Implication de la neuropiline-2 dans la Transition-Epithélio- Mésenchymateuse

Neuropilins (NRPs) are transmembrane non tyrosine-kinase glycoproteins first identified as receptors for class-3 semaphorins. They are particularly involved in neural crest migration and axonal growth during development of thé nervous System. Since many types of tumor and endothelial cells express NRP receptors. various soluble molécules were also found to interact with thèse receptors to modulate cancer progression. Among them, angiogenic factors belonging to thé Vascular Endothelial Growth Factor (VEGF) family seem to be responsible for NRPs-related angiogenesis.While NRP1 was intensively studied from many years and identified as an attractive angiogenesis target for cancer therapy. NRP2 signaling pathway has just recently been studied. NRP2 may regulate tumor progression by several concurrent mechanisms, not only angiogenesis but lymphangiogenesis, epithelial-mesenchymal transition and metastasis. In view of their multiples functions in cancer promotion, NRP2 fulfills ail thé criteria of a therapeutic target for innovative anti-tumor thérapies. Our thesis focuses on NRP2-specific rôles in tumor progression and subséquent development of a NRP2-neutralizing monoclonal antibody.Les Neuropilines (NRPs) sont des récepteurs transmembranaires non tyrosine-kinase identifiés à l'origine comme des récepteurs pour les sémaphorines de la classe 3. Ces glycoprotéines sont particulièrements impliquées dans la migration de la crête neurale et dans la croissance axonale au cours du développement embryonnaire du système nerveux. En outre, les NRPs sont exprimées par une large variété de tumeurs et de nombreuses molécules solubles semblent interagir avec ces protéines pour moduler la progression tumorale. Parmi elles, les facteurs angiogéniques de la famille du facteur de croissance de l'endothélium vasculaire (VEGF) semblent être à l'origine d'une angiogénèse médiée par les NRPs.Tandis que la NRP1 a été largement étudiée et reconnue comme une cible intéressante dans le cadre du développement de thérapies anti-angiogéniques, très peu d'études s'étaient intéressées au rôle de la NRP2 dans la progression tumorale jusqu'à présent. La NRP2 semble réguler la progression tumorale par de nombreux mécanismes, non seulement l'angiogénèse mais aussi la lymphangiogénèse, la Transition-Epithélio-Mésenchymateuse (TEM) et la formation de métastases. A la vue de ces multiples rôles dans la progression tumorale, la NRP2 apparaît donc comme une cible intéressante dans le cadre du développement de thérapies ciblées innovantes en cancérologie. Au cours de notre thèse, nous nous sommes attachés à caractériser le rôle de la NRP2 dans la progression tumorale dans le cadre du cancer colorectal puis nous avons développé un anticorps monoclonal thérapeutique ciblant spécifiquement cette protéine

Development of ISB 1442, a CD38 and CD47 bispecific biparatopic antibody innate cell modulator for the treatment of multiple myeloma

Antibody engineering can tailor the design and activities of therapeutic antibodies for better efficiency or other advantageous clinical properties. Here we report the development of ISB 1442, a fully human bispecific antibody designed to re-establish synthetic immunity in CD38+ hematological malignancies. ISB 1442 consists of two anti-CD38 arms targeting two distinct epitopes that preferentially drive binding to tumor cells and enable avidity-induced blocking of proximal CD47 receptors on the same cell while preventing on-target off-tumor binding on healthy cells. The Fc portion of ISB 1442 is engineered to enhance complement dependent cytotoxicity, antibody dependent cell cytotoxicity and antibody dependent cell phagocytosis. ISB 1442 thus represents a CD47-BsAb combining biparatopic targeting of a tumor associated antigen with engineered enhancement of antibody effector function to overcome potential resistance mechanisms that hamper treatment of myeloma with monospecific anti-CD38 antibodies. ISB 1442 is currently in a Phase I clinical trial in relapsed refractory multiple myeloma

Neuropiline-2 role in Epithélio to Mésenchymal Transition

Les Neuropilines (NRPs) sont des récepteurs transmembranaires non tyrosine-kinase identifiés à l'origine comme des récepteurs pour les sémaphorines de la classe 3. Ces glycoprotéines sont particulièrements impliquées dans la migration de la crête neurale et dans la croissance axonale au cours du développement embryonnaire du système nerveux. En outre, les NRPs sont exprimées par une large variété de tumeurs et de nombreuses molécules solubles semblent interagir avec ces protéines pour moduler la progression tumorale. Parmi elles, les facteurs angiogéniques de la famille du facteur de croissance de l'endothélium vasculaire (VEGF) semblent être à l'origine d'une angiogénèse médiée par les NRPs.Tandis que la NRP1 a été largement étudiée et reconnue comme une cible intéressante dans le cadre du développement de thérapies anti-angiogéniques, très peu d'études s'étaient intéressées au rôle de la NRP2 dans la progression tumorale jusqu'à présent. La NRP2 semble réguler la progression tumorale par de nombreux mécanismes, non seulement l'angiogénèse mais aussi la lymphangiogénèse, la Transition-Epithélio-Mésenchymateuse (TEM) et la formation de métastases. A la vue de ces multiples rôles dans la progression tumorale, la NRP2 apparaît donc comme une cible intéressante dans le cadre du développement de thérapies ciblées innovantes en cancérologie. Au cours de notre thèse, nous nous sommes attachés à caractériser le rôle de la NRP2 dans la progression tumorale dans le cadre du cancer colorectal puis nous avons développé un anticorps monoclonal thérapeutique ciblant spécifiquement cette protéine.Neuropilins (NRPs) are transmembrane non tyrosine-kinase glycoproteins first identified as receptors for class-3 semaphorins. They are particularly involved in neural crest migration and axonal growth during development of thé nervous System. Since many types of tumor and endothelial cells express NRP receptors. various soluble molécules were also found to interact with thèse receptors to modulate cancer progression. Among them, angiogenic factors belonging to thé Vascular Endothelial Growth Factor (VEGF) family seem to be responsible for NRPs-related angiogenesis.While NRP1 was intensively studied from many years and identified as an attractive angiogenesis target for cancer therapy. NRP2 signaling pathway has just recently been studied. NRP2 may regulate tumor progression by several concurrent mechanisms, not only angiogenesis but lymphangiogenesis, epithelial-mesenchymal transition and metastasis. In view of their multiples functions in cancer promotion, NRP2 fulfills ail thé criteria of a therapeutic target for innovative anti-tumor thérapies. Our thesis focuses on NRP2-specific rôles in tumor progression and subséquent development of a NRP2-neutralizing monoclonal antibody

Ca²⁺ flux in gated NK cells in response to odorants.

<p><b>A)</b> Total splenocytes from naive mice were stained with mAbs to CD3 and DX5 followed by cell loading with the Ca²⁺ sensitive dye Indo-1-AM. Gated CD3^- DX5⁺ NK cells were analyzed for the ratio of Indo-1 violet to blue fluorescence emission (Ratio Indo-1V/Indo-1B) over time before and after (indicated by an arrow) the addition of Bourgeonal. In the middle panel each dot represents an individual cell and the mean Indo-1V/Indo-1B ratio is plotted in the right hand panel. The indicated concentrations of several odorants, but not their solvent (DMSO), induce a Ca²⁺ flux response in NK cells from naive mice (<b>B</b>) the human NK cell line NKL (<b>C</b>) and the human T cell line Jurkat (<b>D</b>). No response is observed in the human embryonic kidney cell line HEK293 (<b>E</b>). Data are representative for 2–3 independent experiments for each condition and cell type.</p

Hapten recognition by TRP channels.

<p><b>(A</b>) Relative to untransfected control HEK293 cells (red line) stable hTRPA1 transfectants (blue line) respond to Bourgeonal, Oxa and DNFB and AITC. <b>(B</b>) The TRPA1 inhibitor HC0300301 (100 μM) inhibits Bourgeonal, Oxa and DNFB-induced Ca²⁺ flux response in HEK293 cells expressing hTRPA1 (green line). <b>(C</b>) The TRP1A inhibitor HC0300301 (100 μM) does not inhibit Oxa and DNFB-induced Ca²⁺ flux in NK cells. <b>(D)</b> Response of wild type (<i>TRPA1</i>^<i>+/+</i>) and <i>TRPA1</i>^<i>-/-</i> NK cells to Bourgeonal, Oxa and DNFB. n = 4 from 2 independent experiments.</p

Bourgeonal-, Oxa- and DNFB-induced Ca²⁺ flux in the presence of 2-APB and BTP2.

<p><b>(A</b>) Ca²⁺ flux induced by Bourgeonal, Oxa and DNFB in the presence of 2-APB (100 μM) and BTP2 (10 μM) in NK cells. The response by the odorant/hapten is shown in red, that in the presence of 2-APB is in blue, and that of BTP2 in yellow. The bar graph depicts the mean fold change (±SEM) in Indo-1 emission (i.e. the Indo-1V/Indo-1B ratio observed at the peak response relative to that of the baseline) induced by the stimulus alone (black bar) or the stimulus plus 2-APB (dark grey bar) or BTP2 (light grey) in NK cells from naive mice. n = 3 independent experiments, statistics as compared to stimulus alone using t-test ** p<0.01, * p<0.05, ns not significant (p>0.05). (<b>B</b>) Ca²⁺ flux induced by Bourgeonal, Oxa and DNFB in the presence of 2-APB (100 μM) and BTP2 (10 μM) in Jurkat cells (<b>B</b>). Representative of 2–3 independent experiments. <b>(C</b>) HEK293 cells were stably transfected with hSTIM1 without or with hORAI1, hORAI2 or hORAI3. Transfectants were kept in Ca²⁺-free medium and intracellular Ca²⁺ stores were depleted using Thapsigargin (TG) (1 μM). Ca²⁺ entry was measured following the addition of extracellular CaCl₂ (1 mM). Maximal Ca²⁺ entry was detected when HEK293 cells co-expressed hSTIM1 plus hORAI1, hORAI2 or hORAI3. <b>(F</b>) Oxa (0.4 mM) and DNFB (0.25 mM) fail to induce Ca²⁺ flux in hSTIM1/hORAI transfected HEK293 cells.</p

Odorant and hapten responses by primary lymphocytes and cell lines.

<p>Odorant and hapten responses by primary lymphocytes and cell lines.</p

Partial TRPC3-dependence of the hapten response.

<p>(<b>A</b>) Ca²⁺ flux response by gated NK cells induced by Bourgeonal, Oxa or DNFB (red lines) in the presence of Pyr3 (10 μM) (blue lines). The bar graph depicts the mean fold change (±SEM) in Indo-1 emission (i.e. the Indo-1V/Indo-1B ratio observed at the peak response relative to that of the baseline) by the indicated compound alone (black bar) and in the presence of Pyr3 (open bar). n = 3, Statistics as compared to compound alone using t-test ** p<0.01, * p<0.05, ns not significant. (<b>B</b>) Ca²⁺ flux response by Jurkat cells induced by Bourgeonal, Oxa or DNFB (red lines) in the presence of Pyr3 (10 μM) (blue lines). <b>(C</b>) Ca²⁺ entry into <i>TRPC3</i> ko (purple line) or wild type Jurkat cells (red line) induced by OKT3 antibody (2 μg/mL), Ionomycin (1 μg/mL), Bourgeonal (100 μM), Oxa (400 μM) or DNFB (250 μM). The bar graph depicts the mean fold change (±SEM) in Indo-1 emission (i.e. the Indo-1V/Indo-1B ratio observed at the peak response relative to that of the baseline) by the indicated stimulus in TRPC3 wild type (wt) (black bar) and a TRPC3 mutant clone (open bar). Data are compiled from 3–4 independent experiments (n = 3–7). Statistical significance ** p<0.01, * p<0.05, ns not significant based on unpaired t-test (p>0.05).</p

Hapten-induced Ca²⁺ flux depends on plasma membrane Ca²⁺channels.

<p><b>A</b>) Pre-treatment with Undecanal (2.5 μM) does not inhibit Bourgeonal-induced Ca²⁺ flux in NK cells, NKL or Jurkat cells. Rather Undecanal enhanced Ca²⁺ flux. Data are representative of two independent experiments. <b>(B</b>) Chelation of extracellular Ca²⁺ using EGTA (1mM) blocks the Ca²⁺ flux response induced by Bourgeonal (0.1 mM), Oxa (0.4 mM) and DNFB (0.5 mM) in NK cells. <b>(C</b>) Ca²⁺ release from intracellular stores induced by Thapsigargin (TG) (1 μM) in NK cells when extracellular Ca²⁺ is chelated by EGTA (1mM).</p

Ca²⁺ flux in NK cells in response to haptens.

<p>Ca²⁺ flux responses induced by indicated concentrations of the haptens Oxazolone (Oxa), 1-fluor-2,4 dinitrobenzene (DNFB), dinitrobenzene sulfonic acid (DNBS) and 2,4,6 trinitrobenzene sulfonic acid (TNBS) or the respective solvents (aceton, DMSO, H₂O) in NK cells from naive mice (<b>A</b>), the human NK cell line NKL (<b>B</b>), and the human T cells line Jurkat (<b>C</b>). No response is observed in the human embryonic kidney cell line HEK293 (<b>D</b>). Data are representative of at least 2–3 independent experiments for each condition and cell type.</p