In vivo knockdown of TAK1 accelerates bone marrow proliferation/differentiation and induces systemic inflammation.

TAK1 (TGF-β Activated Kinase 1) is a MAPK kinase kinase, which activates the p38- and JNK-MAPK and NF-κB pathways downstream of receptors such as Toll-Like-, cytokine- and T-cell and B-cell receptors. Representing such an important node in the pro-inflammatory signal-transduction network, the function of TAK1 has been studied extensively. TAK1 knock-out mice are embryonic lethal, while conditional knock-out mice demonstrated either a pro- or anti-inflammatory function. To study the function of TAK1 protein in the adult immune system, we generated and characterized a transgenic mouse expressing TAK1 shRNA under the control of a doxycycline-inducible promoter. Following treatment of TAK-1 shRNA transgenic mice with doxycycline an effective knockdown of TAK1 protein levels was observed in lymphoid organs and cells in the peritoneal cavity (>50% down regulation). TAK1 knockdown resulted in significant changes in leukocyte populations in blood, bone marrow, spleen and peritoneal cavity. Upon TAK1 knockdown mice demonstrated splenomegaly, signs of systemic inflammation (increased levels of circulating cytokines and increase in cellularity of the B-cell areas and in germinal center development in the follicles) and degenerative changes in heart, kidneys and liver. Not surprisingly, TAK1-Tg mice treated with LPS or anti-CD3 antibodies showed enhanced cytokine/chemokine secretion. Finally, analysis of progenitor cells in the bone marrow upon doxycycline treatment showed increased proliferation and differentiation of myeloid progenitor cells. Given the similarity of the phenotype with TGF-β genetic models, our data suggest that in our model the function of TAK1 in TGF-β signal-transduction is overruling its function in pro-inflammatory signaling

TAK1 knockdown shows more activated T- and B-cells in the spleen and bone marrow.

<p>Wildtype (WT) and TAK1 shRNA transgenic mice (TAK1-Tg) were treated with 0.2 mg/ml doxycycline in the drinking water for five weeks. Mice (n = 10 in each group) were sacrificed and spleens and bone marrow were harvested. Characterization of T- and B-cells was performed by flow cytometry. The percentage of naive T-cells (CD4⁺CD62L⁺CD44^int), effector/memory T-cells (CD4⁺CD62L⁻CD44⁺) and regulatory T-cells (CD4⁺CD25⁺Foxp3⁺) are presented as % of total CD4⁺ cells in panel <i>A</i>. The percentage of mature B-cells (B220⁺AA4.1⁻CD23⁺IgM^low), Marginal zone B-cells (B220⁺AA4.1⁻CD23⁻IgM^high) and MHCII-expression on B220⁺ cells (in fluorescence intensity) are depicted in panel <i>B</i>. Statistical significance was determined using the Mann-Whitney test (p<0.05). Data are representative of at least two independent experiments.</p

TAK1 knockdown affects the homeostasis of the immune system.

<p>n.s. = not significant;</p>1<p>mass of subpopulation = % of sub-population x weight of spleen.</p

TAK1 knockdown affects cellular composition of different immunological compartments.

<p>Wildtype (WT) and TAK1 shRNA transgenic mice (TAK1-Tg) were treated with 0.2 mg/ml doxycycline in the drinking water for five weeks. Mice were sacrificed and organs harvested. Weights of the spleen and mice for WT and TAK1 mice is presented (n = 10 for each group). Statistical significance was determined using the Mann-Whitney test (p<0.05). Data are representative of at least two independent experiments.</p

Induction of TAK1 knock-down in vivo.

<p>Transgenic mice expressing TAK1 shRNA under the control of a doxycycline-inducible promoter were generated. Expression of TAK1 shRNA was induced in adult mice by addition of 0.2 mg/ml doxycycline hydrochloride in the drinking water for two weeks. Mice were sacrificed and organs of interest were harvested. Knock-down of TAK1 was determined in wild type mice treated with doxycycline (WT +Dox), TAK1 shRNA transgenic mice treated with doxycycline (TAK1-Tg +Dox) or without doxycycline (TAK1-Tg –Dox). <i>A.</i> TAK1 mRNA levels were detected in liver, spleen and thymus by Q-PCR. The levels of HP1 mRNA was used for normalization. The average of the mRNA levels detected in samples derived from TAK1-Tg that were not treated with doxycycline were used as reference (100%). <i>B</i>. TAK1 protein levels were determined in liver, spleen, thymus, skin, lymph nodes and cells isolated from the peritoneal cavity using western blotting. β-actin levels were used to check for loading efficiency. Data are representative of at least three independent experiments.</p

TAK1 knockdown induces circulating levels of cytokines and chemokines.

<p>Wildtype (WT) and TAK1 shRNA transgenic mice (TAK1-Tg) were treated with 0.2 mg/ml doxycycline in the drinking water for five weeks. Mice (n = 8 each group) were sacrificed and blood was harvested. Circulating levels of cytokines and chemokines were determined by Multiplex and are represented in pg/ml. Statistical significance was determined using the Mann-Whitney test (p<0.05). Data are representative of at least two independent experiments.</p

TAK1 knockdown causes histopathological changes in spleen, heart, kidneys and liver.

<p>Wild type (WT) and TAK1 shRNA transgenic mice (TAK1-Tg) were treated with 0.2 mg/ml doxycycline in the drinking water for five weeks. At autopsy, anaesthetized mice were sacrificed and organs and tissues were dissected and processed for histopathological examination (Formalin-fixed and HE-stained). <i>A.</i> Spleen (Obj. 10x). <i>Left,</i> WT: normal histological appearance of the spleen with white pulp (WP) comprising germinal centers (GC) and red pulp (RP). <i>Right,</i> TAK1-Tg: increase in cellularity of the B-cell areas and in germinal center development in the follicles. <i>B.</i> Spleen. Obj. 10x (<i>top</i>) and Obj. 40x (<i>bottom</i>). <i>Left</i>, WT: normal histological appearance of the spleen with white pulp (WP) and red pulp (RP); no extramedullary erythropoiesis. <i>Right,</i> TAK1-Tg: moderate increase in extramedullary erythropoiesis in the red pulp (arrows, <i>top</i>) showing increased numbers of erythroblasts (white arrows, <i>bottom</i>) and megakaryocytes (black arrows, <i>bottom</i>). <i>C.</i> Heart. Obj. 10x. <i>Left</i>, WT: normal histological appearance of the heart with intact muscle fibers. <i>Right,</i> TAK1-Tg: scattered slight muscle fiber degeneration and necrosis accompanied with inflammatory cells (arrows). <i>D.</i> Kidney. Obj. 40x. <i>Left</i>, WT: normal histological appearance of the kidney (cortex) with glomeruli (G), proximal (PT) and distal (DT) tubules. <i>Right,</i> TAK1-Tg: diffuse marked degeneration (arrows) and regeneration (asterisks) of the proximal tubular cells. <i>E.</i> Liver. Obj. 40x. <i>Left</i>, WT: normal histological appearance of the liver in the centrilobular region next to the central vein (CV). <i>Right</i>, TAK1-Tg: scattered several to many hepatocytes showing single cell necrosis (arrows).</p

TAK1 knockdown exacerbates acute inflammation.

<p>Wildtype (WT) and TAK1 shRNA transgenic mice (TAK1-Tg) were treated with 0.2 mg/ml doxycycline in the drinking water for five weeks. Acute inflammation was induced by administration of LPS (n = 5 each group) (<i>A</i>) or anti-CD3 antibodies (n = 8 each group) (<i>B</i>) in the peritoneal cavity. After 1.5 or 3 hours, respectively mice were sacrificed and circulating levels of cytokines and chemokines were detected by Multiplex and are represented in pg/ml. Statistical significance was determined using the Mann-Whitney test (p<0.05). Data are representative of at least two independent experiments.</p

TAK1 knockdown increases proliferation and differentiation of myeloid precursors in the bone marrow.

<p>Wildtype (WT) and TAK1 shRNA transgenic mice (TAK1-Tg) were treated with 0.2 mg/ml doxycycline in the drinking water for two weeks. Mice (n = 5 each group) were sacrificed and spleens and bone marrow were harvested. (A) Lin⁻c-Kit⁺ progenitors, Lin⁻c-Kit⁺Sca-1⁺ cells (LKS population), LT-HSCs (CD34⁻Flt3⁻), ST-HSC (CD34⁺Flt3⁻), MPPs (CD34⁺Flt3⁺) and myeloid precursors (Lin⁻c-Kit⁺Sca-1⁻) were characterized by flow cytometry using the indicated cell markers. (B) Subpopulation of the myeloid precursors were characterized using the following markers: GMPs (CD34⁺CD16/32⁺), MEPs (CD34⁻CD16/32⁻) and CMPs (CD34⁺CD16/32^low). (C). Using staining with antibodies specifically recognizing BrdU, proliferating cells in the myeloid precursors (Lin⁻c-Kit⁺Sca-1⁻) were identified. Statistical significance was determined using the two-tailed test (p<0.05).</p

BYON4228 is a pan-allelic antagonistic SIRPα antibody that potentiates destruction of antibody-opsonized tumor cells and lacks binding to SIRPγ on T cells

Background Preclinical studies have firmly established the CD47-signal-regulatory protein (SIRP)α axis as a myeloid immune checkpoint in cancer, and this is corroborated by available evidence from the first clinical studies with CD47 blockers. However, CD47 is ubiquitously expressed and mediates functional interactions with other ligands as well, and therefore targeting of the primarily myeloid cell-restricted inhibitory immunoreceptor SIRPα may represent a better strategy.Method We generated BYON4228, a novel SIRPα-directed antibody. An extensive preclinical characterization was performed, including direct comparisons to previously reported anti-SIRPα antibodies.Results BYON4228 is an antibody directed against SIRPα that recognizes both allelic variants of SIRPα in the human population, thereby maximizing its potential clinical applicability. Notably, BYON4228 does not recognize the closely related T-cell expressed SIRPγ that mediates interactions with CD47 as well, which are known to be instrumental in T-cell extravasation and activation. BYON4228 binds to the N-terminal Ig-like domain of SIRPα and its epitope largely overlaps with the CD47-binding site. BYON4228 blocks binding of CD47 to SIRPα and inhibits signaling through the CD47-SIRPα axis. Functional studies show that BYON4228 potentiates macrophage-mediated and neutrophil-mediated killing of hematologic and solid cancer cells in vitro in the presence of a variety of tumor-targeting antibodies, including trastuzumab, rituximab, daratumumab and cetuximab. The silenced Fc region of BYON4228 precludes immune cell-mediated elimination of SIRPα-positive myeloid cells, implying anticipated preservation of myeloid immune effector cells in patients. The unique profile of BYON4228 clearly distinguishes it from previously reported antibodies representative of agents in clinical development, which either lack recognition of one of the two SIRPα polymorphic variants (HEFLB), or cross-react with SIRPγ and inhibit CD47-SIRPγ interactions (SIRPAB-11-K322A, 1H9), and/or have functional Fc regions thereby displaying myeloid cell depletion activity (SIRPAB-11-K322A). In vivo, BYON4228 increases the antitumor activity of rituximab in a B-cell Raji xenograft model in human SIRPαBIT transgenic mice. Finally, BYON4228 shows a favorable safety profile in cynomolgus monkeys.Conclusions Collectively, this defines BYON4228 as a preclinically highly differentiating pan-allelic SIRPα antibody without T-cell SIRPγ recognition that promotes the destruction of antibody-opsonized cancer cells. Clinical studies are planned to start in 2023