From the journal RSC Chemical Biology Peer review history

03-Jun-2020

Dear Dr Weissleder:

Manuscript ID: CB-REV-02-2020-000022
TITLE: THE CHEMICAL BIOLOGY OF IL-12 PRODUCTION

Thank you for considering and submitting your manuscript to RSC Chemical Biology, published by the Royal Society of Chemistry. I have received reviewers reports which are copied below.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after suggested reviewers’ revisions.

Please submit a revised manuscript which fully addresses all of the reviewers’ comments. In particular it is important additional clarification about the cIAP inhibitors and their mechanism of action, as well as covering additional mechanism of IL-2 generation driven from canonical signaling supported by references. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Further peer review of your revised manuscript may be needed. Full details of the files you need to submit are listed at the end of this email.

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Associate Editor, RSC Chemical Biology

************

Reviewer 1

In this manuscript, Koch and colleagues review IL-12 production, regulation, and druggable targets in the pathway, particularly regarding its importance for cancer immunotherapy. The article is well written and provides a concise overview of what is currently known about IL-12 regulatory pathways in relevant cell types (e.g. dendritic cells) as well as current therapeutic approaches for increasing IL-12 production. This should serve as a useful introduction for members of a general audience while summarizing salient facts for those with more expertise in the field. There are a few minor changes that would improve clarity before publication.

In the section on cIAP inhibitors, the development of these agents is a bit confusing. The authors mention cIAP inhibitors turn on the non-canonical NFkB pathway but also mention some IAPs are pro-apoptotic (e.g. xIAP). Are inhibitors able to cleanly target one or the other (for distinct mechanisms of action) or do some inhibitors hit both? If they hit both, is this a problem, or can they achieve dual functions (similar to how some microtubule inhibitors stimulate dendritic cells while killing cancer cells)? Did this result from confusion in the field, where these agents were first developed to induce apoptosis and then discovered to stimulate IL-12? Some clarity on this issue would help.

A more specific term than ‘Spatial Considerations’ for the title of the final section may help avoid some confusion. Prior to this, sections were focused on extracellular biologics or intracellular small molecules, leading me to believe spatial considerations was referring to cellular location of the drug targets at first. Something like “Localizing IL-12 Production” or “Delivering Modulators of IL-12 Production” might work. An introductory sentence could also be used to clarify (e.g. starting with “ideally, IL-12 production would be located exclusively within the tumor microenvironment…”

I’m not sure about space limitations, but a few sentences to conclude would help round out the article rather than ending with the nanoparticle subsection.


Some minor points:

Page 2 – A reference on Coley’s toxins may be helpful. For a general audience, it’s not immediately obvious while IL-12 would be the operative cytokine from injecting dead bacteria.

Please increase the font size in Fig. 1B. The labels (particularly for intracellular proteins) are hard to read at 100% magnification

Reviewer 2

Overall, the review is well done, authoritative, and balanced. It is likely to be of significant interest to the field. However, the major concern is the overall premise of the review. It is not fully established that IL-12 transcription is directly associated with noncanonical NF-kB signaling as presented in this review. In general, noncanonical NF-kB signaling is associated with a narrow range of chemokine transcription. In the review, it is presented as if IL-12 is only generated through the noncanonical pathway. However, the most common mechanism of IL-12 generation is actually driven from canonical signaling, c-REL also plays a significant role. This must be covered significantly better in the review, including references that IL-12 is associated with RelB/p52..

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters.

Dear Dr. Nikolovska-Coleska,

We were pleased to learn that our manuscript, entitled “The Chemical Biology of IL-12

Production (CB-REV-02-2020-000022)” was well received by the reviewers. To address their

questions, we have carefully revised the manuscript by including additional discussions and

clarifying selected portions. We have also addressed all the editorial comments.

We have addressed the reviewer comments in an attached point-by-point response, where our

replies are in blue text and revisions in red. Revisions are similarly highlighted within the

manuscript file. We hope that these revisions satisfactorily address the recommendations made

by the reviewers.

Thank you again for the continued attention to our work, and for the opportunity to publish in

RSC Chemical Biology.

Sincerely,

Ralph Weissleder, MD, PhD

Center for Systems Biology

Simches Research Building, Room 5-206

185 Cambridge Street, Boston, MA, 02114

Tel: 617.726.8226 | Fax: 617.643.6133

E-mail: rweissleder@mgh.harvard.edu

Ralph Weissleder, MD, PhD

Director, Center for Systems Biology

Massachusetts General Hospital

Professor (Radiology and Systems Biology)

Harvard Medical School

Referee 1

Comments to the Author

In this manuscript, Koch and colleagues review IL-12 production, regulation, and

druggable targets in the pathway, particularly regarding its importance for cancer

immunotherapy. The article is well written and provides a concise overview of what is

currently known about IL-12 regulatory pathways in relevant cell types (e.g. dendritic

cells) as well as current therapeutic approaches for increasing IL-12 production. This

should serve as a useful introduction for members of a general audience while

summarizing salient facts for those with more expertise in the field. There are a few

minor changes that would improve clarity before publication.

In the section on cIAP inhibitors, the development of these agents is a bit confusing.

The authors mention cIAP inhibitors turn on the non-canonical NFkB pathway but also

mention some IAPs are pro-apoptotic (e.g. xIAP). Are inhibitors able to cleanly target

one or the other (for distinct mechanisms of action) or do some inhibitors hit both? If

they hit both, is this a problem, or can they achieve dual functions (similar to how some

microtubule inhibitors stimulate dendritic cells while killing cancer cells)? Did this result

from confusion in the field, where these agents were first developed to induce apoptosis

and then discovered to stimulate IL-12? Some clarity on this issue would help.

Thank you for the comments. We agree that the current presentation could be clarified

and which we have now done as outlined below.

Are inhibitors able to cleanly target one or the other (for distinct mechanisms of

action) or do some inhibitors hit both?

As with most small molecules, cIAP inhibitors exhibit polypharmacology, and different

drugs have varying degrees of selectivity against cIAP and xIAP. Of the molecules

discussed, LCL161 and birinapant exhibit preferential activity against cIAP compared to

xIAP, but nonetheless are still reasonably potent inhibitors of xIAP. To our knowledge,

no inhibitor solely inhibits cIAP, without any activity against xIAP. We have highlighted

various cIAP inhibitors along with commercially published IC50s against various targets

in Table 1. Whether this polypharmacology is relevant for the drug mechanism

remains to be determined.

Table 1: IC50 (in nM) of small molecule inhibitors against cIAP1/2 and xIAP. Data taken from multiple

references 1–4

The new text reads as follows:

“As with most small molecules, cIAP inhibitors exhibit polypharmacology, and different

drugs have varying degrees of selectivity against cIAP and xIAP. In table 1, we highlight

the affinities of various cIAP inhibitors against selected members of the IAP family.

LCL161 and birinapant, two cIAP inhibitors in clinical trials, exhibit preferential activity

against cIAP compared to xIAP, but nonetheless are still reasonably potent inhibitors of

xIAP. Other inhibitors, such as AZD5582 and GDC-0152, exhibit less preferential

activity against cIAP. Whether and how this polypharmacology manifests in a clinical

setting remains to be determined.

If they hit both, is this a problem, or can they achieve dual functions (similar to how

some microtubule inhibitors stimulate dendritic cells while killing cancer cells)?

Both cIAP1/2 and xIAP have roles in regulation of apoptosis, but only cIAP1/2 is

involved in production of IL-12 via non-canonical NFkB signaling. How these functions

depend on cell type and model system needs further biological exploration. We would

expect that dual mechanisms, of activating IL-12 production and promoting apoptosis,

would be beneficial, similar to microtubule inhibitors referenced above in the reviewer’s

comment. In fact, given that dying tumor cells could be a source of antigens from the

immune system, we would expect these mechanisms to potentially synergize. This is

still an unresolved question in the field and more research is needed. There is some

evidence that in certain model systems, the mechanism of drug is solely due to antitumor immunity5, 6.

We have incorporated discussion of these points in the main text, shown below:

It also remains to be determined whether the therapeutic mechanism of cIAP inhibitors

in humans is due to anti-tumor immunity, pro-apoptotic effects, or both. We expect both

mechanisms to be relevant. In fact, it is reasonable that they may be synergistic, since

promoting apoptosis would lead to dying tumor cells that could be a source of antigens

for the immune system7. Conceptually, such a dual mechanism is similar to how

increasing numbers of other cytotoxic drugs, such as PARP inhibitors, both kill tumor

cells and prime immune cells for activation8. Nonetheless, it is likely that the mechanism

is highly dependent on the context of the model system.One study, using the cIAP1 and

xIAP inhibitor, LCL161, found that the drug was curative in mouse models of multiple

myeloma5. Response was associated with an innate immune signature, and in fact was

independent of direct cytotoxic effects of the drug on the tumor.”

Did this result from confusion in the field, where these agents were first developed to

induce apoptosis and then discovered to stimulate IL-12?

Most of the early cIAP inhibitor literature focused on their pro-apoptotic effects in cell

lines. For example, the publication characterizing the discovery of GDC-0152 focused

solely on this mechanism2. Many clinical trials were initiated with this mechanism in

mind (e.g. NCT02098161 for LCL161). Shortly after, it became increasingly recognized

that cIAP inhibitors had immunomodulatory effects, and further research clarified that

activation of non-canonical NFkB signaling mediates this effect.

A more specific term than ‘Spatial Considerations’ for the title of the final section may

help avoid some confusion. Prior to this, sections were focused on extracellular

biologics or intracellular small molecules, leading me to believe spatial considerations

was referring to cellular location of the drug targets at first. Something like “Localizing

IL-12 Production” or “Delivering Modulators of IL-12 Production” might work. An

introductory sentence could also be used to clarify (e.g. starting with “ideally, IL-12

production would be located exclusively within the tumor microenvironment…”

Thank you for the suggestion. We have renamed the section, “Localized Delivery of

IL-12 Modulators”, and have added the introductory sentence.

I’m not sure about space limitations, but a few sentences to conclude would help

round out the article rather than ending with the nanoparticle subsection.

We have added concluding remarks, as detailed below.

CONCLUDING REMARKS

It is increasingly clear that local tumoral production of IL-12 is an attractive option for

cancer immunotherapy, and has particular promise in synergizing with checkpoint

blockade. In this review, we have highlighted the non-canonical NFkB pathway as one

source of targets that could be pharmacologically modulated for IL-12 production. At

the time of writing, therapeutics targeting this pathway include agonists of TNFSFRs as

well inhibitors of cIAP. Clinical trials are ongoing to evaluate these therapeutics but

more research is needed to clarify how these drugs work in man. In parallel, it is

important to further study non-canonical NFkB signaling to identify new promising

approaches, as well as analyze whether any existing drugs have effects on this pathway.

Finally, advances in localized delivery of drugs to the tumor microenvironment will be

pertinent for IL-12 therapeutics, as it can aid efforts in mitigating immunotoxicity.

Page 2 – A reference on Coley’s toxins may be helpful. For a general audience, it’s

not immediately obvious while IL-12 would be the operative cytokine from injecting dead

bacteria.

We have included a reference. In response to a point brought up by reviewer 2, we

have also included a discussion of how bacteria can trigger IL-12 production (see

below).

Please increase the font size in Fig. 1B. The labels (particularly for intracellular

proteins) are hard to read at 100% magnification

The font size has been increased.

Referee: 2

Overall, the review is well done, authoritative, and balanced. It is likely to be of

significant interest to the field. However, the major concern is the overall premise of the

review. It is not fully established that IL-12 transcription is directly associated with

noncanonical NF-kB signaling as presented in this review. In general, noncanonical NFkB signaling is associated with a narrow range of chemokine transcription. In the review,

it is presented as if IL-12 is only generated through the noncanonical pathway. However,

the most common mechanism of IL-12 generation is actually driven from canonical

signaling, c-REL also plays a significant role. This must be covered significantly better in

the review, including references that IL-12 is associated with RelB/p52..

Thank you for the comments. The reviewer is absolutely correct that IL-12 can be

induced via multiple other pathways as well, including canonical NFkB signaling as well

as IRF (interferon-regulatory-factor) signaling. Canonical signaling can be activated via

many upstream pathways, including TLR2/4/7/8 (Toll-like receptors 2, 4, 7, or 8), STING

(Stimulator of Interferon Genes), and RIG-I (Retinoic acid-inducible gene I).

However, we focused our discussion on IL-12 through non-canonical NFkB signaling for

multiple reasons. First, non-canonical NFkB signaling is arguably much less explored

than the aforementioned ones, so we felt a review on this topic would be more novel.

Next, one of the key motivators in studying of IL-12 for immunotherapy is that it

synergized with checkpoint blocked 9. This study found that the IL-12 generated in

tumor associated dendritic cells was via the non-canonical NFkB pathway, as indicated

by concomitant upregulation of NIK. Lastly, we felt that inclusion of the other pathways

would make the scope of our review too broad.

In response to the reviewer’s points, we have clarified the text to emphasize that while

our focus is on IL-12 from activated non-canonical NFkB signaling, IL-12 can also be

produced to other pathways as well. We have added a section discussing other

pathways, pasted below, but we have kept this section comparatively brief in

accordance with our views listed above.

We also propose changing the title of our review to “The Chemical Biology of IL-12

Production via the Non-Canonical NFkB Pathway”, to make it more clear that our review

focuses on non-canonical NFkB signaling.

CANONICAL NFKB AND OTHER IL-12 SIGNALING PATHWAYS

While the primary focus in this review has been on the non-canonical NFkB pathway, it

is important to note that IL-12 can also be produced in response to other stimuli as well.

Signaling through the canonical NFkB pathway or via the interferon-regulatory-factor-3/7

(IRF3/7) pathways can promote IL-12 secretion10–12. Canonical NFkB signaling and

IRF3/7 signaling are both activated by upstream pathways. Examples include the Tolllike-receptor (TLR), STING, and RIG-I pathways13–15. These pathways are all similar in

that they are triggered by pathogen associated molecular patterns (PAMPs), including

bacterial lipopolysaccharide or viral nucleic acid. Each pathway is initiated when a

specific receptor binds to a PAMP; activation then funnels into canonical NFkB and/or

IRF3/7 signaling, which promote IL-12 secretion.

The canonical NFkB pathway in particular, shares many of the same nodes as the noncanonical pathway. In brief, for the canonical NFkB pathway, an IKK kinase

phosphorylates IkB, leading to its degradation by the proteasome. Degradation of the

IkB protein allows translocation of a RelA/p50 dimer to the nucleus where it turns on a

transcriptional response. In contrast to non-canonical NFkB signaling, canonical

pathway responds quickly to stimuli and is transient. For more information on this

signaling pathway, we refer the reader elsewhere11, 12.

Therapeutics acting on the above pathways include PAMPs themselves or synthetic

mimetics. For example, the Baccillus Calmette-Guerin (BCG) vaccine, which contains

attenuated bacteria, is used in bladder cancer16. Poly-IC, a synthetic dsRNA mimetic

that binds RIG-I and TLR3, is being pursued in several cancer immunotherapy

strategies17, 18, and is also in several clinical trials, including one which it is being used

in combination with a CD40 agonist (NCT01008527). Synthetic small molecule agonists

have also been developed, as such therapeutics have improved pharmacokinetic

properties compared to PAMPs, which are large, and often charged. Imiquimod and

resiquimod are imidaquozinolines that binds TLR7 and TLR819–21. There is substantial

interest in developing more improved therapeutics on these pathways, which have

potential to potently induce IL-12.

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13-Jul-2020

Dear Dr Weissleder:

Manuscript ID: CB-REV-02-2020-000022.R1
TITLE: THE CHEMICAL BIOLOGY OF IL-12 PRODUCTION

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

Reviewer 2

The authors have addressed my major concerns with this review. It is substantially improved over the original submission.

Reviewer 1

The authors have adequately addressed my concerns.