SPARC in cancer-associated fibroblasts is an independent poor prognostic factor in non-metastatic triple-negative breast cancer and exhibits pro-tumor activity.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and lacks specific targeted therapeutic agents. The current mechanistic evidence from cell-based studies suggests that the matricellular protein SPARC has a tumor-promoting role in TNBC; however, data on the clinical relevance of SPARC expression/secretion by tumor and stromal cells in TNBC are limited. Here, we analyzed by immunohistochemistry the prognostic value of tumor and stromal cell SPARC expression in 148 patients with non-metastatic TNBC and long follow-up (median: 5.4 years). We also quantified PD-L1 and PD-1 expression. We detected SPARC expression in tumor cells (42.4%), cancer-associated fibroblasts (CAFs; 88.1%), tumor-associated macrophages (77.1%), endothelial cells (75.2%) and tumor-infiltrating lymphocytes (9.8%). Recurrence-free survival was significantly lower in patients with SPARC-expressing CAFs. Multivariate analysis showed that SPARC expression in CAFs was an independent prognostic factor. We also detected tumor and stromal cell SPARC expression in TNBC cytosols, and in patient-derived xenografts and cell lines. Furthermore, we analyzed publicly available single-cell mRNA sequencing data and found that in TNBC, SPARC is expressed by different CAF subpopulations, including myofibroblasts and inflammatory fibroblasts that are involved in tumor-related processes. We then showed that fibroblast-secreted SPARC had a tumor-promoting role by inhibiting TNBC cell adhesion and stimulating their motility and invasiveness. Overall, our study demonstrates that SPARC expression in CAFs is an independent prognostic marker of poor outcome in TNBC. Patients with SPARC-expressing CAFs could be eligible for anti-SPARC targeted therapy.

Biasing human epidermal growth factor receptor 4 (HER4) tyrosine kinase signaling with antibodies: Induction of cell death by antibody-dependent HER4 intracellular domain trafficking.

Human epidermal growth factor receptor 4 (HER4) isoforms have oncogenic or tumor suppressor functions depending on their susceptibility to proteolytic cleavage and HER4 intracellular domain (4ICD) translocation. Here, we report that the neuregulin 1 (NRG1) tumor suppressor mechanism through the HER4 JMa/CYT1 isoform can be mimicked by the agonist anti-HER4 Ab C6. Neuregulin 1 induced cleavage of poly(ADP-ribose) polymerase (PARP) and sub-G1 DNA fragmentation, and also reduced the metabolic activity of HER3- /HER4+ cervical (C-33A) and ovarian (COV318) cancer cells. This effect was confirmed in HER4 JMa/CYT1-, but not JMa/CYT2-transfected BT549 triple-negative breast cancer cells. Neuregulin 1 favored 4ICD cleavage and retention in mitochondria in JMa/CYT1-transfected BT549 cells, leading to reactive oxygen species (ROS) production through mitochondrial depolarization. Similarly, the anti-HER4 Ab C6, which binds to a conformational epitope located on a.a. 575-592 and 605-620 of HER4 domain IV, induced 4ICD cleavage and retention in mitochondria, and mimicked NRG1-mediated effects on PARP cleavage, ROS production, and mitochondrial membrane depolarization in cancer cells. In vivo, C6 reduced growth of COV434 and HCC1187 tumor cell xenografts in nude mice. Biasing 4ICD trafficking to mitochondria with anti-HER4 Abs to mimic NRG1 suppressor functions could be an alternative anticancer strategy.

Quantification of HER1, HER2 and HER3 by time-resolved Förster resonance energy transfer in FFPE triple-negative breast cancer samples.

BACKGROUND: Triple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed. Here, we investigated the expression of human epidermal receptor (HER) family members in TNBC and evaluated their potential as biomarkers of recurrence. METHODS: We developed Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays to quantify HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour tissues. After assessing the performance and precision of our assays, we quantified HER protein expression in 51 TNBC specimens, and investigated the association of their expression with relapse-free survival. RESULTS: The assays were quantitative, accurate, and robust. In TNBC specimens, HER1 levels ranged from ≈4000 to more than 2 million receptors per cell, whereas HER2 levels varied from ≈1000 to 60,000 receptors per cell. HER3 expression was very low (less than 5500 receptors per cell in all samples). Moderate HER2 expression was significantly associated with higher risk of recurrence (HR = 3.93; P = 0.003). CONCLUSIONS: Our TR-FRET assays accurately quantify HER1, HER2 and HER3 in FFPE breast tumour specimens. Moderate HER2 expression may represent a novel prognostic marker in patients with TNBC.

An auristatin-based antibody-drug conjugate targeting HER3 enhances the radiation response in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer characterized by poor response to chemotherapy and radiotherapy due to the lack of efficient therapeutic tools and early diagnostic markers. We previously generated the nonligand competing anti-HER3 antibody 9F7-F11 that binds to pancreatic tumor cells and induces tumor regression in vivo in experimental models. Here, we asked whether coupling 9F7-F11 with a radiosensitizer, such as monomethylauristatin E (MMAE), by using the antibody-drug conjugate (ADC) technology could improve radiation therapy efficacy in PDAC. We found that the MMAE-based HER3 antibody-drug conjugate (HER3-ADC) was efficiently internalized in tumor cells, increased the fraction of cells arrested in G2/M, which is the most radiosensitive phase of the cell cycle, and promoted programmed cell death of irradiated HER3-positive pancreatic cancer cells (BxPC3 and HPAC cell lines). HER3-ADC decreased the clonogenic survival of irradiated cells by increasing DNA double-strand break formation (based on γH2AX level), and by modulating DNA damage repair. Tumor radiosensitization with HER3-ADC favored the inhibition of the AKT-induced survival pathway, together with more efficient caspase 3/PARP-mediated apoptosis. Incubation with HER3-ADC before irradiation synergistically reduced the phosphorylation of STAT3, which is involved in chemoradiation resistance. In vivo, the combination of HER3-ADC with radiation therapy increased the overall survival of mice harboring BxPC3, HPAC cell xenografts or patient-derived xenografts, and reduced proliferation (KI67-positive cells). Combining auristatin radiosensitizer delivery via an HER3-ADC with radiotherapy is a new promising therapeutic strategy in PDAC.

ITCH-dependent proteasomal degradation of c-FLIP induced by the anti-HER3 antibody 9F7-F11 promotes DR5/caspase 8-mediated apoptosis of tumor cells.

BACKGROUND: HER3/ErbB3 receptor deletion or blockade leads to tumor cell apoptosis, whereas its overexpression confers anti-cancer drug resistance through upregulation of protective mechanisms against apoptosis. We produced the anti-HER3 antibody 9F7-F11 that promotes HER3 ubiquitination and degradation via JNK1/2-dependent activation of the E3 ubiquitin ligase ITCH, and that induces apoptosis of cancer cells. Cellular FLICE-like inhibitory protein (c-FLIP) is a key regulator of apoptotic pathways. Here, we wanted to determine the mechanisms underlying the pro-apoptotic effect of 9F7-F11. METHODS: Anti-HER3 antibody-induced apoptosis was assessed by western blot, and by flow cytometry measurement of Annexin V/7-AAD-labelled tumor cells (BxPC3, MDA-MB-468 and DU145 cell lines). c-FLIP/ITCH interaction and subsequent degradation/ubiquitination were investigated by co-immunoprecipitation of ITCH-silenced vs scramble control cells. The relationship between ITCH-mediated c-FLIP degradation and antibody-induced apoptosis was examined by western blot and flow cytometry of tumor cells, after ITCH RNA interference or by pre-treatment with ITCH chemical inhibitor chlorimipramine (CI). RESULTS: Following incubation with 9F7-F11, cancer cell apoptosis occurs through activation of caspase-8, - 9 and - 3 and the subsequent cleavage of poly (ADP-ribose) polymerase (PARP). Moreover we showed that ubiquitination and proteasomal degradation of the anti-apoptotic protein c-FLIP was mediated by USP8-regulated ITCH recruitment. This effect was abrogated by ITCH- and USP8-specific RNA interference (siRNA), or by the ITCH chemical inhibitor CI. Specifically, ITCH silencing or CI blocked 9F7-F11-induced caspase-8-mediated apoptosis of tumor cells, and restored c-FLIP expression. ITCH-silencing or CI concomitantly abrogated HER3-specific antibody-induced apoptosis of Annexin V/7-AAD-labelled BxPC3 cells. 9F7-F11 favored the extrinsic apoptosis pathway by inducing TRAIL-R2/DR5 upregulation and TRAIL expression that promoted the formation of death-inducing signaling complex (DISC), leading to caspase-8-mediated apoptosis. Incubation with 9F7-F11 also induced BID cleavage, BAX upregulation and BIM expression, which initiated the caspase-9/3-mediated mitochondrial death pathway. The anti-HER3 antibody pro-apoptotic effect occurred concomitantly with downregulation of the pro-survival proteins c-IAP2 and XIAP. CONCLUSIONS: The allosteric non-neuregulin competing modulator 9F7-F11, sensitizes tumor cells to DR5/caspase-8-mediated apoptosis through ITCH-dependent downregulation of c-FLIP.

Examination of HER3 targeting in cancer using monoclonal antibodies.

The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells' ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma-tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients.

A novel Fc-engineered cathepsin D-targeting antibody enhances ADCC, triggers tumor-infiltrating NK cell recruitment, and improves treatment with paclitaxel and enzalutamide in triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC. METHODS: Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc+) or prevent (F1M1-Fc-) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc+ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide. RESULTS: Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc+ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc- was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc+ displayed higher antitumor activity than F1M1, whereas F1M1-Fc- was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc+ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc+ antitumor activity, demonstrating their key role. F1M1-Fc+ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc+ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity. CONCLUSIONS: F1M1-Fc+ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.

Anti-cathepsin D immunotherapy triggers both innate and adaptive anti-tumour immunity in breast cancer.

BACKGROUND AND PURPOSE: Triple-negative breast cancer (TNBC) has poorer outcomes than other breast cancers (BC), including HER2+ BC. Cathepsin D (CathD) is a poor prognosis marker overproduced by BC cells, hypersecreted in the tumour microenvironment with tumour-promoting activity. Here, we characterized the immunomodulatory activity of the anti-CathD antibody F1 and its improved Fab-aglycosylated version (F1M1) in immunocompetent mouse models of TNBC (C57BL/6 mice harbouring E0771 cell grafts) and HER2-amplified BC (BALB/c mice harbouring TUBO cell grafts). EXPERIMENTAL APPROACH: CathD expression was evaluated by western blotting and immunofluorescence, and antibody binding to CathD by ELISA. Antibody anti-tumour efficacy was investigated in mouse models. Immune cell recruitment and activation were assessed by immunohistochemistry, immunophenotyping, and RT-qPCR. KEY RESULTS: F1 and F1M1 antibodies remodelled the tumour immune landscape. Both antibodies promoted innate antitumour immunity by preventing the recruitment of immunosuppressive M2-polarized tumour-associated macrophages (TAMs) and by activating natural killer cells in the tumour microenvironment of both models. This translated into a reduction of T-cell exhaustion markers in the tumour microenvironment that could be locally supported by enhanced activation of anti-tumour antigen-presenting cell (M1-polarized TAMs and cDC1 cells) functions. Both antibodies inhibited tumour growth in the highly-immunogenic E0771 model, but only marginally in the immune-excluded TUBO model, indicating that anti-CathD immunotherapy is more relevant for BC with a high immune cell infiltrate, as often observed in TNBC. CONCLUSION AND IMPLICATION: Anti-CathD antibody-based therapy triggers the anti-tumour innate and adaptive immunity in preclinical models of BC and is a promising immunotherapy for immunogenic TNBC.

Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.

Time-resolved fluorescence resonance energy transfer (TR-FRET) to analyze the disruption of EGFR/HER2 dimers: a new method to evaluate the efficiency of targeted therapy using monoclonal antibodies.

In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation.

Vaccination with human anti-trastuzumab anti-idiotype scFv reverses HER2 immunological tolerance and induces tumor immunity in MMTV.f.huHER2(Fo5) mice.

INTRODUCTION: Novel adjuvant therapies are needed to prevent metastatic relapses in HER2-expressing breast cancer. Here, we tested whether trastuzumab-selected single-chain Fv (scFv) could be used to develop an anti-idiotype-based vaccine to inhibit growth of HER2-positive tumor cells in vitro and in vivo through induction of long-lasting HER-specific immunity. METHODS: BALB/c mice were immunized with anti-trastuzumab anti-idiotype (anti-Id) scFv (scFv40 and scFv69), which mimic human HER2. Their sera were assessed for the presence of HER2-specific Ab1' antibodies and for their ability to reduce viability of SK-OV-3 cells, a HER2-positive cancer cell line, in nude mice. MMTV.f.huHER2(Fo5) transgenic mice were immunized with scFv40 and scFv69 and, then, growth inhibition of spontaneous HER2-positive mammary tumors, humoral response, antibody isotype as well as splenocyte secretion of IL2 and IFN-γ were evaluated. RESULTS: Adoptively-transferred sera from BALB/c mice immunized with scFv40 and scFv69 contain anti-HER2 Ab1' antibodies that can efficiently inhibit growth of SK-OV-3 cell tumors in nude mice. Similarly, prophylactic vaccination with anti-Id scFv69 fully protects virgin or primiparous FVB-MMTV.f.huHER2(Fo5) females from developing spontaneous mammary tumors. Moreover, such vaccination elicits an anti-HER2 Ab1' immune response together with a scFv69-specific Th1 response with IL2 and IFN-γ cytokine secretion. CONCLUSIONS: Anti-trastuzumab anti-Id scFv69, used as a therapeutic or prophylactic vaccine, protects mice from developing HER2-positive mammary tumors by inducing both anti-HER2 Ab1' antibody production and an anti-HER2 Th2-dependent immune response. These results suggest that scFv69 could be used as an anti-Id-based vaccine for adjuvant therapy of patients with HER2-positive tumors to reverse immunological tolerance to HER2.

CD4 ligation excludes the Carma1-Bcl10-MALT1 complex from GM1-positive membrane rafts in CD3/CD28 activated T cells.

The antibody 13B8.2, which is directed against the CDR3-like loop on the D1 domain of CD4, induces CD4/ZAP-70 reorganization and ceramide release in membrane rafts. Here, we investigated whether CD4/ZAP-70 compartmentalization could be mediated by an effect of 13B8.2 on the Carma1-Bcl10-MALT1 complex in membrane rafts. We report that treatment of CD3/CD28-activated Jurkat T cells with 13B8.2, but not rituximab, excluded Carma1-Bcl10-MALT1 proteins from GM1(+) membrane rafts and concomitantly decreased NF-κB activation. Fluorescence confocal imaging confirmed that Carma1-Bcl10 and Carma1-MALT1 co-patching, observed in GM1(+) membrane rafts following CD3/CD28 activation, were abrogated after a 24h-treatment with 13B8.2. The CD4/ZAP-70 compartmentalization in membrane rafts induced by 13B8.2 is thus related to Carma1-Bcl10-MALT1 raft exclusion.

Improving Biologics' Effectiveness in Clinical Oncology: From the Combination of Two Monoclonal Antibodies to Oligoclonal Antibody Mixtures.

Monoclonal antibodies have revolutionized the treatment of many diseases, but their clinical efficacy remains limited in some other cases. Pre-clinical and clinical trials have shown that combinations of antibodies that bind to the same target (homo-combinations) or to different targets (hetero-combinations) to mimic the polyclonal humoral immune response improve their therapeutic effects in cancer. The approval of the trastuzumab/pertuzumab combination for breast cancer and then of the ipilimumab/nivolumab combination for melanoma opened the way to novel antibody combinations or oligoclonal antibody mixtures as more effective biologics for cancer management. We found more than 300 phase II/III clinical trials on antibody combinations, with/without chemotherapy, radiotherapy, small molecules or vaccines, in the ClinicalTrials.gov database. Such combinations enhance the biological responses and bypass the resistance mechanisms observed with antibody monotherapy. Usually, such antibody combinations are administered sequentially as separate formulations. Combined formulations have also been developed in which separately produced antibodies are mixed before administration or are produced simultaneously in a single cell line or a single batch of different cell lines as a polyclonal master cell bank. The regulation, toxicity and injection sequence of these oligoclonal antibody mixtures still need to be addressed in order to optimize their delivery and their therapeutic effects.

Anti-Müllerian hormone (AMH) autocrine signaling promotes survival and proliferation of ovarian cancer cells.

In ovarian carcinoma, anti-Müllerian hormone (AMH) type II receptor (AMHRII) and the AMH/AMHRII signaling pathway are potential therapeutic targets. Here, AMH dose-dependent effect on signaling and proliferation was analyzed in four ovarian cancer cell lines, including sex cord stromal/granulosa cell tumors and high grade serous adenocarcinomas (COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN). As previously shown, incubation with exogenous AMH at concentrations above the physiological range (12.5-25 nM) decreased cell viability. Conversely, physiological concentrations of endogenous AMH improved cancer cell viability. Partial AMH depletion by siRNAs was sufficient to reduce cell viability in all four cell lines, by 20% (OVCAR8 cells) to 40% (COV434-AMHRII cells). In the presence of AMH concentrations within the physiological range (5 to 15 pM), the newly developed anti-AMH B10 antibody decreased by 25% (OVCAR8) to 50% (KGN) cell viability at concentrations ranging between 3 and 333 nM. At 70 nM, B10 reduced clonogenic survival by 57.5%, 57.1%, 64.7% and 37.5% in COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN cells, respectively. In the four cell lines, B10 reduced AKT phosphorylation, and increased PARP and caspase 3 cleavage. These results were confirmed in ovarian cancer cells isolated from patients' ascites, demonstrating the translational potential of these results. Furthermore, B10 reduced COV434-MISRII tumor growth in vivo and significantly enhanced the median survival time compared with vehicle (69 vs 60 days; p = 0.0173). Our data provide evidence for a novel pro-survival autocrine role of AMH in the context of ovarian cancer, which was targeted therapeutically using an anti-AMH antibody to successfully repress tumor growth.

Anti-Müllerian hormone concentration regulates activin receptor-like kinase-2/3 expression levels with opposing effects on ovarian cancer cell survival.

Anti­Müllerian hormone (AMH) type II receptor (AMHRII) and the AMH/AMHRII signaling pathway are potential therapeutic targets in ovarian carcinoma. Conversely, the role of the three AMH type I receptors (AMHRIs), namely activin receptor­like kinase (ALK)2, ALK3 and ALK6, in ovarian cancer remains to be clarified. To determine the respective roles of these three AMHRIs, the present study used four ovarian cancer cell lines (COV434­AMHRII, SKOV3­AMHRII, OVCAR8, KGN) and primary cells isolated from tumor ascites from patients with ovarian cancer. The results demonstrated that ALK2 and ALK3 may be the two main AMHRIs involved in AMH signaling at physiological endogenous and supraphysiological exogenous AMH concentrations, respectively. Supraphysiological AMH concentrations (25 nM recombinant AMH) were associated with apoptosis in all four cell lines and decreased clonogenic survival in COV434­AMHRII and SKOV3­AMHRII cells. These biological effects were induced via ALK3 recruitment by AMHRII, as ALK3­AMHRII dimerization was favored at increasing AMH concentrations. By contrast, ALK2 was associated with AMHRII at physiological endogenous concentrations of AMH (10 pM). Based on these results, tetravalent IgG1­like bispecific antibodies (BsAbs) against AMHRII and ALK2, and against AMHRII and ALK3 were designed and evaluated. In vivo, COV434­AMHRII tumor cell xenograft growth was significantly reduced in all BsAb­treated groups compared with that in the vehicle group (P=0.018 for BsAb 12G4­3D7; P=0.001 for all other BsAbs). However, the growth of COV434­AMHRII tumor cell xenografts was slower in mice treated with the anti­AMRII­ALK2 BsAb 12G4­2F9 compared with that in animals that received a control BsAb that targeted AMHRII and CD5 (P=0.048). These results provide new insights into type I receptor specificity in AMH signaling pathways and may lead to an innovative therapeutic approach to modulate AMH signaling using anti­AMHRII/anti­AMHRI BsAbs.

Anti-tumoral activity of the Pan-HER (Sym013) antibody mixture in gemcitabine-resistant pancreatic cancer models.

Chemoresistance, particularly to gemcitabine, is a major challenge in pancreatic cancer. The epidermal growth factor receptor (EGFR) and human epidermal growth factor receptors 2 and 3 (HER2, HER3) are expressed in many tumors, and they are relevant therapeutic targets due to their synergistic interaction to promote tumor aggressiveness and therapeutic resistance. Cocktails of antibodies directed against different targets are a promising strategy to overcome these processes. Here, we found by immunohistochemistry that these three receptors were co-expressed in 11% of patients with pancreatic adenocarcinoma. We then developed gemcitabine-resistant pancreatic cancer cell models (SW-1990-GR and BxPC3-GR) and one patient-derived xenograft (PDX2846-GR) by successive exposure to increasing doses of gemcitabine. We showed that expression of EGFR, HER2 and HER3 was increased in these gemcitabine-resistant pancreatic cancer models, and that an antibody mixture against all three receptors inhibited tumor growth in mice and downregulated HER receptors. Finally, we demonstrated that the Pan-HER and gemcitabine combination has an additive effect in vitro and in mice xenografted with the gemcitabine-sensitive or resistant pancreatic models. The mixture of anti-EGFR, HER2 and HER3 antibodies is a good candidate therapeutic approach for gemcitabine-sensitive and -resistant pancreatic cancer.

A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment.

Rationale: Alternative therapeutic strategies based on tumor-specific molecular targets are urgently needed for triple-negative breast cancer (TNBC). The protease cathepsin D (cath-D) is a marker of poor prognosis in TNBC and a tumor-specific extracellular target for antibody-based therapy. The identification of cath-D substrates is crucial for the mechanistic understanding of its role in the TNBC microenvironment and future therapeutic developments. Methods: The cath-D substrate repertoire was investigated by N-Terminal Amine Isotopic Labeling of Substrates (TAILS)-based degradome analysis in a co-culture assay of TNBC cells and breast fibroblasts. Substrates were validated by amino-terminal oriented mass spectrometry of substrates (ATOMS). Cath-D and SPARC expression in TNBC was examined using an online transcriptomic survival analysis, tissue micro-arrays, TNBC cell lines, patient-derived xenografts (PDX), human TNBC samples, and mammary tumors from MMTV-PyMT Ctsd-/- knock-out mice. The biological role of SPARC and its fragments in TNBC were studied using immunohistochemistry and immunofluorescence analysis, gene expression knockdown, co-culture assays, western blot analysis, RT-quantitative PCR, adhesion assays, Transwell motility, trans-endothelial migration and invasion assays. Results: TAILS analysis showed that the matricellular protein SPARC is a substrate of extracellular cath-D. In vitro, cath-D induced limited proteolysis of SPARC C-terminal extracellular Ca2+ binding domain at acidic pH, leading to the production of SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa). Similarly, cath-D secreted by TNBC cells cleaved fibroblast- and cancer cell-derived SPARC at the tumor pericellular acidic pH. SPARC cleavage also occurred in TNBC tumors. Among these fragments, only the 9-kDa SPARC fragment inhibited TNBC cell adhesion and spreading on fibronectin, and stimulated their migration, endothelial transmigration, and invasion. Conclusions: Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited SPARC proteolysis, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments. Our study will pave the way for the development of strategies for targeting bioactive fragments from matricellular proteins in TNBC.

Anti-HER2 vaccines: new prospects for breast cancer therapy.

Each year, breast cancer accounts for more than 400,000 new cancer cases and more than 130,000 cancer deaths in Europe. Prognosis of nonmetastatic breast cancer patients is directly related to the extent of the disease, mainly nodal spreading and tumor size, and to the molecular profile, particularly HER2 over-expression. In patients with HER2-over-expressing tumors, different studies have shown cellular and/or humoral immune responses against HER2 associated with a lower tumor development at early stages of the disease. These findings have led to the hypothesis that the generation of an anti-HER2 immune response should protect patients from HER2-over-expressing tumor growth. Taken together with the clinical efficiency of trastuzumab-based anti-HER2 passive immunotherapy, these observations allowed to envisage various vaccine strategies against HER2. The induction of a stable and strong immunity by cancer vaccines is expected to lead to establishment of immune memory, thereby preventing tumor recurrence. However, an immunological tolerance against HER2 antigen exists representing a barrier to effective vaccination against this oncoprotein. As a consequence, the current challenge for vaccines is to find the best conditions to break this immunological tolerance. In this review, we will discuss the different anti-HER2 vaccine strategies currently developed; considering the strategies having reached the clinical phases as well as those still in preclinical development. The used antigen can be either composed of tumoral allogenic cells or autologous cells, or specific to HER2. It can be delivered by dendritic cells or in a DNA, peptidic or proteic form. Another area of research concerns the use of anti-idiotypic antibodies mimicking HER2.

Higher Anti-Tumor Efficacy of the Dual HER3-EGFR Antibody MEHD7945a Combined with Ionizing Irradiation in Cervical Cancer Cells.

PURPOSE: The outcome of locally advanced cervical cancer (LACC) is dismal. Biomarkers are needed to individualize treatments and to improve patient outcomes. Here, we investigated whether coexpression of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3) could be an outcome prognostic biomarker, and whether targeting both EGFR and HER3 with a dual antibody (MEHD7945A) enhanced ionizing radiation (IR) efficacy. METHODS AND MATERIALS: Expression of EGFR and HER3 was evaluated by immunohistochemistry in cancer biopsies (n = 72 patients with LACC). The antitumor effects of the MEHD7945A and IR combotherapy were assessed in 2 EGFR- and HER3-positive cervical cancer cell lines (A431 and CaSki) and in A431 cell xenografts. The mechanisms involved in tumor cell radiosensitization were also studied. The interaction of MEHD7945A, IR, and cisplatin was evaluated using dose-response matrix data. RESULTS: EGFR and HER3 were coexpressed in only in 7 of the 22 biopsies of FIGO IVB cervix cancer. The median overall survival was 14.6 months and 23.1 months in patients with FIGO IVB tumors that coexpressed or did not coexpress EGFR and HER3, respectively. In mice xenografted with A431 (squamous cell carcinoma) cells, MEHD7945A significantly increased IR response by reducing tumor growth and increasing cleaved caspase-3 expression. In A431 and CaSki cells, the combotherapy increased DNA damage and cell death, particularly immunogenic cell death, and decreased survival by inhibiting the MAPK and AKT pathways. An additive effect was observed when IR, MEHD7945A, and cisplatin were combined. CONCLUSIONS: Targeting EGFR and HER3 with a specific dual antibody enhanced IR efficacy. These preliminary results and the prognostic value of EGFR and HER3 coexpression should be confirmed in a larger sample.