatic castration- resistant disease. However, overall survival differences were not demonstrable.17 These data suggest that the αv integrin is probably implicated in the biology of prostate cancer progression in bone but that adaptive resistance to monotherapy likely dictates the lack of survival advantage. Rapid membrane recycling of α5β1 in response to αv blockade with celingitide offers evidence of cross-regulation of these two integrins to account for drug-resistance. The enriched expression of α5 and αv integrins in disseminated tumor cells recovered from bone marrow aspirates and in pathological specimens of bone metastases in prostate cancer 18,19 lends additional rationale to the importance and necessity of targeting both integrins.

The potential advantages of a bispecific antibody targeting α5β1 and αv over a combination of MAbs include harmonized pharmacokinetic and pharmacodynamic considerations within one molecule, lack of steric hindrance between two large molecules targeting proximate targets in the cell membrane, cross-linking of targets leading to increased binding affinity and superior on-target time, and the induction of novel biology such as induced degradation of target proteins. More than one of these mechanisms of action likely account for the markedly improved in vitro activity of the BsAbα5β1/αv over MAbs.

In addition, upregulated cellular integrin α5 and/or αv expression noted with either MAbs or cMAbs may represent an autoregulatory cellular adaptive response that could generate resistance to therapy. In VCAP cells, which are α5 membrane negative, cellular integrin α5 expression is robustly upregulated by αv blockade demonstrating that upregulation of integrins emerges despite lack of detectable baseline expression. We have not noted an increase in membrane-specific expression of integrins that

correlates with this cellular upregulation, suggesting that intracellular pooling of these integrins in endosomes from impaired recycling may occur primarily as a result of MAb therapy. Persistent low-level recycling or intracellular signaling from endosomal integrins20 may account for resistance to MAb or cMAb therapy. Such resistance pathways are probably mitigated by the BsAbα5β1/αv which triggers lysosomal degradation, loss of intracellular integrins and membrane recycling.

The precise mechanism by which BsAbα5β1/αv induces degradation is unknown, but we speculate that the steric consequences of BsAbα5β1/αv binding may destabilize the Rab family regulated machinery required for integrin recycling 8. Exceptionally, combined MAbs may also trigger lysosomal degradation of integrins, as demonstrated in C4-2B cells. The BsAbα5β1/αv effect on target integrin expression is slower to develop i.e. over 48 hours but also long-lasting, reversing after 7 days. These remarkably slow pharmacodynamic changes after a single brief exposure of tumor cells to the BsAbα5β1/αv have challenged the accurate tracking of integrin fates with traditional immunofluorescent tagging. When our data are taken together, the BsAbα5β1/αv may be seen to impose an early effect on chemotaxis, clonogenic survival and induction of endothelial migration secondary to highly-effective dual blockade of the membrane integrins on tumor cells followed by a persistent late effect driven by degradation and loss of the tumor integrins.

Integrin-depletion offers a method for pharmacodynamic monitoring of tumor cells in vivo. There is lack of consistency in the impact of BsAbα5β1/αv on downstream signaling intermediates which may reflect FAK-dependent and FAK-independent integrin signaling11 and the varied genetic backgrounds of these prostate cancer cells. Notwithstanding these signaling and pharmacodynamic variations, the functional consequences of the BsAbα5β1/αv have been consistent and justify further translational study of this first-in-class strategy to target integrins in prostate cancer.

The bispecific antibody strategy may also prove relevant as a molecular therapeutics strategy to target other components of the enabling tumor microenvironment regulated by αv and α5β1 integrins including immunosuppressive cancer-associated fibroblasts, bone-remodeling cells and blood vessels. Other neoplastic and non-neoplastic disease states including pathological fibrosis and angiogenesis in which these two integrins have been implicated may also be susceptible.

Acknowledgements: Funding support from the Will and Julie Person Prostate Cancer Research Fund is gratefully acknowledged. Technical advice and assistance from Dr Edi Goihberg and Dr Albert Tsai with the Biacore system is appreciated.

References

1. Mendez-Ferrer S, Michurina TV, Ferraro F, et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 2010;466:829-34. 2. Shiozawa Y, Pedersen EA, Havens AM, et al. Human prostate cancer metastases target the hematopoietic stem cell niche to establish footholds in mouse bone marrow. J Clin Invest 2011;121:1298-312. 3. Joshi R, Goihberg E, Ren W, Pilichowska M, Mathew P. Proteolytic fragments of fibronectin function as matrikines driving the chemotactic affinity of prostate cancer cells to human bone marrow mesenchymal stromal cells via the α5β1 integrin. Cell Adhesion & Migration 2016:1-11. 4. Ren W, Joshi R, Mathew P. Synthetic Lethality in PTEN-mutant Prostate Cancer is Induced by Combinatorial PI3K/Akt and BCL-XL Inhibition. Molecular Cancer Research : MCR 2016. 5. Marshall JF, Rutherford DC, McCartney AC, Mitjans F, Goodman SL, Hart IR. Alpha v beta 1 is a receptor for vitronectin and fibrinogen, and acts with alpha 5 beta 1 to mediate spreading on fibronectin. J Cell Sci 1995;108 ( Pt 3):1227-38. 6. Mitjans F, Sander D, Adan J, et al. An anti-alpha v-integrin antibody that blocks integrin function inhibits the development of a human melanoma in nude mice. Journal of cell science 1995;108 ( Pt 8):2825-38. 7. Ramakrishnan V, Bhaskar V, Law DA, et al. Preclinical evaluation of an anti- alpha5beta1 integrin antibody as a novel anti-angiogenic agent. Journal of experimental therapeutics & oncology 2006;5:273-86. 8. De Franceschi N, Hamidi H, Alanko J, Sahgal P, Ivaska J. Integrin traffic - the update. J Cell Sci 2015;128:839-52. 9. Almokadem S, Belani CP. Volociximab in cancer. Expert Opin Biol Ther 2012;12:251-7. 10. Veine DM, Yao H, Stafford DR, Fay KS, Livant DL. A D-amino acid containing peptide as a potent, noncovalent inhibitor of alpha5beta1 integrin in human prostate cancer invasion and lung colonization. Clin Exp Metastasis 2014;31:379-93. 11. Seguin L, Desgrosellier JS, Weis SM, Cheresh DA. Integrins and cancer: regulators of cancer stemness, metastasis, and drug resistance. Trends in cell biology 2015;25:234-40. 12. van der Horst G, van den Hoogen C, Buijs JT, et al. Targeting of alpha(v)- integrins in stem/progenitor cells and supportive microenvironment impairs bone metastasis in human prostate cancer. Neoplasia (New York, NY) 2011;13:516-25. 13. Bradley DA, Daignault S, Ryan CJ, et al. Cilengitide (EMD 121974, NSC 707544) in asymptomatic metastatic castration resistant prostate cancer patients: a randomized phase II trial by the prostate cancer clinical trials consortium. Investigational new drugs 2011;29:1432-40. 14. Schneider JG, Amend SR, Weilbaecher KN. Integrins and bone metastasis: integrating tumor cell and stromal cell interactions. Bone 2011;48:54-65. 15. Sutherland M, Gordon A, Shnyder SD, Patterson LH, Sheldrake HM. RGD- Binding Integrins in Prostate Cancer: Expression Patterns and Therapeutic Prospects against Bone Metastasis. Cancers (Basel) 2012;4:1106-45.

16. Caswell PT, Chan M, Lindsay AJ, McCaffrey MW, Boettiger D, Norman JC. Rab- coupling protein coordinates recycling of alpha5beta1 integrin and EGFR1 to promote cell migration in 3D microenvironments. The Journal of cell biology 2008;183:143-55. 17. Hussain M, Le Moulec S, Gimmi C, Bruns R, Straub J, Miller K. Differential Effect on Bone Lesions of Targeting Integrins: Randomized Phase II Trial of Abituzumab in Patients with Metastatic Castration-resistant Prostate Cancer. Clin Cancer Res 2016. 18. Putz E, Witter K, Offner S, et al. Phenotypic characteristics of cell lines derived from disseminated cancer cells in bone marrow of patients with solid epithelial tumors: establishment of working models for human micrometastases. Cancer research 1999;59:241-8. 19. Connell B KP, Ren W, Joshi R, Naber S, Mathew P. Aberrant integrin alpha v and alpha 5 expression patterns in prostate adenocarcinomas and bone-metastases from prostate cancer are consistent with a bone-colonizing phenotype. Proceedings of the 110th Annual Meeting of the American Association for Cancer Research 2019:Abstract# 4271. 20. Alanko J, Ivaska J. Endosomes: Emerging Platforms for Integrin-Mediated FAK Signalling. Trends Cell Biol 2016;26:391-8.

Figure Legends

Figure 1. Combinatorial integrin α5 and αv blockade is superior to monospecific blockade and BsAbα5β1/αv is superior to combinatorial integrin blockade in inhibiting prostate cancer cell migration, adhesion and induction of endothelial migration. PC-3, DU-145 or C4-2B cells were harvested and treated with 50µg/mL of MAbs or cMAbs (A) or 10µg/mL of BsAbα5β1/αv or cMAbs (20µg/mL total) (B) for 20 minutes on ice before use in functional assays. *: P< 0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.

Figure 2. Loss of integrin expression induced by BsAbα5β1/αv treatment is correlated with inhibition of prostate cancer chemotaxis and clonogenic survival. A) In PC-3 cells, BsAbα5β1/αv treatment results in loss of integrin alpha 5 and alpha v expression from baseline, compared to MAbs and cMAbs. In other lines, upregulation of alpha 5 (C4-2B) or both alpha 5 and alpha v (DU-145 and VCAP) integrins is observed variably with MsAbs and/or their combination, but in each case this upregulation is mitigated by BsAbα5β1/αv treatment. Prostate cancer cells were treated for 20 min on ice with MAbs (10 µg/mL), cMAbs (20µg/mL total) or BsAbα5β1/αv (10µg/mL) before being plated for culture. After 48h, cell extracts were harvested for WB assessment of integrin expression. B) The migration and clonogenic survival of AR-negative prostate cancer cells assessed 48h after treatment is more significantly impaired by BsAbα5β1/αv compared to cMAbs (PC-3, DU-145). In AR-positive lines e.g. C4-2B, αv-directed treatments universally resulted in a marked loss in migratory and clonogenic capacity. (C) Photomicrograph of LNCaP cells 48h post-treatment exemplifies markedly reduced numbers of filopodia-like projections with BsAbα5β1/αv. *: P< 0.05, **: P<0.01, ***: P<0.001.

Figure 3. Regulation of integrin expression dynamics by BsAbα5β1/αv is post- translational and related to altered trafficking and lysosomal degradation of target integrins. A) In PC-3 cells, BsAbα5β1/αv treatment results in progressive loss (over 24h+) of membrane alpha v expression. B) ITGA5 and ITGAV transcript expression is not significantly altered (fold change > 1.5 or <0.5) in PC-3 cells following MAb, CMAb or BsAbα5β1/αv treatment. Blockade of protein translation with cycloheximide failed to abrogate the differential impact of MsAbs or BsAbαv/α5β1 on integrin expression in PC- 3 cells (C). Lysosomal inhibition with chloroquine blocked the downregulation of integrin expression with BsAbα5β1/αv therapy in PC-3 cells (D). (C: 10µg/mL cycloheximide, D: 50µM chloroquine 50µM, Selleck Chem, Houston TX).

Figure 1

Figure 2

Figure 3

Epithelial-stromal interactions in the tumor microenvironment

Mesenchymal stromal cells

Niche Fibronectin containing matrix

Homing and survival MMP 1 Prostate cancer Legend

2 α5β1 αv

Proliferative escape Endothelial and secondary seeding cells

Advantages of a bispecific antibody strategy

Increased binding precision Increased apparent a nity

1 2 1

α5β1 αv α5β1 αv α5β1 αv α5β1 αv

Physical blocking Cross-linking Two binding events One binding event

Loss of target expression 1 2 3

α5β1 αv α5β1 αv

α5β1 αv

Binding Endocytosis/blocked recycling Lysosomal degradation

© 2019 American Association for Cancer Research Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2019 American Association for Cancer Research. Molecular Cancer Research Rapid Impact Author Manuscript Published OnlineFirst on October 21, 2019; DOI: 10.1158/1541-7786.MCR-19-0442 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A bispecific antibody targeting the αv and α5β1 integrins induces integrin degradation in prostate cancer cells and is superior to monospecific antibodies

Raghav Joshi, Wenying Ren and Paul Mathew

Mol Cancer Res Published OnlineFirst October 21, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-19-0442

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