ATOM INDONESIA JOURNAL
Referee’s Report
Article No. : # 437
Title of Paper : Radiolabeled Antibody Fragment for Preparation of (177Lu-DOTA)m-PAMAM G3.0-F(ab’)2- trastuzumab as a Radiopharmaceutical for Cancer Therapy
Referee Name : Dr. Abdul Mutalib
Comment on Descriptions 1. Title
[√] Appropriate [ ] Should be changed
2. Abstract
Yes[√] No[ ] Is the length reasonable?
Yes[√] No[ ] Is it an appropriate summary of the content?
3. Main Text
Yes[ ] No[√] Is there anything new in this work?
Yes[√] No[ ] Is the relation to previous studies adequately stated?
Yes[ ] No[√] Are the assumption(s) and/or method(s) described comprehensively?
Yes[ ] No[√] Are the new results adequately emphasized?
Line # Referee’s Comments
12-13
Several radiolabeled monoclonal antibodies (mAb’s) have been used as radioimmunotherapy (RIT) agents for cancer therapy. The use of mAb as RIT agent is due to its ability to carry effectors, in the form of radionuclides which emit alpha (α), beta (β) particles or auger electrons, and bind specifically to cancer expressed targeted receptor. This paper reports reported the preparation of radiolabeled trastuzumab in the form of (177Lu)n-(DOTA)m-PAMAM-G3-F(ab')2-trastuzumab, which will be expected as a potential RIT agent for therapy of breast cancer overexpressed over expressed human epidermal growth factor receptor 2 (HER-2). Due to its downsized molecular weight, the use of F(ab')2-trastuzumab on the above–mentioned RIT agent candidate is expected to reaches its target much faster compared to the intact whole trastuzumab. Meanwhile, the use role of PAMAM-G3 is expected to increase the specific activity of the RIT radiotherapeutic agent of Lu-177 due to its ability of agent candidate as PAMAM-G3 which has 32 –NH2 functional groups that are able to bind many DOTA (≤ 31) which in turn would bind a large number of 177Lu. The preparation was initiated by fragmentation of trastuzumab using pepsin enzyme in 0.02 M acetic acid buffer pH 4.5 to give F(ab')2-trastuzumab with purity of 95% after purification with PD-10 column. The F(ab')2-
trastuzumab was then reacted with succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) to give SMCC-F(ab ')2-trastuzumab. The next reaction was to conjugate SMCC-F(ab ')2- trastuzumab with DOTA-PAMAM-G3.0-SH, which was prepared by reaction NHS-DOTA with PAMAMG3.0 and followed by reacting it with 2-iminothiolane to give (DOTA)m-PAMAMG3.0- F(ab')2-trastuzumab. Finally, the (DOTA)m-PAMAM-G3.0-F(ab')2-trastuzumab was radiolabeled with a radionuclide of 177Lu to give (177Lu-DOTA)m-PAMAMG3.0-F(ab')2-trastuzumab (177Lu)n-(DOTA)m-PAMAMG3.0-F(ab')2-trastuzumab resulting in radiochemical purity of 98%
after purification with PD-10 column.
18 ….incidence of death, cancer, and its prevalence for….
25-26 …..(MAb) has long been used for cancer therapy; the target is a tumor antigen.
28 ……..transduction signal, blocking the receptor, reducing…..
32 …..which is loaded with a toxin, radioisotope or drug.
38-39 HER-2 is one of the family members of Epidermal Growth Factor Receptor (EGFR), which consists of HER-1, HER-2, HER-3, and HER-4.
42 ……. target, target because its level is closely……..
44-47 Also, In addition to breast cancer, overexpressions of HER-2 are found in others cancer other cancers, such as the pancreas, ovarian, and colorectal.
49 …..HER-2-positif positive tumor cell
51 Radioimunotherapy Radioimmunotherapy (RIT) can be used as…….
52-53 ……treatment in which a radioisotop radioisotope that is combined with 69-72
mono Mono-N-hydroxysuccinimide ester1,4,7,10-tetraazacyclododecane-N,N’,N’,N’”- tetraacetic acid tetraaceticacid (NHS-DOTA) is an example of bifunctional chelating agent [9] utilized in this research.
74-75 Dendrimers are a monodisperse macromolecule, homogenous, nano-sized [10],…..
77-80 The structure of dendrimer that which includes molecular weight, size, and the amount of surface functional groups can be modified.
80-82 Therefore, dendrimer exists in the form of low to high generation namely dendrimer generation 1 (G1), G2, G3, and so on etc.[11)
85-88 These groups can be used to bind both a complex compounds, such as 177Lu-DOTA, and to a molecular carriers, such as trastuzumab [12].
90-92 This study aims at developing a new radioimmunotherapeutic agent radioimmunotherapy (RIT) of (177Lu-DOTA)m-PAMAM-G3-F(ab')2-trastuzumab.
92-94 (DOTA)m-PAMAM-G3-F(ab')2-trastuzumab is synthesized through four step reaction reaction steps ………..
112 ….. Multi-Purpose Reactor and ……….
114-
115 …..National Nuclear Energy of Indonesia Indonesian National Nuclear Energy Agency ….
118- 120
….. sulfo-SMCC (Thermo Fisher Scientific) and Traut’s reagent of 2-iminothiolane (Thermo Fisher Scientific) from Thermo Fisher Scientific,.
120- 125
Bovine serum albumin, trizma base, ……….and tris-glysin tris glycine (Sigma) were purchased from Sigma, while EDTA, HCl, and NaOH were from E125 Merck (obtained from E
125
Merck),.125 Others were protein standard standart and protein dye (Bio-Rad),………
128- 129
(Sephadex G25 Columns, GE Healthcare) and Instance thin layer silica gel (ITLC-SC) strips (Agilent).
134 Others were thermomixer and micropipette (Eppendorf), 141-
142
After dialysis dyalisis process using dialysis cassette complete, aliquot liquot (100 μL) of digestion buffer was added to 0.75 mg pepsin.
144 …. properly propelly dissolved followed ……….
147 …. 10 mM at mMat pH 8.0 was added to …..
152- 153
The fraction of with F(ab’)2-trastuzumab purity > 95% were then pooled then used for further study.
161- 162
PAMAM G 3.0 in 0.1 M phosphate buffer at pH 7.4 was added into sulfo NHS-DOTA solution in 0.1 M phosphate buffer (mol ratio 1: 96).
168- 172
All purification processes in this work project used PD-10 column which was pre-blocked with BSA, and equilibrated with the desired eluent, and The column was then eluted with 0.01 M
PBS at pH 7.4 (contains 5 mM EDTA).
172- 173
The eluates from a purification step of (DOTA)m-PAMAM 3.0 conjugate were separated into 30 fractions of 0.25 mL each.
185- 192
Aliquot of 2- iminothiolane was added into PAMAM G 3.0 conjugate (DOTA)m-PAMAM G 3.0 cojugate with mol ratio of 2:1 followed by incubationat incubation at room temperature for 1 hr. under nitrogen gas atmosphere to form The DOTA)m-PAMAM G 3.0-SH that formed was then purified form unreacted 2-iminothiolane using PD-10 column in similar to that of Sec. 3.1.
195 …….using the dye-protein.
195- 197
Fractions, which gave a blue colour indicating the existence of (DOTA)m-PAMAM G 3.0-SH, were then retrieved and used for further study.
202- 205
Sulfo-SMCC was dissolved in a small amount of DMSO/DMF, and then the concentration was made to become 1 mg/ mL by addition of 0.1 M PBS at pH 7.4 (containing 5 mM EDTA).
208 ...incubation at incubationat room temperature for 30 minutes.
211 …… (containing 5 mM EDTA) in a similar manner with Section 3.1.
214-
215 ………and tested using the dye-protein dye protein.
223- 225
The activated DOTA- PAMAM G 3.0 [(DOTA)m-PAMAM G 3.0-SH] (DOTA)m-PAMAM G 3.0-SH was added to into the activated trastuzumab (F(ab’)2-trastuzumab-SMCC).
240-
241 …….. in the Multi-Purposes GA Siwabessy Reactor …..
243 …..by addition of hen, 2 mL of 6 M HCl.
247 …… was re-dissolved in with 3 mL of HCl 0.05 M.
251- 253
The solution of 177Lu was diluted with 0.25 M ammonium acetate at pH 7.5 (1:3), and then subsequently added into the (DOTA)m-PAMAM G 3.0- F(ab’)2-trastuzumab.
255-
256 ……… to 5.5 with 0.1 N NaOH, and then it was incubated for 90 min at 37 °C.
259-
261 The reaction mixture from the radiolabelling process was then loaded into a PD-10 column.
261- 262
The column was then eluted using 0.01 M PBS at pH 7.4, and the eluent was collected and separated into 50 fractions (0.25 mL/ fraction).
265-
266 The fractions, which had high radioactivity, were then tested for ….
269-
270 ……. saline solution were used as a stationary and mobile phases, respectively.
276- 294
This paragraph does not report and even discuss the experimental results. The content of this paragraph was mentioned in the experimental method section. If you think this paragraph is important, it is better you move it to the introduction section or to the experimental method section.
295-
299 This statement was mentioned previously in the previous sections. Please remove it.
300- 301
The chromatograms chromatogram of standard proteins that have passed the SEC column is shown in Figure 2.A.
302- 304
This chromatogram shows 5 five peaks of protein standard five standard proteins with different molecular weight as indicated with each its retention time (RT).
304- 306
Figure 2B shows demonstrates the chromatogram of trastuzumab that It has a molecular weight of ~148 kDa [13];.
310 ….. while the Rt RT of the….
312- 314
It indicates is assumed that the data measured using the HPLC in this experiment agree with the reference data. (There is still uncertainty in this statement)
321- 322
…..
between the second peak (_-globulin, MW 158 kDa KDa) and the third peak (Ovalbumin, MW 44 kDa Kda).325 The measured results demonstrates demonstrate that…..
327-
328 a small peak with a percent age percentage area of 5 %...
329- 330
…which is assumed corresponds to be the Fc portion that has not been removed completely separated through the column
374- 375
This synthesis reaction was carried out in consisted of the four stages as described in the previous section. : the first stage was preparation of NHS-DOTA-PAMAM G 3.0.
376- 381
The bifunctional chelating agent (BFCA) selected in this project was NHS-DOTA. The reason of the use of NHS-DOTA instead of p-SCN-Bn-DOTA for preparing the conjugate of NHS- DOTA-PAMAM G 3.0 due to its low solubility of (DOTA-p-SCN-Bn)n PAMAM-G3.0 conjugate after the freeze dry process as evidenced in the previous studies.is more advantageous than p-SCN-Bn-DOTA. Previous studies showed that after freeze dry process, the solubility of conjugate (DOTA-p-SCN-Bn)n PAMAM-G3.0 became low
382- 384
Therefore, it was not able impossible to be well re-constituted completely the conjugate before its labeling with 177Lu [14].
384- 393
The next step stage of the synthesis was activation of conjugate dendrimers dendrimer conjugate, (DOTA)m-PAMAM G 3.0. Activation of this conjugate with 2-iminothiolane was intended to provide a sulfhydryl group on the surface of (DOTA)m-PAMAM G 3.0 which is going to be used for further coupling. The resulted (DOTA)m-PAMAM G 3.0-SH was purified from unreacted 2-iminothiolane and other by-products By using a PD-10 column (eluted with eluent of phosphate buffer 0.05 M at pH 7.4 containing EDTA 5mm).
401- 412
The color changes in this colorimetric assay are is due to the electrostatic interactions between the remaining primary amine groups of dendrimer and protonated one with one sulfo groups of Coomassie blue [12].
414- 432
The further step third stage of the synthesis was activation of F(ab')2-trastuzumab by reacting it with SMCC. maleimide This activation was intended to provide F(ab')2-trastuzumab with maleimide and F(ab')2-trastuzumab-SMCC, which is then coupled specifically with the sulfhydryls from of (DOTA)m-PAMAM G3.0-SH. Purification and testing for (DOTA)m- PAMAM G3.0-SH F(ab')2-trastuzumab-SMCC were was carried out in similar to that of (DOTA)m-PAMAM G3.0-sulfhydril. The results of a colorimetric assay of F(ab')2-trastuzumab- SMCC is shown in Figure 3B. The positive fractions containing trastuzumab-SMCC were fractions numbered 14-19. The results of a colorimetric assay of F(ab')2-trastuzumab-SMCC is shown in Figure 3B. The last step The fourth stage was the conjugation of DOTA-PAMAM G3.0–SH with F(ab')2-trastuzumab-SMCC to form a DOTA-PAMAM G3.0–F(ab')2-
trastuzumab conjugate. The reaction mixture was incubated overnight, which was then dialyzed using slides A-Lyzer (20 K MWCO) with 0.25 M ammonium acetate at pH 7.2 containing 1.2 grams of Chelex-100.
434- 446
The complex formation of (177Lu-DOTA)m-PAMAM G3.0-F(ab')2-trastuzumab was performed by reacting a DOTA-PAMAM G3.0–F(ab')2-trastuzumab conjugate with 177Lu3+ at pH 5 - 5.5 with the incubation time of 90 minutes. At the end of Once the incubation time was complete, EDTA was added to the reacting mixture to bind to the free 177Lu3+, which was not bound to the DOTA-PAMAM G3.0-F(ab')2-trastuzumab [15]. Analysis of the labeling percentage of the (177Lu-DOTA)m-PAMAM G3.0 - F(ab')2-trastuzumab before and after passing through the column was determined using thin layer chromatography ( of ITLC-SG and saline solution as stationary and mobile phases, respectively).
456- 472
Figure 4A is a radiochromatogram of (177Lu-DOTA)m-PAMAM G3.0-F(ab')2-trastuzumab before passing through the PD-10 column. The presence of To determine whether the compound did not contain free 177Lu, (in the form of 177Lu-EDTA), was confirmed with a
radiochromatogram of the177Lu-EDTA,which was prepared separately and was also spotted on ITLC SG strips and developed with saline solution (see Fig. 4B).
Figure 4A and 4B show chromatograms of (177Lu-DOTA)m-PAMAM G3.0 - F(ab’)2-
trastuzumab and free 177Lu (in the form of 177Lu -EDTA respectively. It can be seen that there was free 177Lu (Rf = 1) in the reacting reaction mixture that contain a main product of the (177Lu-DOTA)m-PAMAM G3.0-F(ab')2-trastuzumab (Rf = 0) that has . it is recognized by the presence of 177Lu peaks (Rf = 1) in the chromatogram of the 177Lu- DOTA-PAMAM G3.0- F(ab')2-trastuzumab (Rf = 0). These results are confirmed by calculation of the radiochemical purity of , which is only 76.77%.(please, state the radiochemical purity with its standard deviation).
480- 493
Thus it was necessary to do the purification process. The purification of (177Lu-DOTA)m- PAMAM G3.0-F(ab')2-trastuzumab was carried out using the PD-10 column and the eluatet eluate was collected and separated into 50 fractions (in 0.25 mL each). The radioactivity of each fraction was measured using a dose calibrator [13]. its elution profile Elution profiles of the (177Lu-DOTA)m-PAMAM G3.0-F(ab')2-trastuzumab are is shown in Figure 5. It appears From these profiles, it is understood that based on the 177Lu radioactivity there are two distinct peaks of ; the first peak is the complex of the (177Lu-DOTA)m-PAMAM G3.0-F(ab')2- trastuzumab and the second peak is bonded to a free 177Lu EDTA, respectively. Fractions which have high radioactivity were then tested for their radiochemical purity using the ITLC-SG..
493- 499
What do you mean with this statement “The selected fractions were fraction No. 14 and 15.”??
(see again carefully Fig. 5. Are the fractions No. 14 and 15 relevant?)
Please remove this very common statement “The radiochemical purity of the (177Lu–DOTA)m- PAMAM-F(ab’)2–trastuzumab was calculated based on the ratio between the count rate under the (177Lu–DOTA)m-PAMAM-F(ab’)2–trastuzumab peak and the total count rate”. If you think this statement is very important, please move it to the Experimental Section.
500- 508
Figure 6 shows radiochromatograms an ITLC-SG radiochromatogram of fraction 12 from the elution profile of Fig. 5. The radiochemical purity of fractions fraction No.12 as (177Lu-
DOTA)m-PAMAM G3.0-F(ab')2-trastuzumab in the ITLC-SG radiochromatogram is found to be 98.18%.(please, state the radiochemical purity with its standard deviation as a measure of its uncertainty. Please also specify the impurity species of 1.82%). Experimental results showed that purification of (177Lu-DOTA)m-PAMAM F(ab')2–trastuzumab using a PD-10 column was found to be effective in increasing the radiochemical purity of (177Lu-DOTA)m-PAMAM- F(ab')2–trastuzumab from 76.77% to 98.18%. It means there is a radiochemical purity increase of about 21.41%.
518- 533
The (177Lu)n-(DOTA)m-PAMAM-G3-F(ab')2-trastuzumab (177Lu-DOTA)m-PAMAM-G3- F(ab')2-trastuzumab, which will be expected to be potential as an RIT agent for therapy of breast cancer overexpressed HER-2, has been prepared successfully. The F(ab')2-trastuzumab as a precursor of (177Lu)n-(DOTA)m-PAMAM-G3-F(ab')2-trastuzumab was prepared by enzymatic digestion of intact trastuzumab using pepsin. The radiolabeling of (DOTA)m-PAMAM-G3.0- F(ab')2-trastuzumab with 177Lu at 37°C and incubation time of 90 min results in (177Lu)n- (DOTA)m-PAMAM-G3-F(ab')2-trastuzumab (177Lu-DOTA)m-PAMAM-G3-F(ab')2- trastuzumab with the radiochemical purity of 98% after purification with PD-10 column. The stability and other tests (in vitro and in vivo) of the (177Lu)n-(DOTA)m-PAMAM-G3.0-F(ab')2 (177Lu-DOTA)m-PAMAM-G3-F(ab')2-trastuzumab and other tests (in vitro and in vivo) will be published reported shortly.
Final comments and recommendations:
Comments:
1. The authors put several statements associated with an experimental procedure in the section of results and discussion, besides also use very general statements or statements which are already common knowledge.
2. The authors take a speculative conclusion related to determining the molecular weight of trastuzumab fragment.
3. The authors do not reveal new things that emerge from this research in the section of results and discussion.
4. The authors always show the experimental data with a numeric value that does not have uncertainty. Writing such data also indicates that the data as if it was obtained from one run of the experiment
5. The authors do not attempt to make sure that the primary product of (177Lu-DOTA)m-PAMAM-G3-F(ab')2- trastuzumab is actually free from the impurities of (DOTA)m-PAMAM-G3-F(ab')2-trastuzumab, PAMAM- G3-F(ab')2-trastuzumab, and F(ab')2-trastuzumab, because these impurities will be influential in the process of interaction of (177Lu-DOTA)m-PAMAM-G3-F(ab')2-trastuzumab with her-2 receptors
6. The authors do not mention how to determine the value of m in the compounds of (177Lu-DOTA)m-PAMAM- G3-F(ab')2-trastuzumab and (DOTA)m-PAMAM-G3-F(ab')2-trastuzumab.
Recommendations:
1. Statement or phrase related to the experimental procedures should not be placed on the results and discussion 2. The determination of molecular weight of F(ab')2-trastuzuma should be based on a regression calibration
curve of molecular weight variation of protein standard vs. retention time.
3. Preferably the quantitative data obtained from the measurement results is accompanied by its standard deviation. Standard deviation is a measure of uncertainty.
This paper is recommended to be [ ] Accepted without further revision [√] Accepted with minor revision [ ] Major Revision is required [ ] Rejected
