<?xml version="1.0" encoding="UTF-8"?><rss version="2.0" xmlns:content="http://purl.org/rss/1.0/modules/content/" xmlns:wfw="http://wellformedweb.org/CommentAPI/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:atom="http://www.w3.org/2005/Atom" xmlns:sy="http://purl.org/rss/1.0/modules/syndication/" xmlns:slash="http://purl.org/rss/1.0/modules/slash/" > <channel> <title>ADCs – Creative Biolabs ADC Blog</title> <atom:link href="https://www.creative-biolabs.com/blog/adc/tag/adcs/feed/" rel="self" type="application/rss+xml" /> <link>https://www.creative-biolabs.com/blog/adc</link> <description>Antibody-Drug Conjugate (ADC) - Creative Biolabs</description> <lastBuildDate>Mon, 18 Mar 2024 03:00:49 +0000</lastBuildDate> <language>en-US</language> <sy:updatePeriod> hourly </sy:updatePeriod> <sy:updateFrequency> 1 </sy:updateFrequency> <generator>https://wordpress.org/?v=6.3.1</generator> <image> <url>https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2020/02/cropped-favicon-32x32.png</url> <title>ADCs – Creative Biolabs ADC Blog</title> <link>https://www.creative-biolabs.com/blog/adc</link> <width>32</width> <height>32</height> </image> <item> <title>Several Ways to Sort Out Sulfhydryl-Conjugated ADCs</title> <link>https://www.creative-biolabs.com/blog/adc/several-ways-to-sort-out-sulfhydryl-conjugated-adcs/</link> <dc:creator><![CDATA[bioadc]]></dc:creator> <pubDate>Mon, 18 Mar 2024 03:00:49 +0000</pubDate> <category><![CDATA[Review]]></category> <category><![CDATA[ADCs]]></category> <category><![CDATA[Sulfhydryl-Conjugated]]></category> <guid isPermaLink="false">https://www.creative-biolabs.com/blog/adc/?p=642</guid> <description><![CDATA[The popularity of sulfhydryl groups in ADC preparation can be traced back to three factors: (a) thiols are the most nucleophilic amino acid side-chain functional group (much more nucleophilic than amines); (b)<a class="moretag" href="https://www.creative-biolabs.com/blog/adc/several-ways-to-sort-out-sulfhydryl-conjugated-adcs/">Read More...</a>]]></description> <content:encoded><![CDATA[<p><span style="font-size: 15px;">The popularity of sulfhydryl groups in ADC preparation can be traced back to three factors: (a) thiols are the most nucleophilic amino acid side-chain functional group (much more nucleophilic than amines); (b) the relatively small number of cysteines (8 for IgG1 and IgG4) in the antibody suitable for conjugation; and (c) the fact that stability of the antibody is not critical, allowing for relatively controlled customization of the DAR; Interchain cysteines are present in the hinge region and connect the heavy chain (HC) and light chain (LC), appearing to favor “hiding” hydrophobic <span style="color: #0000ff;"><strong><a style="color: #0000ff;" href="https://www.creative-biolabs.com/adc/classify-adc-toxins-6.htm">payloads</a></strong></span> after conjugation. It must be noted that a reduction step is always required prior to conjugation to release free thiols.</span></p> <p><span style="font-size: 15px;">The vast majority of thiol-conjugated ADCs are maleimide-based (see Figure 1). Alkylation occurs between the primary interstrand cysteine (see Figures 1A and B) and the engineered cysteine (see Figure 1C). The exception is ADCs prepared by nucleophilic aromatic substitution. Other chemistries, such as disulfide exchange, are also being developed for some preclinical assets.</span></p> <p><span style="font-size: 15px;"><a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-1.jpg"><img decoding="async" fetchpriority="high" class="aligncenter wp-image-643" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-1.jpg" alt="" width="334" height="268" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-1.jpg 617w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-1-300x240.jpg 300w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-1-600x480.jpg 600w" sizes="(max-width: 334px) 100vw, 334px" /></a></span></p> <p style="text-align: center;"><span style="font-size: 12px;">Figure 1. Groups based on maleimide chemically coupled ADC thiols<sup>1</sup></span></p> <h6><span style="font-size: 15px;"><strong>Reaction with Maleimide</strong></span></h6> <p><span style="font-size: 15px;">Maleimide is susceptible to alkylation by the nucleophilic Michael addition reaction of the thiol group to form a stable thioether bond (Figure 2). In the pH range of 6.5–7.5, the maleimide reaction is specific for thiols and reacts 1000 times faster with amines than at pH 7.0, so complete transformation can be achieved with only a small amount of excess reagent. The main drawback of the maleimide alkylation reaction is the reversibility of the Michael addition reaction, the rate of which has a lot to do with the pKa of the specific cysteine residue linked. This reverse Michael reaction may lead to the premature release of the linker payload in the bloodstream and may lead to adverse effects, which is also an important driver for the development of more stable maleimide variants.</span></p> <p><span style="font-size: 15px;"> <a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-2.jpg"><img decoding="async" class="aligncenter size-full wp-image-644" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-2.jpg" alt="" width="720" height="113" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-2.jpg 720w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-2-300x47.jpg 300w" sizes="(max-width: 720px) 100vw, 720px" /></a></span></p> <p style="text-align: center;"><span style="font-size: 12px;">Figure 2. Alkylation of various maleimide reagents with cysteine<sup>1</sup></span></p> <p><span style="font-size: 15px;">As mentioned above, cysteines in monoclonal antibodies require a reduction step to release the thiol group prior to conjugation. As a result, a number of free thiol groups (up to 8 for IgG1 and IgG4) can be released by carefully optimizing the amount of reducing agent (typically TCEP or DTT) prior to the addition of maleimide-functionalized payloads. ADCs prepared by conjugation with cysteine include Adcetriss, Polivys, Padcevs, Enhertu, Blenrep, Trodelvys, Zynlontat, and others. Due to the lack of site specificity, these ADCs are typically produced as random mixtures with an average DAR of 2.3–4. However, there are two notable exceptions, Enhertu and Trodelvys, which, due to the relatively low toxicity of the camptothecin toxins carried, are produced by (near) quantitative conjugation of all interchain cysteines, with DARs of 7.7 and 7.6, respectively, and therefore can also be considered so-called site-directed coupling.</span></p> <p><span style="font-size: 15px;">It is important to note that maleimide (mc) linkers are used in the vast majority of cases, although some ADCs use more electron-poor β-aminoethyl or ethylene glycol spacers. Some phenyl-substituted maleimides have also been reported to be more stable and are currently in preclinical development.</span></p> <p><span style="font-size: 15px;">In 2008, it was reported that site-specific conjugation of the cytotoxic <span style="color: #0000ff;"><strong><a style="color: #0000ff;" href="https://www.creative-biolabs.com/adc/classify-drug-linker-complexes-8.htm">linker payload</a></strong></span> to monoclonal antibodies with specific cysteine may improve the therapeutic index of ADCs compared to randomized ADCs. Since then, a variety of site-specific ADCs have entered the clinic, such as SGNCD19B, CD123A, and CD352A (all HC-S239C mutations), RG7861/DSTA4637S (LC-V205C mutations), IMGN632 (S442C mutations), and BAT8003 (A114C mutations), and even bicysteine-based ADCs such as PF-06804103 (LC-K183C+). HC-K290C)。 In addition, two complementary technologies have been developed, namely cysteine insertion (e.g., MEDI2228 (HC-i239C)) and fusion of HC-terminal peptide tags (e.g., ALT-P7 (C-terminal ACGHAACGHA)). Unfortunately, the use of cysteine-engineered antibodies does not guarantee clinical success, as some of these assets have been abandoned (e.g., BAT8003 and all three Seagen ADCs mentioned above).</span></p> <p><span style="font-size: 15px;">The inverse Michael reaction can be inhibited by hydrolyzing the original thioether-succinimide adduct to provide a maleamide acid derivative. This hydrolysis can be achieved by prolonged treatment of ADCs at pH 9. Such conditions may apparently result in additional post-translational modifications of the antibody, such as deamidation or pyroglutamic acid formation. In response to this problem, Seagen has developed an effective solution by adding aminomethyl maleimide, which is based on 2,3-diaminopropionic acid (DPR technology), to the α position of the N-alkyl chain (see figure below). The amino group induces spontaneous autohydrolysis of the succinimide conjugate, thereby blocking the inverse Michael reaction. The ADC product SGN-CD48A (now abandoned) was the first to feature DPR technology, as was SGN-CD228A.</span></p> <p><span style="font-size: 15px;"> <a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-3.jpg"><img decoding="async" class="aligncenter size-full wp-image-645" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-3.jpg" alt="" width="634" height="277" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-3.jpg 634w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-3-300x131.jpg 300w" sizes="(max-width: 634px) 100vw, 634px" /></a></span></p> <p style="text-align: center;"><span style="font-size: 12px;">Figure 3. Cysteine is alkylated with a diaminopropionic acid (DPR)-based aminomethyl maleimide reagent and then autohydrolyzed to obtain maleic acid conjugates.<sup>1</sup></span></p> <p><span style="font-size: 15px;">Our drug-linker complex products:</span></p> <table style="border-style: solid; border-color: #050505; width: 102.814%;"> <tbody> <tr> <td style="border-style: solid; border-color: #050505; text-align: center; width: 17.5497%;" width="94"><span style="font-size: 15px;">Cat.No.</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 43.543%;" width="182"><span style="font-size: 15px;">Product name</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 19.0397%;" width="138"><span style="font-size: 15px;">CAS NO</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 22.5166%;" width="138"><span style="font-size: 15px;">Price</span></td> </tr> <tr> <td style="border-style: solid; border-color: #050505; text-align: center; width: 17.5497%;" width="94"><span style="font-size: 15px;">ADC-S-024</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 43.543%;" width="182"><span style="color: #0000ff;"><strong><span style="font-size: 15px;"><a style="color: #0000ff;" href="https://www.creative-biolabs.com/adc/dbm-mmaf-4658.htm">DBM-MMAF</a></span></strong></span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 19.0397%;" width="138"><span style="font-size: 15px;">–</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 22.5166%;" width="138"><span style="color: #0000ff;"><strong><span style="font-size: 15px;"><a style="color: #0000ff;" href="https://www.creative-biolabs.com/adc/form/order">Inquiry</a></span></strong></span></td> </tr> <tr> <td style="border-style: solid; border-color: #050505; text-align: center; width: 17.5497%;" width="94"><span style="font-size: 15px;">ADC-S-025</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 43.543%;" width="182"><span style="color: #0000ff;"><strong><span style="font-size: 15px;"><a style="color: #0000ff;" href="https://www.creative-biolabs.com/adc/mc-betaglucuronide-mmae-1-4659.htm">MC-betaglucuronide-MMAE-1</a></span></strong></span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 19.0397%;" width="138"><span style="font-size: 15px;">–</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 22.5166%;" width="138"><strong><span style="font-size: 15px; color: #0000ff;"><a style="color: #0000ff;" href="https://www.creative-biolabs.com/adc/form/order">Inquiry</a></span></strong></td> </tr> <tr> <td style="border-style: solid; border-color: #050505; text-align: center; width: 17.5497%;" width="94"><span style="font-size: 15px;">ADC-S-026</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 43.543%;" width="182"><span style="font-size: 15px;"><a href="https://www.creative-biolabs.com/adc/mc-betaglucuronide-mmae-2-4660.htm"><span style="color: #0000ff;"><strong>MC-betaglucuronide-MMAE-2</strong></span></a></span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 19.0397%;" width="138"><span style="font-size: 15px;">–</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 22.5166%;" width="138"><span style="color: #0000ff;"><strong><span style="font-size: 15px;"><a style="color: #0000ff;" href="https://www.creative-biolabs.com/adc/form/order">Inquiry</a></span></strong></span></td> </tr> <tr> <td style="border-style: solid; border-color: #050505; text-align: center; width: 17.5497%;" width="94"><span style="font-size: 15px;">ADC-S-027</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 43.543%;" width="182"><span style="color: #0000ff;"><strong><span style="font-size: 15px;"><a style="color: #0000ff;" href="https://www.creative-biolabs.com/adc/sgd-1910-mc-val-ala-pbd-4661.htm">SGD-1910 (Mc-Val-Ala-PBD)</a></span></strong></span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 19.0397%;" width="138"><span style="font-size: 15px;">1342820-51-2</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 22.5166%;" width="138"><strong><span style="font-size: 15px; color: #0000ff;"><a style="color: #0000ff;" href="https://www.creative-biolabs.com/adc/form/order">Inquiry</a></span></strong></td> </tr> <tr> <td style="border-style: solid; border-color: #050505; text-align: center; width: 17.5497%;" width="94"><span style="font-size: 15px;">ADC-S-028</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 43.543%;" width="182"><span style="color: #0000ff;"><strong><span style="font-size: 15px;"><a style="color: #0000ff;" href="https://www.creative-biolabs.com/adc/mc-pbd-4662.htm">MC-PBD</a></span></strong></span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 19.0397%;" width="138"><span style="font-size: 15px;">–</span></td> <td style="border-style: solid; border-color: #050505; text-align: center; width: 22.5166%;" width="138"><strong><span style="font-size: 15px; color: #0000ff;"><a style="color: #0000ff;" href="https://www.creative-biolabs.com/adc/form/order">Inquiry</a></span></strong></td> </tr> </tbody> </table> <h6><span style="font-size: 15px;"><strong>Other Cysteine Alkylation Reagents</strong></span></h6> <p><span style="font-size: 15px;">For example, the high reactivity of α-haloacetamide reagents is well known, and in the case of 2-iodoacetamide, it is an indisputable choice for blocking free cysteine in proteins or protein mixtures (e.g., cell lysates). Importantly, the resulting thioether bond is completely irreversible and therefore superior to thiol-maleimide ether in terms of stability. In fact, Alley et al. reported in 2008 that the use of a-bromoacetamide hexanoyl linkers could provide a thioether ADC with excellent plasma stability, with no measurable systemic drug release within 2 weeks of mouse dosing and a 25% increase in intratumoral drug exposure within 7 days. However, despite the differences in stability between bromoacetamide and maleimide-based ADCs, there were no significant differences in their potency, activity, or toxicity properties. The reason for this surprising lack of distinction is likely to be that for this particular maleimide-based ADC, the half-life of the ADC’s linker and the half-life of the ADC clearance are similar. Since ADC concentrations decrease over time, prolonging the junction half-life beyond the pharmacokinetic half-life is unlikely to have a significant effect on drug exposure.</span></p> <p><span style="font-size: 15px;">Based on the discovery that methylsulfonylbenzothiazole (MSBT) is a selective thiol blocker, Barbas et al. developed a series of aromatic methylsulfonone reagents for the alkylation of thiol-containing proteins, mainly oxazinazole and benzothiazole. Similarly, methylsulfonate reacts with thiols through a nucleophilic aromatic substitution mechanism (see figure below), a concept that is applied in SKB264/BT001035.</span></p> <p><span style="font-size: 15px;">Researchers at Glythera have reported highly stable thioether ADCs obtained by incubating free cysteine thiols with 4-vinylpyridine, a technique known as PermaLinks. The resulting ADC is significantly stable and is not easily degraded.</span></p> <p><span style="font-size: 15px;">The last technology worth mentioning is the phosphoamide coupling technology developed at the University of Berlin, which is still in its early stages but is currently being commercially used by Tubulis. Studies have shown that aryl phosphonate with electron-deficient triple bonds has excellent cysteine-selective reactivity, and the resulting (Z)-vinyl sulfide has excellent stability compared to traditional maleimide linkages.</span></p> <p><span style="font-size: 15px;"> <a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-4.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-646" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-4.jpg" alt="" width="660" height="305" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-4.jpg 660w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-4-300x139.jpg 300w" sizes="(max-width: 660px) 100vw, 660px" /></a></span></p> <p style="text-align: center;"><span style="font-size: 12px;">Figure 4. Alternative techniques for cysteine alkylation<sup>1</sup></span></p> <h6><span style="font-size: 15px;"><strong>Alkylation/Crosslinking with Bismaleimide or Bismaleamide Acid</strong></span></h6> <p><span style="font-size: 15px;">The ability to alkylate maleimide has also been applied to various cross-linked bismaleimide reagents. Each reagent captures two free cysteine thiol groups, resulting in the formation of DAR4 ADCs after the complete reduction of all eight interchain disulfide bonds. It is important to note that although mixtures are generated due to interchain cross-linking between thiol groups (e.g., from HC-226C to HC-229C), the resulting ADCs have a narrow DAR distribution. NewBio’s NBT828 uses a bismaleimide crosslinker. Bisuccinimide conjugates may also be hydrolyzed to carbamate derivatives upon the nucleophilic addition of a thiol group, providing a conjugate that cannot undergo reverse Michael degradation.</span></p> <p><span style="font-size: 15px;"> <a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-5.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-647" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-5.jpg" alt="" width="660" height="195" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-5.jpg 660w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-5-300x89.jpg 300w" sizes="(max-width: 660px) 100vw, 660px" /></a></span></p> <p style="text-align: center;"><span style="font-size: 12px;">Figure 5. Bismaleimide reagent cross-linked cysteine<sup>1</sup></span></p> <p><span style="font-size: 15px;">Researchers from AstraZeneca reported rebridging cysteine-engineered antibodies with bismaleimide-modified payloads (PBDs) to obtain DAR1 ADCs.</span></p> <h6><span style="font-size: 15px;"><strong>THIObridget Conjugation</strong></span></h6> <p><span style="font-size: 15px;">A novel coupling reagent originally reported by Del Rosario et al. and Wilbur et al., taking advantage of the high nucleophilicity and susceptibility to Michael addition reactions of free thiols, was further developed by Poly-Therics (now known as Abzena, with THIObridget technology) for ADCs. This rebridging technique enhances the stability of the reducing antibody by using the thiol formed by religating the dithiol reaction group, more effectively maintaining the conformational integrity of the final conjugate. Close-to-the-molecular-scale dithiol reactive groups are well suited for this purpose, as they can be seamlessly integrated into the protein structure without disrupting subunit interactions. The a-toluenesulfonyl methyl-a,b-unsaturated ketone reagent was applied to the reducing antibody, which was generated <em>in situ</em> from a precursor of bis-a,a-toluenesulfonyl methyl ketone, triggering a series of reactions including Michael addition, b-elimination toluenesulfonic acid, and subsequent Michael addition to produce a stable conjugated product.</span></p> <p><span style="font-size: 15px;"> <a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-6.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-648" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-6.jpg" alt="" width="816" height="323" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-6.jpg 816w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-6-300x119.jpg 300w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-6-768x304.jpg 768w" sizes="(max-width: 816px) 100vw, 816px" /></a></span></p> <p style="text-align: center;"><span style="font-size: 12px;">Figure 6. THIObridget technology<sup>1</sup></span></p> <h6><span style="font-size: 15px;"><strong>Alkylation/Conjugation by Bisbromomethyl Aromatic Reagents</strong></span></h6> <p><span style="font-size: 15px;">Concortis (now Sorrento) developed a third coupling method for DAR4 ADCs, called C-lockt technology. The C-lockt technique combines the nucleophilic nature of thiols with the propensity of the benzyl bromide population to undergo nucleophilic SN2 substitution reactions. Thus, by reducing the interchain disulfide bonds, which then react with a bisbromomethyl aromatic reagent (e.g., bisbromomethylquinoxaline), an irreversible cross-linking reaction occurs to form a thioether. Interestingly, it was reported that the clinical ADC (CD38-ADC) prepared using the C-lockt technique was not DAR4 but DAR3 (averaged).</span></p> <p><span style="font-size: 15px;"> <a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-7.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-649" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-7.jpg" alt="" width="788" height="139" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-7.jpg 788w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-7-300x53.jpg 300w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-7-768x135.jpg 768w" sizes="(max-width: 788px) 100vw, 788px" /></a></span></p> <p style="text-align: center;"><span style="font-size: 12px;">Figure 7. Cysteine crosslinking by diaalkylation of bisbromomethylquinoxaline<sup>1</sup></span></p> <h6><span style="font-size: 15px;"><strong>Use Dibromopyridine Dione for Conjugation</strong></span></h6> <p><span style="font-size: 15px;">Inspired by the use of dibromomaleimide groups to re-bridging reduced disulfide bonds, Chudasama et al. developed a rebridging coupling technique based on the pyridine dione (PD) core structure. Unlike maleimide derivatives, pyridine dione rings are more stable and do not open the rings by hydrolysis, resulting in a more homogeneous binder. The authors prepared monobromo-PD (MBPD) and dibromo-PD (DBPD) derivatives and demonstrated that both react specifically with thiol groups at pH 8.0, even in the presence of amines. DBPD reactive groups have been used to develop a range of crosslinking and modification reagents that incorporate other reaction sites, such as alkynes for click chemistry and even derivatives containing reducing agents, for one-step antibody reduction and conjugation. The use of DBPD thiols to rebridge chemistry with antibody binding has been applied to create highly fixed-point ADCs, such as trastuzumab for DAR4 MMAE.</span></p> <p><span style="font-size: 15px;"> <a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-8.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-650" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-8.jpg" alt="" width="660" height="319" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-8.jpg 660w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-8-300x145.jpg 300w" sizes="(max-width: 660px) 100vw, 660px" /></a></span></p> <p style="text-align: center;"><span style="font-size: 12px;">Figure 8. Cross-linking of antibody thiol groups with dibromoazadione derivatives<sup>1</sup></span></p> <h6><span style="font-size: 15px;"><strong>Disulfide bond coupling</strong></span></h6> <p><span style="font-size: 15px;">Disulfide bonds have been used since the early days of ADCs, such as in MLN2704, CMD-401, BIWI-1, and IMGN242. A linker design containing a disulfide bond is located somewhere between the cytotoxic payload and the antibody and can facilitate a reversible linkage that can be cleaved by glutathione reduction within tumor cells to release the toxin. However, the reality is that premature severing of circulating disulfide bonds often occurs due to the presence of free cysteine, which results in the release of the payload before the tumor cells enter. The addition of a protective group, such as a methyl or dimethyl group, to the carbon adjacent to the disulfide bond has been used to increase stability in circulation by creating steric hindrance around the disulfide bond, but in many cases, it also reduces the rate of toxin release from tumor cells. In addition, even though the increased steric hindrance improves the stability of disulfide bonds, these ADCs are not as effective as conjugating drugs to engineered cysteine residues at optimal locations in the antibody sequence.</span></p> <p><span style="font-size: 15px;">For the first time, Pillow et al. directly conjugated a small molecule drug to an engineered cysteine antibody at different locations to generate a sterically unhindered disulfide bond-Mertansine ADC. The results were compared with ADCs with different sterically hindered disulfide bonds conjugated to lysine residues. The authors identified the engineered cysteine located at LC-K149C as the most stable binding site for drug attachments containing disulfide bonds. ADCs created using the LC-K149C locus show excellent circulatory stability while allowing potent glutathione (GSH)-mediated cleavage within tumor cells and thus have the highest potency against mouse human non-Hodgkin lymphoma xenografts of all mutants evaluated.</span></p> <p><span style="font-size: 15px;"> <a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-9.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-651" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-9.jpg" alt="" width="800" height="139" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-9.jpg 800w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-9-300x52.jpg 300w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2024/03/Ablog-202403-9-768x133.jpg 768w" sizes="(max-width: 800px) 100vw, 800px" /></a></span></p> <p style="text-align: center;"><span style="font-size: 12px;">Figure 9. Disulfide bonds are produced by engineering cysteines to prepare antibody conjugates<sup>1</sup></span></p> <p><span style="font-size: 15px;">Reference:</span></p> <ol> <li><span style="font-size: 15px;">van Delft, Floris, and John M. Lambert, eds. Chemical Linkers in Antibody-Drug Conjugates (ADCs). Vol. 81. Royal Society of Chemistry, 2021.</span></li> </ol> ]]></content:encoded> </item> <item> <title>How to Optimize the Safety Profile of ADC Drugs?</title> <link>https://www.creative-biolabs.com/blog/adc/how-to-optimize-the-safety-profile-of-adc-drugs/</link> <dc:creator><![CDATA[bioadc]]></dc:creator> <pubDate>Fri, 01 Sep 2023 06:20:15 +0000</pubDate> <category><![CDATA[Uncategorized]]></category> <category><![CDATA[ADC Safety]]></category> <category><![CDATA[ADCs]]></category> <guid isPermaLink="false">https://www.creative-biolabs.com/blog/adc/?p=576</guid> <description><![CDATA[Optimization Strategies for Dose Adjustment Due to the dose-dependent nature of adverse reactions in ADC therapies, optimizing safety can be achieved through dose adjustment strategies. These include setting dose limits, maximum treatment<a class="moretag" href="https://www.creative-biolabs.com/blog/adc/how-to-optimize-the-safety-profile-of-adc-drugs/">Read More...</a>]]></description> <content:encoded><![CDATA[<h6><span style="font-size: 15px;"><strong>Optimization Strategies for Dose Adjustment</strong></span></h6> <p><span style="font-size: 15px;">Due to the dose-dependent nature of adverse reactions in ADC therapies, optimizing safety can be achieved through dose adjustment strategies. These include setting dose limits, maximum treatment duration, optimizing dosing frequency, dose adjustment guided by clinical response, and randomized dose confirmation studies.</span></p> <p><span style="font-size: 15px;">The strategy of dose limits involves determining the maximum dose based on patient weight and pharmacokinetics to avoid excessive drug exposure and unnecessary adverse effects. For example, in populations weighing ≥ 100 kg using Enfortumab Vedotin, three treatment-related deaths prompted the use of a dose limit of 125 mg to control dosing.</span></p> <p><span style="font-size: 15px;">The maximum treatment duration strategy controls drug exposure by limiting the treatment duration to reduce chronic and potential permanent adverse events. In treating relapsed or refractory diffuse large B-cell lymphoma with Polatuzumab Vedotin, an 8-course regimen increased ≥ Grade 2 peripheral neuropathy by over 50% compared to a 6-course regimen. While this strategy has not been verified for solid tumor ADC applications, it remains a consideration for control.</span></p> <p><span style="font-size: 15px;">The dosing frequency optimization strategy adjusts peak blood concentration (Cmax) under the same cumulative dose to mitigate Cmax-driven adverse events. Gemtuzumab Ozogamicin’s original dosing regimen of 9 mg/m² q2w led to high rates of hepatotoxicity and veno-occlusive disease, resulting in its withdrawal in 2010. A revised regimen with doses of 3 mg/m² on days 1, 4, and 7 of each induction cycle reduced adverse reactions and gained approval in 2017.</span></p> <p><span style="font-size: 15px;">Patient response-guided dose adjustment adapts doses based on individual treatment responses to maximize efficacy and minimize toxicity. In blood cancer therapy, Inotuzumab Ozogamicin’s initial dose of 1.8 mg/m² decreased to 1.6 mg/m² upon achieving complete remission, demonstrating a mature application of this strategy.</span></p> <p><span style="font-size: 15px;">Randomized dose confirmation studies prospectively evaluate multiple doses of a drug to determine the optimal dose that maximizes therapeutic index. In exploring T-DXd’s application in HER2-positive non-small cell lung cancer, a comparison of 5.4 mg/kg and 6.4 mg/kg found similar efficacy, with higher doses having more adverse reactions, leading to the determination of a 5.4 mg/kg dose. Such dose exploration studies hold valuable references for other ADC dose development.</span></p> <h6><span style="font-size: 15px;"><strong>Optimization Strategies for ADC Design</strong></span></h6> <p><span style="font-size: 15px;">Every structural innovation in <span style="color: #0000ff;"><strong><a style="color: #0000ff;" href="/adc/one-stop-adc-development-service.htm">ADC drug design</a></strong></span> can fine-tune pharmacological properties, impacting tolerability. Optimizing ADC’s structure is a fundamental approach to addressing adverse reactions.</span></p> <p><span style="font-size: 15px;">At the antibody innovation level, most ADCs currently target antigens with high expression in tumor cells and low expression in normal cells. The design concept of the next-generation antibody-drug conjugates (probody-drug conjugates, PDCs) brings more possibilities. PDCs have an antibody part that acts as a probody, shielded by a cleavable peptide in the antigen-binding region. It selectively activates and exposes the binding region in the tumor microenvironment, avoiding off-target effects and enhancing efficacy and safety. Another strategy involves developing bispecific antibodies to enhance ADC’s tumor cell targeting for reduced adverse reactions.</span></p> <p><span style="font-size: 15px;">In linker technology innovation, the current focus lies in achieving homogeneity by specifically bridging the cytotoxic payload and antibody parts, thereby improving pharmacokinetics. Additionally, adding polyethylene glycol structures to the linker enhances hydrophilicity, reducing non-specific uptake and off-target effects.</span></p> <p><span style="font-size: 15px;">At the<strong><span style="color: #0000ff;"> <a style="color: #0000ff;" href="/adc/classify-adc-toxins-6.htm">payload</a> </span></strong>innovation level, novel ADCs with the same monoclonal antibody binding two different payloads are under development. Combining multiple mechanisms of cytotoxic payloads can enhance tolerability and anti-tumor activity, even when utilizing immune-stimulating agents and tyrosine kinase inhibitors as payloads. Furthermore, introducing neutralizing antibody fragments into payloads can bind free cytotoxic payloads in the circulatory system, reducing off-target effects and improving tolerability.</span></p> <h6><span style="font-size: 15px;"><strong>Optimization Strategies for Safety Monitoring</strong></span></h6> <p><span style="font-size: 15px;">After clinical ADC use, recognizing risk factors for adverse reactions, effectively monitoring patients’ clinical presentations, and preventing, detecting, and treating adverse reactions in a timely manner are effective safety optimization strategies.</span></p> <p><span style="font-size: 15px;">On the one hand, patients’ pharmacogenomic characteristics help predict the risk of adverse reactions after ADC application. Post-analysis of the ASCENT study revealed that UGT1A1 gene polymorphism causes UGT1A1 deficiency and correlates with adverse events of SG. Prior studies showed that UGT1A128 homozygosity is associated with neutropenia induced by irinotecan and SG. While routine testing of UGT1A1 gene polymorphism is not recommended due to the low frequency (<10%), closer toxicity monitoring is advised for known UGT1A128 homozygous patients. Early inclusion of pharmacogenomic profiles in clinical research design provides security for future clinical safety prediction as newer ADCs are developed.</span></p> <p><span style="font-size: 15px;">On the other hand, early monitoring better manages adverse reactions in ADC. Wearable biosensors (WBS) that monitor patient health status are under development. These devices offer real-time, continuous, non-invasive monitoring of metrics like oxygen saturation, heart rate, and respiratory rate, facilitating early detection of adverse reactions.</span></p> ]]></content:encoded> </item> <item> <title>NEJM: ADC Drug for Advanced Breast Cancer Treatment Shows Amazing Effect</title> <link>https://www.creative-biolabs.com/blog/adc/adc-drug-for-advanced-breast-cancer-treatment-shows-amazing-effect/</link> <dc:creator><![CDATA[bioadc]]></dc:creator> <pubDate>Mon, 27 Jun 2022 08:05:44 +0000</pubDate> <category><![CDATA[Antibody-drug Conjugates Research]]></category> <category><![CDATA[ADC toxins]]></category> <category><![CDATA[ADCs]]></category> <category><![CDATA[HER2]]></category> <guid isPermaLink="false">https://www.creative-biolabs.com/blog/adc/?p=429</guid> <description><![CDATA[Breast cancer is the most common cancer among women. In 2020, breast cancer has replaced lung cancer as the number one cancer in the world. The latest global cancer burden data for<a class="moretag" href="https://www.creative-biolabs.com/blog/adc/adc-drug-for-advanced-breast-cancer-treatment-shows-amazing-effect/">Read More...</a>]]></description> <content:encoded><![CDATA[<p><span style="font-size: 15px;">Breast cancer is the most common cancer among women. In 2020, breast cancer has replaced lung cancer as the number one cancer in the world. The latest global cancer burden data for 2020 released by WHO’s International Agency for Research on Cancer (IARC) shows that there will be as many as 2.26 million new cases of breast cancer and 680,000 deaths worldwide in 2020.</span></p> <p><span style="font-size: 15px;">Breast cancer can be divided into HER2-positive breast cancer (about 20%) and HER2-negative breast cancer (about 80%), and about half of these HER2-negative breast cancer patients actually express low levels of HER2. However, the currently available HER2 targeting therapy is ineffective in patients with breast cancer with low expression of HER2 (HER2-low).</span></p> <p><span style="font-size: 15px;">On June 5th, 2022, <em>The New England Journal of Medicine (NEJM)</em> published a phase III clinical trial of the ADC drug Enhertu in patients with advanced breast cancer with low expression of HER2 (HER2-Low), led by Dr. Shanu Modi of MSKCC. The title of the thesis is: Trastuzumab Deruxtecan in Previously Treated HER2-Low Advanced Breast Cancer.</span></p> <p><span style="font-size: 15px;">The phase III clinical trial showed that Enhertu extended progression-free survival by about 50% and overall survival by 40% in advanced breast cancer patients with low HER2 expression compared with patients receiving standard chemotherapy.</span></p> <p><span style="font-size: 15px;">This is also the first time that it has been proven that drugs can target low-expressed HER2 proteins and have a therapeutic effect on cancer. About half of breast cancer patients previously thought to be HER2-negative actually have low HER2 expression (HER2-low) breast cancer, which means they can benefit from the ADC drug. This opens up new treatment possibilities for thousands of patients with advanced breast cancer.</span></p> <p><span style="font-size: 15px;">The Phase III clinical trial involved 557 patients with metastatic advanced breast cancer who had previously received first or second-line chemotherapy with low expression of HER2. The definition of low expression of HER2 was that the score of IHC analysis was 1+, or the score of IHC was 2+, but the result of in situ hybridization was negative.</span></p> <p><span style="font-size: 15px;">Enhertu (Trastuzumab Deruxtecan), an <span style="color: #0000ff;"><a style="color: #0000ff;" href="/adc/one-stop-adc-development-service.htm"><strong>antibody-drug conjugate</strong></a></span> (ADC) developed by AstraZeneca and Daiichi Sankyo, was approved by FDA in 2019 for the treatment of unresectable or metastatic HER2-positive breast cancer. This ADC drug connects HER2 monoclonal antibody Trastuzumab with topoisomerase 1 inhibitor exatecan derivative (DXd) to block HER2 protein and exert the anticancer effect of <span style="color: #0000ff;"><a style="color: #0000ff;" href="/adc/classify-adc-toxins-6.htm"><strong>chemical toxins</strong></a></span>.</span></p> <p><span style="font-size: 15px;">The patients were randomly assigned to receive ADC drug Enhertu (Trastuzumab Deruxtecan) or standard chemotherapy according to the proportion of 2:1. Of the 557 metastatic advanced breast cancer patients with low expression of HER2, 494 (88.7%) were hormone receptor positive (HR+) and 63 (11.3%) were hormone receptor negative (HR-).</span></p> <p><span style="font-size: 15px;">In the hormone receptor positive (HR+) cohort, the median progression-free survival was 10.1 months in the ADC drug Enhertu treatment group and 5.4 months in the standardized treatment group, and the overall survival time was 23.9 months and 17.5 months, respectively.</span></p> <p><span style="font-size: 15px;">In all patients, the median progression-free survival was 9.9 months in the ADC drug Enhertu group and 5.1 months in the standardized treatment group, and the overall survival time was 23.4 months and 16.8 months, respectively.</span></p> <p><span style="font-size: 15px;">52.6% of patients in the ADC drug Enhertu group had grade 3 or higher adverse events compared to 67.4% in the standardized treatment group. In addition, 12.1% of patients in the ADC drug Enhertu group developed interstitial lung disease or pneumonia associated with drug treatment, of which 3 (0.8%) died as a result.</span></p> <p><span style="font-size: 15px;">Overall, this phase III trial involving metastatic/advanced breast cancer patients with low expression of HER2 (HER2-low) showed that ADC drug Enhertu significantly prolonged progression-free survival and overall survival compared with standard chemotherapy.</span></p> ]]></content:encoded> </item> <item> <title>Analytical methods of average drug to antibody ratio (DAR) of antibody-drug conjugates</title> <link>https://www.creative-biolabs.com/blog/adc/analytical-methods-of-average-drug-to-dar-of-antibody-drug-conjugates/</link> <dc:creator><![CDATA[bioadc]]></dc:creator> <pubDate>Thu, 08 Jul 2021 03:53:01 +0000</pubDate> <category><![CDATA[Review]]></category> <category><![CDATA[ADCs]]></category> <category><![CDATA[DAR analysis]]></category> <category><![CDATA[HIC]]></category> <category><![CDATA[HILIC]]></category> <category><![CDATA[LC-MS]]></category> <category><![CDATA[Ultraviolet spectrophotometry]]></category> <guid isPermaLink="false">https://www.creative-biolabs.com/blog/adc/?p=394</guid> <description><![CDATA[Antibody-drug conjugate (ADC) is a new type of biological targeting drug for the cancer treatment, which perfectly combines the high specificity of antibody and the strong lethal power of cytotoxin. With the<a class="moretag" href="https://www.creative-biolabs.com/blog/adc/analytical-methods-of-average-drug-to-dar-of-antibody-drug-conjugates/">Read More...</a>]]></description> <content:encoded><![CDATA[<p><span style="font-size: 15px;">Antibody-drug conjugate (ADC) is a new type of biological targeting drug for the cancer treatment, which perfectly combines the high specificity of antibody and the strong lethal power of cytotoxin. With the targeting ability of antibodies, cytotoxins can be accurately delivered to the target cells, effectively increasing the drug concentration in tumor tissues, and greatly reducing drug exposure to other tissues and organs, to achieve synergism and reduce toxicity, thereby achieving “accurate treatment” at the cellular level.</span></p> <p><span style="font-size: 15px;">Drug to antibody ratio (DAR) is an important attribute that affects the efficacy and safety of ADC. Low drug loading will weaken the efficacy of ADC, while high drug loading will have a negative effect on the pharmacokinetics and <span style="color: #0000ff;"><a style="color: #0000ff;" href="/adc/adc-in-vitro-cytotoxicity-assay.htm"><strong>toxicity of ADC</strong></a></span>.</span></p> <p><span style="font-size: 15px;">At present, there are many methods to determine the <span style="color: #0000ff;"><a style="color: #0000ff;" href="/adc/dar-and-payload-distribution-analysis.htm"><strong>DAR value</strong></a></span> of ADC, and the commonly used ones are ultraviolet spectrophotometry (UV), hydrophobic interaction chromatography (HIC), liquid chromatography-mass spectrometry (LC-MS), reversed-phase high performance liquid chromatography (RP), hydrophilic interaction chromatography (HILIC), cathepsin B (Cathepsin B) digestion, and so on.</span></p> <h6>Ultraviolet spectrophotometry (UV)</h6> <p>Ultraviolet spectrophotometry is the simplest method to determine DAR based on Lambert Beer’s law.</p> <p>Three conditions must be met to adopt this method.</p> <ol> <li>Small molecular drugs have chromogenic groups in the ultraviolet/visible region.</li> <li>Small molecular drugs and antibodies show obvious and different maximum absorption values in their UV/visible spectra.</li> <li>The presence of small molecular drugs do not affect the optical absorption properties of the antibody part of the ADC sample, and vice versa.</li> </ol> <p><span style="font-size: 15px;">If these conditions are met, the ADC sample can be used as a two-component mixture, for Lambert’s law to determine the concentrations of antibodies and small molecular drugs respectively, and calculate the average DAR value accordingly.</span></p> <p><span style="font-size: 15px;">The advantage of ultraviolet spectrophotometry is simple and does not need to be separated, but the shortage is that it is not accurate enough, and only the average coupling rate can be obtained, not including the information of small molecular drug distribution.</span></p> <h6>Hydrophobic interaction chromatography (HIC)</h6> <p>Hydrophobic interaction chromatography is the mostly used method for DAR value analysis.</p> <p><span style="font-size: 15px;">According to the difference in hydrophobicity of small molecular drugs, the antibodies linked to different numbers of small molecular drugs can be separated through the increase of hydrophobicity. The antibodies of unconjugated small molecular drugs are the weakest in hydrophobicity and are eluted first. The more the number of small molecular drugs, the stronger the hydrophobicity. The weighted average DAR value is calculated by the percentage of chromatographic peak area and the number of coupling drugs.</span></p> <p><span style="font-size: 15px;">The percentage of peak area represents the relative distribution of different quantities of small molecular drug antibodies. The attribution of each peak can be determined by referring to the peak distribution of LC-MS-Ms. After collecting the peaks in the HIC map, the specific peak can be identified by determining the molecular weight of each peak. Hydrophobic interaction chromatography can be used to analyze not only the DAR, but also the distribution of small molecular drugs. The advantage of HIC is that the conditions for elution are mild, which ensures that the biomolecules remain undenatured.</span></p> <h6>Liquid chromatography-mass spectrometry (LC-MS)</h6> <p>LC-MS can not only calculate the DAR value, but also give the distribution information of different numbers of small molecular drugs and the distribution of air-linked joints of by-products in the reaction process, which is very important for identifying different drug loading forms of ADC. The results can be verified by hydrophobic interaction chromatography.</p> <p><span style="font-size: 15px;">The premise of determining DAR by LC-MS is that all substances have the same ionization efficiency as uncoupled antibodies regardless of drug loading. Therefore, its application is based on the assumption that all substances have the same recovery and ionization. But this is not always the case, because the coupling of drugs with positively charged amines will lead to changes in charge and hydrophobicity.</span></p> <h6>Reversed-phase high performance liquid chromatography (RP)</h6> <p><span style="font-size: 15px;">RP functions according to the polarity of the substance to be tested, which generally needs to dissociate the light chain and heavy chain of antibody by reduction reaction, and then separate the light chain and heavy chain containing different numbers of small molecular drugs by high performance liquid chromatography. By calculating the peak area percentage of each light and heavy chain, combined with the number of small molecular drugs coupled with each peak, the weighted average DAR value is calculated.</span></p> <p><span style="font-size: 15px;">These two methods can not only calculate the DAR value, but also analyze the distribution of small molecular drugs at the light chain and heavy chain level, and the structure information of ADC is more abundant. For some ADCs, whose heavy chains having larger polarity than that of light chains coupled with one small molecule are more likely to be eluted, LC-MS is recommended for peak identification.</span></p> <h6>Hydrophilic interaction chromatography (HILIC)</h6> <p>Hydrophilic interaction chromatography is the mostly used method for the determination of antibody N-sugar spectrum, which functions according to the hydrophilicity of the substances to be tested.</p> <p><span style="font-size: 15px;">In recent years, some scholars used hydrophilic interaction chromatography to analyze ADC. The pretreatment was the same as reversed-phase high performance liquid chromatography. ADC was digested by IdeS and then reduced to get Fc/2 and light chain L0, the light chains L1 conjugating one small molecule drug, Fd, and Fd1-Fd3 conjugating one to three small molecule drugs, which were analyzed by hydrophilic interaction chromatography.</span></p> <p><span style="font-size: 15px;">Hydrophilic interaction chromatography can not only analyze the DAR value and drug distribution of ADC, but also analyze the distribution of N-sugar. The identification of each peak is recommended to use LC-MS. This method is confirmed by reversed-phase high performance liquid chromatography (RP-HPLC).</span></p> <h6>Cathepsin B enzyme digestion method (Cathepsin B)</h6> <p><span style="font-size: 15px;">Cathepsin B is a lysosomal cysteine proteolytic enzyme whose catalysis is realized by Cys and His. It is easily inhibited by sulfhydryl reagents, which also called sulfhydryl enzyme and belongs to the papain family. In the Cathepsin B digestion method, the ADC was cut under the hinge region with IdeS enzyme, and then reduced to make the small molecular drugs fully exposed. This pretreatment allows Cathepsin B to enter the cleavage site without restriction, ensuring adequate restriction endonuclease digestion. The pyrolyzed drug is separated from the protein component by reversed-phase high performance liquid chromatography (RP-HPLC), and its concentration is determined according to UV absorption.</span></p> <p><span style="font-size: 15px;">Cathepsin B digestion method is suitable for ADCs with cleavable linker and Cathepsin B digestion site. This method overcomes the limitations in the analysis of ADC by UV spectrophotometry, hydrophobic interaction chromatography, and LC-MS. For example, ultraviolet spectrophotometry needs to meet three basic conditions, hydrophobic interaction chromatography requires different hydrophobicity of different coupling components, LC-MS requires that the ionization efficiency of each component should be the same, and so on. The Cathepsin B enzyme digestion method can complement the above methods which is reliable, accurate, and general, and can be used to measure the DAR value of any ADC with similar chemical properties.</span></p> <p><span style="font-size: 15px;">In addition to the above methods, there are capillary electrophoresis, radiation measurement, iso-point focusing capillary electrophoresis, and so on. In practical use, appropriate analysis methods should be selected according to the ADC coupling characteristics.</span></p> ]]></content:encoded> </item> <item> <title>Clinical Results of immunogenicity of Multiple ADC Molecules</title> <link>https://www.creative-biolabs.com/blog/adc/clinical-results-of-immunogenicity-of-multiple-adc-molecules/</link> <dc:creator><![CDATA[bioadc]]></dc:creator> <pubDate>Sun, 31 Jan 2021 14:48:16 +0000</pubDate> <category><![CDATA[Antibody-drug Conjugates Research]]></category> <category><![CDATA[News]]></category> <category><![CDATA[ADA]]></category> <category><![CDATA[ADCs]]></category> <category><![CDATA[Anti-drug antibody]]></category> <guid isPermaLink="false">https://www.creative-biolabs.com/blog/adc/?p=356</guid> <description><![CDATA[Protein drugs have high risks of inducing immunogenicity, which may affect the efficacy or even be life-threatening. Most biotherapies, such as antibody-drug conjugates (ADCs), fusion proteins, and polyethylene glycolization, carry unnatural human<a class="moretag" href="https://www.creative-biolabs.com/blog/adc/clinical-results-of-immunogenicity-of-multiple-adc-molecules/">Read More...</a>]]></description> <content:encoded><![CDATA[<p class="MsoNormal"><span lang="EN-US">Protein drugs have high risks of inducing immunogenicity, which may affect the efficacy or even be life-threatening. Most biotherapies, such as <a href="https://www.creative-biolabs.com/adc/one-stop-adc-development-service.htm"><span style="color: #0000ff;"><strong>antibody-drug conjugates</strong></span></a> (ADCs), fusion proteins, and polyethylene glycolization, carry unnatural human protein sequences or structural motifs that may increase the risk of immunogenicity. Comprehensive risk assessment strategies and analytical methods are required to monitor and characterize the immunogenicity of ADC and other biotherapies in order to understand and predict potential clinical effects. </span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US">The monoclonal antibody (mAb) in an ADC is covalently linked to the cytotoxic agent through a stable conjugate, which combines the specificity of the mAb to tumor cell surface target antigens and the high efficacy of cytotoxic drugs. Although current ADC uses humanized mAbs and small molecular payloads, its hapten structure may increase the risk of immunogenicity compared with therapeutic mAbs. </span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US"><span style="color: #0000ff;"><a style="color: #0000ff;" href="https://www.creative-biolabs.com/adc/classify-anti-drug-abs-4.htm"><strong>Anti-drug antibody</strong></a></span> (ADA) can target different domains of an ADC, such as mAb epitopes, novel mAb epitopes, connectors, and cytotoxic agents. If large ADC-ADA immune complexes are ingested by non-targeted immune cells, resulting in cell death, then anti-cytotoxic ADAs have a safety risk. </span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US">It is generally believed that the practices and regulatory guidelines of the industry used to evaluate the immunogenicity of biotherapeutic drugs can be applied to ADC. The key to the evaluation of ADC immunogenicity is risk assessment, appropriate detection, and additional characteristics of ADA domain specificity. Immunogenicity risk assessment covers a variety of known factors related to patients and products, which may affect the immunogenicity of drugs and the potential consequences of immune responses. The safety risk of ADC is generally considered higher than that of therapeutic monoclonal antibodies. </span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US">Based on risk assessment, the immunogenicity data of 8 ADC drugs were analyzed in 11 clinical trials involving a range of solid and hematological tumor indications. All ADCs’ incidences of ADA at baseline, the incidences after treatment, and other characteristics of immune response was evaluated. </span></p> <p class="MsoNormal"><a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/A-2021-01-1.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-363" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/A-2021-01-1.jpg" alt="" width="796" height="333" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/A-2021-01-1.jpg 796w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/A-2021-01-1-300x126.jpg 300w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/A-2021-01-1-768x321.jpg 768w" sizes="(max-width: 796px) 100vw, 796px" /></a></p> <ol> <li> <h6>Baseline incidence of ADAs</h6> </li> </ol> <p>According to the research data, the baseline incidence of ADAs is between 1.4% and 8.1%, which is within the range reported by other monoclonal antibody-derived biotherapies, including the ADC drug—<span lang="EN-US">Kadcyla.</span></p> <p><a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-2.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-364" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-2.jpg" alt="" width="827" height="375" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-2.jpg 827w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-2-300x136.jpg 300w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-2-768x348.jpg 768w" sizes="(max-width: 827px) 100vw, 827px" /></a></p> <p class="MsoNormal"><span lang="EN-US">In the clinical studies of those 8 ADC antibodies, they are unlikely to be related to other components of ADC. In addition, of the 8 ADC evaluated, the background signal in the untreated patient population was similar to the mixed serum control signal from healthy volunteers.</span></p> <h6 class="MsoListParagraph" style="margin-left: 18.0pt; text-indent: -18.0pt; mso-char-indent-count: 0; mso-list: l0 level1 lfo1;"><!-- [if !supportLists]--><span lang="EN-US">2.<span style="font-variant-numeric: normal; font-variant-east-asian: normal; font-stretch: normal; font-size: 7pt; line-height: normal; font-family: 'Times New Roman';"> </span></span><span lang="EN-US">Incidence of ADAs after ADC treatment</span></h6> <p class="MsoNormal"><span lang="EN-US">The incidence of ADAs after baseline was between 0 and 35.8%. There were fewer ADC patients targeting hematological tumors than ADA patients targeting solid tumors, which can be attributed to the immune killing of patients with hematological tumors.</span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US">Overall, those data indicate that the hapten structure of these ADCs is unlikely to increase the risk of immunogenicity in most patients compared with traditional therapeutic monoclonal antibodies.</span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US">As for the ADA level, the total titer of those ADCs ranged from less than 1.30 to 4.92, and the average titer of all ADCs exceeded 2.50. </span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US">It showed that the time of occurrence of treatment-induced ADA was variable in all ADCs, usually between 3 and 42 weeks. In addition, 6 of the 8 ADCs developed ADAs in more than 60% of patients within 3 to 6 weeks after treatment.</span></p> <p class="MsoNormal"><span lang="EN-US"> <a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-3.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-365" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-3.jpg" alt="" width="827" height="271" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-3.jpg 827w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-3-300x98.jpg 300w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-3-768x252.jpg 768w" sizes="(max-width: 827px) 100vw, 827px" /></a></span></p> <p class="MsoNormal"><span lang="EN-US">Most of the responses of all ADCs were persistent, ADC H and I had the highest transient responses (43.8% and 34.1%, respectively). However, classifying the ADA response as persistent or transient in oncology studies may be misleading because the treatment time is usually short. In fact, a more detailed assessment of the ADA positive response data showed that the three ADCs (F, H, and I) had higher ADA positive rates. </span></p> <p class="MsoNormal"><span lang="EN-US"> <a href="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-4.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-366" src="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-4.jpg" alt="" width="354" height="253" srcset="https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-4.jpg 354w, https://www.creative-biolabs.com/blog/adc/wp-content/uploads/2021/01/a-2021-01-4-300x214.jpg 300w" sizes="(max-width: 354px) 100vw, 354px" /></a></span></p> <p class="MsoNormal"><span lang="EN-US">Of the 652 patients, 33 had antibodies at baseline, and 3 had increased ADA response. The ADA titers of the three patients varied over time, with baseline titers ranging from 1.89 to 2.71 and post-baseline titers from 2.50 to 4.10. 2 of the 3 patients had peak ADA at the time point after the first baseline after treatment, and the time range of enhanced response was 3 to 9 weeks. The level of ADA in all patients at the last time point was lower than the peak level. The domain specificity of ADA remained unchanged at baseline and post-baseline time points. </span></p> <h6 class="MsoListParagraph" style="margin-left: 18.0pt; text-indent: -18.0pt; mso-char-indent-count: 0; mso-list: l0 level1 lfo1;"><!-- [if !supportLists]--><strong><span lang="EN-US">3.<span style="font-variant-numeric: normal; font-variant-east-asian: normal; font-stretch: normal; font-size: 7pt; line-height: normal; font-family: 'Times New Roman';"> </span></span></strong><span lang="EN-US"><strong>The impact of ADA</strong></span></h6> <p class="MsoNormal"><span lang="EN-US">Immunogenicity is an important part of the clinical characteristics of protein drugs, of which the data should be evaluated in the context of other factors such as efficacy, competition, and safety. Here we focus on the three ADCs, F, H, and I, with a high number of patients producing ADA, with 19, 16, and 43 post-baseline ADA positive patients, respectively. </span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US">The incidence of ADAs in ADC F was 35.8% (19/53), and 18 patients were treatment-induced ADAs. 1 and other patients were treatment-enhanced ADAs. Overall, no differences were observed in terms of competition, safety, or efficacy outcomes compared with patients who did not produce ADC F antibodies. In addition, the titer of ADA has an effect on the competition of these patients, but it has been previously reported in cynomolgus monkeys. </span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US">As for ADC-H, the incidence of ADAs is 25% (16/64). All 16 patients had treatment-induced ADAs. Similar to ADC-F, the trough level of total antibody in some patients with higher ADA titer was lower than that in patients with lower ADA titer, but the patients with higher total ADA titer did not correspond to those with lower total antibody titer.</span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US">A total of 43 patients with ADC-I had ADAs, 41 induction, and 2 patients enhancement. It is worth noting that the total antibody levels of 7 patients (including 2 patients with ADAs enhancement) were lower than the detected levels at one or more trough points. However, a comprehensive analysis is still needed to conclude that ADA titers have an impact on the competition of these patients. However, it is worth noting that the high titer of ADA (~4.00) has a significant effect on the competition curve of trastuzumab ADC in cynomolgus monkeys. </span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US">Limited data obtained from listed ADCs show that ADAs has little impact on clinical outcomes. The incidence of ADAs of Adcetris was 37%, and the infusion response frequency of patients with persistent positive ADAs was higher, resulting in the withdrawal of treatment in two patients. The incidence of ADAs of Kadcyla is 5.3%. The development of Perry ADA seems to have no effect on safety, competition, or efficacy. In the phase I study, there were some ADA-positive patients treated with Mylotarg, including one with transient shortness of breath associated with ADAs. Under the currently recommended dose regimen, no additional immunogenicity data for Mylotarg was generated in clinical trials. In the clinical trial of Besponsa, the incidence of ADAs was 3%, which had no effect on the clearance rate of ADC. </span></p> <h6 class="MsoNormal"><strong><span lang="EN-US"> </span></strong><span lang="EN-US"><strong>Summary</strong></span></h6> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US">The above experimental results show that the hapten-like structure plays a very small role in the immune response of ADC. These results reinforce the fact that molecular structure is an important but not the only factor in the immune response to biotherapy. </span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US">Although data from the eight ADCs indicate that the risk may be reduced as the project enters a later stage of clinical development, a conservative approach is still recommended for these new ADCs. In addition, as predictive immunogenicity tools become more common, they will provide additional information for inclusion in risk assessment and immunogenicity strategies for ADCs and new treatments. </span></p> <p class="MsoNormal"><span lang="EN-US"> </span><span lang="EN-US" style="font-size: 10pt;">References:</span></p> <p class="MsoNormal"><span style="font-family: Calibri, sans-serif; font-size: 10pt;">Immunogenicity of antibody-drug conjugates: observations across 8molecules in 11 clinical trials. Bioanalysis. 2019 Sep;11(17):1555-1568</span></p> ]]></content:encoded> </item> </channel> </rss>