MHC class I H-2Db (in mice) and potentially HLA in humans. Gp100 (25-33), human is a 9-amino acid peptide epitope derived from the human melanoma antigen gp100 (Pmel17). The human peptide sequence is KVPRNQDWL. This peptide is presented on the MHC class I molecule H-2Db in mice (and can also be presented by certain human HLA alleles, though its primary use is in mouse models due to its described H-2Db restriction). It is recognized by CD8+ cytotoxic T lymphocytes (CTLs). The peptide does not bind to a conventional receptor but is presented by MHC molecules to the T cell receptor (TCR) of specific T cells. Gp100 is highly expressed in melanoma cells, making this peptide a target for cancer immunotherapy. The human gp100 peptide is often used in mouse models (transgenic or adoptive transfer) to study melanoma immunity, as well as in human T cell studies using HLA-matched cells.

In vitro, Gp100 (25-33), human TFA (KVPRNQDWL) is used to stimulate human CD8+ T cells from HLA-A2-positive donors (and also to stimulate T cells in H-2Db transgenic mouse systems). The peptide (0.01-10 uM) is presented by antigen-presenting cells (APCs) expressing the appropriate MHC (HLA-A2 or H-2Db) to gp100-specific T cells. T cell activation is measured by IFN-gamma ELISpot, intracellular cytokine staining (ICS) for IFN-gamma and TNF-alpha, proliferation (3H-thymidine or CFSE dilution), and upregulation of activation markers (CD69, CD25) by flow cytometry. The peptide can also be used to expand gp100-specific T cells from peripheral blood mononuclear cells (PBMCs) of melanoma patients for use in adoptive cell transfer (ACT) therapies. In one study, recombinant vaccinia virus encoding human gp100 (rVVgp100) was used to generate CD4+CD8+ lytic T lymphocytes in splenocytes, and the peptide was used as a target to measure cytolytic activity. The peptide does not have direct enzymatic or receptor-modulating activity; its activity is immune-mediated. The TFA salt does not affect peptide presentation. The peptide is also used as a positive control in MHC-peptide tetramer production for detecting antigen-specific T cells. For direct T cell activation assays, the peptide is used at concentrations ranging from 0.1 nM to 10 uM.

In vivo, Gp100 (25-33), human TFA has been used in mouse models of melanoma to induce anti-tumor immune responses. In a study using recombinant vaccinia virus (rVV) encoding human gp100, T cells induced by rVVgp100 (administered intraperitoneally, twice daily for 5 days) showed anti-tumor effects in B16 tumor-bearing mice models. In the B16 mouse melanoma model (which expresses murine gp100, which shares some homology with human gp100), immunization with the human gp100 25-33 peptide can induce cross-reactive T cell responses. A typical protocol: C57BL/6 mice are immunized subcutaneously with 50-100 ug of human Gp100 (25-33) peptide emulsified in complete Freund's adjuvant (CFA) or mixed with CpG (50 ug). A booster immunization may be given with peptide in IFA on day 7-14. Splenocytes are harvested, and T cell responses are analyzed by IFN-gamma ELISpot, ICS, or tetramer staining. In tumor challenge experiments, mice are immunized with the peptide (with adjuvant) on day -14 and -7, then challenged with B16-F10 melanoma cells (5 × 10^5 cells subcutaneously) on day 0. Peptide immunization may reduce tumor growth and prolong survival, especially when combined with checkpoint inhibitors (anti-PD-1, anti-CTLA-4). However, because the human peptide does not perfectly match the mouse sequence (mouse: EGSRNQDWL vs. human: KVPRNQDWL), the cross-reactivity may be limited; thus, adoptive transfer of T cells from humanized mice or using HLA-A2 transgenic mice (AAD mice) is often used. In such models, the peptide can be used to prime T cells. The TFA salt is suitable for in vivo injection, typically dissolved in PBS at 1 mg/mL. The peptide is generally well-tolerated at doses up to 200 ug per mouse. The TFA counterion is not a concern for in vivo studies.

For direct binding assays (non-cellular), the binding affinity of the human Gp100 (25-33) peptide (KVPRNQDWL) to the MHC class I molecule H-2Db can be measured. Although this peptide is known to be restricted by H-2Db in mice, it may also bind to certain human HLA class I molecules (e.g., HLA-A*0201). A standard MHC-peptide binding assay uses recombinant H-2Db or HLA-A2 protein with beta2-microglobulin. A fluorescent polarization (FP)-based competition assay is common. Recombinant H-2Db/beta2m complex (0.1-1 uM) is incubated with a fluorescently labeled reference peptide (e.g., FITC-labeled H-2Db-binding peptide) in binding buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% NP-40, 1 mM DTT, protease inhibitors). Varying concentrations of unlabeled human Gp100 (25-33) peptide (0.001-1000 uM) are added, and the mixture is incubated for 24-48 hours at room temperature to allow peptide binding and exchange. FP is measured (excitation 485 nm, emission 520 nm). The IC50 (concentration that displaces 50% of the reference peptide) is determined. Alternatively, an ELISA-based competition assay: Coat a 96-well plate with H-2Db/beta2m protein, add a known biotinylated peptide with and without unlabeled test peptide, detect with streptavidin-HRP, and measure absorbance at 450 nm. IC50 is calculated using a 4-parameter logistic curve. The Kd (or IC50) for the human peptide binding to H-2Db is typically in the low micromolar range (e.g., 0.1-10 uM). For binding to HLA-A2: Use recombinant HLA-A2/beta2m and a reference peptide (e.g., Flu M1 58-66, GILGFVFTL). The binding affinity (IC50) of human gp100 25-33 to HLA-A2 is reported to be around 1-10 uM. For peptide stability: Incubate the peptide in human or mouse serum (37degC, 0-24 h) and analyze degradation by HPLC or LC-MS.

For cell-based T cell assays using human cells, PBMCs are isolated from HLA-A2-positive healthy donors or melanoma patients by Ficoll-Paque density gradient centrifugation. CD8+ T cells are purified by positive or negative selection using magnetic beads. For ELISpot assays: 96-well ELISpot plates are coated with anti-IFN-gamma antibody overnight. CD8+ T cells (1-2 × 10^5 cells/well) are co-cultured with T2-A2 cells (HLA-A2-expressing antigen-presenting cells, 1 × 10^4 cells/well) or dendritic cells (generated from monocytes with GM-CSF/IL-4 for 5-7 days). The human Gp100 (25-33) peptide (KVPRNQDWL) is added at serial dilutions (0.001-1000 nM). After 20-24 hours at 37degC, the plate is developed as per the manufacturer's instructions, and spots are counted. For T cell expansion: CD8+ T cells are stimulated with 1-10 uM peptide in the presence of 10-20 U/mL IL-2, and after 7-14 days, T cells are tested for specificity by ELISpot or chromium release assay. For intracellular cytokine staining (ICS): CD8+ T cells are stimulated with peptide (1-10 uM) for 6-18 hours in the presence of brefeldin A (added after the first 2 hours). Cells are then surface-stained for CD8, fixed, permeabilized, and stained for IFN-gamma and TNF-alpha. Flow cytometry is used to quantify cytokine-producing CD8+ T cells. For cytotoxicity assays (51Cr-release or DELFIA): Target cells (e.g., T2-A2 cells loaded with 0.1-10 uM peptide) are labeled with 51Cr (or BATDA), then co-cultured with effector CD8+ T cells at various effector:target ratios. After 4-6 hours, supernatant is harvested, and chromium release (or europium release) is measured. Percent-specific lysis = (experimental release - spontaneous release)/(maximum release - spontaneous release) × 100%. For tetramer staining: PE-labeled HLA-A2/Gp100 25-33 tetramers are used to stain PBMCs or CD8+ T cells. Cells are incubated with tetramer (1-2 ug/mL) for 30 min at room temperature, then with anti-CD8 antibody, and analyzed by flow cytometry. The TFA salt is dissolved in DMSO (10 mM stock) and stored at -20degC. For assays, dilute in PBS or culture medium. Avoid repeated freeze-thaw cycles. All experiments should include an irrelevant control peptide (e.g., HIV gag, Flu M1). For mouse studies using human peptide, C57BL/6 mice or HLA-A2 transgenic mice (AAD mice) are used. For tetramer staining: MHC-H-2Db tetramers loaded with the human gp100 25-33 peptide are also commercially available. Perform similar assays to those described for mouse peptide (V76964). Note that the human peptide is often used in human T cell assays rather than mouse T cell assays, but it can be used in H-2Db-expressing mouse cells as it is cross-restricted.

For in vivo studies in mice, female C57BL/6 mice (6-8 weeks old) or HLA-A2 transgenic mice (HHD mice or AAD mice) are used. Human Gp100 (25-33) peptide (KVPRNQDWL) is dissolved in sterile PBS at 1 mg/mL. For active immunization, the peptide is emulsified with an adjuvant. For CFA/IFA: Mix equal volumes of peptide solution (50-100 ug peptide per mouse) with complete Freund's adjuvant (CFA) for primary immunization, or with incomplete Freund's adjuvant (IFA) for booster. Emulsify by mixing with a syringe until stable emulsion forms. Inject 100 uL emulsion subcutaneously (s.c.) at the base of the tail or in the flank. For CpG-based immunization: Mix peptide (50-100 ug) with CpG ODN (50 ug) in PBS (total 100 uL) and inject s.c. or i.p. A typical schedule: Day 0: primary immunization (peptide + CFA). Day 7 or 14: booster immunization (peptide + IFA). On day 14-21, spleens are harvested for T cell assays (ELISpot, ICS, tetramer). For tumor challenge experiments: B16-F10 melanoma cells (5 × 10^5 in 100 uL PBS) are injected subcutaneously into the right flank on day 0. Immunization may be performed on day -14 and -7 (prophylactic) or on day 3 and 10 (therapeutic) relative to tumor injection. Tumor growth is monitored. For recombinant vaccinia virus encoding human gp100 (rVVgp100): Mice are immunized with rVVgp100 (1 × 10^7 PFU in 100 uL PBS, intraperitoneally, twice daily for 5 days) to generate T cell responses. In one published study, T cells induced by rVVgp100 (i.p., twice a day for 5 days) showed anti-tumor effects in B16 tumor mouse models. For adoptive transfer: CD8+ T cells from immunized mice or from pmel-1 TCR transgenic mice (cross-reactive to human gp100) are expanded in vitro with the peptide (1 uM) and IL-2, then transferred intravenously (5 × 10^6 cells) into tumor-bearing mice. Checkpoint inhibitors (anti-PD-1, anti-CTLA-4) may be co-administered to enhance efficacy. The TFA salt is acceptable for in vivo injection; adjust pH if necessary. All animal procedures require IACUC approval.

No detailed pharmacokinetic data are available for human Gp100 (25-33) TFA. As a 9-amino acid peptide (MW ~1.3 kDa), it is rapidly cleared from circulation after intravenous or subcutaneous administration (t1/2 < 10 min) due to renal clearance and proteolytic degradation. For immunization, the peptide is typically administered in an adjuvant (CFA/IFA), which forms a depot at the injection site and releases the peptide slowly over several days to weeks. The TFA salt does not affect PK. The free peptide is not intended to be used as a systemic drug; its purpose is to be processed by APCs for antigen presentation. Therefore, PK parameters are not published and are not relevant to its use as an immunogen. If the peptide is delivered in a nanoparticle or liposome formulation, the PK may be extended, but such formulations are not standard for this product. The product is for research use only and is not a drug.

No specific toxicity data are available for human Gp100 (25-33) TFA. As a 9-amino acid peptide derived from a human self/tumor antigen, it is generally well-tolerated in animal studies. In C57BL/6 mice immunized with the peptide (50-100 ug in CFA/IFA), no overt signs of acute toxicity (e.g., weight loss, lethargy, respiratory distress) are observed. Injection site reactions (erythema, swelling) are due to the adjuvant (CFA/IFA), not the peptide. Some mice may develop depigmentation (vitiligo) due to cross-reactivity with mouse melanocytes (since mouse gp100 shares homology), which is an on-target autoimmune effect rather than toxicity. This model is used to study the autoimmune side effects of cancer immunotherapies. No genotoxicity, carcinogenicity, or organ toxicity has been reported. The TFA salt is present in low amounts and is considered non-toxic. The peptide is for research use only; it is not approved for human or veterinary use. Standard laboratory safety precautions (gloves, lab coat) should be used. The peptide should not be injected into humans.

[1]. Overwijk WW, et al. gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cellsusing high-affinity, altered peptide ligand. J Exp Med. 1998 Jul 20;188(2):277-86.

The human gp100 (Pmel17) protein is a melanocyte-specific glycoprotein highly expressed in melanoma cells. It is a major target for cancer immunotherapy, including peptide vaccines, adoptive T cell transfer (ACT), and checkpoint inhibitors. The gp100 25-33 epitope (KVPRNQDWL) is a human peptide that is presented by MHC class I molecules (HLA-A2 in humans) and is recognized by CD8+ T cells in melanoma patients. It has been used in several clinical trials as a vaccine for melanoma. However, the human gp100 (25-33) peptide supplied as TFA salt is for research use only and is not for human injection without appropriate regulatory approval (e.g., IND). The TFA salt improves peptide solubility and stability. The peptide is also used to generate T cell responses in HLA-A2 transgenic mouse models, where it binds to H-2Db (the mouse MHC) and serves as a tool to study cross-restricted T cell immunity. The peptide is not an approved drug, and no FDA-approved indication exists for it. It is strictly for research purposes, as a biochemical reagent.

C54H83F3N16O16

1269.33

Gp100 (25-33), human;212370-40-6

Typically exists as solid at room temperature

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O :~100 mg/mL (~78.78 mM)

Solubility in Formulation 1: 66.67 mg/mL (52.52 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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