Targeting GD2-Positive Tumor Cells by Pegylated scFv Fragment–Drug Conjugates Carrying Maytansinoids DM1 and DM4

Round 1

Reviewer 1 Report

Here, Kalinovsky et al report the results that validate the production and functionality of a pegylated and drug-conjugated single-chain fragment variable (scFv) targeting the cancer cell associated antigen disialoganglioside GD2. They used the scFv fragment of the FDA-approved antibody ch14.18 (Dinutuximab). By using a two-steps conjugation method with a 10 kDa tetra-functional 4-arm polyethylene glycol (PEG) maleimide, they produced oligomers of pegylated-scFv conjugated to maytansine derivatives (DM1 and DM2). They reported a stoichiometry of PEG/scFv/drug in one conjugation. This was about 1.5 scFv and 2.5 drug per PEG molecule. They analyzed the conjugate capacity to bind GD2 by ELISA and found no noticeable change induced by the scFv-drug pegylation compared to non conjugated scFv. They investigated the influence of such pegylation (PEG4: with 4 arm) on the recognition of GD2 on cell surface by the scFv fragment by generating a fluorescein-coupled scFv-PEG4. They found that conjugation of scFv-drug-PEG4 did not affect the capacity of the antibody fragment to bind GD2 on cell surface. The authors also evaluated the cytotoxicity of the PEG4-scFv-drug conjugates for different cell lines and determined how specific they are for GD2-expressing cells. Interestingly, the pegylated scFv-drug conjugates presented toxicity that were specific to and potent for GD2-expressing tumor cells.

General comment and questions:

The report is of interest and is a good contribution for the development of targeted cancer therapy. Pegylation of scFv is known to increase these biologics half life in blood circulation thereby enhancing their uptake my solid tumors.

1/To what extent is pegylation with PEG4 maleimide influencing scFv half life in blood? This could be interesting to discuss. Here the authors used PEG of 10 kDa to generate the conjugates. In a recent work by Li et al J Con Rel 2017, it appeared that PEG of higher size conjugated to diabodies had better half life, recognition capacity and drug delivery capacity.

2/The authors found that scFv-PEG4 was toxic at high doses compared to non conjugated scFv. What is the toxicity of PEG4 alone for B78-D14 cells?

3/ The conjugates scFv-Peg-DM1 and scFv-Peg-DM4 were specifically toxic for GD2-expressing cells. The conjugate with DM4 was two fold more potent than that with DM1. What could be the molecular mechanisms supporting the specific activity of these drug conjugates? Could it be by endocytosis of drug conjugates? Can the drug be released in the cells? This can be interesting to discuss. It would be very interesting to analyze microtubule polymerization state of the cells treated by these drug conjugates. A discussion also about the structural difference between DM1 and DM4 could be interesting. The appearance of a dramatic difference between these two drugs only when conjugated to GD2-specific scFv-PEG4 is intriguing. How the structural difference between DM1 and DM4 could help in better understanding the mechanisms of action of scFv-Peg-DMs would be an important question.

4/ Based on the electrophoresis, the authors concluded that most bands were pegylated monomers and dimers of scFv conjugated through PEG4. Taking into account the size of the monomeric non pegylated scFv (above 26 kDa), the most pegylated complexes seem to be rather conjugates of two (~52) and  three (between 72 and 96 kDa) scFv fragments. Is this possible?

Minor point:

 

The authors state at line 316: "the IC50 of DM1 and DM4 for both cell lines was roughly the same: 2.4±0.6 and 4.3±0.8 nM, respectively (Figure 4С, D, Table S2)".

It is the opposite. IC50 of DM1 =4.3 and IC50 of DM4 = 2.4nM.

Please read carefully, there are some typos

Author Response

We thank the Reviewers and the Editors for the detailed analysis of our manuscript and valuable suggestions. We have extensively revised the manuscript and hope that its quality has improved. Please, find our point-by-point response (AU) to your questions and comments below.

Key changes and additions to the manuscript are indicated by highlighted text in the revised manuscript files submitted for review. Additionally, a series of minor changes regarding language grammar and style, as well as typos were made, which are not highlighted in the text.

Response to Reviewer 1.

Point 1.

1/ To what extent is pegylation with PEG4 maleimide influencing scFv half life in blood? This could be interesting to discuss. Here the authors used PEG of 10 kDa to generate the conjugates. In a recent work by Li et al J Con Rel 2017 , it appeared that PEG of higher size conjugated to diabodies had better half life, recognition capacity and drug delivery capacity.

 

AU:

We thank the reviewer for an important question and for highlighting an interesting publication relevant to the topic of the manuscript. Following the reviewer’s suggestion, we decided to discuss the influence of the PEG chain length and branching on the pharmacokinetic profile of the antibody fragments at length, in the Discussion section of the manuscript (see highlighted text starting from line 426 of the manuscript). The work by Li et al. [https://doi.org/10.1016/j.jconrel.2018.04.013], as well as a number of other works, have been taken into consideration.

Point 2.

2/ The authors found that scFv-PEG4 was toxic at high doses compared to non conjugated scFv. What is the toxicity of PEG4 alone for B78-D14 cells?

AU:

It is generally considered that that polyethylene glycol is not toxic and safe for use pharmaceuticals, foods, and cosmetics. Specifically, at least 10 pegylated proteins have been approved for clinical use, and we have included a link to an extensive review on the topic [doi: 10.1007/s12079-018-0492-0] in the Discussion section.

 

Following the reviewer’s question, we evaluated toxicity of PEG4 in the GD2-positive B78-D14 (see updated Figure 4E) and GD2-negative B16 (updated Figure 4F) cell lines in the MTT-assay. PEG4 is not toxic for these cell lines within the concentration range evaluated in our study.

Point 3.

3/ The conjugates scFv-Peg-DM1 and scFv-Peg-DM4 were specifically toxic for GD2-expressing cells. The conjugate with DM4 was two fold more potent than that with DM1. What could be the molecular mechanisms supporting the specific activity of these drug conjugates? Could it be by endocytosis of drug conjugates? Can the drug be released in the cells? This can be interesting to discuss. It would be very interesting to analyze microtubule polymerization state of the cells treated by these drug conjugates. A discussion also about the structural difference between DM1 and DM4 could be interesting. The appearance of a dramatic difference between these two drugs only when conjugated to GD2-specific scFv-PEG4 is intriguing. How the structural difference between DM1 and DM4 could help in better understanding the mechanisms of action of scFv-Peg-DMs would be an important question.

AU:

The primary mechanism of action of both DM1 and DM4 is the inhibition of tubulin polymerization. Tubulin is a key protein that forms microtubules. By preventing tubulin polymerization, maytansinoids halt the formation of the mitotic spindle, leading to cell cycle arrest at the G2/M phase and eventually causing apoptotic cell death. We have provided some detail in the Discussion section of the manuscript on the key difference of DM1 and DM4 – an additional dimethyl group next to the terminal cysteine of the DM4 molecule – that is considered by some to be the reason of its increased cytotoxicity. We observed such increased cytotoxicity of DM4 compared to DM1 for all cell lines used in the work.

Two high-quality reviews on the topic have also been provided. It might be important to add that the focus of research of the USA-based company Immunogen, a pioneer in the field responsible for the development of the only two clinically approved maytansinoid-based ADCs, trastuzumab emtansine in 2013 and mirvetuximab soravtansin in 2022, has over the years shifted from DM1 to DM4.

The appearance of an obvious difference between our scFv-PEG4-DM1 and scFv-PEG4-DM4 conjugates in the GD2-positive B78-D14 cell line and not in the GD2-negative B16 cell line can be explained specifically by a more pronounced bystander cell killing of DM4. DM4 metabolites are considered to cross the cell membrane more efficiently than DM1 metabolites due to increased hydrophobicity, upon antibody-mediated delivery into target GD2-specific cells, and induce killing of all surrounding cells. At the same time, the cytotoxic effect of both DM1 and DM4 within the intact conjugates is considerably lower. The  cytotoxic activity of free DM1 and DM4 and as part of small molecule drug conjugates (SMDC) comparable to that observed in our work were shown by Lo et al. [doi.org/10.3390/pharmaceutics14071316] in the triple-negative breast cancer cell line HCC1806. The IC50 of free DM4 was 3 times lower than IC50 of DM1, and it was reduced for both drugs when they were part of the SMDCs . These data are is in the good correlation with our results. The mechanisms of the observed effects are interesting and important for cancer therapy since DM1 and DM4 are widely used as payloads in ADC and require a distinct and more profound study.

Point 4.

4/ Based on the electrophoresis, the authors concluded that most bands were pegylated monomers and dimers of scFv conjugated through PEG4. Taking into account the size of the monomeric non pegylated scFv (above 26 kDa), the most pegylated complexes seem to be rather conjugates of two (~52) and three (between 72 and 96 kDa) scFv fragments. Is this possible?

AU:

Both from our own experience and from multiple other studies, the apparent molecular weight of PEGylated proteins in gel electrophoresis is almost always bigger than what is calculated by summing the weight of the protein and the PEG. The typical explanation of this phenomenon lies in a much bigger hydrodynamic radius of PEG compared to proteins. Each ethylene glycol subunit in solution is tightly associated with two or three water molecules, and this binding of water molecules makes pegylated compounds function as though they are much larger than a corresponding soluble protein of similar molecular mass, both in size-exclusion chromatography and gel electrophoresis [https://doi.org/10.1038/nrd1033].

Pegylation leads to a much larger increase in the hydrodynamic radius of the modified molecule compared to the addition of a protein with an equivalent molecular weight. The correspondence of the hydrodynamic radius of pegylated vs non-pegylated proteins of the same molecular weight to the observed peaks in size-exclusion chromatography was elegantly shown in this work [doi: 10.1002/biot.200900218].

Regarding gel electrophoresis, a somewhat extreme example [doi: 10.1002/elps.201400373] demonstrates that conjugation of a recombinant human ciliary neurotrophic factor (~20 kDa) modified with a free cysteine and conjugated to a 40kDa PEG with one maleimide group results in a product with observable molecular weight on the gel of ~150 kDa. Interestingly for our topic, this specific work shows that PEG chain may be lost in routine SDS-PAGE analysis of PEG-Maleimide proteins, increasing the observed relative quantity of original non-pegylated protein on the gel.  

In our work with a series of monovalent and polyvalent PEG-maleimides, we always observed increased molecular weight of pegylated protein. 

Two links to the works discussed above were added to the manuscript in line 263.

Point 5.

Minor point: The authors state at line 316: "the IC50 of DM1 and DM4 for both cell lines was roughly the same: 2.4±0.6 and 4.3±0.8 nM, respectively (Figure 4С, D, Table S2)".

It is the opposite. IC50 of DM1 =4.3 and IC50 of DM4 = 2.4nM.

AU: Corrected.

Point 6.

Comments on the Quality of English Language. Please read carefully, there are some typos

AU: Extensive revision of the language was performed.

Reviewer 2 Report

The authors present conjugation of an anti-GD2 scFv with PEG equipped with cytotoxic drugs DM1 and DM4. The conjugates were able to bind to the cognate antigen in ELISA similarly to the parental scFv, and also recognized the GD2-positive cell line, while there was no reactivity with the antigen-negative cell line. They exhibited stronger toxicity towards the antigen positive cell line than scFv and scFv-PEG compound without bound toxin, and also decreased its viability more efficiently than was measured for the negative cell line.

My main comment to the manuscript is that the authors mention statistical analysis of the data and also comment on the significance of the results, but the analysis itself is not shown. Both Figures and the results section should be corrected.

Biophysical characterization of the conjugate should be performed, at minimum size exclusion chromatography in native conditions, to evaluate the composition of the PEGylated FDC. Further, single fractions should be gel purified, as the authors currently evaluate the effect of a mixture of compounds. This is also important for determination of cytotoxicity of scFv-PEG4, which in their experience is dependent on multimers of scFv (and this cannot be studied if a mixture of products is applied).

Further, the authors show that the conjugates bind to GD2-positive cells, but a titration should be performed to quantitatively estimate cell surface binding of all scFv-compounds used for cytotoxicity assays (maybe over the FLAG tag used for ELISA detection). The EL-4 cell line should also be characterized better in this aspect.

As there is also a toxic effect of the conjugates for the antigen-negative cell line, an estimation should be made of how many antigen copies are expressed on the antigen-positive cells, to be able to judge on the window of specific toxicity.

Please find below a list of remarks which I hope you will find helpful.

Line 66: are being studied

Line 71: …were generated based on minibodies and scFv fragments, and both of these carried…

Line 112: °C

Line 173: B16 cell line

Line 273: The dependence of the optical density (OD) on the concentration

Line 274: “moles of scFv“: molar concentration of scFv

Line 283: to bind to GD2-positive cells

Line 295: …conjugates with such structure can specifically bind to CD2-positive cells.

Line 296: Сytotoxic effects

Line 301: Remarkably

Line 302: The presentation of IC50 is unusual. This value should be calculated from the slope of the inhibitory curve. Taking 50% viability as IC50 is not that good because even at the highest concentration of FDCs used, there is some signal indicating viability in Figure 4A (maybe 5-10%, although this is typical for this assay and probably all cells are dead).

Line 303: for the cell line

Line 304: Word order: “Thus the difference in effectiveness of the obtained conjugates clearly depended on the drug“

Line 306: This sentence is not clear: The obtained pegylated conjugates had less effect, not even reaching IC50. Please reword to: decreased the viability to xxx% at the highest concentration used, or compare the viability for both cell lines at IC50 for the positive one, or similar.

Line 308: the assumption of the selective activity

Line 326: “As the toxicity of scFv-PEG4 itself was not significant“: please provide the  information on significance in the Figure and the method of significance estimation in the Figure Legend.

Line 331: EL-4 is sometimes written EL4

Line 372: and 1.4

Lines 374-380: evaluation of significance is not determined.

Line 387: “blood circulation time“: serum half-life, or half-life in vivo is a better expression

Line 390: “The proposed format of the antibody fragment drug conjugates will potentially exhibit reduced side effects compared to the classical ADCs due to the absence of Fc fragments in their structure“: this comment is not appropriate regarding the success of ADCs as therapeutic compounds (and all approved ones are full-length antibodies).

Supplementary Figure 2: I would recommend adding the results of the effect of scFv alone and scFv-PEG4 also for the EL-4 cell line. Description of the cell line EL-4, cultivation conditions, and assay procedure should be given. Also the expression of GD2 antigen should be examined.

Author Response

We thank the Reviewers and the Editors for the detailed analysis of our manuscript and valuable suggestions. We have extensively revised the manuscript and hope that its quality has improved. Please, find our point-by-point response (AU) to your questions and comments below.

Key changes and additions to the manuscript are indicated by highlighted text in the revised manuscript files submitted for review. Additionally, a series of minor changes regarding language grammar and style, as well as typos were made, which are not highlighted in the text.

Response to Reviewer 2.

 Point 1.

 My main comment to the manuscript is that the authors mention statistical analysis of the data and also comment on the significance of the results, but the analysis itself is not shown. Both Figures and the results section should be corrected.

Biophysical characterization of the conjugate should be performed, at minimum size exclusion chromatography in native conditions, to evaluate the composition of the PEGylated FDC. Further, single fractions should be gel purified, as the authors currently evaluate the effect of a mixture of compounds. This is also important for determination of cytotoxicity of scFv-PEG4, which in their experience is dependent on multimers of scFv (and this cannot be studied if a mixture of products is applied).

AU:

We thank the reviewer for the detailed study of the manuscript and for important relevant comments.

Regarding the reviewer’s main comment, we totally acknowledge the limitation of the study that stems from the fact that the product of our reaction is a mixture of several compounds. The product of the two-stage thiol-maleimide reaction between the scFv fragment with a C-terminal cysteine, the PEG molecule with four functional maleimide groups, and the thiol-containing drug DM1 (DM4) is, as observed on protein gel electrophoresis, predominantly a mix of the pegylated scFv monomer and the pegylated scFv dimer, and also includes significant amounts of the non-conjugated scFv fragment, and the pegylated scFv trimer and tetramer. It is important to stress that this mixture does not contain both the non-conjugated DM1 (DM4) and the PEG4-DM1 (DM4) conjugate, since the purification of the product mixture, specifically its step regarding filtration through 30 kDa filters, ensures that these products are discarded; and also non-conjugated DM1 and DM4 have very low solubility in aqueous solutions.

UV-Vis spectroscopy is an accurate quantitative method to evaluate the drug (DM1/DM4) to scFv monomer ratio in the product mix, since the drug and the scFv fragment are the only two components of the solution that absorb within the evaluated wavelength range (DM1/DM4 have a characteristic absorption peak at around 253 nm, protein absorption in typically analyzed at 280 nm, and polyethylene molecules do not absorb). Our analysis routinely gave a DM1 : scFv monomer ratio of approx. 1.5 for the product generated according to the reaction protocol described in the manuscript, and the purification ensured that all the drug present in the final mix is conjugated to scFv.

On the basis of the results from gel electrophoresis (the product is predominantly a mix of the pegylated scFv monomer and the pegylated scFv dimer, that is, the typical product as observed in gel electrophoresis will contain 1.5 scFv molecules) and the drug/scFv ratio, we provided a rough estimate that “in the resulting scFv-PEG4-DM1 and scFv-PEG4-DM4 conjugates, on average, 1.5 scFv molecules and 2.5 drug molecules are attached to one PEG4 molecule”. Following the reviewer’s comments, we moved this point from the Results section to the Discussion section (line 410).

Next, we performed a revision of the manuscript and corrected all instances in the text where the product of the two-stage thiol-maleimide reaction was presented as an exclusively pegylated product (for example, line 157 in Materials and methods, line 249 in Results, line 275 in Results).

Also, several instances in the manuscript, in which the product of the one-step conjugation between the scFv fragment and tetravalent PEG-maleimide generated according to our earlier work [doi:10.3390/molecules24213835] was named the “scFv tetramer”, were edited; for instance, in line 31 of the abstract, a change was made to “the product of scFv and PEG4 conjugation consisting predominantly of pegylated scFv multimers and monomers”.

We fully agree with the reviewer that a chromatographic purification of the reaction product and the following evaluation of the single fractions by gel chromatography would considerably add to the characterization of the product. However, such a purification, that we earlier attempted to perform by size-exclusion and ion-exchange chromatography among other methods, with varying success and typically not yielding homogeneous fractions, is unfortunately not within the scope of the current work. The main task of this work was to demonstrate in principle that generation of FDCs carrying GD2-directed scFvs and DM1/DM4 bridged by a multivalent PEG molecule is possible, and to present a general in vitro characterization of the heterogeneous product.

In our future work, we plan to evaluate PEG molecules with other reaction groups, including heterofunctional PEG molecules, for conjugation with antibody fragments, which may allow us to increase the efficiency of the reaction and the homogeneity of its product, as well as to simplify the method of purifying PEGylated products.

 Point 2.

The authors show that the conjugates bind to GD2-positive cells, but a titration should be performed to quantitatively estimate cell surface binding of all scFv-compounds used for cytotoxicity assays (maybe over the FLAG tag used for ELISA detection). The EL-4 cell line should also be characterized better in this aspect.

AU:

Answering the reviewer’s question, we assessed the binding of all scFv-containing molecules used in this study to the surface of GD2-positive B78-D14 cells using FITC-labeled anti-FLAG antibodies. In an earlier work [https://doi.org/10.3390/molecules24213835], we carried out a similar analysis on different GD2-positive lines, EL-4 and IMR-32, so data on the EL-4 cell line was not presented in this work. Additional experiments with the EL-4 line are included in supplementary files.

Point 3.

As there is also a toxic effect of the conjugates for the antigen-negative cell line, an estimation should be made of how many antigen copies are expressed on the antigen-positive cells, to be able to judge on the window of specific toxicity.

AU:

For gangliosides, estimation of antigen copies is complicated. Therefore, we usually calculate the selectivity ratio - determined as the ratio between the IC50 values for GD2-negative cells to GD2-positive cells. Since we did not reach IC50 values for GD2-negative cell line by induction with pegylated conjugates, such a calculation was not performed.

Point 4.

A list of remarks on typos as well as language grammar and style.

AU: Corrected.

Point 5.

Line 302: The presentation of IC50 is unusual. This value should be calculated from the slope of the inhibitory curve. Taking 50% viability as IC50 is not that good because even at the highest concentration of FDCs used, there is some signal indicating viability in Figure 4A (maybe 5-10%, although this is typical for this assay and probably all cells are dead).

AU:

According to the reviewer's recommendation, we reconstructed the cytotoxicity graphs, in which we calculated the inhibition curves by replacing the “Cell viability” parameter with “Growth inhibition”.

Point 6.

Line 306: This sentence is not clear: The obtained pegylated conjugates had less effect, not even reaching IC50. Please reword to: decreased the viability to xxx% at the highest concentration used, or compare the viability for both cell lines at IC50 for the positive one, or similar.

AU:

Changed to “Both pegylated FDCs manifested considerably weaker effects in the B16 cell line that does not express GD2, inducing growth inhibition by 43.4±2.1% and 41.4±2.9% for scFv-PEG4-DM1 and scFv-PEG4-DM4, respectively”.

Point 7.

Line 326: “As the toxicity of scFv-PEG4 itself was not significant“: please provide the information on significance in the Figure and the method of significance estimation in the Figure Legend.

Lines 374-380: evaluation of significance is not determined.

AU:

Within the manuscript, when speaking of significantly different values between two groups, we refer to analysis by unpaired Student's two-sample t-test with significance level of P<0.05. Following the Reviewer’s comment, we added additional details on sample sizes and the p-value used in the text for the cases where experimental groups were considered significantly different.

Significance levels of P<0.05 were considered statistically reliable.

As stated in Statistical analysis in Methods, most data in our experiments are presented as mean ± SEM of at least three independent experiments. We double checked all cases involving presentation of means and number of replicates. The number of replicates for specific cases is either presented in the Methods section, or presented in line with the description of the results of the Results section.

Point 8.

Line 390: “The proposed format of the antibody fragment drug conjugates will potentially exhibit reduced side effects compared to the classical ADCs due to the absence of Fc fragments in their structure“: this comment is not appropriate regarding the success of ADCs as therapeutic compounds (and all approved ones are full-length antibodies).

AU:

We paraphrased the sentence so that it is clear that potential reduction of side effects is mentioned in contrast to the approved GD2-specific antibodies. One of the key mechanisms of their side effects is considered to be the Fc-dependent complement-mediated antibody-induced allodynia. A relevant link [DOI: 10.1016/j.pain.2010.01.024] was added.

Point 9.

Supplementary Figure 2: I would recommend adding the results of the effect of scFv alone and scFv-PEG4 also for the EL-4 cell line. Description of the cell line EL-4, cultivation conditions, and assay procedure should be given. Also the expression of GD2 antigen should be examined.

AU:

Both in the work mentioned earlier [https://doi.org/10.3390/molecules24213835], and in several other of our works [http://dx.doi.org/10.1136/jitc-2022-004646; https://doi.org/10.1186/1471-2407-14-295], we performed a complete analysis of the EL-4 cell line regarding GD2 expression and binding to PEGylated scFv fragments. Therefore, in this work we do not present data on this line in the main part of the article, and additional experiments on the EL-4 line are included in supplementary files.

Round 2

Reviewer 2 Report

The authors have corrected the manuscript and answered to reviewer's questions. Unfortunately, they did not perform additional experiments to improve the level of knowledge on the studied molecules, but they have explained what the rationale behind the experiments is and modified the discussion and comments to those data. They also included more references to support the value of their present work.