松田豊（Yutaka Matsuda)-ADC Chemist-

Living in LALA land? Forty years of attenuating Fc effector functions The Fc region of antibodies is vital for most of their physiological functions, many of which are engaged through binding to a range of Fc receptors. However, these same interactions are not always h...

Living in LALA land? Forty years of attenuating Fc effector functions
onlinelibrary.wiley.com/doi/10.1111/...

7. AG Mutation:
A330L and G331S mutations decrease binding to FcγRIIIa and FcγRIIb in the IgG1 Fc region.

These mutations play a crucial role in modulating antibody function for therapeutic applications.

5. TM Mutation:
T256E and T307E mutations affect glycosylation in the Fc region, reducing Fcγ receptor binding.

6. E333A Mutation:
E333A reduces Fcγ receptor binding, thereby suppressing antibody effector functions.

4. N297A Mutation:
The N297A mutation alters the glycosylation site in the Fc region, preventing glycan attachment. This reduces Fcγ receptor binding and ADCC activity. N297G has a similar effect.

3. LED Mutation:
A variant of LALA that includes L234A, L235A, and D265A, providing even stronger suppression of Fcγ receptor binding and effector functions.

2. LALAPG Mutation:
A combination of L234A, L235A, D265A, P238S, and G239D mutations. These modifications further reduce Fcγ receptor and C1q binding, effectively suppressing effector functions.

1. LALA Mutation:
The most well-known mutation, involving L234A and L235A. It reduces binding to Fcγ receptors, thereby suppressing effector functions such as ADCC and CDC. This mutation has already been applied in multiple therapeutic antibodies.

Several well-known mutations are used to silence the Fc region. Below is a summary of representative mutations.

Among the 756 antibodies containing an Fc domain:
- 339 consist of an IgG2 or IgG4 Fc domain or a mutated IgG1 Fc domain, leading to Fc silencing.
- Multispecific antibodies are more likely to be silenced, with 51 out of 67 being silenced when limited to those containing an Fc domain.

As a result, 57 antibody formats and 90 constant region variants were categorized. The discussion on Fc is particularly interesting, as follows:

Out of 804 antibodies, 48 completely lack the Fc domain, likely aiming for Fc silencing, reduced half-life, or improved tumor penetration.

Systematic analysis of the varied designs of 819 therapeutic antibodies and Fc fusion proteins assigned international nonproprietary names By combining and cross-checking data from the World Health Organization’s lists of international nonproprietary names with three other databases, we assembled a dataset of amino acid sequences of 8...

This review analyzes therapeutic mAbs and Fc-fusion proteins with International Nonproprietary Names (INN) by collecting and examining data from the WHO INN list and three other databases.
www.tandfonline.com/doi/full/10....

Fc-engineered antibodies with immune effector functions completely abolished Elimination of the binding of immunoglobulin Fc to Fc gamma receptors (FcγR) is highly desirable for the avoidance of unwanted inflammatory responses to therapeutic antibodies and fusion proteins. Man...

The L234S/L235T/G236R mutation can achieve nearly complete Fc silencing and has been reported to exhibit lower affinity compared to LALA, LALAPG, and aglycosyl variants. (PLOS ONE)
journals.plos.org/plosone/arti...

Late-Stage Desulfurization Enables Rapid and Efficient Solid-Phase Synthesis of Cathepsin-Cleavable Linkers for Antibody–Drug Conjugates The synthesis of linker-payloads is a critical step in developing antibody-drug conjugates (ADCs), a rapidly advancing therapeutic approach in oncology. The conventional method for synthesizing cathep...

A **Bioconjugate Chem** paper presents a solid-phase synthesis of Val-Ala-PAB-MMAE, simplifying high-potency compound handling. Using a disulfide-tethered resin and alanine-based elongation, the method eliminates purification steps, enhancing efficiency and safety. (pubs.acs.org/doi/10.1021/...)

PEGylated therapeutics in the clinic The covalent attachment of polyethylene glycol (PEG) to therapeutic agents, termed PEGylation, is a well-established and clinically proven drug delivery approach to improve the pharmacokinetics and p....

PEGylation improves drug solubility, extends circulation, and reduces immunogenicity. This review summarizes PEGylation strategies, clinical applications, and impacts. Open access.
aiche.onlinelibrary.wiley.com/doi/10.1002/...

Taurine, a Naturally Occurring Amino Acid, as a Physical Stability Enhancer of Different Monoclonal Antibodies - The AAPS Journal Degradation of therapeutic monoclonal antibodies (mAbs) is a major concern as it affects efficacy, shelf-life, and safety of the product. Taurine, a naturally occurring amino acid, is investigated in ...

Taurine Stabilizer
link.springer.com/article/10.1...

Comparison of Cyclic and Linear PEG Conjugates Bioconjugation of polymers to proteins is a method to impart improved stability and pharmacokinetic properties to biologic systems. However, the precise effects of polymer architecture on the resultin...

Another open access article on PEG (Bioconjugate Chem). Cyclic and linear PEG were introduced into proteins (using lysozyme as a model) and compared. The main finding: cyclic PEG has a smaller hydrodynamic radius and lower viscosity.
pubs.acs.org/doi/full/10....

Strategies for UF/DF-Based Impurity Removal in the Post-conjugation Purification of Antibody–Drug Conjugates Antibody–drug conjugates (ADCs) are increasingly prevalent as investigational and marketed treatments for a variety of cancers and other diseases. The structures of most ADCs comprise a small-molecule component (the drug-linker) chemically conjugated to a monoclonal antibody, and this hybrid construct presents a number of challenges for Chemistry, Manufacturing, and Controls (CMC) development. A Small Molecule Considerations for ADC Development Working Group (WG) has been established within the IQ Consortium to serve as a forum for biopharmaceutical industry companies to discuss development strategies for ADCs, with a focus on the drug-linker portion. The preceding paper from the WG presented results from a benchmarking survey of IQ member companies on a number of topics relating to drug-linker development. One of the key findings was that ultrafiltration/diafiltration (UF/DF) is used by all responding companies for purification of the ADC following the conjugation step and is a critical operation for control of small-molecule impurities in the ADC drug substance. UF/DF is well established in monoclonal antibody processing, but to date, there are relatively few literature reports detailing its application to ADCs. To help address this gap, this manuscript presents results and analysis from a more focused survey of IQ member companies on the application of UF/DF for post-conjugation purification of ADCs. Insight is provided into practical considerations such as common operating parameters, relative efficiency of removal of different types of impurities, technical challenges, and application of emerging technologies. In addition, recommendations are offered on where to start when developing a UF/DF process for a new ADC. The overall goals are to provide an overview of current industry practices for UF/DF purification of ADCs and to spur further communication and innovation in this area.

This paper covers UF/DF in ADC purification, including key parameters, impurity removal, challenges, and new technologies. It’s a great resource on industry standards and a valuable read since ADC process studies are limited.
pubs.acs.org/doi/10.1021/...

Design and Evaluation of ZD06519, a Novel Camptothecin Payload for Antibody Drug Conjugates Abstract. In recent years, the field of antibody drug conjugates (ADC) has seen a resurgence, largely driven by the clinical benefit observed in patients treated with ADCs incorporating camptothecin-b...

A comprehensive paper from Zymeworks on the SAR of their Topo1 inhibitor and the discovery of lead compound ZD06519, including ADC synthesis. It's an excellent resource for understanding CPT-based compounds.
aacrjournals.org/mct/article/...

Thank you for sharing this! I absolutely love your beautiful cartoon of the mushroom and amanitin-ADCs—such a creative and insightful depiction!

The "NIH BioArt Source" is a public domain illustration site that offers bio-related illustrations for free use. A great resource for anyone in need of high-quality visuals for scientific presentations, publications, or educational materials!
bioart.niaid.nih.gov

Effects of arginine in therapeutic protein formulations: a decade review and perspectives Statement of Significance: Beneficial and detrimental effects of arginine in therapeutic protein formulations are reviewed and perspectives are provided.

Interestingly, guanidine often fails where arginine succeeds, emphasizing the unique importance of arginine in these applications.
academic.oup.com/abt/article/...

An open-access review summarizing the role of arginine in protein formulation. It highlights arginine's ability to enhance solubility, reduce buffer viscosity (critically important in TFF processes), and solve various formulation challenges.

Trace levels of the CHO host cell protease cathepsin D caused particle formation in a monoclonal antibody product Chinese hamster ovary (CHO) cells are often used to produce therapeutic monoclonal antibodies (mAbs). CHO cells express many host cell proteins (HCPs) required for their growth. Interactions of HCPs ...

aiche.onlinelibrary.wiley.com/doi/10.1002/...

If Cathepsin D remains as a CHO host cell protein (HCP), it can cleave antibodies even at trace levels. Since Protein A purification may not completely remove it, optimizing the washing steps and identifying residual HCPs are crucial for ensuring product quality.

Click Chemistry: Reaction Rates and Their Suitability for Biomedical Applications Click chemistry has become a commonly used synthetic method due to the simplicity, efficiency, and high selectivity of this class of chemical reactions. Since their initial discovery, further click chemistry methods have been identified and added to the toolbox of click chemistry reactions for biomedical applications. However, selecting the most suitable reaction for a specific application is often challenging, as multiple factors must be considered, including selectivity, reactivity, biocompatibility, and stability. Thus, this review provides an overview of the benefits and limitations of well-established click chemistry reactions with a particular focus on the importance of considering reaction rates, an often overlooked criterion with little available guidance. The importance of understanding each click chemistry reaction beyond simply the reaction speed is discussed comprehensively with reference to recent biomedical research which utilized click chemistry. This review aims to provide a practical resource for researchers to guide the selection of click chemistry classes for different biomedical applications.

This is a review on click chemistry, published in *Bioconjugate Chemistry* (Open Access).
pubs.acs.org/doi/10.1021/...

A particularly valuable feature is its comprehensive coverage of reaction rates, making it a highly useful reference for reaction development introductions and various applications. It's a must-read for chemists working in reaction development or bioconjugation, and broadly recommended.

The impact of E3 ligase choice on PROTAC effectiveness in protein kinase degradation Proteolysis targeting chimera (PROTACs) provide a novel therapeutic approach that is revolutionizing drug discovery. The success of PROTACs largely de…

A comprehensive review summarizing E3 ligases used in PROTACs. It's intriguing to explore which of these ligases could be adaptable for DACs (linkerable ones), offering potential directions for further development.
www.sciencedirect.com/science/arti...

Reversible Chemical Modification of Antibody Effector Function Mitigates Unwanted Systemic Immune Activation Antibody effector functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) are mediated through the interaction of the antibody Fc region with Fcγ receptors present on immune cells. Several approaches have been used to modulate antibody Fc-Fcγ interactions with the goal of driving an effective antitumor immune response, including Fc point mutations and glycan modifications. However, robust antibody-Fcγ engagement and immune cell binding of Fc-enhanced antibodies in the periphery can lead to the unwanted induction of systemic cytokine release and other dose-limiting infusion-related reactions. Creating a balance between effective engagement of Fcγ receptors that can induce antitumor activity without incurring systemic immune activation is an ongoing challenge in the field of antibody and immuno-oncology therapeutics. Herein, we describe a method for the reversible chemical modulation of antibody-Fcγ interactions using simple poly(ethylene glycol) (PEG) linkers conjugated to antibody interchain disulfides with maleimide attachments. This method enables dosing of a therapeutic with muted Fcγ engagement that is restored in vivo in a time-dependent manner. The technology was applied to an effector function enhanced agonist CD40 antibody, SEA-CD40, and experiments demonstrate significant reductions in Fc-induced immune activation in vitro and in mice and nonhuman primates despite showing retained efficacy and improved pharmacokinetics compared to the parent antibody. We foresee that this simple, modular system can be rapidly applied to antibodies that suffer from systemic immune activation due to peripheral FcγR binding immediately upon infusion.

pubs.acs.org/doi/10.1021/...

This study addresses the dual nature of Fcγ interactions in antibodies—providing benefits like ADCC activity but also drawbacks like Fcγ-mediated uptake. The authors successfully reduced interchain disulfide bonds in the antibody and introduced PEG to suppress Fcγ interactions.

Payload-Binding Fab Fragments Increase the Therapeutic Index of MMAE Antibody–Drug Conjugates Abstract. Monomethyl auristatin E (MMAE) is a potent tubulin inhibitor that is used as the payload for four FDA-approved antibody–drug conjugates (ADC). Deconjugated MMAE readily diffuses into untarge...

Using anti-payload antibodies to mitigate the toxicity of ADCs. Published in Molecular Cancer Therapeutics
aacrjournals.org/mct/article/...