Currently clinically used ADCs rely in most cases on efficient internalization and intracellular lysosomal transport for bio-cleavage of the linker and thus release of the drug. In addition, the cleaved drug must escape from the lysosome to achieve its therapeutic effect. However, not all tumor antigens ensure efficient ADC treatment as described above, especially in solid tumors. Furthermore, currently internalized Antibody-drug Conjugates are also sensitive to acquired tumor resistance mechanisms.

Research Status of ADC Therapies Extracellular ADC cleavage in the tumor microenvironment (TME) could serve as a valuable alternative to traditional ADCs. In this case, once released, the drug can passively diffuse into the tumor mass, penetrate and kill adjacent antigen-negative tumor cells, thereby maximizing the bystander effect. Specific targeting of the TME in solid tumors can be achieved through the use of weak or non-internalized antigens (e.g. CAIX, VEGFR, cadherins), components of the extracellular matrix (e.g. splice variants of fibronectin and Tenascin-C, fibrin, collagen type IV, αvβ3 integrin) or proteins secreted by tumor cells in the TME (e.g. VEGF). All of these would be excellent targets for ADC therapy if the drug could be selectively released from the TME.

Mechanism of ADC Click Chemistry Currently, significant therapeutic efficacy of bio-cleavable ADCs based on disulfide linkers or peptide linkers against non-internalizing tumor targets has been found. Extracellular cleavage of linkers containing disulfide bonds is thought to be due to the release of reducing agents (such as glutathione) by dying cells leading to more cell death and thus release of more reducing agents. However, extracellular bio-cleavage is less ubiquitous and less efficient than intracellular bio-cleavage. Researchers have thus recently explored ways in which ADC linkers can be chemically triggered (Fig. 1).

In this approach, the ADC binds to the extracellular tumor target, and the unbound ADC is cleared from the blood. Then, an intravenously injected exogenous chemical probe (activator) selectively and rapidly reacts with the ADC linker to release the drug, thereby bypassing the dependence on tumor biology for drug release. Due to the high antigen density of ADCs and the fast pharmacokinetics typical of activators, in vivo reagent concentrations and reaction times are low, this approach requires the use of fast and highly selective reactions such as bio-orthogonal.

Classical Click-Release Reaction-IEDDA Pyridazine Elimination Orthogonal Cleavage Reaction The fastest bio-orthogonal chemical reaction is the inverse electron demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine, which is the basis for the orthogonal cleavage reaction of pyridazine elimination. It introduces a carbamate-linked payload at the TCO allylic position. In the first step (click), TCO reacts rapidly and selectively with a 1,2,4,5-tetrazine derivative (activator) to generate several DHP tautomers. The 1,4-DHP isomer then rapidly releases the amine-containing payload and carbon dioxide in a second electron-cascade elimination step.

Tetrazine-triggered pyridazine elimination is a robust and broadly applicable chemical reaction with good TCO linker and tetrazine activator stability coupled with rapid and high-yield release in vitro and in vivo. One disadvantage of using TCOs embedded in the cleavable linker of ADCs compared to typical TCOs for bioconjugation is the reduced reactivity towards tetrazine due to steric hindrance from the allyl substituent. To further enhance click responsiveness and potentially reduce in vivo dose levels, the researchers developed a novel click-release strategy. This strategy is still based on the robust pyridazine elimination reaction between TCO and tetrazine, but uses TCO as the activator and embeds the tetrazine into the linker (Fig. 3). In such a system, derivatives of sTCO can be used to release payloads with three orders of magnitude greater click reactivity relative to tetrazine-triggered pyridazine elimination.

Application of Click Chemistry ADC The first click-cleavable ADC was based on the CC49 mAb with TCO-Dox (DAR ~2), targeting the non-internalized tumor antigen TAG72 (Fig. 4). The ADC is very stable, showing similar PK properties to the parental CC49 antibody in tumor-bearing mice. However, the low click-binding response of activators limits their further applications.

The second-generation click-cleavable ADC selected TAG72-targeted Diabody with a shorter half-life. This Diabody-TCO-linked MMAE payload was attached to an engineered Cys residue via a PEG linker, resulting in a click-cleavable ADC (tc-ADC) (Fig. 5). It has high tumor uptake and very low levels in blood and other non-target tissues. Pharmacokinetic studies in mice showed that ADC was almost completely cleared from blood when there was a two-day interval between ADC and activator administration. The activators are small molecules containing a high-release 3,6-dialkyltetrazine motif and a clearance-modulating PEG11-DOTA. In addition, studies have shown that when the dosage is 0.33 mmol/kg, it can completely react with tumor-bound TCO.

In addition to pyridazine elimination reactions, some other bio-orthogonal cleavage reactions were explored for ADC applications. Recently, Chen’s group set out to establish a metal-based bio-orthogonal cleavage reaction into a linker cleavage reaction. The speed of these cleavage reactions can be as fast as pyridazine elimination reactions, or even faster. To this end, the group conducted a systematic survey of 24 different species containing copper, palladium, ruthenium, nickel, cobalt, and iron. Among all the compounds tested, the copper(I) complexes had a positive effect on the presence of propargyloxyacetyl or propargyl functional linkers were shown to cleave efficiently and rapidly and release amine- or phenol-containing payloads.

Liu’s group developed a bio-orthogonal cleavage reaction derived from an organic deprotection reaction rather than a click ligation reaction. The group synthesized an aromatic linker comprising an ortho-carbamoylmethylene silyl-phenol ether system, and removed the silicon group using fluoride or a fluoride transfer agent, followed by electronic rearrangement, resulting in an amino-containing payload and release of carbon dioxide. In PBS, 90% of the payload was released within 24 h in the presence of trifluoroboron phenylalanine (Phe-BF3). In the presence of hydrogen peroxide, glutathione, and cysteine, only little payload release was observed. Mechanistically, Phe-BF3 mimics natural phenylalanine and is actively taken up by tumor cells through LAT-1.

Based on this, the research group developed a linker containing tert-butyldimethylsilyl-functionalized phenol (TBSO). This linker links trastuzumab to MMAE, resulting in a chemically cleavable internalized ADC (Fig. 7). In a proof-of-concept study in HER2-positive gastric cancer xenografts (BGC823), significant free MMAE was present in the tumors of mice injected with ADC and activator, demonstrating efficient release of MMAE.

Click chemistry has great application potential in ADCs. Click-chemistry ADCs can activate them independent of tumor biology, thus allowing expansion to non-endocytic cancer targets. In addition, click chemistry ADCs can achieve stronger bystander effects by selecting appropriate payloads. In heterogeneous solid tumors, extracellular cleavage may provide more uniform drug distribution, thereby increasing therapeutic efficacy. However, the clinical application of click chemistry puts high demands on the safety and sufficient in vivo stability and reactivity of the reagents. So far, only the pyridazine elimination reaction developed by Tagworks has been shown to have clinical potential. However, it is believed that with the accumulation and maturity of the technology, the new generation of click chemistry ADC is expected to be more widely used in the patient population.

Source: https://adc.bocsci.com/resource/application-of-click-chemistry-in-the-next-generation-of-novel-antibody-drug-conjugation-adcs.html

Before the emergence of bioorthogonal chemistry, fluorescent protein labeling was the most popular and extensive method at the protein level. The gene-edited protein, combined with the green fluorescent protein or its variant to track the structure and function of the labeled object. However, this method has an obvious drawback: the marker proteins are often bulky, which can easily affect the labeled biomolecules and alter the experimental results.

Bertozzi's bioorthogonal chemistry approach is much more convenient. Two small molecules interact in a living cell environment to form a covalent bond of a single type of reaction. Then, the combination acts like a "bridge": one of the two small molecules is integrated into the target sugar molecule by a metabolic marker, while the other binds to a chemical marker. In this process, the integration of small molecules and targets takes advantage of existing biochemical reactions in the organism without affecting the reactions themselves. In 2005, Bertozzi's team detailed the experimental strategy in a paper published in Nature Chemical Biology.

The initial bioorthogonal chemistry mainly referred to coupling reactions. In 2000, Bertozzi's team developed the Staudinger ligation, namely the azide-phosphine conjugation, based on the Staudinger reduction, and applied it to the chemical modification of cell surface. Since then, a variety of bioorthogonal reactions developed rapidly.

In 2002, organic chemists Karl Barry Sharpless (USA) and Morten P. Meldal (Denmark) together with their teams independently reported a copper-catalyzed azide-alkyne cycloaddition (CuAAC). The reactions followed the selectivity principle of click chemistry and had the advantages of high yield and wide applications, which was very suitable for bioorthogonal chemistry. The two small functional groups used in the CuAAC reaction were easy to bind to target molecules while not affecting natural biochemical reactions.

Despite the obvious features, the CuAAC reaction had a defect in that copper catalysts reacting in cells will produce toxic reactive oxygen species (ROS). Therefore, Bertozzi's team found that an organic compound called cyclooctyne could react with azide to perform a strain-promoted [3+2] azide-alkyne cycloaddition (SPAAC) under physiological conditions. In a few months, the team built the reactive molecule and selectively modified it first on proteins and then on cells. The results were satisfactory and non-toxic. Later in 2004, the team published a paper in the Journal of the American Chemical Society demonstrating the application potential of copper-free click chemical. Subsequently, the team further improved the cyclooctyne reagent for faster reaction kinetics, and successfully imaged the membrane-associated glycans in developing living zebrafish to identify and track the expression patterns of glycans.

While Bertozzi's team was making leaps and bounds in bioorthogonal chemistry, other teams were actively exploring new reaction strategies. In 2008, Joseph Fox's team at the University of Delaware published a paper in the Journal of the American Chemical Society, first proposed the cycloaddition reaction of S-tetrazine and trans-Cyclooctene (TCO) without catalyst under physiological conditions. The tetrazine ligation has been one of the fastest known bioorthogonal reactions so far. Fox said, "with the most powerful TCO, the reaction rate exceeds 3,300,000 M-1 S-1".

In 2010, Chen's team from the School of Chemistry and Molecular Engineering at Peking University, first put forward the concept of bioorthogonal cleavage reaction. Before that, bioorthogonal reactions often followed the bond reaction, but Chen's team broke the bonds in bioorthogonal reactions to activate proteins and even deliver drugs.

As a result, Chen's team developed a unique protein in which a key residue at the active site was "caged" by a protective functional group. The bioorthogonal cleavage reaction was then used to "uncaging", removing the protection and activating key residue. The team achieved in situ deprotection of protein side chains (conversion of proc-lysine to natural lysine) in living cells. The advantage of this strategy was that unnatural amino acids were directly inserted into the catalytic active site of the target protein enzyme, making it completely "off". In the process of activation, a small number of proteins in the "on" state were enough to study its function and related biological functions. In 2019, in the JACS, the team proposed another bioorthogonal cleavage reaction of "dual-substituted propargyl (dsPra) or propargyloxycarbonyl (dsProc)/copper complexes", extending from terminal uncaging to intramolecular cleavage. Based on this, the team designed and prepared controlled-release drug conjugated antibody (cleavable ADCs) based on amino and phenol hydroxyl groups, which successfully achieved selective killing of cancer cells.

Summary Bioorthogonal chemistry was innovatively pioneered by Bertozzi and over the last 20 years, scientists have been expanding its implications and implications. Although bioorthogonal reactions have more or fewer problems in terms of efficiency, rate, substrate stability, accessibility, biological compatibility, mutual orthogonality, and ease of operation, the technique has grown independently and now become one of the core fields of biochemistry from the supplement to the coupling reaction. Bioorthogonal chemistry not only promotes the development of glycobiology but also the potential scope of drug targeting is likely to be further expanded in the future. For example:

In situ synthesis of pharmaceutical agents: Bioorthogonal chemistry may facilitate the assembly of drugs from smaller precursors. By creating drugs in the time needed, they are more effective and less toxic and the range of drug interventions could also be expanded.

Glycan labeling: Scientists have used folic acid ligands to generate lipid nanoparticles containing azide-labeled galactosamine. Tumor membranes incorporated azide-functionalized dibenzocyclooctyne, triggering an immune response when tumor cells were exposed to human sera.

Click to Release: This method uses bioorthogonal chemistry to control the timing and location of drug release to produce drugs with selective toxicity to target cells.

Source: https://bioconjugation.bocsci.com/resources/the-2022-nobel-prize-in-chemistry-bioorthogonal-chemistry.html

What are antibody-drug conjugates (ADCs)? Antibody-drug Conjugate (ADC) is a chemotherapeutic drug with strong cytotoxicity, which has both the powerful lethality of small molecule drugs and the high targeting of pure monoclonal antibodies, so it has become a hot issue in the research and development of tumor targeted therapy.

What are the technologies used in ADC? The production technologies of ADC mainly includes monoclonal antibody preparation, linker preparation, small molecule drug preparation, ADC conjugation, purification and finished product assembly.

What are the mechanism of action of ADCs? Mechanistically, an ADC acts by binding to the target antigen on the cell surface, followed by its internalization via antigen-mediated endocytosis, trafficking into the lysosome, and the release of the payload through the proteolytic degradation of the antibody moiety and/or cleavage of the linker.

What are the principles of ADC development? The rationale for developing an ADC is that linking a cytotoxic agent to a tumor-targeting antibody will enable its selective targeting to cancer cells, leading to their eradication while sparing cells in normal tissues.

What are the manufacturing steps of ADCs? ADCs are relatively complicated products which require careful assembly. In some cases, preparation of antibody and drug--linker and the final conjugation are done at different sites by specialist companies. ADC manufacturing is a multistep process that can be divided into three distinct stages: cGMP production of the antibody, cGMP synthesis of the drug-linker complex, and conjugation to form the ADC.

What are drug antibody ratio (DAR)? DAR is defined as the number of small drug molecules attached to an antibody, which can be obtained by testing methods such as HPLC-MS. A clear DAR value is essential for the later stages of ADC drug development. ADC drugs are taken up by tumor cells in limited quantities during circulation in vivo, so a higher DAR is generally beneficial for increased potency.

Are ADCs immunotherapy? Antibody drug conjugates (ADCs) are a form of targeted immunotherapy. They are composed of three components: a monoclonal antibody (mAb) and a cytotoxic payload made from a chemotherapy agent, which are connected together using a chemical linker.

What are the next generation ADCs? The key of next generation ADC drugs is site-specific binding that can ensure a clear DAR. Through the specific binding of small molecule drugs and monoclonal antibodies, the stability and pharmacokinetics of the drugs are significantly improved, and relative drug activity and binding activity to cells at lower antigen levels are enhanced as well. In addition, antibody optimization, linkers, and small molecule drugs can significantly improve the therapeutic effect of ADC drugs.

How many antibody drug conjugates are approved? After decades of research and troubleshooting , appreciable technological advances and an improved mechanistic understanding of ADC activity has culminated in the FDA approval of 14 ADCs, each providing demonstrable therapeutic benefit to cancer patients.

How to design a good ADC drug? To design a good ADC drug, choosing the right combinations of antibody backbone, linker chemistry (conjugation technology), and payloads is crucial. Each of these components must be adjusted and optimized to obtain the correct balance between effectiveness and safety. For example, the ideal affinity of an antibody depends on the density of the antigen on the target; bystander activity may or may not be required; the optimal DAR depends on the effectiveness of the payload.

Source: https://adc.bocsci.com/resource/frequently-asked-questions.html

More recently, the potential therapeutic benefits of deuterium have been exploited to alter the pharmacokinetic properties of drug candidates and their associated metabolic profiles. Interest in deuterium drugs has increased the need to enable precise deuteration, the installation of deuterium atoms in controlled molecular locations. In particular, the incorporation of deuterium (2H) atoms at the chiral centers of compounds is becoming an increasingly desired target for pharmaceutical and analytical chemists.

BOC Sciences has extensive experience in deuterium, especially in the field of asymmetric synthesis. Our DCL™ technology platform greatly expands the toolbox available to organic chemists, providing near-perfect selectivity and mild operating conditions for the synthesis of deuterated drugs.

Our Capabilities Due to the increasing importance of NADH-dependent reductases in chiral fine chemical synthesis, advanced enzyme evolution techniques have greatly expanded the scope of biocatalysis. However, the deuterated reducing agents currently used, such as deuterated ethanol, deuterated isopropanol, deuterated glucose or deuterated formate, are not only expensive to prepare, but also difficult to recover, which increases the cost and complexity of the process, severely limited their application in deuterated chemistry.

BOC Sciences has developed a method for biocatalytic deuteration reactions that installs deuterium while generating chiral centers with high chemical, stereo, and isotopic selectivity. The method cites the inherent safety, cost-effectiveness, availability, and ease of handling of 2H2O as a solvent and isotopic source, combined with an appropriate reductase to reduce a series of C=O, C=N, and C=C bonds. All under mild reaction conditions and 2H can be incorporated directly from the heavy water reaction medium.

In conclusion, our best asymmetric deuteration technology can be used in many drug molecules, such as tasalidine, cevimeline and solifenacin. We can also customize services according to customer needs.

Intellectual Property Protection BOC Sciences has always regarded intellectual property as the most valuable asset of the company and its customers. We have signed non-disclosure agreements with customers and employees before the project starts, and provide synthetic route design and synthesis services in strict accordance with the terms of the non-disclosure agreement, striving to provide customers with target compounds in the shortest time possible.

For more info: https://deuterated.bocsci.com/applications/asymmetric-deuteration-based-on-dcl.html

This analysis method requires five representative random production batches to be analyzed for the identification and quantification of all components present in the substance as manufactured. The 5-batch analysis is performed to account for at least 98% of the composition of a technical grade active ingredient, and this characterization data is necessary to support TGAI registration. The study requires the analysis of five representative random production batches for the presence of significant impurities (≥ 0.1% w/w) and borderline impurities (0.1% x ≥ 0.06%) to generate the product specification for global regulatory needs.

Application of 5-Batch Analysis Identify and quantify impurities within the production batches of substances Provide crucial information that is used to generate an agrochemical product specification A core requirement for any registration of the technical material

Five-Batch Analysis Process BOC Sciences works with our clients to advise the best 5-batch testing approach for a new arochemical product or for an existing product that has undergone a change in the manufacturing process or manufacturing site.

Selection of five manufacturing batches Our experts follow many guidelines when performing analytical methods to provide information on toxicological relevant impurities. The five randomly selected batches must be at a point in the manufacturing process, after which no further chemical reactions are intended (aimed at producing or purifying the substance). Analytical method development A comprehensive study of the manufacturing process help identify the nature of the impurities and select the appropriate analytical technique for analysis, therefore, before initiating the method development, our analytical chemists fully consider the country of submission of the dossier for the five-batch analysis. Once the analytical technique is identified, we then optimize a series of parameters such as the polarity or phase of the column, column temperature, etc. The combined set of optimized parameters will be defined as a suitable method for the next step of preliminary screening of five batches. Prior to method validation, the active ingredients of all known and unknown impurities are characterized by using 1H NMR, Mass and FTIR to demonstrate the respective structures. Method validation For the method validation process, the parameters to be covered are linearity, LOQ, LOQ, precision and accuracy. After the validation of the method, the methodology is used to analyze the target five batches. Results Analysis We provide the reports generated for 2D or 3D analysis including impurity identification and synthesis, active ingredient and impurity content determination. 2D and 3D Techniques of Chromatography BOC Sciences supports a set of analytical methods or transfer and validates existing methodology using a wide range of equipment:

Scanning of 5 batches using suitable techniques like GC-MS / LC-MS / LC-MS-MS / 3D HPLC / Ion Chromatography Identification and characterizations of actives and impurities by GC-MS / LC-MS / LC-MS-MS / 1H-NMR / 13C NMR Method validation in compliance with SANCO/OPPTS guideline parameters Quantitative analysis of all components by GC/HPLC/GC-MS/ LC-MS

BOC Sciences Advantages Highly specialized technical and analytical services for the worldwide registration and regulatory compliance of agrochemicals Robust analytical testing programs that span from research and product development through the production process to the final product Relies on broad industrial experience, ensuring that all of our work meets the high standards expected by our clients Our regulatory experts, toxicology consultants, scientists and inspectors will ensure that you receive the maximum levels of guidance, testing and inspection you need.

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