From the journal RSC Chemical Biology Peer review history

06-Oct-2021

Dear Dr Chudasama:

Manuscript ID: CB-REV-04-2021-000082
TITLE: Enabling the next steps in cancer immunotherapy: from antibody-based bispecifics to multispecifics, with an evolving role for bioconjugation chemistry

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Associate Editor, RSC Chemical Biology

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

Reviewer 1

The review paper by Thoreau and Chudasama gives an interesting perspective of bi- multi-specific Abs in Immunotherapy. This review actually does not intend to compare the possibilities offered by molecular biology and bioconjugation based methods in order to to produce multi-specify Abs, nor does it discuss in depth the pros and cons of the different Ab format, but it rather describes in a clear manner the various therapeutic targets that could potently be combined in a lego-like chemical approach. In that sense the chapter describing chemical approaches presented at the end of the manuscript take all its dimension.
I have personally learned a lot upon reading this manuscript which brought me some hint on the choice of possible target combinations to be chose for exemplification of novel chemical-based protein linkage process. I must however admit that I am not an expert in cancer biology and cannot judge the exhaustivity or the relevance of the immunooncology regulatory processes and the actors described.
As a summary I believe that this review will be of use and interest for many chemists and biologist who wants to get at a glance a synthetic presentation of cancer immunotherapy from the perspective of understanding the different potent targets and to get a general overview of the chemical approaches allowing to combine different Abs and Ab formats into a single entity addressing them.Hence I would recommend publication of this review in RSC Chemical Biology.

Reviewer 2

This review focuses on key developments of bi-specific and multi-specific cancer immunotherapies by providing an overview of the clinical landscape of immune checkpoint modulators and immune cell engagers, and their advantages and disadvantages (e.g., toxicity and resistance). The review is comprehensible, succinct, well-articulated, and thus suitable for the RSC Chemical Biology journal and its general readership.

Comments to the author:

1. The authors noted that “the trend in clinical trials is currently shifting to the use of anti-PD-1/PD-L1 antibodies in combination therapies”, which is fascinating. Please comment on why this trend exist? why are other immunotherapies (e.g., anti-CTLA-4 antibodies) not as prevalent? Is it primarily driven by regulatory T cell depletion and/or other factors (e.g., on-target, off-site toxicity)?

2. Conditional activation of these biologics may also enhance selectivity and reduce systemic toxicity. These biomolecules are activated by specific environmental conditions in the tumor microenvironment (e.g., enzymes or pH) and some have been in the clinic (e.g., by companies such as CytoMx or BioAlta). Please comment on these new developments in the review.

3. Other chemistries can also be utilized to introduce clickable handles such as chemo-enzymatic or unnatural amino acids [see works by Peter Schultz (e.g., Proc Natl Acad Sci, 2012; 2 :16101-6) or ADCs developed by Ambrx]. Please comment on these advancements in the review.

4. Please cite the following reviews that highlight site-specific, chemical and enzymatic methods to conjugate native proteins/antibody that have been in the clinic and readily be used to construct multi-specific antibody conjugates (e.g., via click chemistry).

Antibody Therapeutics, 2020; 3, 4: 271–284, DOI: 10.1093/abt/tbaa027.
Chembiochem, 2019; 20: 2729–37, DOI: 10.1002/cbic.201900178.

5. Please cite seminal work by Sharpless (Angew Chem, 2001; 40: 2004–21) on click chemistry and Bertozzi (J Am Chem Soc, 2004; 126: 15046–7) on copper-free click chemistry, which lay the groundwork for subsequent click chemistries.

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

Response to reviewers’ comments
Referee 1
We would like to thank referee 1 for their very positive comments. There were no corrections or
amendments requested, as such we have not made any changes in response to this referee.
Referee 2
We would like to thank referee 2 for their very positive review and kind comments, we really
appreciate their comment on the review being “comprehensible”, “succinct” and “well-articulated”.
Their individual corrections have been addressed in full below:
1. The authors noted that “the trend in clinical trials is currently shifting to the use of anti-PD-1/PD-L1
antibodies in combination therapies”, which is fascinating. Please comment on why this trend
exist? why are other immunotherapies (e.g., anti-CTLA-4 antibodies) not as prevalent? Is it
primarily driven by regulatory T cell depletion and/or other factors (e.g., on-target, off-site toxicity)?
We thank the referee for this comment. This sentence is from a paragraph that covers
PD1/PD-L1 axis immunotherapy. The sentence was probably not clear enough, but it was
trying to point out the fact that anti-PD1 and anti-PD-L1 antibodies are currently more
employed in the context of combination therapies (with other immunotherapy, targeted therapy
or chemotherapy) rather than as a single therapy. Thus, it was not particularly opposing the
PD1/PD-L1 immunotherapies to other immune checkpoint therapies. For a clearer
comprehension, however, the original sentence “the trend in clinical trials is currently shifting
to the use of anti-PD-1/PD-L1 antibodies in combination therapies” has been replaced by
“However, if approved anti-PD-1/PD-L1 antibodies are mainly used as monotherapy, the trend
in clinical trials is currently shifting to their use in the context of combination therapies (76% of
active trials in 2019 were testing them as combination regimen in 2019).”
The questioning about “why this trend exists” can also be applied to the observation of a shift
from monotherapies to combination therapies. This question is addressed in a subsequent
paragraph (“III.4. The limitations of Monoclonal antibodies). But the following sentence has
also been added to provide further explanation: “An explanation for this trend might be their
lack of cancer-targeting moiety, potentially resulting in on-target, off-cancer activation of T
cells and the related toxicity through cytokine release syndrome. Apparition of resistance
mechanism against anti-PD-L1/PD-1 antibodies is another argument in the favour of
combination therapies”
2. Conditional activation of these biologics may also enhance selectivity and reduce systemic
toxicity. These biomolecules are activated by specific environmental conditions in the tumor
microenvironment (e.g., enzymes or pH) and some have been in the clinic (e.g., by companies
such as CytoMx or BioAlta). Please comment on these new developments in the review.
We thank the referee for this suggestion. Indeed, probodies (or masked antibodies) represent
a very exciting alternative to alleviate some of the current breaks of immunotherapies and
improve the therapeutic window. The fact that some have been to the clinic is an additional
good argument to include these developments in the review, even though the initial purpose of
the review was to focus on the improvement of immunotherapies through the combination of
various effectors (multispecific species). To address the useful comment, we have added a
small description of the probody concept, referred to a recent review in the area and
mentioned relevant clinical trials:
“To tackle the on-target, off-tumour toxicity, a monoclonal antibody can be converted to a
probody format. This strategy confers a conditional activation to the antibody by masking its
binding site with a connected substrate that will be released only in specific conditions found in
the tumour micro-environment, such as low pH or high protease activity. This concept offering
better selectivity and safety has been reviewed elsewhere,98 and proved promising since
probody formats of anti-CTLA-4 activated by protease cleavage (BMS-986249, CytomX) or
reversibly activated by acidic pH (BA3071, BioAtla) have notably been to the clinic.99
”
3. Other chemistries can also be utilized to introduce clickable handles such as chemo-enzymatic or
unnatural amino acids [see works by Peter Schultz (e.g., Proc Natl Acad Sci, 2012; 2 :16101-6) or
ADCs developed by Ambrx]. Please comment on these advancements in the review.
We thank the referee for this suggestion. Indeed, in order to combine proteins with each other
to produce a MuTICEM format, either bioengineering or chemical ligations can be used. The
purpose of the review was, however, to review the chemical methods, which require
introduction of click handles on native (or digested) proteins through site-selective organic and
bioorganic reactions. These methods are the focus of the review and we hope we were clear
enough about this choice (“organic/bioconjugation chemistry might partially alleviate these
obstacles, by allowing the production of MuTICEMs directly from native (or facilely engineered
from native) proteins”; “Starting from native proteins, the most recent procedures to chemically
generate a multispecific antibody format rely on digesting parent antibodies and isolate the
binding motifs of interest (Fab fragments and eventually Fc fragment) to subsequently
assemble them directly with each other or on a chemical platform by using fast, orthogonal
and chemo- and/or site-selective ligation reactions. Smaller units such as scFvs or small
binding sites can also be produced or purchased and chemically modified in order to include
them in a multispecific antibody format”).
However, it is perfectly true that clickable handles can be introduced via chemo-enzymatic
methods or by unnatural amino acids. As such, to stay in line with the more “native protein and
organic chemistry-oriented focus” of our review but address the point, and if the Editor agrees,
we will also briefly comment on chemo-enzymatic and unnatural amino acids methods. We do
agree that they are valuable methods to mention and thank the referee again for this
suggestion, but we also hope the Editor/referee understand why we will not go into this in
more detail due to the scope of the review. The review did already contain some references to
chemo-enzymatic methods (“However, native proteins generally have to be chemically or
enzymatically modified to introduce click chemistry functionalities” ; “a wide range of strategies
such as […] enzymatic and chemo-enzymatic modification of antibody glycans,
224
”), but we
have added the following sentences to give more detail and further address the referee’s
comment:
“However, native proteins generally have to be modified to introduce click chemistry
functionalities. This can be done through protein engineering that allows the site-selective
introduction of unnatural amino acids in the protein sequence, providing bio-orthogonal
handles such as azide, alkyne or tetrazine to the non-native generated protein.217–219
”
“…enzymatic and chemo-enzymatic modification of either antibody glycans,
224 or antibody
amino-acid residues (with enzymes such as sortase A or transglutaminase, which allow to
site-selectively connect peptide-containing substrates on the antibody, or the formylglycine
generating enzyme which introduces an aldehyde group exploited in subsequent coupling
reactions),
225
”
4. Please cite the following reviews that highlight site-specific, chemical and enzymatic methods to
conjugate native proteins/antibody that have been in the clinic and readily be used to construct
multi-specific antibody conjugates (e.g., via click chemistry).
Antibody Therapeutics, 2020; 3, 4: 271–284, DOI: 10.1093/abt/tbaa027.
Chembiochem, 2019; 20: 2729–37, DOI: 10.1002/cbic.201900178.
We thank the referee for this suggestion. These references have been added to the relevant
paragraph.
5. Please cite seminal work by Sharpless (Angew Chem, 2001; 40: 2004–21) on click chemistry and
Bertozzi (J Am Chem Soc, 2004; 126: 15046–7) on copper-free click chemistry, which lay the
groundwork for subsequent click chemistries.
We thank the reviewer for this suggestion. These references have been added to the relevant
paragraph.

22-Oct-2021

Dear Dr Chudasama:

Manuscript ID: CB-REV-04-2021-000082.R1
TITLE: Enabling the next steps in cancer immunotherapy: from antibody-based bispecifics to multispecifics, with an evolving role for bioconjugation chemistry

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 addressed all the comments. Well done!