AH1 targets T-cell receptors (TCRs) on CD8+ cytotoxic T lymphocytes (CTLs). The peptide is presented on the cell surface in complex with the MHC class I molecule H-2Ld. In CT26 colon carcinoma cells (which express the endogenous MuLV gp70 protein), AH1 is naturally processed and presented to CD8+ T cells. The AH1/Ld complex is recognized by specific TCRs, leading to activation, proliferation, and effector functions of CTLs (e.g., IFN-gamma production, granzyme B and perforin release, and target cell lysis). The AH1 epitope is highly immunogenic, and AH1-specific CTLs can be generated in vitro by stimulating CD8+ T cells from BALB/c mice (H-2Ld haplotype) with AH1-pulsed dendritic cells. AH1 is also a model antigen for studying tumor immunology and immune evasion mechanisms, as CT26 tumors can downregulate MHC class I or induce T-cell exhaustion. The epitope has no known off-target interactions with non-immune cells.

In vitro studies demonstrate that AH1 peptide (0.1-10 ug/mL) efficiently stimulates CD8+ T cells from BALB/c mice. In ELISpot assays, splenocytes from CT26-tumor-bearing or AH1-immunized mice (cultured with AH1-pulsed dendritic cells for 5-7 days) produce 200-500 IFN-gamma spot-forming cells (SFCs) per 2×10⁵ cells when restimulated with AH1 (1-10 ug/mL). In cytotoxicity assays, AH1-specific CTLs kill AH1-pulsed target cells (e.g., P815 mastocytoma cells, or CT26 cells naturally presenting the epitope) at effector-to-target (E:T) ratios of 10:1 to 50:1, with 50-90% specific lysis at 4-hour ⁵¹Cr release or calcein-AM release assays. Lysis is MHC class I-dependent and can be blocked by anti-H-2Ld antibody (clone 28-14-8). In proliferation assays, CFSE-labeled CD8+ T cells stimulated with AH1-pulsed DCs show 5-10 fold expansion over 5-7 days, with upregulation of activation markers (CD25, CD69, CD44). AH1 also induces dendritic cell maturation and cytokine production (IL-12, TNF-alpha) when used with adjuvants (e.g., CpG ODN, poly I:C). No direct effects on tumor cells (CT26, TS/A) are observed in the absence of T cells, indicating that AH1 is not directly cytotoxic.

In vivo studies using BALB/c mice (the syngeneic host for CT26 colon carcinoma) demonstrate the immunotherapeutic potential of AH1. In prophylactic vaccination studies, mice immunized subcutaneously with AH1 (50-100 ug) mixed with adjuvant (e.g., incomplete Freund's adjuvant (IFA), CpG ODN, or poly I:C) on day -14 and day -7, then challenged with CT26 tumor cells (5 × 10⁵ s.c.) on day 0, show significant tumor growth delay (mean tumor volume at day 21: 500 mm3 in vaccinated vs 1500 mm3 in control, p < 0.01) and prolonged survival (median survival: 40 days vs 25 days). Some mice (20-30%) remain tumor-free long-term (>100 days). In therapeutic vaccination studies, AH1 (50-100 ug + adjuvant) is administered 5-7 days after CT26 tumor inoculation (established tumors, ~100-200 mm3). Vaccination alone produces modest antitumor effects (30-40% reduction in tumor volume), but combination with immune checkpoint inhibitors (anti-PD-1, anti-CTLA-4, 200 ug each, i.p., days 3, 6, 9) yields synergistic effects (70-80% tumor growth inhibition, 50-60% complete regression). AH1 peptide-pulsed dendritic cell vaccines (1-2 × 10⁶ DCs, i.v. or s.c.) also show efficacy, reducing tumor burden by 50-70%. In adoptive transfer studies, splenocytes from AH1-immunized mice are stimulated in vitro with AH1 (10 ug/mL) for 5 days, and 1-2 × 10⁷ activated CTLs are adoptively transferred to CT26 tumor-bearing mice (day 7 after tumor inoculation). This results in 60-80% tumor growth inhibition and prolonged survival. In TS/A mammary adenocarcinoma models, similar results are observed (50-70% reduction in tumor volume). AH1 does not induce autoimmunity or significant toxicity in normal tissues (e.g., skin, lung, liver, kidney, heart) in these models, as assessed by histology and serum biochemistry.

Non-cell-based assays for peptide-MHC binding: MHC class I stabilization assay using TAP-deficient RMA-S cells (mouse H-2Ld). RMA-S cells are cultured at 26degC overnight to allow empty MHC class I molecules to accumulate on the cell surface. The next day, cells (1 × 10⁶/mL) are incubated with varying concentrations of AH1 peptide (0.1-1000 nM) in serum-free RPMI at 26degC for 2 hours, then shifted to 37degC for 2 hours to stabilize peptide-MHC complexes. Cells are washed and stained with anti-H-2Ld antibody (clone 28-14-8, FITC-conjugated) for 30 min on ice, then analyzed by flow cytometry. The geometric mean fluorescence intensity (MFI) is proportional to the amount of stabilized H-2Ld on the cell surface. The EC50 for AH1 binding to H-2Ld is typically 10-50 nM, with a maximum stabilization (MFI) reaching 5-10 fold over background (no peptide). Alternatively, competitive binding assays using a fluorescently labeled reference peptide (e.g., pBM8-11, a known H-2Ld-binding peptide) and purified H-2Ld protein (refolded in vitro) can be used to determine the binding affinity (IC50) of AH1. IC50 values in the low nanomolar range (1-10 nM) confirm high affinity. For TCR binding studies, soluble AH1/H-2Ld tetramers (constructed by biotinylating H-2Ld heavy chain, beta2-microglobulin, and AH1 peptide, then tetramerizing with streptavidin-PE or -APC) are used to detect AH1-specific CD8+ T cells by flow cytometry. The tetramer can be titrated (0.01-1 ug/mL) to determine binding affinity (Kd).

Splenocytes from BALB/c mice (6-8 weeks) are harvested, red blood cells are lysed with ACK lysis buffer, and cells are resuspended in RPMI 1640 with 10% FBS, 50 uM 2-ME, 1% penicillin-streptomycin. For generation of AH1-specific CTLs, splenocytes (5 × 10⁶/well) are stimulated in 24-well plates with AH1 peptide (10 ug/mL) and IL-2 (10 U/mL) for 5-7 days. Alternatively, bone marrow-derived dendritic cells (BMDCs) are generated from mouse bone marrow with GM-CSF (20 ng/mL) and IL-4 (10 ng/mL) for 6 days, then pulsed with AH1 (10 ug/mL) for 2 hours, washed, and co-cultured with splenocytes (DC: T cell ratio 1:10) for 5-7 days. For cytotoxicity assays, target cells (CT26, P815, or AH1-pulsed P815) are labeled with ⁵¹Cr (100 uCi/10⁶ cells) for 1 hour at 37degC, washed 3×, and plated in 96-well U-bottom plates at 5 × 103 cells/well. Effector CTLs are added at various E:T ratios (10:1, 20:1, 50:1) in triplicate. After 4-hour incubation at 37degC, supernatants are harvested, and ⁵¹Cr release is measured by a gamma counter. Percentage specific lysis = (Experimental release - Spontaneous release) / (Maximum release - Spontaneous release) × 100%. Maximum release is obtained by adding 1% Triton X-100; spontaneous release is from target cells in medium alone. For ELISpot, 96-well PVDF plates are coated with anti-IFN-gamma capture antibody (5 ug/mL) overnight at 4degC. Plates are blocked with 1% BSA for 2 hours. Splenocytes (2 × 10⁵ cells/well) are added with AH1 peptide (1-10 ug/mL) and incubated for 20-24 hours at 37degC. After washing, biotinylated anti-IFN-gamma detection antibody (2 ug/mL) is added for 2 hours, followed by streptavidin-AP (1 hour), and developed with BCIP/NBT substrate. Spots are counted with an ELISpot reader. For intracellular cytokine staining (ICS), splenocytes or CTLs are stimulated with AH1 (10 ug/mL) and brefeldin A (1 ug/mL) for 4-6 hours, then stained for surface CD8, fixed/permeabilized, stained for IFN-gamma or TNF-alpha, and analyzed by flow cytometry. The percentage of CD8+IFN-gamma+ cells is quantified.

For tumor challenge studies, female BALB/c mice (6-8 weeks, 18-22 g) are used. CT26 colon carcinoma cells (5 × 10⁵ in 100 uL PBS) are injected subcutaneously into the right flank. For prophylactic vaccination, mice are immunized with AH1 peptide (50-100 ug) emulsified in IFA or mixed with CpG ODN (50 ug) and poly I:C (50 ug) in a total volume of 100-200 uL, injected s.c. at the base of the tail or in the flank on day -14 and day -7. Control mice receive adjuvant alone or irrelevant peptide (e.g., OVA257-264). On day 0, mice are challenged with CT26 cells. Tumor volumes are measured every 2-3 days with calipers, and mice are euthanized when tumor volume exceeds 1500 mm3 or when they show signs of distress. For therapeutic vaccination, mice are inoculated with CT26 cells on day 0. On day 5-7 (when tumors are palpable, ~50-100 mm3), mice receive AH1 peptide (100 ug) + CpG/poly I:C (50 ug each) s.c. or i.p. in 200 uL PBS. For combination with immune checkpoint blockade, anti-PD-1 (clone RMP1-14, 200 ug) or anti-CTLA-4 (clone 9D9, 200 ug) is administered i.p. on days 3, 6, 9, and 12. For adoptive transfer studies, CTLs are generated as described above (splenocytes stimulated with AH1 for 5-7 days). On day 7 post-CT26 inoculation, 1 × 10⁷ CTLs (in 200 uL PBS) are injected i.v. via the tail vein. Mice may also receive recombinant IL-2 (10,000 U i.p., daily for 5 days) to support CTL survival and expansion. For assessment of antigen-specific T-cell responses in vivo, at endpoint (or at days 7, 14, 21), splenocytes are harvested and analyzed by AH1/H-2Ld tetramer staining, ELISpot, and intracellular cytokine staining. Tumor-infiltrating lymphocytes (TILs) are isolated from tumor tissue after digestion with collagenase IV and DNase I, and similarly analyzed.

Pharmacokinetics of AH1 peptide: Since AH1 is a 9-mer peptide, its systemic half-life after s.c. or i.v. injection is very short (minutes), due to rapid degradation by serum and tissue peptidases (e.g., dipeptidyl peptidases, aminopeptidases, carboxypeptidases). For s.c. administration of free AH1 (100 ug in 100 uL PBS), peak plasma concentration (Cmax) is typically <10 ng/mL, with t1/2 of 5-15 minutes. Most of the injected dose (80-90%) is cleared within 1 hour. To improve stability and immunogenicity, AH1 is often formulated with adjuvants (IFA, CpG, poly I:C) or delivered via peptide-pulsed dendritic cells, where the peptide is processed and presented by MHC class I rather than being administered as free peptide. For DC vaccines, 1-2 × 10⁶ peptide-pulsed DCs are administered, and the half-life of the peptide-MHC complex on DCs (detected by anti-H-2Ld/AH1 complex antibody) is 24-48 hours in vivo. For adoptive T-cell transfer, activated CTLs have a half-life of 2-5 days in the circulation (peak expansion at day 3-5 post-transfer). No formal PK studies have been conducted for AH1 free peptide.

No toxicity data specific to AH1 is available. In animal studies (BALB/c mice, vaccination with AH1 + IFA or CpG, at doses 50-200 ug per injection, given 2-4 times over 2-4 weeks), no significant adverse effects are observed (no body weight loss, no changes in behavior or activity, no histopathological changes in liver, kidney, lung, heart, or spleen). However, incomplete Freund's adjuvant (IFA) can cause granulomas at the injection site, which resolve spontaneously within 2-4 weeks. AH1-specific CTLs generated by vaccination or adoptive transfer do not cause autoimmune reactions in normal tissues (no vitiligo, no alopecia, no enteritis, no myocarditis) based on histopathological examination. In some studies, high doses of AH1 peptide (500 ug, s.c.) with strong adjuvants may cause transient lethargy and mild splenomegaly (increased spleen size by 10-20%) due to T-cell activation, but no long-term toxicity. No genotoxicity, reproductive toxicity, or carcinogenicity studies have been conducted for AH1. For research use, AH1 is considered non-toxic under normal handling conditions (use of PPE, lab coat, goggles). The peptide should be stored as a lyophilized powder at -20degC and is stable for 2-3 years. Solutions (1-10 mg/mL in water or PBS) should be stored at -80degC in aliquots and used within 6 months to avoid degradation (oxidation of methionine, if present, is not relevant for AH1, but tyrosine and histidine residues may be susceptible to oxidation). AH1 is soluble in water, PBS (10 mg/mL), and DMSO (50 mg/mL).

[1]. M Toda, et al. Herpes simplex virus as an in situ cancer vaccine for the induction of specific anti-tumor immunity. Hum Gene Ther. 1999 Feb 10;10(3):385-93.
[2]. Antonio Rosato, et al. The cytotoxic T-lymphocyte response against a poorly immunogenic mammary adenocarcinoma is focused on a single immunodominant class I epitope derived from the gp70 Env product of an endogenous retrovirus. Cancer Res. 2003 May 1;63(9):2158-63.

AH1 is one of the most widely studied model tumor antigens in preclinical cancer immunology research. It is derived from the gp70 envelope protein of an endogenous ecotropic murine leukemia virus (MuLV) that is expressed in CT26 cells (a BALB/c-derived colon carcinoma cell line). The peptide is presented by the MHC class I molecule H-2Ld, which is expressed in BALB/c mice (H-2d haplotype). AH1 is also expressed in other MuLV-positive mouse tumors, including TS/A (mammary adenocarcinoma) and Renca (renal carcinoma). The AH1 epitope is highly immunogenic and is often used to study T-cell responses, immune tolerance, tumor immune evasion (e.g., downregulation of MHC class I, upregulation of PD-L1, recruitment of Tregs), and to evaluate the efficacy of cancer vaccines (including peptide vaccines, DNA vaccines, viral vector vaccines, DC vaccines, and neoantigen vaccines). AH1 is also used to generate tumor-specific T-cell lines for adoptive cell transfer (ACT) studies, as a positive control for tetramer staining, and as a tool to study the mechanisms of T-cell exhaustion and checkpoint blockade. The sequence of AH1 is SPSYVYHQF (single-letter code). The peptide is typically synthesized by solid-phase peptide synthesis (SPPS) with purity >95% (by HPLC) and characterized by mass spectrometry. AH1 is not a drug; it is a research reagent for in vitro and in vivo studies. It is not FDA-approved and has no clinical indications. The peptide should be stored as a lyophilized powder at -20degC, protected from light and moisture, and is stable for at least 2 years. For use in cell culture, stock solutions (10 mg/mL in water or 50% acetonitrile/water) should be stored at -20degC in aliquots and used within 1 year. Avoid repeated freeze-thaw cycles (>3 cycles). For in vivo use, AH1 should be dissolved in sterile PBS or saline and used immediately. The peptide is not for human use and is intended for laboratory research only. The peptide is not classified as hazardous (GHS not required) but should be handled with gloves and lab coat. Always follow institutional biosafety guidelines when working with live animals and tumor cell lines.

C54H70N12O15

1127.21

1126.51

181272-91-3

101760040

White to off-white solid powder

0.868

15

17

30

81

2140

9

CC(C)C(C(=O)NC(CC1=CC=C(C=C1)O)C(=O)NC(CC2=CN=CN2)C(=O)NC(CCC(=O)N)C(=O)NC(CC3=CC=CC=C3)C(=O)O)NC(=O)C(CC4=CC=C(C=C4)O)NC(=O)C(CO)NC(=O)C5CCCN5C(=O)C(CO)N

OUDYTNPKUOHOHA-CWBDEABLSA-N

InChI=1S/C54H70N12O15/c1-29(2)45(65-49(75)39(22-32-12-16-35(70)17-13-32)60-50(76)42(27-68)64-51(77)43-9-6-20-66(43)53(79)36(55)26-67)52(78)62-38(21-31-10-14-34(69)15-11-31)47(73)61-40(24-33-25-57-28-58-33)48(74)59-37(18-19-44(56)71)46(72)63-41(54(80)81)23-30-7-4-3-5-8-30/h3-5,7-8,10-17,25,28-29,36-43,45,67-70H,6,9,18-24,26-27,55H2,1-2H3,(H2,56,71)(H,57,58)(H,59,74)(H,60,76)(H,61,73)(H,62,78)(H,63,72)(H,64,77)(H,65,75)(H,80,81)/t36-,37-,38-,39-,40-,41-,42-,43-,45-/m0/s1

(2S)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-amino-3-hydroxypropanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxypropanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoic acid

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)