The therapy assay - T UMOR GROWTH - MATERIALS AND METHODS

CHAPTER 2 MATERIALS AND METHODS

3.10 T UMOR GROWTH

3.10.2 The therapy assay

Mice were inoculated with 1 x 10⁵ CT26/CEA and four days later they were orally immunized with 1 x 10⁸ Salmonella typhimurium transformed with pAAV-CEA and pAAV-SARS in 20 μl PBS, respectively. The negative group was fed with 20 μl PBS. Every week mice were re-immunized and tumor volume was measured once every two or three days.

Mice in the SARS group had the smallest tumor volume, indicating the SARS fragment provided enough protection against CT26/CEA (Figure 38). The tumor volume was significantly different between the SARS group and the CEA group on day 44 (Figure 38).

H2-Kd nonamers go to top

Position 1 2 3 4 5 6 7 8 9 score

78 (within SARS) W Y V W L G F I A 21

59 (within SARS) R L Q S L Q T Y V 19

89 (within SARS) I A I V M V T I L 18

83 (within SARS) G F I A G L I A I 17

5 (within CEA) S A P P H R W C I 16

36 (within CEA) W G L L G R T G L 16

42 (within SARS) T G L K V E A E V 16

65 (within SARS) T Y V T Q Q L I R 16

Table 1. The epitope scores of the CEA-SARS sequence calculated by the Internet software, SYFPEITHI. Epitopes that score 16 and above are listed. Epitope starting at the 78^th amino acid in the CEA-SARS sequence has the highest score at 21, which serves as the template for affinity mutation.

H2-Kd

Table 2. The CEA and CEA-SARS epitopes compared with other known epitopes that have been proved to elicit immunity in Balb/c mice. The score of the CEA and CEA-SARS sequences are calculated by the Internet software, SYFPEITHI. The score of CEA is lower than the well-known epitopes of Listeria (LLO91-99 and p60217-225) and EGFP200-208. The epitope score of CEA-SARS is closer to these well-known epitopes and therefore, may be expected to elicit the immune response.

KVVEEAAEEVVQQIIDDRRLLIITTGGRRLLQQSSLLQQTTYYVVTTQQQQLLIIRRIIKKWWPPWWYYVVWWLLGGFFIIAAGGLLIIAAIIVVMMVVTTIILLLLCCCCMMTT

WYYVVWWLLGGFFIIII

WWYYVVWWLLGGTTIIII

WWYYVVPPLLGGTTIIII

SSYYFFPPEEIITTHHII:: ppooiinntt mmuuttaattiioonn

SSYYFFPPEEIITTHHII:: ppooiinntt mmuuttaattiioonn m1

Table 3. The flow chart of mutagenesis. First, the epitope sequence is predicted. Then, every amino acid within the predicted epitope is substituted to gain higher affinity between the MHC molecule (H2-Kd) and the epitope. Among all, the epitope that has the highest score was chosen to be our first mutation construct and served as the template for the next round of mutation. It was named m1. The same approach was applied to obtain the two accumulative mutations in the m2 construct and three accumulative mutations in the m3 construct.

H2-Kd nonamers go to top

Pos 1 2 3 4 5 6 7 8 9 score

(Template) 34 W Y V W L G F I A 21

1 W Y V W L G F E A 22

1 W Y V W L G F I V 23

1 W Y V W L G F I L 25

(m1) 1 W Y V W L G F I I 25

Table 4. The candidate sequence of the m1 construct. The epitope (WYVWLGFIA) in the CEA-SARS sequence was substituted by the approach described in the text. After substitution, two epitopes scored highest (at 25) were randomly chosen to be the first mutation construct (m1) and served as the template for the next round of mutation.

m3 m2

Table 5. The generation of m2 and m3. The construct of m2 (WYVWLGTII) was mutated from the m1 and the m3 (WYVPLGTII) construct was mutated from m2.

1 2 3 4 5 6 7 8 M 9 10 11 12 13 14 15 16 17

400 bp 300 bp

Figure 3. The construct of pAAV-CEA (with a stop codon). The CEA sequence with a stop codon was cloned into pAAV-MCS. The PCR with β–globin intron and hGH poly(A) primers proves that the CEA sequence with a stop codon was constructed into the plasmids (336 bp).

Clone #1, #3, #4, and #5 were picked and sequenced. Clone #1 was chosen for plasmid DNA extraction. M: 100 bp ladder marker. #1~#17: clones.

M 1 2 3 4 5 6 M

200 bp 100 bp

Figure 4. Restriction enzyme digestion of the pAAV-CEA construct without a stop codon.

The plasmid was digested by EcoRI and XbaI into a 135 bp fragment. Clones #1, #2, and #3 were picked and sequenced. Clone #1 was chosen for plasmid DNA extraction. M: 100 bp ladder marker; #1~#6: clones.

M 1 2 3 4 5 6 7 8 9 10 M

200 bp 100 bp

Figure 5. Restriction enzyme digestion of the pAAV-CEA-SARS construct. The plasmid DNA was digested by XbaI and HindIII into a 174 bp fragment. Clone #1, #2, and #3 were picked and sequenced. #2 was picked. M: 100 bp ladder marker; #1~#10: clones.

1 2 M

200 bp 100 bp

Figure 6. Restriction enzyme digestion of the pAAV-CEA-m1 construct. The plasmid was digested by XbaI and HindIII into a 174 bp fragment. Clone #1 and #2 were picked and sequenced. #2 was picked. M: 100 bp ladder marker; #1~#2: clones.

M 1 2 3 4 5 M

400 bp 300 bp

Figure 7. Restriction enzyme digestion of the pAAV-CEA-m2 construct. The plasmid was digested by EcoRI and XhoIII into a 309 bp fragment. Clone #2 picked and sequenced. M:

100 bp ladder marker; #1~#5: clones.

M 1 2 3 4 5 6 7 8 9 10

400 bp 300 bp

Figure 8. Restriction enzyme digestion of the pAAV-CEA-m3 construct. The plasmid was digested by EcoRI and XhoIII into a 309 bp fragment. Clone #9 and #10 were picked and sequenced. M: 100 bp ladder marker; #1~#10: clones.

CEA

P CMV

CEA SARS

P CMV

CEA SARS-m1

P CMV

CEA SARS-m2

P CMV

CEA SARS-m3

P CMV

Figure 9. The diagram of pAAV-CEA, pAAV-CEA-SARS, pAAV-CEA-m1, pAAV-CEA-m2, and pAAV-CEA-m3 for immunization.

3’ 5’

3’

5’

XbaI SalI

Figure 10. Primer annealing of the CEA sequence without its leader sequence. The primers were annealed at 95℃ and was cooled down to room temperature. The 5’ and 3’ end of primers have been designed as the stick end of XbaI (CTAGT) and SalI (TCGAC) restriction sites. Therefore, the annealing product did not undergo PCR before ligation.

1 2 3 4 5 M

800 bp 500 bp

Figure 11. The construct of pAAV-CEA-B7.1. The CEA sequence without its leader sequence was cloned into pAAV-B7.1 The PCR withβ–globin intron and hGH poly(A) primers shows that a fragment with the predicted size (830 bp) was obtained. #1 and #2 were picked and sequenced to prove the existence of the CEA sequence without its leader sequence.

M: 100 bp ladder marker; #1~#5: clones.

1 2 3 4 5 M 6 7 8 9 10

500 bp 800 bp

Figure 12. The construct of pAAV-CEA-B7.1-IVH3H. IVH3H was obtained from primer annealing and was cloned into pAAV-CEA-B7.1. The PCR with primers β–globin intron and IVH3H 3’ shows that t that a fragment with the predicted size (870 bp) was obtained. Clone

#3 was picked and sequenced to prove the existence of the CEA-B7.1-IVH3H sequence.

CEA B7.1 IVH3H

P CMV

Figure 13. The diagram of pAAV-CEA-B7.1-IVH3H for CEA antibody production.

M 1 2 3 M

200 bp 100 bp

Figure 14. Restriction enzyme digestion of pMSCVneo-CEA. The restriction digestion by EcoRI and XhoI produced a 135 bp fragment in clone #1 and #2, the latter of which was picked and sequenced. M: 100 bp ladder marker; #1~#3: clones.

CEA

5’ LTR

Ψ

Figure 15. The diagram of pMSCVneo-CEA for CT26 infection.

Figure 16. The fluorescence expression of hrGFP in P338D1. pAAV-hrGFP was transformed in Salmonella typhimurium and bacteria were incubated with P338D1 overnight.

The expression of hrGFP was detected by cytometry (FL1). The pAAV-hrGFP transfected bacteria (dark line) had a higher fluorescence expression than its negative counterpart (dotted line). Dotted line: negative control (P338D1 + Salmonella w/o any plasmid); Dark line:

P338D1 + Salmonella w/ plasmid.

100 101 102 103 104

FL1-H

a) b)

Figure 17. Selection of PT67 transfected with pMSCVneo-CEA. PT67 was selected at 500 μg/ml G418 on Day 9. a) Neg, PT67 without pMSCVneo-CEA transfection. Most of the PT67 cells had been killed. b) PT67 with pMSCVneo-CEA transfection. The transfected PT67 cells became confluent even under drug-resistance selection.

a) b)

c) d)

e) f)

Figure 18. The G418 resistance test of CT26. Before infection, the G418 resistance test was performed at different concentrations. 400 μg/ml G418 was chosen for infection. On day 7, the cell condition at each concentration is shown. a) 0 μg/ml, confluent b) 100 μg/ml, partially dead c) 200 μg/ml, mostly dead d) 300 μg/ml, all dead e) 400 μg/ml, all dead f) 500 μg/ml, all dead.

a) b)

Figure 19. Infection of CT26 at 400 μg/ml G418 on Day 7. a) Neg, CT26 without infection b) CT26 with infection, which became confluent in the 24-well plate.

Figure 20. The CEA antibody in the sera of Balb/c mice. Balb/3T3 was transfected with pAAV-CEA-B7.1 and pAAV-B7.1-IVH3H. The pAAV-CEA-B7.1-IVH3H transfected Balb/3T3 had a higher expression of fluorescence than its negative control and pAAV-B7.1 transfected counterpart by cytometry. The percentage in the M1 region is: a) 1.10% b) 51.17%

c) 72.92%, respectively.

S1 S2 N

Figure 21. The secretion of CEA from CT26. CEA is secreted by CT26 by dot blotting. N:

negative control (PBS alone); S1: cell lysis; S2: cell soup collected after 48 hr incubation at 37℃.

Figure 22. The mortality rate and surface fluorescence of CT26 after overnight DIOC18 staining. Before the killing assay, the effect DIOC18 on CT26 condition was tested. DIOC18 did not affect CT26 after overnight staining as revealed by 50 μg/ml propidium iodide (PI).

a) Neg, w/o DIOC18 staining b) DIOC18 staining c) DIOC18 staining + 7% formaldehyde.

The mortality rate = (the number of cells of PI staining and DIOC18 staining) / (the number of cells of DIOC18 staining).

Figure 23. The mortality rate and surface fluorescence of CT26/CEA after overnight DIOC18 staining. Before the killing assay, the effect DIOC18 on CT26/CEA condition was tested. DIOC18 did not affect CT26/CEA after overnight staining as revealed by 50 μg/ml propidium iodide (PI). a) Neg, w/o DIOC18 staining b) DIOC18 staining c) DIOC18 staining + 7% formaldehyde. The mortality rate = (the number of cells of PI staining and DIOC18 staining) / (the number of cells of DIOC18 staining).

Figure 24. The mortality rate and surface fluorescence of YAC-1 after overnight DIOC18 staining. DIOC18 did not affect YAC-1 after overnight staining as revealed by 50 μg/ml propidium iodide (PI). a) Neg, w/o DIOC18 staining b) DIOC18 staining c) DIOC18 staining + 7% formaldehyde. The mortality rate = (the number of cells of PI staining and DIOC18 staining) / (the number of cells of DIOC18 staining).

ct26/cea 100:1

Figure 25. The CT26/CEA killing assay. CT26/CEA was incubated with splenocytes for 4 hr at 37℃. E/T ratios at a) 100/1, b) 50/1, and c) 25/1 are shown. Specific lysis (%) = (the number of cells of PI staining and DIOC18 staining) / (the number of cells of DIOC18 staining). *p < 0.05, when compared with the negative group; #p < 0.05, when compared with the CEA group.

CT26 100:1

Figure 26. The CT26 killing assay. CT26 was incubated with splenocytes for 4 hr at 37℃.

E/T ratios at a) 100/1, b) 50/1, and c) 25/1 are shown. Specific lysis (%) = (the number of cells of PI staining and DIOC18 staining) / (the number of cells of DIOC18 staining). *p <

0.05, when compared with the negative group.

-10 -8 -6 -4 -2 0 2 4 6 8 10

neg cea sars m1 m2 m3

Specific killing (%)

Figure 27. CT26/CEA specific killing at an E/T ratio = 25/1. Specific killing was calculated by the formula below: CT26/CEA specific killing = (% of CT26/CEA killing in Figure 25c) – (% of CT26 killing in Figure 26c).

Yac-1 100:1

Figure 28. The YAC-1 killing assay. CT26 was incubated with splenocytes for 4 hr at 37℃.

E/T ratios at a) 100/1, b) 50/1, and c) 25/1 are shown. Specific lysis (%) = (the number of cells of PI staining and DIOC18 staining) / (the number of cells of DIOC18 staining). *p <

0.05, when compared with the negative group; #p < 0.05, when compared with the CEA group.

NA-S CA-S SA-S M1A-S M2A-S M3A-S

TNF-alpha (pg/ml)

NC-S CC-S SC-S M1C-S M2C-S M3C-S

TNF-alpha (pg/ml)

Figure 29. In vitro TNF-αexpression after CT26/CEA soup or CT26 soup stimulations.

Splenocytes were stimulated by either a) CT26/CEA soup or b) CT26 soup for 24 hr. Groups are shown in the order: negative, CEA, SARS, m1, m2, and m3. *p < 0.05, when compared with the negative group; #p < 0.05, when compared with the CEA group.

NA-S CA-S SA-S M1A-S M2A-S M3A-S

IL-10 (pg/ml)

NC-S CC-S SC-S M1C-S M2C-S M3C-S

IL-10 (pg/ml)

Figure 30. In vitro IL-10 expression after CT26/CEA soup or CT26 soup stimulations.

-5 0 5 10 15

NA-S CA-S SA-S M1A-S M2A-S M3A-S

IL-4 (pg/ml)

* *

0 5 10 15

NC-S CC-S SC-S M1C-S M2C-S M3C-S

IL-4 (pg/ml)

N.D.

Figure 31. In vitro IL-4 expression after CT26/CEA soup or CT26 soup stimulations.

-5 0 5 10 15

NAS CAS SAS M1A-S M2A-S M3A-S

IL-12 (pg/ml)

NCS CCS SCS M1C-S M2C-S M3C-S

IL-12 (pg/ml)

* *

# #

Figure 32. In vitro IL-12 expression after CT26/CEA soup or CT26 soup stimulations.

0 5 10 15 20

NAS CAS SAS M1A-S M2A-S M3A-S

IFN-gamma (pg/ml)

N.D.

0 5 10 15 20

NCS CCS SCS M1C-S M2C-S M3C-S

IFN-gamma (pg/ml)

* *

N.D.

Figure 33.In vitro IFN-γ expression after CT26/CEA soup or CT26 soup stimulations.

IL-2

Figure 34. In vivo cytokine expression. Sera were collected for the in vivo cytokine detection, including a) IL-2, b) TNF-α, c) IFN-γ, d) IL-4, and e) IL-5. Groups are shown in the order: negative, CEA, SARS, m1, m2, and m3.

Tumor-free rate

0 20 40 60 80 100 120

17 19 21 23 25 27 29 31 33 35

Day

% of tumor-free

neg cea sars m1 m2 m3

Figure 35. The tumor-free rate of Balb/c mice in the protection assay. Mice were immunized with Salmonella typhimurium, which had been transformed into each plasmid construct three times in two weeks and were inoculated with 5 x 10⁵ CT26/CEA.

Tumor Volume

0.0 300.0 600.0 900.0 1200.0

20 21 22 23 24 25 26 27 (d)

Volume (mm3 )

Neg CEA SARS 1' 2' 3'

* *

Figure 36. The tumor volume of Balb/c mice in the protection assay. Mice were immunized with Salmonella typhimurium, which had been transformed into each plasmid construct three times in two weeks and were inoculated with 5 x 10⁵ CT26/CEA. Tumor volume = length x width x height. *p < 0.05, when compared with the negative group.

Survival

0 20 40 60 80 100 120

20 22 24 26 28 30 32 34

Day

% of survival

neg CEA SARS m1 m2 m3

Figure 37. The survival rate of Balb/c mice in the protection assay. Mice were immunized with Salmonella typhimurium, which had been transformed into each plasmid construct three times in two weeks and were inoculated with 5 x 10⁵ CT26/CEA.

therapy

0 1000 2000 3000 4000 5000 6000 7000

5 10 15 20 25 30 35 40 45 50

tumor volume (mm3 )

Neg CEA SARS

Figure 38. The tumor volume of Balb/c mice in the therapy assay. Mice were inoculated with 1 x 10⁵ CT26/CEA and were immunized 4 days later with Salmonella typhimurium, which had been transformed into each plasmid construct. Mice were re-boosted once every week and the tumor volume was measured every two to three days. *p < 0.05, when compared with the negative group.

在文檔中融合一段SARS片段至腫瘤來源的胜肽可提高免疫反應 (頁 66-107)