(Z)-Leukadherin-1 (ADH-503 free base; 4 μM; 8 days) decreases the overall number of CD11b+ cells that infiltrate tumors as well as the subsets of CD11b+ monocytes, granulocytes, eosinophils, and macrophages [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.

Z-leukadherin-1 (ADH-503 free base; orally administered; doses of 30, 60, or 120 mg/kg; administered twice daily for 60 days) slows the growth of the tumor, which reduces the number of tumors in the time point analysis burden and increases overall survival [1]. The maximal concentration of (Z)-Leukadherin-1 is 1716 and 2594 ng/ml, with a mean half-life of 4.68 and 3.95 hours (oral gavage; 30, 100 mg/kg; twice daily; days 1 and 5). 30 The AUC0-t in plasma were 6950 ng.h/ml and 13962 ng.h/ml at the mg/kg and 100 mg/kg dosages, respectively [1].

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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, respectively, 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]

C22H15NO4S2

421.48880314827

421.044

2055362-72-4

Leukadherin-1;344897-95-6;ADH-503;2055362-74-6

5342077

Light yellow to yellow solid powder

4.8

1

6

5

29

680

0

S1C(N(C(/C/1=C/C1=CC=C(C2C=CC(C(=O)O)=CC=2)O1)=O)CC1C=CC=CC=1)=S

AEZGRQSLKVNPCI-UNOMPAQXSA-N

InChI=1S/C22H15NO4S2/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)26/h1-12H,13H2,(H,25,26)/b19-12-

4-[5-[(Z)-(3-benzyl-4-oxo-2-sulfanylidene-1,3-thiazolidin-5-ylidene)methyl]furan-2-yl]benzoic acid

ADH-503 free base ADH-503 ADH 503 ADH503 Leukadherin-1 choline LA1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~4.55 mg/mL (~10.80 mM)
Methanol :< 1 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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