Biomedical Engineering Reference
In-Depth Information
TABLE 18.2 Synthesis Method and Formulations of Chemically Conjugated Immunotoxins Tested in Clinical Trials
Chemically Conjugated
Immunotoxin
Target
Antigen
Conjugation
Reagents
Formulation, Concentration,
and Storage
Conjugate Purification
References
Anti-CD3 IgG
(SPV-T3a)-dgA
CD3
SMPT
Blue affinity/S-300
GF/ultrafiltration
PBS, pH 7.4, 0.05% Tween 20;
1mg/mL; 20 C
[76]
PBS, pH 7.4; 1mg/mL; 4 C
Anti-CD5 IgG
(H65)-RTA
CD5
SPDP
Protein A affinity/
ultrafiltration
[77,78]
Anti-CD7 IgG
(WT1)-dgA
CD7
SMPT
Blue affinity/S-300
GF/ultrafiltration
PBS, pH 7.4, 0.05% Tween 20;
1mg/mL; 20 C
[76]
Anti-CD22 Fab 0
(RFB4)-dgA
CD22
DTNB
Blue affinity/S-200 GF
0.145 M NaCl, pH 6.5; 1mg/mL;
70 C
[79,80]
Anti-CD22 IgG
(RFB4)-dgA
CD22
SMPT
Blue affinity/S-200
GF/ultrafiltration
0.15M NaCl/5mM lysine, pH 9;
0.5mg/mL; 70 C
[81]
Anti-CD19 IgG
(HD37)-dgA
CD19
SMPT
S-200 GF/blue affinity
0.15M NaCl, pH 6.5; 0.5mg/mL;
70 C
[81]
Anti-CD19 IgG
(anti-B4)-bRicin
CD19
SMCC
S cation/anti-bRicin affinity/
S-300 GF
PBS, 1mg/mL HSA, pH 7.3;
0.1mg/mL; 4 C
[61,82]
Anti-CD19 IgG
(B43)-PAP
CD19
SPDP and
Traut
TSK-3000-SW GF/CM
cation/Affi-Prep polymyxin
40mM sodium phosphate, 150mM
NaCl, pH 7.5; 1mg/mL; 70 C
[83]
Anti-CD19 IgG
(BU12)-saporin
CD19
SPDP
CM cation/dialysis
PBS, pH 7.2; 80 C
[84]
Anti-CD25 IgG
(RFT5)-dgA
CD25
SMPT
S-200 GF/blue affinity
0.85% NaCl, 5mM lysine; 0.5mg/mL;
[85,86]
70 C
0.9% PBS, pH 7.0; 1.0mg/mL; 4 C
Antimelanoma IgG
(XMMME-001)-RTA
Melanoma SPDP
S-200 GF
[87-89]
Anti-breast carcinoma
IgG (260F9)-rRA
55 kDa
antigen
SPDP
TSK-300 GF
0.1M sodium sulfate, 0.02M sodium
phosphate, pH 6.8
[90,91]
PBS, pH 7.4; 0.21mg/mL; 150 C
Anti-CD30 IgG
(Ber-H2)-saporin
CD30
Traut
S-200 GF
[92,93]
Anti-CD33 IgG
(HuM195)-rGel
CD33
SPDP and
Traut
S-300 GF/blue affinity
PBS, pH 7.4
[94,95]
B3(IgG)-NLysPE38 or
LMB-1
LeY
Traut and
SMCC
Mono Q anion/TSK 250 GF
PBS; 1mg/mL
[50,96]
Transferrin-CRM107
TfR
Traut and
MBS
TSK3000 GF
Unpublished
[97]
dgA, deglycosylated ricin A chain; bRicin, blocked ricin; RTA, ricin A chain; rRA, recombinant ricin A chain; PAP, pokeweed antiviral protein; rGel,
recombinant gelonin; TfR, transferrin receptor; HSA, human serum albumin; DTNB, 5,5 0 -dithiobis(2-nitorbenzoic acid); SPDP, N-succinimidyl 3-(2-pyridyl-
dithio)-propionate; SMCC, N-succinimidyl 4-(N-maleimidomethyl) cyclohexane carboxylate; SMPT, N-succinimidyl-oxycarbonyl- a -methyl-(2-pyridyl-
dithio) toluene; GF, gel filtration; Traut, 2-iminothiolane; MBS, m-maleimidobenzoyl-N-hydrosuccinimide ester.
ion exchange and gel filtration chromatography according
to protein characteristics such as isoelectric point (pI)
and molecular weight. For DAB 389 IL2, DAB 486 IL2, and
DAB 389 EGF, these recombinant immunotoxins are
expressed in periplasm, lysed and purified by affinity, anion
exchange, and size-exclusion chromatography. As discussed
earlier, the second-generation immunotoxins have only one
ligand and relatively simple structure so that these molecules
can be produced in E. coli with reasonable yields. However,
production of the third-generation immunotoxins with two
ligands by in vitro refolding from E. coli is very inefficient
because of the multidomain structure [98]. Good refolding
could be achieved in a toxin-resistant CHO cell line, but
production capacity is limited to 5 m g/mL [99]. P. pastoris
is an ideal host strain to express recombinant immunotox-
ins via the secretory route, because secretory expression
minimizes exposure of immunotoxins to the cytosol where
the catalytic domain inactivates protein synthesis [98,100].
Further, a DT-resistant P. pastoris strain has been devel-
oped by a point mutation (G710R) of elongation factor 2
[101]. The DT-resistant strain can produce an anti-CD3
DT immunotoxin at three times higher concentrations
(120mg/L) than wild-type strain (37mg/L) after optimi-
zation of fermentation conditions [102,103]. Production
method and formulation of recombinant immunotoxins are
summarized in Table 18.3.
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