Protocol to prepare MUC1 glycopeptide vaccines and evaluate immunization effects in mice

Summary

The tumor-associated mucin MUC1 is overexpressed in almost all types of epithelial tumor tissues, making it an attractive target antigen for cancer immunotherapy. Here we present a protocol to prepare MUC1 glycopeptide vaccines and to evaluate immunization effects in mice. We describe steps for synthesizing glycopeptide antigen and conjugating it with carrier protein to make vaccine candidates. We then detail procedures for mice immunization, antibody response evaluation, and cellular immune response.

For complete details on the use and execution of this protocol, please refer to Cai et al.^1,2

Graphical abstract

Highlights

• •Prepare tumor-associated glycopeptide antigen by SPPS
• •Prepare vaccine candidates using protein carrier to increase glycopeptide immunogenicity
• •Evaluate vaccine effect by antibody potency and cellular immune response

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The tumor-associated mucin MUC1 is overexpressed in almost all types of epithelial tumor tissues, making it an attractive target antigen for cancer immunotherapy. Here we present a protocol to prepare MUC1 glycopeptide vaccines and to evaluate immunization effects in mice. We describe steps for synthesizing glycopeptide antigen and conjugating it with carrier protein to make vaccine candidates. We then detail procedures for mice immunization, antibody response evaluation, and cellular immune response.

Before you begin

MUC1 is a type I transmembrane protein that is widely expressed in epithelial cells of various tissues and organs. Compared with normal cells, the expression level of MUC1 on tumor cells is greatly increased and the glycan profile is significantly changed, making it an attractive target for cancer immunotherapy.³ The tumor-associated carbohydrate antigens comprise the Thomsen-Friedenreich antigen (T antigen), its precursor (Tn antigen), and their respective sialylated derivatives STn, 2,3-ST and 2,6-ST.⁴ The protocol below describes the specific steps for preparing MUC1 glycopeptide (HGVTSAPDTRPAPGSTAPPA) bearing Tn antigen and conjugating it with bovine serum albumin (BSA) as the carrier protein.^1,2 This protocol also applies to conjugate MUC1 glycopeptide bearing different carbohydrate antigens with other carrier proteins such as bacteriophage Qβ carrier,⁵ keyhole limpet hemocyanin (KLH),⁵ tetanus toxoid (TTox)^6,7 and diphtheria toxoid (DT). In addition, the protocol can be further refined and expanded for drug discovery of MUC1 monoclonal antibodies.

Institutional permissions

All animal handling and procedures were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University under license numbers: 2023002447. Anyone who is interested in conducting described experiments needs to acquire permission from the relevant institutions before.

Synthesis of threonine monomer of Tn antigens modified by protecting groups

Timing: 2 weeks

This protocol describes the synthesis of MUC1 glycopeptide using glycosylated amino acids by combining the building block strategy with Fmoc solid-phase peptide synthesis strategy. Threonine monomer of Tn antigen modified by protecting groups need to be synthesized before glycopeptide synthesis taken place (Figure 1).^8,9

• 1.
  Prepare N-(9H-Fluoren-9-yl)-methoxycarbonyl-L- threonin-tert-butylester 3.

  • a.
    Add 110.7 g dicyclohexylcarbodiimide (537 mmol, 3.3 equiv), 22.6 g tert-butanol (698 mmol, 4.3 equiv) and 1.2 g Copper(I) chloride into a flask and stir for 4 days protected from light and argon.
  • b.
    Dilute the resulting mixture with 100 mL of absolute anhydrous dichloromethane and cool it in an ice bath.
  • c.
    Dissolve 55.0 g N-(9H-Fluoren-9-yl)-methoxycarbonyl-L-threonine 2 (161 mmol, 1.0 equiv) in 250 mL absolute anhydrous dichloromethane solution.

    Note: Add it slowly and dropwise into the cooled mixture from the previous step.

    • i.
      Stir for 90 min.
    • ii.
      Monitor by electrospray ionization mass spectrometry (ESI-MS).
  • d.
    After completion of the reaction, filter it through diatomaceous earth or Celite and wash with cold dichloromethane.
  • e.
    Wash the resulting filtrate with 50 mL of saturated sodium bicarbonate for three times, dry it with anhydrous magnesium sulfate and then remove the organic solvent through reduced-pressure (vacuum).
  • f.
    Dissolve 60 g residue in 80 mL ethyl acetate.

    • i.
      Keep at −30°C for 3 h.
    • ii.
      Filter to remove the precipitated solid insoluble material, and remove the solvent by reduced-pressure (vacuum).
  • g.
    Purify the crude product by silica gel column (80 × 800 mm, Synthware) filled with 100 g silica gel. R_f = 0.50 (cyclohexane:ethyl acetate = 3:1).
  • h.
    Dissolve 35.0 g purified product in 50 mL ether, and then precipitate it by dropwise addition of petroleum ether for further purification.
• 2.
  Prepare N-(9H-Fluoren-9-yl)-methoxycarbonyl-O-(3,4,6-tri-O-acetyl-2-azido-2-desoxy-α- D-galactopyranosyl)-L-threonine-tert-butylester 5 (Fmoc-Thr(αAc3GalN3)-OtBu).

  • a.
    Dissolve 12.8 g of compound 3 (37.5 mmol, 1.0 equiv) in 140 mL of anhydrous dichloromethane and 120 mL of anhydrous toluene.

    • i.
      Add 40 g activated molecular sieve 4 Å.
    • ii.
      Stir for 1 h at 25°C under argon and exclusion of light.
    • iii.
      Cool to 0°C with an ice bath.
  • b.
    Add 14.8 g silver carbonate (53.7 mmol, 1.4 equiv) and 1.9 g silver perchlorate (9.0 mmol, 0.24 equiv, dissolve in 60 mL of absolute anhydrous toluene), and stir for 30 min at 0°C in the dark.
  • c.
    Dissolve 14.8 g (37.5 mmol, 1.0 equiv) of acetylated galactosyl bromide 4 in 300 mL of anhydrous toluene and dichloromethane (1:1), and add it to the above reaction system slowly, stir the reaction mixture for 20 h at 25°C.
  • d.
    Upon completion of the reaction, dilute the reaction mixture with 300 mL of dichloromethane and filter through diatomaceous earth or Celite.

    • i.
      Wash the resulting filtrate twice with 300 mL of saturated sodium bicarbonate and twice with 300 mL of saturated sodium chloride.
    • ii.
      Dry the organic phase with anhydrous magnesium sulfate and remove the organic solvent by reduced-pressure (vacuum).
  • e.
    Purify the crude product by silica gel column chromatography (40 × 200 mm, Synthware) with the eluent of dichloromethane:ethyl acetate = 10:1, and obtain the target product 5. R_f = 0.63.

Note: The synthesis of acetylated galactosyl bromide 4 can refer to the method reported by Lemieux and Ratcliffe.⁸

• 3.
  Prepare N-(9H-Fluoren-9-yl)-methoxycarbonyl-O-(2-actamido-3,4,6-tri-O-acetyl-2-desoxy-α-D-galactopyranosyl)-L-threonine-tert-butylester 6 (Fmoc-Thr(αAc3GalNAc)-OtBu).

  • a.
    Dissolve 11.0 g compound 5 (15.5 mmol) in 1000 mL of a mixture of tetrahydrofuran, acetic anhydride, and acetic acid (3:2:1), add 10.1 g zinc powder (dissolved in 400 mL 2% cupric sulfate), and stir the mixture for 16 h at 25°C.
  • b.
    Filter the mixture through diatomaceous earth or Celite to remove insoluble solids and wash with 300 mL of tetrahydrofuran.
  • c.
    Remove the solvent by reduced-pressure (vacuum), and then dissolve the resulting residue in 200 mL of dichloromethane.

    • i.
      Wash the residue three times with 150 mL of saturated sodium bicarbonate solution and once with 150 mL of saturated saline.
    • ii.
      Dry the organic phase with anhydrous magnesium sulfate, and then remove the solvent by reduced-pressure (vacuum).
  • d.
    Purify the crude product by silica gel column chromatography (40 × 200 mm, Synthware) with the eluent of cyclohexane:ethyl acetate = 1:3, and obtain the target product 6. R_f = 0.41.
• 4.
  Prepare N-(9H-Fluoren-9-yl)-methoxycarbonyl-O-(2-actamido-3,4,6-tri-O-acetyl-2-desoxy-α-D-galactopyranosyl)-L-threonine 1 Fmoc-Thr(αAc3GalNAc)-OH.

  • a.
    Dissolve 2.5 g compound 6 (3.4 mmol) in 30 mL of trifluoroacetic acid and 1.8 mL of anisole and stir under argon protection at 25°C for 2 h.
  • b.
    Remove the solvent by reduced-pressure (vacuum) and add appropriate amount of toluene in three portions to bring out the residual solvent.
  • c.
    Purify the crude product by silica gel column chromatography (40 × 200 mm, Synthware) using ethyl acetate as eluent, and then obtain the target product 1. R_f = 0.51.

Figure 1.

Synthesis of threonine monomer of Tn antigens modified by protecting groups

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Dicyclohexylcarbodiimide	BIDE	Cat#BD220228
Tert-butanol	Macklin	Cat#T819475
Copper(I) chloride	Sigma-Aldrich	Cat#224332
Dichloromethane	Titan	Cat#1019
Sodium bicarbonate	Macklin	Cat#S818079
Anhydrous magnesium sulfate	BIDE	Cat#BD137048
Ethyl acetate	Titan	Cat#1024
Cyclohexane	Macklin	Cat#C804201
Toluene	XIHUA	Cat#1105
Sodium chloride	XIHUA	Cat#1069
Tetrahydrofuran	Macklin	Cat#T818769
Acetic anhydride	XIHUA	Cat#zhyl-14
Acetic acid	Macklin	Cat#A801295
Zinc powder	XIHUA	Cat#zhyl-18
Cupric sulfate	BIDE	Cat#BD122482
Trifluoroacetic acid	Macklin	Cat#T818778
Anisole	Macklin	Cat#A800314
N, N-dimethyl formamide	YINLI	Cat#SHYL-DMF
Triisopropylsilane	Macklin	Cat#T819181
Ethanol	XIHUA	Cat#1001
Fmoc-Ala-2-Chlorotrityl resin	GL Biochem	Cat#44001
N-hydroxybenzotriazole (HOBT)	GL Biochem	Cat#00602
O-(1H-benzotriazol-1-yl)-N, N, N′, N′-tetramethyluronium hexa-fluoro-phosphate (HBTU)	GL Biochem	Cat#00702
N-hydroxy-7-azabenzotria-zole (HOAT)	GL Biochem	Cat#00601
O-(7-azabenzotriazole-1-yl)-N, N, N′, N′-tetramethyl-uronium hexafluoro-phosphate (HATU)	GL Biochem	Cat#00703
N, N-Diisopropylethylamine (DIEA)	Macklin	Cat#N807281
1-(9H-Fluoren-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azahexadecan-16-oic acid	BIDE	Cat#BD250099
D-biotin	GL Biochem	Cat#10703
Acetonitrile	Tedia	Cat#01284488-AS1122-801
Sodium methylate	Macklin	Cat#S817797
Methanol	Thermo Fisher Scientific	Cat#A452
Sodium tetraborate	Macklin	Cat#S817467
Potassium bicarbonate	Macklin	Cat#P816192
3,4-diethoxy-3-cyclobutene-1,2-dione	Meryer	Cat#M32760
Bovine serum albumin	Sigma	Cat#B2064
Rehydragel@LV alum adjuvant	Bioss	Cat#C5084
pNPP one component ELISA substrate	DuraStab	Cat#PNST-0500
pNPP Stop Solution 405	DuraStab	Cat#STPY-0500
Casein blocking buffer	Macklin	Cat#P917797
Phosphate-buffered saline with Tween-20 (PBST)	Biosharp	Cat#BL345A
Phosphate-buffered saline (PBS)	Biosharp	Cat#BL302A
Tween-20	Selleck	Cat#Tween-20
Fetal bovine serum (FBS)	Gibco	Cat#C0235
Penicillin-streptomycin solution (100x)	Gibco	Cat#15140122
0.25% trypsin-EDTA	Gibco	Cat#25200056
DMEM, high glucose, pyruvate	Gibco	Cat#C11995500BT
RPMI 1640 medium	Gibco	Cat#11875093
Brefeldin A	MCE	Cat#HY-16592
Antibodies
Anti-mouse IgG (whole molecule)-alkaline phosphatase antibody produced in goat (1:3,000)	Sigma	Cat#A3562
Anti-mouse IgG1 (heavy chain specific), antibody produced in goat (1:1,000)	Sigma	Cat#M5532
Anti-mouse IgG2a (heavy chain specific), antibody produced in goat (1:1,000)	Sigma	Cat#M5657
Anti-mouse IgG2b (heavy chain specific), antibody produced in goat (1:1,000)	Sigma	Cat#M5782
Anti-mouse IgG3 (heavy chain specific), antibody produced in goat (1:1,000)	Sigma	Cat#M5907
Anti-mouse IgM (μ-chain specific), antibody produced in goat (1:1,000)	Sigma	Cat#M6157
Anti-mouse IgA (α-chain specific), antibody produced in goat (1:1,000)	Sigma	Cat#M6032
Donkey anti-goat IgG Alk, phosphatase conjugate (1:3,000)	Alpha Diagnostic International	Cat# 30350-200
Goat-anti-mouse (IgG H&L) (Alexa Fluor 488) (1:1,000)	Abcam	Cat#ab150113
Experimental models: Organisms/strains
BALB/c, female, 6–8 weeks	Zhuhai BesTest Bio-Tech Co,.Ltd.
Other
6-well plate	NEST	Cat#CM400035-703001
Cell culture flask, T-25	NEST	Cat#CM400040-707003
Cell culture flask, T-75	NEST	Cat#CM400040-708003
Centrifuge tubes, conical 50 mL	LABSELECT	Cat#CT-002-50A
Centrifuge tubes, conical 15 mL	LABSELECT	Cat#CT-002-15A
70-μm cell strainer	Biosharp	Cat#BS-70-CS-N1

Materials and equipment

DMEM complete medium

Reagent	Final concentration	Amount
DMEM, High Glucose, Pyruvate	N/A	500 mL
Fetal Bovine Serum	10%	50 mL
Penicillin-Streptomycin (100 x)	1 x	5 mL
Total	N/A	555 mL

The medium can be stored for up to 6 months at 4°C.

1640 complete medium

Reagent	Final concentration	Amount
RPMI-1640 Medium	N/A	500 mL
Fetal bovine serum	10%	50 mL
Penicillin-Streptomycin (100 x)	1 x	5 mL
Total	N/A	555 mL

The medium can be stored for up to 6 months at 4°C.

Other solution

Name	Reagent
PBST	PBS + 0.05% Tween-20 (v/v)
Flow cytometry buffer	PBS + 1% FBS

Step-by-step method details

Preparation of MUC1 glycopeptide bearing Tn antigen

Timing: 4 days

In this part, we describe the preparation of MUC1 glycopeptide based on microwave-assisted solid-phase peptide synthesis by an automated peptide synthesizer (CEM Liberty Blue-Microwave Peptide Synthesizer) (Figure 2).

• 1.
  Synthesize MUC1 glycopeptide 8.

  • a.
    Place Fmoc-Ala-2-Cl-Trityl resin (0.1 mmol, 0.163 mmol/g, 1.0 equiv), Fmoc amino acids (0.5 mmol, 5.0 equiv), O-(1H-benzotriazol-1-yl)-N, N, N′, N′-tetramethyluronium hexa-fluoro-phosphate (HBTU) (0.5 mmol, 5.0 equiv), N-hydroxybenzotriazole (HOBt) (0.5 mmol, 5.0 equiv), and N, N-diisopropylethylamine (DIEA) (1.0 mmol, 10.0 equiv) in the corresponding position of the automated peptide synthesizer.
  • b.
    Initiate the peptide synthesis cycle.

    • i.
      Use 20% piperidine in 4 mL N, N-dimethyl formamide (DMF) to remove Fmoc groups from the resin at 50°C for 2 min, repeat the deprotection step twice.
    • ii.
      Wash the resin with 4 mL DMF four times.
    • iii.
      Add the second Fmoc amino acid (0.5 mmol, 5.0 equiv) at the C-terminus of the peptide, HBTU (0.5 mmol, 5.0 equiv), HOBt (0.5 mmol, 5.0 equiv), and DIEA (1.0 mmol, 10.0 equiv) automatically, and couple the Fmoc amino acid to the resin at 50°C for 10 min.
    • iv.
      Wash the resin with 4 mL DMF four times.
    • v.
      Repeat steps i-iv until all the amino acids and the N-terminal linker 1-(9H-Fluoren-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azahexadecan-16-oic acid are coupled to the resin one by one in the order from C-terminal to N-terminal.

Note: You can activate the protected Tn glycosyl Thr building blocks by O-(7-azabenzotriazole-1-yl)-N, N, N′, N’ -tetramethyluronium hexafluorophosphate (HATU)/ N-hydroxy-7-azabenzotriazole (HOAt) using DIEA in DMF instead of HBTU/HOBt. Dissolve glycosyl amino acid building blocks (0.3 mmol, 3.0 equiv), HATU (0.3 mmol, 3.0 equiv), HOAt (0.3 mmol, 3.0 equiv), and DIEA (0.6 mmol, 6.0 equiv) in 3 mL DMF, mix them manually with the resin and react at 50°C for 45 min. Couple the linker to the resin in the same way.

• 2.Transfer the resin from the peptide synthesizer into a flask.
• 3.Treat the resin with 4 mL mixture of trifluoroacetic acid/triisopropylsilane/water (90/5/5, v/v/v) for 2 h to detach the peptide.
• 4.Confirm the structure and purity of glycopeptide by analytical reversed-phase high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry (ESI-MS) analysis, respectively.

Note: The MUC1 glycopeptide is run on a C18 column (YMC-Triart C18, 4.6 × 250 mm, 5 μm) at a flow rate of 1 mL/min using a linear gradient of 10%–30% acetonitrile containing 0.1% trifluoroacetic acid over 20 min, detection at 215 nm. RP-HPLC retention time, t_R = 20 min.

• 5.Purify the crude glycopeptides by RP-HPLC on a preparative C-18 column (Waters SymmetryPrepTM, 19 × 300 mm, 7 μm) with a flow rate of 20 mL/min, using a linear gradient of 10%–50% acetonitrile containing 0.1% trifluoroacetic acid over 40 min, detection at 215 nm. RP-HPLC retention time, t_R = 18 min.
• 6.After lyophilization, dissolve the glycopeptide in methanol and add 0.5% sodium methylate / methanol solution dropwise carefully until a pH 11.0 is reached. Stir for 16 h at 25°C (controlled by RP-HPLC and ESI-MS).
• 7.Neutralize the reaction mixture with acetic acid, and the solvent is removed by reduced-pressure (vacuum).
• 8.Confirm the structure of glycopeptide by analytical RP-HPLC and ESI-MS analysis, respectively.

Note: The MUC1 glycopeptide is run on a C18 column (YMC-Triart C18, 4.6 × 250 mm, 5 μm) at a flow rate of 1 mL/min using a linear gradient of 10%–30% acetonitrile containing 0.1% trifluoroacetic acid over 20 min, detection at 215 nm. RP-HPLC retention time, t_R = 18 min.

• 9.Purify the left residue by RP-HPLC on a preparative C-18 column (Waters SymmetryPrepTM, 19 × 300 mm, 7 μm) with a flow rate of 20 mL/min, using a linear gradient of 10%–50% acetonitrile containing 0.1% trifluoroacetic acid over 40 min, detection at 215 nm. RP-HPLC retention time, t_R = 15 min.
• 10.Obtain the target glycopeptides 8 after lyophilization.

Figure 2.

Graphical representation of the synthesis, purification and characterization of MUC1 glycopeptide bearing Tn antigen 8 and squaric acid monoamide of MUC1 glycopeptide 9

(A) Schematic diagram of MUC1 glycopeptide bearing Tn antigen 8 synthesis.

(B) Schematic diagram of squaric acid monoamide of MUC1 glycopeptide 9 synthesis. After synthesizing, all the MUC1 glycopeptides are analyzed and confirmed using RP-HPLC (C) and ESI-MS (D). Created with BioRender.com.

Preparation of squaric acid monoamides of MUC1 glycopeptide

Timing: 2 days

This step describes in detail the preparation of squaric acid monoamide of the glycopeptide. Couple 3, 4-diethoxy-3-cyclobutene-1, 2-dione to the N-terminus of the MUC1 glycopeptide to facilitate the next step of coupling to the protein carrier (Figure 2B).

• 11.
  Prepare squaric acid monoamide 9 of the glycopeptide.

  • a.
    Dissolve the deprotected glycopeptide 8 (0.0046 mmol, 1.0 equiv) in ethanol/water (1/1) in a flask. Add 3, 4-diethoxy-3-cyclobutene-1, 2-dione (0.0046 mmol, 1.0 equiv) in the flask. Then add 5 μL solution of saturated sodium carbonate in intervals of 5 min, until reach a pH of 8.0. Stir for 1.5 h at 25°C.
  • b.
    Add acetic acid to neutralize the reaction mixture. Remove the organic solvents in vacuum, and remove the water by lyophilization.
  • c.
    Purify the product by RP-HPLC on analytical C-18 column (YMC-Triart C18, 4.6 × 250 mm, 5 μm) with a flow rate of 1 mL/min, using a linear gradient of 10%–50% acetonitrile containing 0.1% trifluoroacetic acid over 40 min, detection at 215 nm. RP-HPLC retention time, t_R = 20 min.
  • d.
    Obtain the product 9 after lyophilization.

Preparation of MUC1 vaccine candidates

Timing: 3 days

This step describes in detail the preparation of glycopeptide-BSA conjugate. The squaric acid monoamide of MUC1 glycopeptide 9 is coupled to the protein carrier BSA and the product is characterized by MALDI-TOF mass spectrometry (Figure 3).

• 12.
  Prepare glycopeptide-BSA conjugate 10.

  • a.
    Dissolve the glycopeptide squaric acid monoamide 9 (1.5 μmol, 25 equiv) and BSA (0.06 μmol, 1.0 equiv) in 600 μL 0.07 M sodium tetraborate /0.035 M potassium bicarbonate buffer solution. React for 24 h at 25°C.
  • b.
    Dialyze the glycopeptides-BSA conjugate 10 with de-ionized water for 48 h. And remove the left water by lyophilization.
  • c.
    Confirm the loading of the product 10 by MALDI-TOF mass spectrometry.

Figure 3.

Preparation of glycopeptide-BSA conjugate 10

(A) Schematic diagram of preparation of glycopeptide-BSA conjugate 10.

(B) The synthesis product 10 is analyzed and confirmed using MALDI-TOF mass spectrometry. Created with BioRender.com.

Evaluation of antibody response

Timing: 5 weeks

This step describes in detail the immunization of mice with the glycopeptide vaccine and blood collection, and evaluates the immune response to the glycopeptide vaccine by detecting total antibody titers and antibody subtypes in plasma by enzyme-linked immunosorbent assays (ELISA) (Figure 4).

• 13.
  Inject BSA conjugated vaccine or mixture of vaccine and Alum into BALB/c mice (n = 4 per group) subcutaneously for immunization for three times at intervals of two weeks. Each immune injection contains 10 nmol MUC1 glycopeptide (Figure 4A).

  • a.
    Dilute glycopeptide-BSA conjugate (containing 10 nmol MUC1 glycopeptide) in PBS and mix it with Alum adjuvant (Bioss) 1:1.

    Note: Filter glycopeptide-BSA conjugate solution by 0.22 μm sterile filter unit (Merck Millipore) for liquid sterilization.

  • b.
    Immunize each BALB/c mouse with 100 μL MUC1-BSA conjugate or mixture of MUC1-BSA conjugate/Alum adjuvant (1/1, v/v) subcutaneously for three times on days 0, 14 and 28.
  • c.
    After induction of isoflurane gas anesthesia to minimize pain/discomfort, collect the retro-orbital blood in containers with an anticoagulant on days 7, 21 and 35.

    • i.
      Centrifuge the blood sample at 4°C and 4000 g for 8 min.
    • ii.
      Collect the upper plasma.
    • iii.
      Store the plasma sample at −80°C.
• 14.
  Analyze antibody titer by ELISA (Figure 4B).

  • a.
    Coat 96-Well ELISA plates with 100 μL/well streptavidin (5 μg/mL in PBS) and incubate for 12–18 h at 4°C.
  • b.
    Wash the plates with phosphate buffered saline with tween-20 (PBST) (Biosharp) three times.

    Alternatives: The washing buffer can also be prepared by ourselves. Prepare PBST by adding 500 μL of Tween-20 to 1000 mL of PBS.

  • c.
    Block the plate by adding 200 μL/ well of casein blocking buffer (Macklin) for 1 h at 37°C.
  • d.
    Wash the plates as above.
  • e.
    Coat the plates with 100 μL/well Biotin-MUC1 glycopeptide (2 μg/mL diluted in PBS) and incubate at for 60 min at 37°C.
  • f.
    Wash the plates as above after incubation.
  • g.
    Dilute the mouse plasma in PBST (Biosharp) at appropriate dilutions (1:100/200/400/800/1600/3200/6400/12800/25600/51200/102400/204800), add to the plates and incubate for 90 min at 37°C.
  • h.
    Wash the plates as above.
  • i.
    Incubate the plates with 100 μL/well alkaline phosphate conjugated goat anti-mouse antibodies (Sigma, 1:3000 dilution) for 1 h at 25°C.
  • j.
    Wash the plates as above after incubation.
  • k.
    Add 100 μL/well pNPP substrate (DuraStab) and incubate at 37°C for 5–20 min. Then add 100 μL/well pNPP stop solution (DuraStab).
  • l.
    Read the absorbance at 405 nm on an ELISA plate reader.

    Note: Biotin-MUC1 glycopeptide is obtained by modifying D-biotin on MUC1 glycopeptide. Dissolve the MUC1 glycopeptide obtained in step 1 (0.1 mmol,1 equiv), D-Biotin (0.5 mmol, 5 equiv), HBTU (0.5 mmol), HOBt (0.5 mmol, 5 equiv), and DIEA (1.0 mmol, 10 equiv) in an appropriate volume of DMF and react at 25°C for 2 h. Then execute step 2–7 to obtain the biotinylated MUC1 glycopeptide.

• 15.
  Analyze antibody isotype by ELISA (Figure 4C).

  • a.
    Coat 96-Well ELISA plates with 100 μL/well streptavidin (5 μg/mL in PBS) and incubate for 12–18 h at 4°C.
  • b.
    Wash the plates with PBST (Biosharp) three times.
  • c.
    Block the plate by adding 200 μL/ well of casein blocking buffer (Macklin) for 1 h at 37°C.
  • d.
    Wash the plates as above.
  • e.
    Coat the plates with 100 μL/well Biotin-MUC1 glycopeptide (2 μg/mL diluted in PBS) and incubate at for 60 min at 37°C.
  • f.
    Wash the plates as above after incubation.
  • g.
    Dilute the mouse plasma in PBST (Biosharp) at appropriate dilutions (1:100/200/400/800/1600/3200/6400/12800/25600/51200/102400/204800), add to the plates and incubate for 90 min at 37°C.
  • h.
    Wash the plates as above.
  • i.
    Add 100 μL/well IgG1, IgG2a, IgG2b, IgG3, IgM and IgA of goat anti-mouse isotype antibodies (Sigma, 1:1000 dilution) and incubate for 90 min at 25°C.
  • j.
    Wash the plates as above.
  • k.
    Incubate the plates with 100 μL/well alkaline phosphate conjugated donkey anti-goat antibodies (Sigma, 1:3000 dilution) for 60 min at 25°C.
  • l.
    Wash the plates as above after incubation.
  • m.
    Add 100 μL/well pNPP substrate (DuraStab) and incubate at 37°C for 5–20 min. Then add 100 μL/well pNPP stop solution (DuraStab).
  • n.
    Read the absorbance at 405 nm on an ELISA plate reader.

Figure 4.

Evaluation of antibody response

(A) Experimental schematic showing subcutaneous immunization and blood collection in mice (n = 4 per group).

(B) Schematic representation of the detection of total antibody titers in plasma by ELISA.

(C) Schematic representation of the detection of antibody subtypes in plasma by ELISA.

(D) Total antibody titer of the last collected plasma as measured by the ELISA method of Figure B.

(E) The total antibody titers of three collections of plasma detected according to the method of Figure B. Antibody levels increase as the number of immunizations increases.

(F) Detection of antibody subtypes in plasma performed by the method shown in Figure C. Created with BioRender.com.

Determination of antibody binding to tumor cells by FACS analysis

Timing: 1 day

This step describes the detection of the reactivity of the antibody binding induced by vaccine candidate towards MUC1-positive tumor cells MCF-7, B16-MUC1 by fluorescence-activated cell sorting (FACS) analysis in detail. MUC1-negative tumor cells B16-F10 serve as control (Figure 5).

• 16.Culture B16-MUC1, B16-F10 and MCF-7 cells in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin solution (100x) (Gibco).
• 17.Detach these cancer cells from the culture flask with 0.25% Trypsin-EDTA (Gibco) at 37°C for 3 min, then transfer to 50 mL conical centrifuge tube (LABSELECT) and centrifuge at 210 g for 3 min.
• 18.After removing the culture medium, wash cancer cells with 500 μL PBS, then centrifuge at 210 g for 5 min, and remove the supernatant.
• 19.Incubate cancer cells with 100 μL of mouse plasma (1:50 dilution) in flow cytometry buffer (1% FBS in PBS) at 0°C for 1 h.
• 20.Wash three times with flow cytometry buffer.
• 21.Incubate cancer cells with Alexa Fluor 488-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) secondary antibody in flow cytometry buffer (Abcam, 1:1000 dilution) (100 μL per tube) at 0°C for 1 h.
• 22.Wash cells three times as above.
• 23.Add 300 μL of flow cytometry buffer to resuspend the cells, and detect the cells using a flow cytometry.

Note: In addition to flow cytometry, the binding of plasma antibodies to tumor cells can also be observed by confocal fluorescence microscopy. Detail protocol can refer to Guo et al.¹⁰

Figure 5.

Determination of antibody binding to cancer cells by FACS analysis

(A) Experimental schematic showing determination of antibody binding to cancer cells by FACS analysis.

(B) Flow cytometry gating strategy for determination of antibody binding to cancer cells. Histograms (C) and mean fluorescence intensity plots (D) for flow cytometry analysis of the binding of vaccinated mouse plasma samples to cancer cells including B16-MUC1 and MCF-7 cells. (n = 4 per group) Incubation with B16-F10 cells serve as a control. The data are expressed as the mean ± SEM. Asterisks show significant difference compared with each other based on one-way analysis of variance (ANOVA) (no significant difference, ns; ∗∗, p<0.01; ∗∗∗∗, p<0.0001). Created with BioRender.com.

Cellular immune response

Timing: 1 day

This step describes the detection of cellular immune response induced by vaccine candidate by intracellular cytokine staining (ICS) assay (Figure 6).

• 24.On day 42 (14 days after the third immunization), euthanize the mice and isolate the spleens.
• 25.Using the plunger end of the syringe, mash or crush the spleen and press through a 70-μm cell strainer (Biosharp), and then wash with large amount of PBS, centrifuge at 500 g for 5 min and remove the supernatant.
• 26.Add 2–5 mL Erythrocytes Lysate (Biosharp) in each conical centrifuge tube, incubate on ice for 5 min to lyse the red cells.
• 27.After 5 min, add 10–20 mL PBS in each tube, centrifuge at 500 g for 5 min and remove the supernatant. And then wash the cells with 10–20 mL PBS each tube.
• 28.Resuspend the splenocytes in RPMI-1640 medium (Gibco) containing 10% FBS (Gibco) and 1% penicillin-streptomycin solution (100x) (Gibco)at 1 × 10⁶ cells/mL and transfer 2 mL of cells per tube to six-well plates.
• 29.Incubate splenocytes with MUC1 glycopeptide at a concentration of 10 μg/mL at 37°C for 4 h.
• 30.Then Incubate cells with brefeldin A at a concentration of 10 μg/mL (MCE) at 37°C for another 4 h.
• 31.After incubation, wash the cells with flow cytometry buffer and incubate with anti-mouse CD16/CD32 antibody (BD, 1:100 dilution) for 15 min at 4°C to block Fcγ receptors.
• 32.Centrifuge at 500 g for 5 min and remove the supernatant. Stain the cells with APC-Cy7 anti-mouse CD45, FITC anti-mouse CD3, APC anti-mouse CD4 and PerCP-Cy5.5 anti-mouse CD8 (BD, 1:100 dilution) at 4°C for 30 min in the dark.
• 33.Wash the cells with flow cytometry buffer, centrifuge at 500 g for 5 min and remove the supernatant.
• 34.Thoroughly resuspend cells and add 250 μL/tube Fixation/Permeabilization solution (BD)for 20 min at 4°C.
• 35.Wash cells two times with 1 mL/tube 1 x BD Perm/Wash buffer (BD).
• 36.Thoroughly resuspend fixed/permeabilized cells in 100 μL of 1 x BD Perm/Wash buffer containing PE anti-mouse IFN-γ (BD). Incubate at 4°C for 30 min in the dark.
• 37.Wash cells two times with 1 mL/tube 1x BD Perm/Wash buffer (BD).
• 38.Resuspend cells with flow cytometry buffer prior to flow cytometric analysis.

Figure 6.

Detection of cellular immune response by intracellular cytokine staining (ICS) assay

(A) Experimental schematic showing ICS assay by FACS analysis.

(B) Flow cytometry gating strategy for ICS assay.

(C) The spleens are stimulated with the MUC1 glycopeptide, then the results are analyzed by intracellular cytokine staining (ICS) assay to detect the cellular immune response (n = 4 per group). The data are expressed as the mean ± SEM. Asterisks show significant difference compared with each other based on one-way analysis of variance (ANOVA) (no significant difference, ns; ∗∗, p ≤ 0.01; ∗∗∗, p ≤ 0.001). Created with BioRender.com.

Expected outcomes

MUC1 glycopeptide bearing Tn antigen is obtained by microwave-assisted solid-phase peptide synthesis, and the synthesis of glycopeptide is confirmed by analytical liquid chromatography and electrospray ionization mass spectrometry (Figure 2). After modification using 3, 4-diethoxy-3-cyclobutene-1, 2-dione, the glycopeptide is coupled to protein carrier BSA (Figures 2 and 3). The coupling efficiency of the glycopeptide to BSA can be detected by MALDI-TOF mass spectrometry, and an average of 8–14 MUC1 glycopeptides can be coupled to each BSA.

After three subcutaneous immunizations, the antibody titers in mice are measured by ELISA. The serum shows high dilution multiplicity, indicating that a large number of antibodies against the corresponding MUC1 glycopeptide antigens have been produced in mice after three immunizations and the MUC1 glycopeptide vaccine induce a strong immune response in mice (Figures 4D and 4E). Furthermore, plasma antibodies from mice immunized with MUC1-BSA+Alum can effectively recognize and bind MUC1-positive tumor cells including MCF-7 and B16-MUC1 cells (Figures 5C and 5D). And plasma antibodies from all groups could not significantly bind B16-F10 cells, indicating that the binding of antibodies to MCF-7 and B16-MUC1 cells are MUC1-targeted. According to the antibody subtype assay, the MUC1 glycopeptide vaccine produce a Th2-biased immune response in mice (Figure 4F). In order to evaluate cellular immune responses after vaccination, lymphocytes in the spleens are stimulated with the MUC1 glycopeptide, then the results are analyzed by ICS assay. The combination of adjuvant and MUC1 glycopeptide vaccine can increase IFN-γ secretion by CD8⁺T cells to a certain extent (Figure 6).

Limitations

This protocol describes the synthesis of MUC1 glycopeptide using glycosylated amino acids by combining the building block strategy with Fmoc solid-phase peptide synthesis strategy. In this way, it is easy to control specific glycosylation sites, and can easily prepare to obtain glycopeptides with high overall synthetic yield, convenient and rapid operation, and has been widely used in the synthesis of O-glycopeptides. However, the synthesis of complex glycopeptides carrying large and complex glycans is still challenging because the glycosylated amino acid fragments need to be chemically prepared, which involves the stereoselective construction of sialic acid bonds, cumbersome protection-deprotection operations, and β-elimination or cleavage of acid-sensitive sialic acid glycosides and fucosyl glycosides that may occur during the glycopeptide deprotection process.¹¹

Troubleshooting

Problem 1

Unsuitable resins and coupling procedures etc. can lead to lower yields in glycopeptide synthesis.

Potential solution

• •Use dichloro resin to synthesize MUC1 glycopeptide. The second amino acid at the C-terminus of the MUC1 glycopeptide is proline, and synthesis using Wang resin is prone to side reactions and low yields.
• •When the polypeptide sequence is relatively long, set up the program of coupling each amino acid twice, which can improve the yield of the polypeptide.
• •When the side chain protection group of glycan is large, it will also affect the yield and purity of polypeptide, when coupling glycosylated amino acid, you can appropriately increase the equivalent amount of glycosylated amino acid or coupling it twice.
• •Resins not from CEM do not provide the same yield when performing microwave assisted SPPS. If possible, use resins from CEM to get higher yields.

Problem 2

Resin and peptide degradation after prolonged placement.

Potential solution

• •Resin is usually kept at 4°C, and should not be left at 25°C for more than one week at most.
• •Peptides should be purified as soon as possible after synthesis.
• •Crude product is more prone to degradation than pure product, and after purification, peptides should be kept at −20°C.

Problem 3

Different animal models exhibit distinct genetically determined differences in their immune systems under physiological conditions, such as differences in antibody subtypes. The IgG2a gene is deleted in C57BL/6, C57BL/10, SJL and NOD mice, and the IgG2c gene is deleted in BALB/c mice. Incorrect use of animal models can lead to inaccurate analysis.

Potential solution

• •When immunizing BALB/c mice, we should use commercial antibodies against the IgG2a isotype, and analyze the polarization of responses in the Th1/Th2 paradigm according to the ratio of IgG2a to IgG1 or IgG2b to IgG1. While choosing C57BL/6 mice, use commercial antibodies against the IgG2c isotype due to the lack of IgG2a, and analyze Th1/Th2 response according to the ratio of IgG2b to IgG1.

Problem 4

Non-specific binding results in high OD values and high background value. Related to steps 14–15.

Potential solution

• •Ensure that the correct blocking solution is used. Immunizing mice with BSA as an antigen may result in the production of antibodies against BSA, therefore, a blocking solution containing BSA or FBS should be avoided.
• •Long incubation time of serum may lead to elevated background. The incubation time of serum can be shortened appropriately.
• •Ensure that the substrate incubation process is protected from light.
• •Read the OD value in time after adding the stop solution.

Problem 5

The ELISA OD value is low. Related to steps 14–15.

Potential solution

• •Use the correct corresponding biotin-linked glycopeptides.
• •Make sure to use the correct enzyme-labeled antibody which could react with the murine antibody.
• •Extend the incubation time or increase the incubation temperature appropriately.

Problem 6

When carrying out a multicolor flow cytometry experiment, the emission spectra of the various fluorophores can overlap, resulting in detection in a different channel. The unreasonable staining protocol and the setting of control groups will increase the difficulty of experiment and data analysis.

Potential solution

• •Before starting the flow cytometry experiment, select appropriate fluorescent antibodies and design a reasonable staining protocol to reduce the overlap between the spectrum of fluorescence.
• •The setting of technical control groups is used to help adjust detector settings, generate the unmixing matrix, and set boundaries for positive expression. Technical controls consist of unstained cells, single stain controls, isotype controls, and Fluorescence Minus One (FMO) controls. Such technical control samples are required in flow cytometry as they help ensure the integrity of the instrument and facilitate the accurate interpretation of results.

Problem 7

Some harmful chemicals such as trifluoroacetic acid and toluene are used during the synthesis process, which may affect the health of the body.

Potential solution

• •Trifluoroacetic acid is a strong acid that is corrosive and toxic. When using trifluoroacetic acid, necessary safety measures need to be taken such as wearing personal protective equipment like protective gloves, goggles and protective clothing. Be sure to operate in a well-ventilated area and avoid direct contact with skin and eyes. When handling waste, dispose of it properly in accordance with relevant regulations and protocols.
• •Toluene is a volatile liquid, toxic, harmful, for pregnant women’s central nervous system will have a certain anesthetic effect, and easily lead to abortion or fetal deformity. Conduct experiments in a good fume hood; try not to perform toluene-related experiments during pregnancy to protect the mother and fetus, or ask a colleague to help with the experiment.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by prof.Hui Cai (caihui5@mail.sysu.edu.cn).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by prof. Hui Cai (caihui5@mail.sysu.edu.cn).

Materials availability

This work has not released any new products.

Data and code availability

The data reported in this manuscript can be shared by the corresponding author upon reasonable request.