CD200 is an immune checkpoint aberrantly expressed in 70% of newly diagnosed patients with multiple myeloma (MM) and associated with a poor outcome. CD200 expression by tumor cells inhibits immune response through interaction with CD200R and participates in resistance to immune based therapies. However, its role in tumorigenesis remains poorly understood. In this study, we show that MM cells overexpressing CD200 present a significant proliferative advantage in serum free culture medium and clonogenic assays. The cell growth advantage mediated by CD200 in MM cells is dependent on the presence of IGF1R but independent on the presence of CD200R. Of interest, we reported that high expression of CD200 and IGF-1R is associated with a poor outcome in two independent cohorts of MM patients. Moreover, immuno-precipitation and mass spectrometry experiment identifies CD200 in the IGF1R-associated protein complex in association with AKT phosphorylation at Threonine 308 and activation. In conclusion, we demonstrate, for the first time, that CD200 supports myeloma cell growth through interaction with IGF-1R. Furthermore, combined approaches targeting CD200 and IGF-1R may provide new therapeutic avenues to target CD200 mediated cell growth function and its immunosuppressive functions associated with resistance to immune based therapies in MM.

Background/Objectives: Tumor-associated antigens are not tumor-specific antigens but proteins that are overexpressed by tumor cells and also weakly expressed at the surface of healthy tissues. Therefore, some side effects are observed when targeted by therapeutic antibodies, a phenomenon named “on-target, off-tumor toxicity”. As tumors generate an acidic microenvironment, we investigated whether we could generate pH-dependent antibodies to increase their tumor specificity. For this proof-of-concept study, we selected the tyrosine kinase receptor AXL because we already developed several antibodies against this target. Methods: To generate a pH-dependent anti-AXL antibody, we performed classical panning of a single-chain variable fragment (scFv) library using phage display at an acidic pH throughout the process. Results: After the third round of panning, 9 scFvs, among the 96 picked clones, bound to AXL at acidic pH and showed very low binding at a neutral pH. After reformatting them into IgG, two clones were selected for further study due to their strong pH-sensitive binding. Using molecular docking and alanine scanning, we found that their binding strongly depended on two histidine residues present on AXL at positions 61 and 116. Conclusions: To conclude, we set-up an easy process to generate pH-dependent antibodies that may increase their tumor-binding specificity and potentially decrease toxicity towards healthy tissues.

The involvement of calcium signaling in tumor progression has been well documented in the literature. In particular, the calcium channel TRPV6 has been identified as a key player in the physiopathology of solid tumors. At the transcriptomic level, TRPV6 is overexpressed in many cancers such as prostate, pancreatic and ovarian cancers underscoring its potential as a new therapeutic target. While some TRPV6-targeting antagonistic peptides have reached clinical stage, no antibody candidate is currently in development. Leveraging its phage display technology and unique proprietary antibody libraries, Mabqi (France), in collaboration with SATT Nord and PhyCell laboratory (France), has discovered and characterized a first-in-class human anti-TRPV6 antibody "20H3” with high clinical potential. A comprehensive series of vitro and vivo preclinical studies demonstrated the safety profile, efficacy and clinical relevance of 20H3 antibody, as a new candidate for the treatment of prostate cancer. In vitro studies demonstrated the high affinity and specificity of 20H3 antibody against TRPV6 target. Exploration of antigen-antibody interaction was performed through epitope and paratope mapping and antagonistic activity of 20H3 antibody was validated via calcium flux modulation assays. The excellent developability properties were also demonstrated through thermostability, aggregation and stability assays. In vivo, the 20H3 safety profile was confirmed in mice model. After repeated doses up to 64 mg/kg, no effect on mice body weight and no sign of toxicity in main organs collected were detected. The antitumoral potential of 20H3 antibody was evaluated in three vivo cancer models. As initial positioning is prostate cancer indication, efficacy studies in two xenografted prostate models (LNCaP and VCaP) were performed. In both models, specific tumor targeting and tumor penetration were confirmed and a strong antitumoral efficacy was demonstrated at 4-8 mg/kg with a tumor growth inhibition superior to 60 % and an increased mice survival of 62 %. Interestingly a strong efficacy was also shown in MCA205 sarcoma model. To further elucidate antibody’s mode of action, a comparison between Fc-WT and Fc-silencing format was carried out in immunocompetent and immunodeficient mice models. All together, these results demonstrated that antitumor efficacy is mediated by both paratopic region and Fc region of the antibody that potentializes the effect of the treatment. Finally, the 20H3 clinical relevance was demonstrated by immunohistochemistry studies with positive immunoreactivity in a large proportion of prostate cancer patient samples. The 20H3 antibody is the first anti-TRPV6 antibody under development, with a strong potential to advance rapidly into clinic. The CMC process of 20H3 drug candidate is planned in 2025 and Phase Ia clinical trial is expected to start in early 2027. Lindsay B. Alcaraz, Vincent Roux-Portalez, Aurélien Haustrate, Gautier Robin, Alexia Parmentier, Clément Cordier, Florence Boissière-Michot, Amélie Gudin-de-Vallerin, Evelyne Lopez-Crapez, Gaelle Fromont-Hankard, Tristan Mangeat, Margaux Maurel, Marianne Le Gall, Thierry Chardès, Bruno Robert, Yoann Sottejeau, Diego Tosi, Natacha Prevarskaya, V'yacheslav Lehen'kyi, Bernard Pau, Johanna Marines. First-in-class anti-TRPV6 antibody as a new therapeutic agent in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4770.

Background Natural killer (NK) cell therapy is considered an attractive and safe strategy for anticancer therapy. Nevertheless, when autologous or allogenic NK cells are used alone, the clinical benefit has been disappointing. This is partially due to the lack of target specificity. Recently, CD19-specific chimeric antigen receptor (CAR)-NK cells have proven to be safe and potent in patients with B-cell tumors. However, the generation of CAR-NK cells is a complicated manufacturing process. We aim at developing a targeted NK cell therapy without the need for cellular genetic modifications. We took advantage of the natural expression of the IgG Fc receptor CD16a (FcγRIIIa) to induce strong antigen-specific effector functions through antibody-dependent cell-mediated cytotoxicity (ADCC). We have generated the new technology “Pin”, which enables the arming of modified monoclonal antibodies (mAbs) onto the CD16a of ex vivo expanded NK (eNK) cells.MethodsEx vivo eNK were prepared from umbilical cord blood cells and expanded using interleukin (IL)-2/IL-15 and Epstein-Barr virus (EBV)-transformed B-lymphoblastoid feeder cells. mAbs were engineered with four substitutions called Pin mutations to increase their affinity to CD16a. eNK were incubated with anti-CD20 or anti-CD19 Pin-mAbs to generate “armed” eNK and were used to assess effector functions in vitro on cancer cell lines, lymphoma patient cells and in vivo.Results CD16a/Pin-mAb interaction is stable for several days and Pin-mAb eNK inherit the mAb specificity and exclusively induce ADCC against targets expressing the cognate antigen. Hence, Pin-mAbs confer long-term selectivity to eNK, which allows specific elimination of the target cells in several in vivo mouse models. Finally, we showed that it is possible to arm eNK with at least two Pin-mAbs simultaneously, to increase efficacy against heterogenous cancer cell populations.Conclusions The Pin technology provides an off-the-shelf NK cell therapy platform to generate CAR-like NK cells, without genetic modifications, that easily target multiple tumor antigens.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal solid tumor with an unfavorable prognosis, often resistant to conventional treatments due to its dense stroma rich in Cancer- Associated Fibroblasts (CAFs). Limited T cell infiltration and prevalence of immunosuppressive cells within the tumor hinder the efficacy of immune checkpoint inhibitors (ICIs). The objective of our project is to modify immune landscape of PDAC tumor microenvironment (TME) to induce ICIs sensitization. We thus develop novel therapeutic strategies combining an immunomodulatory cytokine Interleukine-15 (IL15), with conventional chemotherapies (Gemcitabine or Folfirinox). To accurately replicate the complex cancer-stroma interactions within the pancreatic tumor microenvironment, we constructed a three-dimensional in vitro heterospheroid model composed of xenograft-derived tumor cells from PDAC patients, both primary or immortalized CAFs, and peripheral blood mononuclear cells (PBMCs) from healthy donors. Human 3D models were first set up and characterized by cytometry, imaging mass spectrometry and immunohistochemistry. We showed that, CAFs produce components of the extracellular matrix, promote tumor cell growth, improve resistance to chemotherapy and are able to down-regulate immune cell infiltration and modulate the nature of infiltrated immune cells. Spatial analysis of spheroids using imaging mass cytometry show that CAF influences the distribution of immune cells in our models. Interestingly, Interleukine 15 treatment prompted robust infiltration of PBMCs into heterospheroids. Immunophenotyping experiments revealed a substantial shift in the nature of immune infiltration, marked by a pronounced increase in CD4+ and CD8+ T lymphocytes, gDelta T cell and NK cell populations. When combined with Gemcitabine and IL15, immune effector cell infiltration is remarkably increased, resulting in antitumoral effects and control of tumor cell growth. Moreover, therapeutic combinations induce activation of infiltrated immune effector cells with an increase of activation, degranulation and cytotoxic markers, such as IFNγ, CD107a, and Granzyme B respectively. Thus, the heterotypic spheroids described in our study are a suitable in vitro model to both characterize the influence of CAF on therapeutic effects and the mechanisms that drives immune suppressive microenvironment. Moving forward, the next steps will consist to conduct in vivo experiments using a syngeneic PDAC orthotopic model to assess whether this combined therapeutic approach sensitize the PDAC microenvironment to anti-PD1 therapies. Citation Format: Thomas Bessede, Clara Freixinos, Jennifer Barrat, Véronique Garambois, Nadia Vie, Henri-Alexandre Michaud, Julien Faget, Nathalie Bonnefoy, Céline Gongora, Bruno Robert, Laurent Gros, Christel Larbouret. Sensitizing the PDAC tumor microenvironment to immune checkpoint therapies: characterization of a PDAC 3D model to decipher immune infiltration [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B045.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal solid tumor with an unfavorable prognosis, often resistant to conventional treatments due to its dense stroma rich in Cancer- Associated Fibroblasts (CAFs). Limited T cell infiltration and prevalence of immunosuppressive cells within the tumor hinder the efficacy of immune checkpoint inhibitors (ICIs). The objective of our project is to modify immune landscape of PDAC tumor microenvironment (TME) to induce ICIs sensitization. We thus develop novel therapeutic strategies combining an immunomodulatory cytokine Interleukine-15 (IL15), with conventional chemotherapies (Gemcitabine or Folfirinox). To accurately replicate the complex cancer-stroma interactions within the pancreatic tumor microenvironment, we constructed a three-dimensional in vitro heterospheroid model composed of xenograft-derived tumor cells from PDAC patients, both primary or immortalized CAFs, and peripheral blood mononuclear cells (PBMCs) from healthy donors. Human 3D models were first set up and characterized by cytometry, imaging mass spectrometry and immunohistochemistry. We showed that, CAFs produce components of the extracellular matrix, promote tumor cell growth, improve resistance to chemotherapy and are able to down-regulate immune cell infiltration and modulate the nature of infiltrated immune cells. Spatial analysis of spheroids using imaging mass cytometry show that CAF influences the distribution of immune cells in our models. Interestingly, Interleukine 15 treatment prompted robust infiltration of PBMCs into heterospheroids. Immunophenotyping experiments revealed a substantial shift in the nature of immune infiltration, marked by a pronounced increase in CD4+ and CD8+ T lymphocytes, gDelta T cell and NK cell populations. When combined with Gemcitabine and IL15, immune effector cell infiltration is remarkably increased, resulting in antitumoral effects and control of tumor cell growth. Moreover, therapeutic combinations induce activation of infiltrated immune effector cells with an increase of activation, degranulation and cytotoxic markers, such as IFNγ, CD107a, and Granzyme B respectively. Thus, the heterotypic spheroids described in our study are a suitable in vitro model to both characterize the influence of CAF on therapeutic effects and the mechanisms that drives immune suppressive microenvironment. Moving forward, the next steps will consist to conduct in vivo experiments using a syngeneic PDAC orthotopic model to assess whether this combined therapeutic approach sensitize the PDAC microenvironment to anti-PD1 therapies. Citation Format: Thomas Bessede, Clara Freixinos, Jennifer Barrat, Véronique Garambois, Nadia Vie, Henri-Alexandre Michaud, Julien Faget, Nathalie Bonnefoy, Céline Gongora, Bruno Robert, Laurent Gros, Christel Larbouret. Sensitizing the PDAC tumor microenvironment to immune checkpoint therapies: characterization of a PDAC 3D model to decipher immune infiltration [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B046.

Amorphous Ge25Se55Te20 chalcogenide thin films have been functionalized with three organosilanes: (3-ami-nopropyl)triethoxysilane, tetraethoxysilane and a mixture of tetraethoxysilane and octyl-trimethoxysilane. The functionalized films have been studied by X-ray photoelectron spectrometry, providing an interesting insight of the surface modification process and chemistry. First, the calculated stoichiometric ratios oxygen/silicon, car-bon/silicon and nitrogen/silicon are in good agreement with the theory, demonstrating the effectiveness of the functionalization and relative precursors homocondensation. Secondly, the deconvolution of the 3d5/2 and 3d3/2 tellurium orbitals before and after functionalization show new oxidized environments. More specifically, the simultaneous disappearance of the bands at 573.0 eV and 583.4 eV attributed to Te-Ge and Te-Te bonds (resp.) and appearance of a Te-O environment (around 576.5 eV and 587.0 eV) strongly suggest covalent anchorage of the three precursors thanks to the formation of new Te-O-Si groups. This is the first report on tellurium anchorage of organosilane precursors onto chalcogenide surface.

Supplementary Table 1 from Quantitative Phosphoproteomics Reveals a Cluster of Tyrosine Kinases That Mediates Src Invasive Activity in Advanced Colon Carcinoma Cells

The annual "Antibody Industrial Symposium", co-organized by LabEx MAbImprove and MabDesign, held its 10th anniversary edition in Montpellier, France, on June 28-29, 2022. The meeting focused on new results and concepts in antibody engineering (naked, mono- or multi-specific, conjugated to drugs or radioelements) and also on new cell-based therapies, such as chimeric antigenic receptor (CAR)-T cells. The symposium, which brought together scientists from academia and industry, also addressed issues concerning the production of these molecules and cells, and the necessary steps to ensure a strong intellectual property protection of these new molecules and approaches. These two days of exchanges allowed a rich discussion among the various actors in the field of therapeutic antibodies.

Suppl.Table 1: Analysis of the cellular localization, pathways and gene expression modification of the 100 genes most correlated with AXL expression in 254 basal-like breast cancers.

Supplementary Table 2 from Quantitative Phosphoproteomics Reveals a Cluster of Tyrosine Kinases That Mediates Src Invasive Activity in Advanced Colon Carcinoma Cells

Radiolabeled-antibodies usually display non-specific liver accumulation that may impair image analysis and antibody biodistribution. Here, we investigated whether Fc silencing influenced antibody biodistribution. We compared recombinant 89Zr-labeled antibodies (human IgG1 against different targets) with wild-type Fc and with mutated Fc (LALAPG triple mutation to prevent binding to Fc gamma receptors; FcγR). After antibody injection in mice harboring xenografts of different tumor cell lines or of immortalized human myoblasts, we analyzed antibody biodistribution by PET-CT and conventional biodistribution analysis. Accumulation in liver was strongly reduced and tumor-specific targeting was increased for the antibodies with mutated Fc compared with wild-type Fc. Antibodies with reduced binding to FcγR display lower liver accumulation and better tumor-to-liver ratios. These findings need to be taken into account to improve antibody-based theragnostic approaches.

Acute pain has been associated with persistent pain sensitization of nociceptive pathways increasing the risk of transition from acute to chronic pain. We demonstrated the critical role of the FLT3- tyrosine kinase receptor, expressed in sensory neurons, in pain chronification after peripheral nerve injury. However, it is unclear whether injury-induced pain sensitization can also promote long-term mood disorders. Here, we evaluated the emotional and sensorial components of pain after a single (SI) or double paw incision (DI) and the implication of FLT3. DI mice showed an anxiodepressive-like phenotype associated with extended mechanical pain hypersensitivity and spontaneous pain when compared to SI mice. Behavioral exaggeration was associated with peripheral and spinal changes including increased microglia activation after DI versus SI. Intrathecal microglial inhibitors not only eliminated the exaggerated pain hypersensitivity produced by DI but also prevented anxiodepressive-related behaviors. Behavioral and cellular changes produced by DI were blocked in Flt3 knock-out animals and recapitulated by repeated intrathecal FL injections in naive animals. Finally, humanized antibodies against FLT3 reduced DI-induced behavioral and microglia changes. Altogether our results show that the repetition of peripheral lesions facilitate not only exaggerated nociceptive behaviors but also induced anxiodepressive disorders supported by spinal central changes that can be blocked by targeting peripheral FLT3.

Supplementary Figure 1. AXL, MER and TYRO-3 expression in Triple Negative Breast Cancer (TNBC) cell lines and PDXs by Western blot analysis. Supplementary Figure 2. AXL mRNA expression in a large dataset of breast cancer tumors. Supplementary Figure 3. The 100 genes most correlated with AXL expression in 254 basal-like breast cancers. Supplementary Figure 4. Effect of the anti-AXL monoclonal antibody 20G7-D9 on matrix degradation by breast cancer cell lines. Supplementary Figure 5. GAS6 regulates expression of EMT markers in TNBC - Western Blot quantifications. Supplementary Figure 6. E-cadherin expression in TNBC cell lines

CRISPR/Cas9 gene-editing technology allows researchers to study protein function by specifically introducing double-stranded breaks in the gene of interest then analyze its subsequent loss in sensitive biological assays. To help characterize one of a series of highly potent, conditionally active, T cell engaging bispecific molecules called COBRA™, the human EpCAM gene was disrupted in HT29 cells using CRISPR/Cas9 and guide RNA targeting its Exon 2. Although a commercially available antibody indicated loss of cell-surface expression, the EpCAM targeting bispecific COBRA was still able to lyse these cells in a T cell dependent cellular cytotoxicity assay. RT-PCR sequence analysis of these cells showed a major alternative transcript generated after CRISPR/Cas9, with Exon 1 and 3 spliced together in-frame, skipping Exon 2 completely, to express a truncated cell-surface receptor recognized by the EpCAM-COBRA. Researchers who use CRISPR/Cas9 must be cognizant of this potential to express alternative versions of their proteins and use sensitive orthogonal detection methods to ensure complete gene disruption.

We report on two selective functionalization strategies to create a chemical contrast between the active rib waveguides and the passive surrounding areas of a chalcogenide-based optical sensing system. In such configuration, the analyte could be concentrated on the waveguides and interact with the evanescent field, producing a stronger optical signature. The rib waveguides are obtained by photolithography and subsequent ion beam etching of amorphous Ge-Se-Te thin films that allow residual resist to remain above the waveguides. The first functionalization strategy consists in the reuse of the resist as a mask during the following surface modification process. It allows the functionalization of all areas around the waveguides. The second strategy consists of depositing a new sacrificial metal layer, leading to a perfect negative functionalization contrast, modifying only the waveguides. For the two strategies developed, three precursors were used. The use of a silylated fluorescein derivative allowed the validation of the protocols, with the observation of a fluorescence contrast between the functionalized and non-functionalized areas. The use of tetraethoxysilane as a hydrophilic precursor and the mixture of tetraethoxysilane and octyl-trimethoxysilane as a hydrophobic precursor created a clear contrast in wettability between the rib waveguides and the surrounding areas. A spore deposition was performed on functionalized components according to the two proposed strategies, with the two hydrophilic/hydrophobic precursors. The spore immobilization rate was increased by making the waveguides more hydrophobic, as well as by making the areas surrounding the guides more hydrophilic, demonstrating the effectiveness of our two strategies.

BACKGROUND:COBRA™ (COnditional Bispecific Redirected Activation) T-cell engagers are designed to target solid tumors as a single polypeptide chain prodrug that becomes activated by proteolysis in the tumor microenvironment. One COBRA molecule comprises seven Ig domains: three single-domain antibodies (sdAbs) recognizing a tumor target or human serum albumin (HSA), and CD3ε-binding variable fragment heavy chain (VH) and variable fragment light chain (VL) and their inactivated counterparts, VHi and VLi. Pairing of VH and VL, and VLi and VHi into single-chain variable fragments (Fv) is prevented by shortened inter-domain linkers. Instead, VH and VL are expected to interact with VLi and VHi, respectively, thus making a diabody whose binding to CD3ε on the T-cells is impaired.METHODS:We analyzed the structure of an epidermal growth factor receptor (EGFR) COBRA in solution using negative stain electron microscopy (EM) and small-angle X-ray scattering (SAXS).RESULTS:We found that this EGFR COBRA forms stable monomers with a very dynamic interdomain arrangement. At most, only five domains at a time appeared ordered, and only one VH-VL pair was found in the Fv orientation. Nonenzymatic posttranslational modifications suggest that the CDR3 loops in the VL-VHi pair are exposed but are buried in the VH-VLi pair. The MMP9 cleavage rate of the prodrug when bound to recombinant EGFR or HSA is not affected, indicating positioning of the MMP9-cleavable linker away from the EGFR and HSA binding sites.CONCLUSION:Here, we propose a model for EGFR COBRA where VH and VLi form an Fv, and VL and VHi do not, possibly interacting with other Ig domains. SAXS and MMP9 cleavage analyses suggest that all COBRA molecules tested have a similar structural architecture.

The positive effects of union canvassing on individual-level union member voter turnout within union-friendly environments have been well documented. Yet, whether unions increase turnout among their membership under constrained circumstances has remained unexamined. Furthermore, there is little consensus on whether union canvassing effects are generalizable to populations with heterogeneous political attributes and individual characteristics. This paper identifies the mechanisms that might explain how union canvassing can be effective under conditions characterized by anti-union legislative actions, adversarial judicial decisions, and right-wing populist rhetoric. We use canvassing and turnout data taken from the 2016 Democratic state and Cook County primary election in Illinois, and our results show that, despite constrained political circumstances relative to those found in previous studies, union canvassing achieved positive union membership turnout effects. This study also tests the moderating effects of individual political attributes (ideology and vote propensity) and voter characteristics (income and ethnicity). The most salient finding is that the effects are more potent for ideologically conservative registered Democrat voters, highlighting the imperative of recognizing the ideological heterogeneity among union members and suggesting specific resource allocation strategies under politically constrained conditions.