Integrin CD11b/CD18

The number of CD11b+ monocytes, granulocytes, eosinophils, and macrophage subsets as well as the total number of tumor-infiltrating CD11b+ cells are decreased by ADH-503 ((Z)-Leukadherin-1 choline; 4 μM; 8 days) [1].

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ADH-503 binds CD11b and reduces myeloid cell recruitment to PDAC tissues [1]
To overcome the dosing limitation of CD11b blockade in previous studies, we developed a small molecule agonist, ADH-503, whose binding achieves a partially active CD11b conformation. ADH-503 directly alters the cytokine profile of PDAC-activated macrophages.

Reducing tumor burden increases overall survival. ADH-503 ((Z)-Leukadherin-1 choline; oral gavage; 30, 60, or 120 mg/kg; twice daily for 60 days) delayed tumor progression, leading to substantial differences in time-point analyses and therapy [1]. ADH-503 (oral gavage; 30, 100 mg/kg; twice daily; days 1 and 5) has an AUC0-t 30 at the mg/kg and 100 mg/kg doses, with plasma concentrations of 6950 ng.h/mL and 13962 ng.h/mL, respectively. Its highest concentration is 1716 and 2594 ng/mL. The mean half-life is 4.68 and 3.95 hours.

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ADH-503 treatment induces the accumulation of CD103+ cDCs in the tumor [1]
Due to the increase in intratumoral T cell numbers and proliferation, we explored whether these effects are driven by changes in DCs. As expected, based on their CD11b expression, we observed reduced numbers of tumor-infiltrating CD11b+ cDC2s and monocyte-derived DCs after 12 days of ADH-503 treatment (Figs. 4H and S4D). In contrast, in ADH-503-treated mice, tumor-infiltrating CD103+ cDC1s (which express CD11b at extremely low levels) were markedly increased in both number and MCH-I and MHC-II expression (Fig. 4H). These data suggest that ADH-503 reduces the numbers of potentially tolerogenic and/or CD4+ T cell-priming DCs, while enhancing cross-presenting by CD103+ cDC1s. The identity of these cDC populations was confirmed using Zbtb46gfp/+ reporter mice (Fig. S1D). To determine whether the changes in cDC1s were necessary for the increased CTL response observed in ADH-503-treated mice, we used BATF3−/− mice, which lack functional cDC1s. In contrast to wild-type controls, treatment with ADH-503 had no effect on T cell infiltration in BATF3-deficient mice (Fig. 4I). Taken together, these findings suggest that myeloid cell reprograming by ADH-503 drives cDC1 infiltration and function, leading to a reinvigorated anti-tumor T cell response.

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ADH-503 impairs tumor growth and improves survival in orthotopic models and KPC GEMMs [1]
To determine the impact of a CD11b agonist on tumor progression, we evaluated three syngeneic orthotopic PDAC models and KPC GEMMs (Fig. 5A–D). In all models, ADH-503 delayed tumor progression, leading to a significantly decreased tumor burden in time point analysis and improved overall survival (Fig. 5C and D). Importantly, ADH-503 had no direct effects on PDAC cell growth in vitro (Fig. S5A). To further confirm the specificity of ADH-503 for CD11b, we utilized CD11b-null (ITGAM-null) mice and found that unlike in wild-type mice, CD11b-null mice had similar tumor growth, regardless of treatment (Fig. 5E).

Leukadherin-1, also known as LA1, is a novel and specific agonist of Complement receptor 3 (CR3) and the leukocyte surface integrin CD11b/CD18 that enhances leukocyte adhesion to ligands and vascular endothelium and thus reduces leukocyte transendothelial migration and influx to the injury sites. Complement receptor 3 (CR3, CD11b/CD18) is a multi-functional receptor expressed predominantly on myeloid and natural killer (NK) cells. Leukadherin-1 (LA1) does not modulate signal transducer and activator of transcription (STAT)-4 phosphorylation. Leukadherin-1 modulates TLR-2 and TLR-7/8-induced monocyte cytokine secretion. Targeting leukocyte trafficking using LA1, an integrin agonist, is beneficial in preventing lung inflammation and protecting alveolar and vascular structures during hyperoxia. Thus, targeting integrin-mediated leukocyte recruitment and inflammation may provide a novel strategy in preventing and treating BPD in preterm infants.

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αA domain ligand-binding assay [2]
MaxiSorp 96-well plates were coated overnight with fibrinogen (1 μg per well) in 10 mM phosphate-buffered saline (PBS, pH 7.4) and blocked with 1% BSA in PBS. Binding of purified, GST-tagged αA domain (50 μl per well of a 5 μg/ml solution) to immobilized fibrinogen was performed in TBS-based assay buffer (TBS containing 0.1% BSA, 1 mM MgCl2, 1 mM CaCl2, and 0.05% Tween 20) (TBS-Ca/Mg buffer) for 1 hour at room temperature. The αA domain was also added to uncoated wells on the plate to estimate the maximum amount of protein that could be captured and detected in each well for data normalization. Unbound αA domain was removed by washing the wells twice with TBS-Ca/Mg buffer. Subsequently, the amount of bound protein was determined by incubation with horseradish peroxidase–conjugated antibody against GST (GE, 1:2000 dilution) for 1 hour. Unbound antibody was removed by washing the wells twice with TBS-Ca/Mg buffer. Detection of bound protein was performed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate kit according to the manufacturer’s protocol. Absorbance was read with a SpectraMax M5 spectrophotometer). Absorbance values were normalized such that the mean absorbance from the input αA domain wells was set at 100%, and the results are presented as the percentage of the total input amounts of the wild-type αA domain. Assays were performed in triplicate wells, and the data shown are from one of at least three independent experiments.

Phagocytosis assay with complement iC3b-coated sheep erythrocytes (EiC3bs) [2]
Sheep erythrocytes coated with complement iC3b were prepared and used in the phagocytosis assay as described previously. Coated erythrocytes (EiC3bs) were diluted to a concentration of 1.5 × 107 to 6 × 107 cells/ml. K562 CD11b/CD18 cells were washed twice in TBS and resuspended to 1 × 106/ml, of which 40 μl (4 × 104 cells) was incubated in suspension with EiC3bs (1.2 × 106) in a total volume of 100 μl at 37°C for 25 min in the presence of 1 mM each of CaCl2 and MgCl2 (in the absence or presence of 50 to 100 μM Leukadherin-1 (LA1), LA2, or LA3), in 1 mM MnCl2, or in 10 mM EDTA. Binding was detected by visually analyzing the formation of rosettes [the binding of multiple erythrocytes (EiC3bs) to individual K562 cells] by phase-contrast microscopy, as has been described previously. For scoring, only those K562 cells that were bound to ≥3 EiC3bs were scored as positive, and >200 cells were examined in multiple fields under each condition. Binding results, showing the percentages of all cells showing rosettes in a field, are reported as histograms representing the mean ± SEM of triplicate experiments; the data shown are from one of at least three independent experiments.

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Cell viability assays [2]
Cell viability assays were performed with commercially available reagents and kits. Briefly, 1 × 104 K562 CD11b/CD18 cells or wild-type B6 neutrophils were incubated in each well of a 96-well plate with increasing amounts of the indicated compounds, and the number of viable cellswas determined with the MTS reagent, according to the manufacturers’ instructions, after 4 hours (neutrophils) or 24 hours (K562 cells) of incubation. A SpectraMaxM5 spectrophotometer was used to read the assay plates. Data are representative of at least two independent experiments.

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Western blotting analysis [2]
K562 CD11b/CD18 cells were incubated with Leukadherin-1 (LA1), LA2, or LA3 (15 μM) or fibrinogen (200 μg) in serum-free medium for 1 hour at 37°C. Cell lysates were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane by means of established protocols. Membranes were incubated with a 1:1000 dilution of an antibody against phosphorylated extracellular signal–regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204), stripped with Reblot mild stripping solution, and then incubated, first with an antibody against total ERK1/2 and then with an antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and developed according to the manufacturer’s instructions. Data presented are representative of at least three independent experiments.

Animal/Disease Models: KPC mouse [p48-CRE/Lox-stop-Lox(LSL)-KrasG12D/p53flox/flox][1]
Doses: 30, 60 or 120 mg/kg
Route of Administration: po (oral gavage); 60-day
Experimental Results: Delays tumor progression, results in Dramatically lower tumor burden in time point analysis, and improves overall survival.

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Animal/Disease Models: Male rat[1]
Doses: 30, 100 mg/kg (pharmacokinetic/PK/PK analysis)
Route of Administration: po (oral gavage), twice (two times) daily; results on days 1 and 5: 30 mg and 100 mg, the average half-lives were 4.68 and 3.95 hrs (hrs (hours)), respectively, the maximum concentrations were 1716 and 2594 ng/mL, and the AUC0-t in plasma were 6950 and 13962 ng.h/mL/kg respectively.

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For animal experiments, ADH-503 was given at 30, 60, or 120 mg/kg and is specified in the text when not at 60mg/kg. ADH-503 was formulated for treatment in 0.5% carboxymethyl cellulose and 0.1 % Tween-80 in sterile water and administered by oral gavage twice a day (BID).

In rats the mean half-life of ADH503 at 30 and 100mg/kg dosing was 4.68 and 3.95 hours with a Cmax and AUC0-t in the plasma of 1716 and 2594 ng/mL and 6950 and 13962 ng.h/mL, respectively. Repeat dosing in rats was similar for these parameters as doses progressed (Fig. 2C, S2C–E). Dosing in C57/B6 mice has similar PK properties (Fig. S2D).[1]

Studies showed that ADH503 is well tolerated and displayed no adverse effects or toxicity after single dose or after repeated-dose for 28 days at doses up to 1500 mg/kg/d in rats and up to 1359 mg/kg/d in dogs. There was no mortality, clinical signs or body weight changes associated with ADH503 administrations and the compound was well-tolerated.[1]

[1]. Agonism of CD11b reprograms innate immunity to sensitize pancreatic cancer to immunotherapies. Sci Transl Med. 2019 Jul 3;11(499).

[2]. Small molecule-mediated activation of the integrin CD11b/CD18 reduces inflammatory disease. Sci Signal. 2011;4(189):ra57.

CD11b Agonist GB1275 is an orally bioavailable small molecule agonist of CD11b (integrin alpha-M; ITGAM; integrin alpha M chain), with potential immunomodulating activity. Upon administration, CD11b agonist GB1275 targets and binds to CD11b, thereby activating CD11b. This leads to CD11b-mediated signaling and promotes pro-inflammatory macrophage polarization while suppressing immunosuppressive macrophage polarization. This reduces influx of tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME), promotes anti-tumor immune responses, induces cytotoxic T-lymphocytes (CTLs) and suppresses tumor growth. CD11b, a member of the integrin family of cell adhesion receptors highly expressed on immune system cells, is a negative regulator of immune suppression and activates anti-tumor innate immunity.

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Although checkpoint immunotherapies have revolutionized the treatment of cancer, not all tumor types have seen substantial benefit. Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy in which very limited responses to immunotherapy have been observed. Extensive immunosuppressive myeloid cell infiltration in PDAC tissues has been postulated as a major mechanism of resistance to immunotherapy. Strategies concomitantly targeting monocyte or granulocyte trafficking or macrophage survival, in combination with checkpoint immunotherapies, have shown promise in preclinical studies, and these studies have transitioned into ongoing clinical trials for the treatment of pancreatic and other cancer types. However, compensatory actions by untargeted monocytes, granulocytes, and/or tissue resident macrophages may limit the therapeutic efficacy of such strategies. CD11b/CD18 is an integrin molecule that is highly expressed on the cell surface of these myeloid cell subsets and plays an important role in their trafficking and cellular functions in inflamed tissues. Here, we demonstrate that the partial activation of CD11b by a small-molecule agonist (ADH-503) leads to the repolarization of tumor-associated macrophages, reduction in the number of tumor-infiltrating immunosuppressive myeloid cells, and enhanced dendritic cell responses. These actions, in turn, improve antitumor T cell immunity and render checkpoint inhibitors effective in previously unresponsive PDAC models. These data demonstrate that molecular agonism of CD11b reprograms immunosuppressive myeloid cell responses and potentially bypasses the limitations of current clinical strategies to overcome resistance to immunotherapy.[1]

C27H28N2O5S2

524.651624679565

524.143

C, 61.81; H, 5.38; N, 5.34; O, 15.25; S, 12.22

2055362-74-6

Leukadherin-1;344897-95-6;(Z)-Leukadherin-1;2055362-72-4; 2055362-74-6 (choline)

145711124

Brown to reddish brown solid powder

1

7

6

36

721

0

S1C(N(C(/C/1=C/C1=CC=C(C2C=CC(C(=O)[O-])=CC=2)O1)=O)CC1C=CC=CC=1)=S.OCC[N+](C)(C)C

GOWDQYRMBCOOJR-JHMJKTBASA-M

InChI=1S/C22H15NO4S2.C5H14NO/c24-20-19(29-22(28)23(20)13-14-4-2-1-3-5-14)12-17-10-11-18(27-17)15-6-8-16(9-7-15)21(25)261-6(2,3)4-5-7/h1-12H,13H2,(H,25,26)7H,4-5H2,1-3H3/q+1/p-1/b19-12-

2-hydroxy-N,N,N-trimethylethan-1-aminium (Z)-4-(5-((3-benzyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)furan-2-yl)benzoate

ADH-503 choline; ADH-503; ADH 503; ADH503; Leukadherin-1 choline; 2-Hydroxy-N,N,N-trimethylethan-1-aminium (Z)-4-(5-((3-benzyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)furan-2-yl)benzoate; THN3VQ67CA; UNII-THN3VQ67CA; 4-[5-[(Z)-(3-benzyl-4-oxo-2-sulfanylidene-1,3-thiazolidin-5-ylidene)methyl]furan-2-yl]benzoate;2-hydroxyethyl(trimethyl)azanium; LA1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

MEthanol : ~100 mg/mL (~190.60 mM)
DMSO : ~21.43 mg/mL (~40.85 mM)
Ethanol : ~3.33 mg/mL (~6.35 mM)

Solubility in Formulation 1: ≥ 2.14 mg/mL (4.08 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.4 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.14 mg/mL (4.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.4 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 2.08 mg/mL (3.96 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)