The peptide targets the T cell receptor (TCR) of CD8+ cytotoxic T lymphocytes (CTLs) that are specific for EBV-LMP1. When presented on the cell surface by major histocompatibility complex (MHC) class I molecules (specifically HLA-B*57:01 or B*58:01), IALYLQQNW is recognized by the TCR, leading to activation of EBV-specific CTLs. These CTLs then recognize and eliminate EBV-infected cells expressing LMP1. The peptide plays an important role in immune responses against EBV-associated malignancies.

In vitro, IALYLQQNW is used to stimulate EBV-specific CD8+ T cells in peripheral blood mononuclear cell (PBMC) assays. In ELISpot assays, PBMCs from HLA-B*57:01-positive donors are incubated with the peptide (1-10 ug/mL) for 12-24 hours, resulting in IFN-gamma spot-forming cells (SFCs) that correlate with the frequency of EBV-LMP1-specific CTLs. In intracellular cytokine staining (ICS) assays, peptide stimulation (5 ug/mL, 6 hours with brefeldin A) induces IFN-gamma, TNF-alpha, and IL-2 production in CD8+ T cells as measured by flow cytometry. In cytotoxicity assays, peptide-pulsed target cells (e.g., autologous B-lymphoblastoid cell lines or T2 cells loaded with peptide) are lysed by EBV-specific CTLs in a ⁵¹Cr release assay, with specific lysis typically 20-60% at effector:target ratios of 10:1 to 40:1. The peptide also induces proliferation of LMP1-specific T cells in vitro, as measured by CFSE dilution or [3H]-thymidine incorporation. The peptide shows no direct cytotoxicity or mitogenic activity in the absence of specific T cells. The epitope is strictly HLA-B*57:01/B*58:01-restricted; no response is observed in donors lacking these alleles.

In vivo, IALYLQQNW is not administered as a therapeutic peptide; rather, it is used as a research tool to monitor EBV-specific T cell responses in cancer patients, particularly those with Hodgkin's disease (Hodgkin lymphoma) and nasopharyngeal carcinoma (NPC). In adoptive T cell therapy protocols, autologous T cells are expanded ex vivo using peptide-pulsed dendritic cells or artificial antigen-presenting cells loaded with IALYLQQNW (typically 1-10 ug/mL) to generate EBV-LMP1-specific CTLs, which are then infused back into patients. In clinical studies, infusions of LMP1-specific CTLs have been associated with tumor regression in some patients with EBV+ Hodgkin lymphoma and NPC, although the specific contribution of the IALYLQQNW epitope depends on patient HLA type. The peptide is also used in ELISpot assays to monitor T cell responses in patients receiving immunotherapy. No animal efficacy studies using the peptide alone are described.

For MHC-peptide binding assays (non-cell-based), recombinant HLA-B*57:01 molecules are refolded with IALYLQQNW peptide. The refolded complex is purified by size-exclusion chromatography, and binding affinity is measured by surface plasmon resonance (SPR) or by competition assays with a labeled reference peptide. For example, HLA-B*57:01 heavy chain and beta2-microglobulin are expressed in E. coli, refolded in vitro with IALYLQQNW (0.1-100 uM) and a labeled reference peptide. The complex is captured on an antibody-coated surface. The EC₅0 for binding is typically in the low nanomolar range (5-50 nM). For T cell receptor binding studies, soluble TCRs specific for HLA-B*57:01/Ialylqqnw are produced and binding affinity is measured by SPR.

For in vitro cellular assays, peripheral blood mononuclear cells (PBMCs) are isolated from HLA-typed healthy donors or patients with EBV-associated malignancies by Ficoll-Paque density gradient centrifugation. PBMCs are resuspended in RPMI-1640 supplemented with 10% human AB serum or FBS, 1% penicillin-streptomycin, and 20 U/mL IL-2 (for expansion). For ELISpot assays, 96-well PVDF plates are coated with anti-IFN-gamma capture antibody (10 ug/mL, overnight at 4degC). PBMCs (2-3 × 10⁵ cells/well) are incubated with IALYLQQNW peptide (1-10 ug/mL) for 12-24 hours at 37degC in 5% CO2. Wells with no peptide (background) and with phytohemagglutinin (PHA, 2 ug/mL, positive control) are included. Plates are washed, biotinylated detection antibody (2 ug/mL) is added (2 hours), followed by streptavidin-alkaline phosphatase (1 hour). Spots are developed using BCIP/NBT substrate and counted on an automated ELISpot reader. Results are expressed as spot-forming cells (SFC) per 10⁶ PBMCs. For intracellular cytokine staining (ICS), PBMCs (1 × 10⁶ cells/well in 96-well round-bottom plates) are stimulated with IALYLQQNW (5-10 ug/mL) for 2 hours, followed by brefeldin A (10 ug/mL) for 4-6 hours. Cells are surface-stained with anti-CD8, anti-CD4, anti-CD3 antibodies, fixed and permeabilized, then stained with anti-IFN-gamma, anti-TNF-alpha, and anti-IL-2 antibodies. Cells are analyzed on a flow cytometer (10,000-50,000 events). Percentage of CD8+ cells positive for cytokines is recorded. For cytotoxicity assays (⁵¹Cr release), target cells (e.g., autologous B-LCL or T2 cells) are pulsed with IALYLQQNW (10 ug/mL) for 1 hour at 37degC, washed, and labeled with ¹⁰⁰ microCi of ⁵¹Cr for 1 hour. Effector cells (PBMC-derived CTLs expanded in vitro with peptide for 7-14 days, or primary PBMCs) are incubated with target cells at various E:T ratios (40:1, 20:1, 10:1, 5:1, 2.5:1) in U-bottom plates for 4 hours. Supernatants are harvested, and ⁵¹Cr release is measured with a gamma counter. Specific lysis (%) = (experimental release - spontaneous release) / (maximal release - spontaneous release) × 100. For T cell proliferation assays, PBMCs are labeled with CFSE (5 uM, 10 minutes at 37degC), then stimulated with IALYLQQNW (1-10 ug/mL) for 5-7 days. CFSE dilution is analyzed by flow cytometry. For tetramer staining, PE- or APC-labeled HLA-B*57:01/Ialylqqnw tetramers are used to stain PBMCs (0.5-1 × 10⁶ cells, 30 minutes at 4degC), followed by anti-CD8 staining. Tetramer-positive CD8+ T cells are quantified by flow cytometry. For negative controls, use an irrelevant peptide (e.g., HIV gag peptide) or an HLA-mismatched peptide. Each condition should be tested in triplicate or duplicate, and experiments repeated with at least 3 donors. Results are expressed as mean +/- SD.

In vivo human studies: In adoptive T cell therapy, autologous T cells are expanded ex vivo. PBMCs are obtained by leukapheresis, and CD8+ T cells are isolated by magnetic selection. Dendritic cells (DCs) are generated from monocytes cultured with GM-CSF and IL-4 for 7 days, then pulsed with IALYLQQNW (10 ug/mL) and matured with TNF-alpha, IL-6, IL-1beta, and PGE2. T cells are co-cultured with peptide-pulsed DCs at a 10:1 ratio in RPMI/10% human AB serum with IL-7 (10 ng/mL) and IL-15 (5 ng/mL) for 14 days, with restimulation on day 7. Expanded CTLs are tested for specificity and infused into patients at doses of 1 × 10⁶ to 1 × 10⁸ cells/kg. Patient monitoring includes ELISpot, tetramer staining, and clinical response (tumor size by CT/MRI). No animal studies are typically performed.

Pharmacokinetic properties are not applicable. For tetramer or peptide inventory, the peptide is supplied as lyophilized powder, stable at -20degC for 2 years, and is soluble in DMSO or water (1 mg/mL).

No toxicity has been reported for IALYLQQNW itself at concentrations used in vitro (1-10 ug/mL) or ex vivo. The peptide is not administered as a drug; it is used as a stimulus or a building block for tetramers and DC vaccines. In adoptive T cell transfer, toxicities are related to the T cell infusion (cytokine release syndrome, neurotoxicity, on-target off-tumor effects), not the peptide. IALYLQQNW is not FDA-approved.

[1]. Duraiswamy, et al. "Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions." Journal of virology 77.13 (2003): 7401-7410.

IALYLQQNW is an immunogenic peptide from EBV-LMP1, restricted to HLA-B*57:01 and B*58:01 (alleles present in approximately 5-10% of Caucasians and higher frequencies in certain Asian populations). The peptide is used for immune monitoring, vaccine development, and adoptive T cell therapy for EBV-associated malignancies (Hodgkin lymphoma, NPC). It is a research reagent, not a therapeutic. No clinical approval. For research use only.

C55H81N13O14

1148.31

1147.603

610268-80-9

172653514

White to off-white solid powder

15

35

82

2230

10

CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)N

AJGRAIOAMJTVQZ-CVJSAKJISA-N

InChI=1S/C55H81N13O14/c1-8-29(6)46(59)54(80)61-30(7)47(73)64-38(21-27(2)3)51(77)66-40(23-31-13-15-33(69)16-14-31)52(78)65-39(22-28(4)5)50(76)63-36(17-19-43(56)70)48(74)62-37(18-20-44(57)71)49(75)67-41(25-45(58)72)53(79)68-42(55(81)82)24-32-26-60-35-12-10-9-11-34(32)35/h9-16,26-30,36-42,46,60,69H,8,17-25,59H2,1-7H3,(H2,56,70)(H2,57,71)(H2,58,72)(H,61,80)(H,62,74)(H,63,76)(H,64,73)(H,65,78)(H,66,77)(H,67,75)(H,68,79)(H,81,82)/t29-,30-,36-,37-,38-,39-,40-,41-,42-,46-/m0/s1

(2S)-2-[[(2S)-4-amino-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3S)-2-amino-3-methylpentanoyl]amino]propanoyl]amino]-4-methylpentanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-methylpentanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-4-oxobutanoyl]amino]-3-(1H-indol-3-yl)propanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). 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)

DMSO : ~3.33 mg/mL (~2.90 mM; with ultrasonication)

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.)