PD-1 signaling pathway

AUNP-12 inhibits by 44% tumor growth of B16F10 mouse melanoma cells injected subcutaneously in mice (5 mg/kg, subcutaneously once daily, 14 days); it reduces lung metastasis of B16F10 cells injected iv. in mice (5 mg/kg, subcutaneously, once daily, 11 days); it inhibits by 44% tumor growth of 4T1 cells injected orthotopically to mammary fat pad in mice (3 mg/kg, subcutaneously, once daily, 40 days). 10% of the animals treated with AUNP-12 showed complete regression and another 10% showed partial regression of tumor growth. AUNP-12 treated animals showed a mean reduction in lung metastasis, measured after euthanasia, to the extent of >60%.

AUNP-12 displays an EC50 = 0.72 nM in the inhibition of binding PD1 to PD-L2 using hPDL2 expressing HEK293 cells, and an EC50 = 0.41 nM in a rat peripheral blood mononuclear cells (PBMC) proliferation assay using hPDL1 expressing MDA-MB231 cells. This corresponds well to the ‘sub-nanomolar potency in disruption of PD1-PDL1/2 interaction’ reported for AUNP-012.

AUNP-12 displays an EC50 = 0.72 nM in the inhibition of binding PD1 to PD-L2 using hPDL2 expressing HEK293 cells, and an EC50 = 0.41 nM in a rat peripheral blood mononuclear cells (PBMC) proliferation assay using hPDL1 expressing MDA-MB231 cells.

AUNP-12 is active in vivo in a lung metastasis model of B16F10 melanoma in mice, showing a 64% reduction in metastasis at 5 mg/kg (subcutaneous, once daily, 14 days).[2]

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Pharmacokinetics of AUNP-12 in Balb/c mice[3]
All animal experimental procedures used in these studies including pharmacokinetic, pharmacodynamic, and efficacy experiments were approved by the Institutional Animal Ethical Committee based on the Committee for the Purpose of Control and Supervision on Experiments on Animals (India) guidelines. AUNP-12 was administered either intravenously or subcutaneously to the animals at a dose of 3 mg/kg to determine the pharmacokinetic parameters using 5% dextrose water as formulation. After administration, blood samples were collected at regular intervals until 24 hours and centrifuged to obtain the plasma fraction. The plasma samples were processed by SPE method and the eluent were analyzed by LC/MS-MS to determine the plasma concentration of the compound. From intravenous administration, plasma concentration after injection (C0 minutes), the area under the concentration−time curve from time zero to infinity (AUC 0−∞), the mean residence time, volume of distribution (Vdss), and clearance (CL) for each mouse were obtained. The maximum plasma concentration (Cmax), time to reach maximum plasma concentration (Tmax), and AUC 0−∞ were obtained from subcutaneous administration of AUNP-12 . On the basis of the intravenous and subcutaneous parameters, bioavailability of AUNP-12 was calculated.

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Syngeneic mouse studies[3]
In all in vivo tumor growth inhibition (TGI) studies, tumor volumes were measured two times weekly using digital calipers and the volume was expressed in mm3 using the formula V = 0.5a × b2, where a and b are the long and short diameters of the tumor, respectively. Body weights and clinical signs were monitored twice a week. AUNP-12 was dissolved in 5% dextrose water for all the in vivo studies, except for B16F10 mouse melanoma and Renca tumor models where 1 × PBS was used. Fresh formulation was prepared every day. Compound and vehicle controls were dosed subcutaneously once a day at a dosing volume of 10 mL/kg body weight.

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[1]. US 2011/0318373 A1
[2]. Cancer Research, vol. 72, no. 8 Suppl. 1, Abstract 2850, 2012.
[3]. A Rationally Designed Peptide Antagonist of the PD-1 Signaling Pathway as an Immunomodulatory Agent for Cancer Therapy. Mol Cancer Ther. 2019 Jun;18(6):1081-1091.

Further in vivo studies revealed that AUNP-12/AUR-012 exhibits an excellent PK-PD correlation with sustained PD for >24 h. In preclinical models of melanoma, breast and kidney cancers, AUR012/AUNP-12 showed superior efficacy compared to therapeutic agents currently used in the clinic in inhibition of both primary tumor growth and metastasis. Interestingly, dosing once in three days was equally efficacious as once a day dosing with no signs of overt toxicity and generation of neutralizing activity.[9] Rescue of proliferation of immune cells analyzed upon stimulation with anti-CD3/anti-CD-28 indicated a complete rescue of CD4+ and CD8+ T cells. Interestingly, the proliferation of CD4+, Foxp3+ T cells was completely abolished with AUR-012/AUNP-12 treatment indicating a complete suppression of regulatory T cells. Sustained activation of circulatory immune cells and their ability to secrete IFN-γ up to 72 h indicate that pharmacodynamic effects persist even after the clearance of the compound in animal models, thus supporting a dosing interval of up to 3 days. In models of melanoma, breast, kidney and colon cancers, AUR-012/AUNP-12 showed efficacy in inhibition of both primary tumor growth and metastasis. Additionally, anti-tumor activity of the compound in a pre-established CT26 model correlated well with pharmacodynamic effects as indicated by intratumoral recruitment of CD4+ and CD8+ T cells, and a reduction in PD1+ T cells (both CD4+ & Page 7/12 CD8+) in tumor and blood. In 14-day repeated dose toxicity studies, AUR-012/AUNP-12 was well tolerated at 100 times the efficacious doses. [2]

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AUNP-12, likely to be identical to the compound previously known under the codenames Aur-012, Aurigene-012, or Aurigene NP-12, is an inhibitor of the so-called PD-1 pathway, and will be in development for several cancer indications. It is so far the only peptide therapeutic in this pathway and could offer more effective and safer combination opportunities compared to current approaches,[2-4] e.g. antibodies such as Nivolumab (BMS), Lambrolizumab (Merck-3475), CT-011 (Curetech), MDX-1105 (BMS), MPDL3280 (GNE) and MEDI-4736 (Medimmune-AZ), or Amplimmune’s PD-L2-FC fusion protein. PD-1, or Programmed cell death 1, is an immunoreceptor belonging to the CD28 family, and plays an important role in negatively regulating immune responses. The amino acid protein structure includes an extracellular amino acid IgV domain followed by a transmembrane region and an intracellular tail. PD-1 is expressed on the surface of activated T cells, B cells, and macrophages, and has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 is expressed on almost all murine tumor cell lines, whereas PD-L2 expression is more restricted and is expressed mainly by DCs and a few tumor lines. Blocking of PD-1 signaling pathways has been shown to result in restoration of defective immune cell functions in cancer and chronic infections. Recent advances in achieving highly durable clinical responses via inhibition of immune checkpoint proteins including PD-1 using antibodies or fusion proteins have revolutionized the outlook for cancer therapy. However, along with impressive clinical activity, severe immune-related adverse events (irAEs) due to the breaking of immune selftolerance are becoming increasingly evident. Sustained target inhibition as a result of a long halflife (>15-20 days) and >70% target occupancy for months are likely contributing to severe irAEs observed in the clinic with antibodies targeting immune checkpoint proteins.[2]

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Pioneering success of antibodies targeting immune checkpoints such as PD-1 and CTLA4 has opened novel avenues for cancer immunotherapy. Along with impressive clinical activity, severe immune-related adverse events (irAE) due to the breaking of immune self-tolerance are becoming increasingly evident in antibody-based approaches. As a strategy to better manage severe adverse effects, we set out to discover an antagonist targeting PD-1 signaling pathway with a shorter pharmacokinetic profile. Herein, we describe a peptide antagonist NP-12 that displays equipotent antagonism toward PD-L1 and PD-L2 in rescue of lymphocyte proliferation and effector functions. In preclinical models of melanoma, colon cancer, and kidney cancers, NP-12 showed significant efficacy comparable with commercially available PD-1-targeting antibodies in inhibiting primary tumor growth and metastasis. Interestingly, antitumor activity of NP-12 in a preestablished CT26 model correlated well with pharmacodynamic effects as indicated by intratumoral recruitment of CD4 and CD8 T cells, and a reduction in PD-1+ T cells (both CD4 and CD8) in tumor and blood. In addition, NP-12 also showed additive antitumor activity in preestablished tumor models when combined with tumor vaccination or a chemotherapeutic agent such as cyclophosphamide known to induce "immunologic cell death." In summary, NP-12 is the first rationally designed peptide therapeutic targeting PD-1 signaling pathways exhibiting immune activation, excellent antitumor activity, and potential for better management of irAEs.[3]

C142H226N40O48

3261.55

3259.65

C, 52.29; H, 6.98; N, 17.18; O, 23.55

1353563-85-5

AUNP-12 TFA

154701623

H-Ser-Asn-Thr-Ser-Glu-Ser-Phe-Lys(1)-Phe-Arg-Val-Thr-Gln-Leu-Ala-Pro-Lys-Ala-Gln-Ile-Lys-Glu-NH2.H-Ser-Asn-Thr-Ser-Glu-Ser-Phe-(1)

SNTSESFK(SNTSESF-NH)FRVTQLAPKAQIKE-NH2

White to off-white solid powder

-23.2

51

53

112

230

7520

33

CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)N)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CCCCNC(=O)[C@H](CC3=CC=CC=C3)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)NC(=O)[C@H](CC4=CC=CC=C4)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N

ZBJUUYIGBAQYBN-QKLNNLIKSA-N

InChI=1S/C142H226N40O48/c1-12-70(6)109(137(226)165-84(37-23-26-52-144)119(208)158-81(113(151)202)43-48-105(196)197)178-125(214)87(42-47-102(148)193)159-114(203)71(7)156-118(207)82(36-22-25-51-143)164-135(224)100-40-29-55-182(100)141(230)72(8)157-126(215)90(56-68(2)3)169-121(210)86(41-46-101(147)192)166-138(227)112(75(11)191)181-136(225)108(69(4)5)177-124(213)85(39-28-54-155-142(152)153)161-127(216)92(58-77-32-18-14-19-33-77)171-120(209)83(160-128(217)93(59-78-34-20-15-21-35-78)172-134(223)97(65-186)174-123(212)89(45-50-107(200)201)163-132(221)99(67-188)176-140(229)111(74(10)190)180-130(219)95(61-104(150)195)168-116(205)80(146)63-184)38-24-27-53-154-117(206)91(57-76-30-16-13-17-31-76)170-133(222)96(64-185)173-122(211)88(44-49-106(198)199)162-131(220)98(66-187)175-139(228)110(73(9)189)179-129(218)94(60-103(149)194)167-115(204)79(145)62-183/h13-21,30-35,68-75,79-100,108-112,183-191H,12,22-29,36-67,143-146H2,1-11H3,(H2,147,192)(H2,148,193)(H2,149,194)(H2,150,195)(H2,151,202)(H,154,206)(H,156,207)(H,157,215)(H,158,208)(H,159,203)(H,160,217)(H,161,216)(H,162,220)(H,163,221)(H,164,224)(H,165,226)(H,166,227)(H,167,204)(H,168,205)(H,169,210)(H,170,222)(H,171,209)(H,172,223)(H,173,211)(H,174,212)(H,175,228)(H,176,229)(H,177,213)(H,178,214)(H,179,218)(H,180,219)(H,181,225)(H,196,197)(H,198,199)(H,200,201)(H4,152,153,155)/t70-,71-,72-,73+,74+,75+,79-,80-,81-,82-,83-,84-,85-,86-,87-,88-,89-,90-,91-,92-,93-,94-,95-,96-,97-,98-,99-,100-,108-,109-,110-,111-,112-/m0/s1

(4S)-5-amino-4-[[(2S)-6-amino-2-[[(2S,3S)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2,6-bis[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-4-amino-2-[[(2S)-2-amino-3-hydroxypropanoyl]amino]-4-oxobutanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-4-carboxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-phenylpropanoyl]amino]hexanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxybutanoyl]amino]-5-oxopentanoyl]amino]-4-methylpentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]hexanoyl]amino]propanoyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]amino]hexanoyl]amino]-5-oxopentanoic acid

Aur-012; AUNP-12; 1353563-85-5; AUNP-12?; CHEMBL4635204; AUNP-12, AUR-012; EX-A7438; NONYLPHENOL POLYOXYETHYLENE ETHER; G13071; Aurigene-012

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)

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.)