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Background: The main challenges of conventional chemotherapy lie in its lack of selectivity and specificity, leading to significant side effects. Using a small-molecule drug conjugate (SMDC) ensures specific delivery of a cytotoxic drug to the tumor site by coupling it to a targeting vector. This promising strategy can be applied to neuroendocrine tumors (NETs) by choosing a targeting vector that binds specifically to somatostatin receptor subtype 2 (SSTR2). Additionally, incorporation of a bifunctional chelate into the molecule enables complexation of both diagnostic and therapeutic radionuclides. Thus, it facilitates monitoring of the distribution of the SMDC in the body and allows for the implementation of combination therapy. In our study, we designed eSOMA-DM1, a SMDC combining the SSTR2-targeted octreotate peptide and the cytotoxic agent DM1 via a chelate-bridged linker (N 3 -Py-DOTAGA). This approach warrants conjugation of the targeting vector and the drug at opposite sites to avoid undesired steric hindrance effects. Methods: Synthesis of the DM1 moiety ( 4 ) involved a three-step synthetic route, followed by the conjugation to the cyclic peptide, N 3 -Py-DOTAGA-d-Phe-cyclo[Cys-Tyr-d-Trp-Lys-Thr-Cys]-Thr-OH, through a copper-free click reaction, resulting in eSOMA-DM1. Subsequent labeling with [ 111 In]InCl 3 gave a high radiochemical yield and purity. In vitro assessments of eSOMA-DM1 binding, uptake, and internalization were conducted in SSTR2-transfected U2OS cells. Ex vivo biodistribution and fluorescence imaging were performed in H69-tumor bearing mice. Results: eSOMA-DM1 exhibited an IC 50 value for SSTR2 similar to the gold standard DOTA-TATE. The uptake of [ 111 In]In-eSOMA-DM1 in U2OS.SSTR2 cells was 1.2-fold lower than that of [ 111 In]In-DOTA-TATE. Tumor uptake in H69-xenografted mice was higher for [ 111 In]In-eSOMA-DM1 at all-time points compared to [ 111 In]In-DOTA-TATE. Prolonged blood circulation led to increased accumulation of [ 111 In]In-eSOMA-DM1 in highly vascularized tissues, such as the lungs, skin, and heart. Excretion through the kidneys, liver, and spleen was also observed. Conclusion: eSOMA-DM1 is a SMDC developed for NET showing promising characteristics in vitro. However, the in vivo results obtained with [ 111 In]In-eSOMA-DM1 suggest the need for adjustments to optimize its distribution.

Antibody-drug conjugate (ADC) consists of engineered antibodies and cytotoxic drugs linked via a chemical linker, and the stability of ADC plays a crucial role in ensuring its safety and efficacy. The stability of ADC is closely related to the conjugation site; however, no method has been developed to assess the stability of different conjugation sites due to the low response of conjugated peptides. In this study, an integrated strategy was developed and validated to assess the stability of different conjugation sites on ADC in serum. Initial identification of the conjugated peptides of the model drug ado-trastuzumab emtansine (T-DM1) was achieved by the proteomic method. Subsequently, a semiquantitative method for conjugated peptides was established in liquid chromatography-hybrid linear ion trap triple quadrupole mass spectrometry (LC-QTRAP-MS/MS) based on the qualitative information. The pretreatment method of the serum sample was optimized to reduce matrix interference. The method was then validated and applied to evaluate the stability of the conjugation sites on T-DM1. The results highlighted differences in stability among the different conjugation sites on T-DM1. This is the first study to assess the stability of different conjugation sites on the ADC in serum, which will be helpful for the design and screening of ADCs in the early stages of development.

In targeted cancer therapy, antibody-drug-conjugates using mertansine (DM1)-based cytotoxic compounds rely on covalent bonds for drug conjugation. Consequently, the cytotoxic DM1 derivative released upon their proteolytic digestion is up to 1000-fold less potent than DM1 and lacks a bystander effect. To overcome these limitations, we developed a DM1 derivative (keto-DM1) suitable for bioconjugation through an acid-reversible hydrazone bond. Its acid-reversible hydrazone conjugate with biotin (B-Hz-DM1) was generated and tested for efficacy using the cetuximab-targeted Avidin-Nucleic-Acid-NanoASsembly (ANANAS) nanoparticle (NP) platform. NP-tethered B-Hz-DM1 is stable at neutral pH and releases its active moiety only in endosome/lysosome mimicking acidic pH. In vitro, the NP/Cetux/B-Hz-DM1 assembly showed high potency on MDA-MB231 breast cancer cells. In vivo both B-Hz-DM1 and NP/Cetux/B-Hz-DM1 reduced tumor growth. A significantly major effect was exerted by the nanoformulation, associated with an increased in situ tumor cell death. Keto-DM1 is a promising acid-reversible mertansine derivative for targeted delivery in cancer therapy., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In this work, we conceived and developed antibody-drug conjugates (ADCs) that could efficiently release the drug after enzymatic cleavage of the linker moiety by tumoral proteases. The antibody-drug linkers we used are the result of a rational optimization of a previously reported PEGylated linker, PUREBRIGHT® MA-P12-PS, which showed excellent drug loading capacities but lacked an inbuilt drug discharge mechanism, thus limiting the potency of the resulting ADCs. To address this limitation, we chose to incorporate a protease-sensitive trigger into the linker to favor the release of a "PEGless" drug inside the tumor cells and, therefore, obtain potent ADCs. Currently, most marketed ADCs are based on the Val-Cit dipeptide followed by a self-immolative spacer for releasing the drug in its unmodified form. Here, we selected two untraditional peptide sequences, a Phe-Gly dipeptide and a Val-Ala-Gly tripeptide and placed one or the other in between the drug on one side (N-terminus) and the rest of the linker, including the PEG moiety, on the other side (C-terminus), without a self-immolative group. We found that both linkers responded to cathepsin B, a reference lysosomal enzyme, and liberated a PEG-free drug catabolite, as desired. We then used the two linkers to generate ADCs based on trastuzumab (a HER2-targeting antibody) and DM1 (a microtubule-targeted cytotoxic agent) with an average drug-to-antibody ratio (DAR) of 4 or 8. The ADCs showed restored cytotoxicity in vitro, which was proportional to the DM1 loading and generally higher for the ADCs bearing Val-Ala-Gly in their structure. In an ovarian cancer mouse model, the DAR 8 ADC based on Val-Ala-Gly behaved better than Kadcyla® (an approved ADC of DAR 3.5 used as control throughout this study), leading to a higher tumor volume reduction and more prolonged median survival. Taken together, our results depict a successful linker optimization process and encourage the application of the Val-Ala-Gly tripeptide as an alternative to other existing protease-sensitive triggers for ADCs., Competing Interests: Declaration of competing interest YM, AS and HY are employees of NOF CORPORATION. GP has been scientific advisor for NOF CORPORATION. The other authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The notable characteristics of recently emerged Antibody-Drug Conjugates (ADCs) encompass the targeting of Human Epidermal growth factor Receptor 2 (HER2) through monoclonal antibodies (mAbs) and a high ratio of drug to antibody (DAR). The achievements of Kadcyla® (T-DM1) and Enhertu® (T-Dxd) have demonstrated that HER2-targeting antibodies, such as trastuzumab, have shown to be competitive in terms of efficacy and price for development. Furthermore, with the arrival of T-Dxd and Trodelvy®, high-DAR (7-8) ADCs, which differ from the moderate DAR (3-4) ADCs that were formerly regarded as conventional, are being acknowledged for their worth. Following this trend of drug development, we endeavored to develop a high-DAR ADC using a straightforward approach involving the utilization of DM1, a highly potent substance, in combination with the widely recognized trastuzumab. To achieve a high DAR, DM1 was conjugated to reduced cysteine through the simple design and synthesis of various dimaleimide linkers with differing lengths. Using LC and MS analysis, we have demonstrated that our synthesis methodology is uncomplicated and efficacious, yielding trastuzumab-based ADCs that exhibit a remarkable degree of uniformity. These ADCs have been experimentally substantiated to exert an inhibitory effect on cancer cells in vitro, thus affirming their value as noteworthy additions to the realm of ADCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This paper outlines a novel drug delivery system for highly cytotoxic mertansine (DM1) by conjugating to an albumin-binding Evans blue (EB) moiety through a tuneable responsive disulfide linker, providing valuable insights for the development of effective drug delivery systems toward cancer therapy.

Cryptophycins are microtubule-targeting agents (MTAs) that belong to the most potent antimitotic compounds known to date; however, their exact molecular mechanism of action remains unclear. Here, we present the 2.2 Å resolution X-ray crystal structure of a potent cryptophycin derivative bound to the αβ-tubulin heterodimer. The structure addresses conformational issues present in a previous 3.3 Å resolution cryo-electron microscopy structure of cryptophycin-52 bound to the maytansine site of β-tubulin. It further provides atomic details on interactions of cryptophycins, which had not been described previously, including ones that are in line with structure-activity relationship studies. Interestingly, we discovered a second cryptophycin-binding site that involves the T5-loop of β-tubulin, a critical secondary structure element involved in the exchange of the guanosine nucleotide and in the formation of longitudinal tubulin contacts in microtubules. Cryptophycins are the first natural ligands found to bind to this new "βT5-loop site" that bridges the maytansine and vinca sites. Our results offer unique avenues to rationally design novel MTAs with the capacity to modulate T5-loop dynamics and to simultaneously engage multiple β-tubulin binding sites., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Antibody drug conjugates (ADCs) with twelve FDA approved drugs, known as a novel category of anti-neoplastic treatment created to merge the monoclonal antibody specificity with cytotoxicity effect of chemotherapy. However, despite many undeniable advantages, ADCs face certain problems, including insufficient internalization after binding, complex structures and large size of full antibodies especially in targeting of solid tumors. Camelid single domain antibody fragments (Nanobody®) offer solutions to this challenge by providing nanoscale size, high solubility and excellent stability, recombinant expression in bacteria, in vivo enhanced tissue penetration, and conjugation advantages. Here, an anti-human CD22 Nanobody was expressed in E.coli cells and conjugated to Mertansine (DM1) as a cytotoxic payload. The anti-CD22 Nanobody was expressed and purified by Ni-NTA resin. DM1 conjugated anti-CD22 Nanobody was generated by conjugation of SMCC-DM1 to Nanobody lysine groups. The conjugates were characterized using SDS-PAGE and Capillary electrophoresis (CE-SDS), RP-HPLC, and MALDI-TOF mass spectrometry. Additionally, flow cytometry analysis and a competition ELISA were carried out for binding evaluation. Finally, cytotoxicity of conjugates on Raji and Jurkat cell lines was assessed. The drug-to-antibody ratio (DAR) of conjugates was calculated 2.04 using UV spectrometry. SDS-PAGE, CE-SDS, HPLC, and mass spectrometry confirmed conjugation of DM1 to the Nanobody. The obtained results showed the anti-CD22 Nanobody cytotoxicity was enhanced almost 80% by conjugation with DM1. The binding of conjugates was similar to the non-conjugated anti-CD22 Nanobody in flow cytometry experiments. Concludingly, this study successfully suggest that the DM1 conjugated anti-CD22 Nanobody can be used as a novel tumor specific drug delivery system., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The therapeutic modalities that involve the endocytosis pathway, including antibody-drug conjugates (ADCs), have recently been developed. Since the drug escape from endosomes/lysosomes is a determinant of their efficacy, it is important to optimize the escape, and the cellular evaluation system is needed. SLC46A3, a lysosomal membrane protein, has been implicated in the pharmacological efficacy of trastuzumab emtansine (T-DM1), a noncleavable ADC used for the treatment of breast cancer, and the cellular uptake efficacy of lipid-based nanoparticles. Recently, we identified the SLC46A3 function as a proton-coupled steroid conjugate and bile acid transporter, which can directly transport active catabolites of T-DM1. Thus, the rapid and convenient assay systems for evaluating the SLC46A3 function may help to facilitate ADC development and to clarify the physiological roles in endocytosis. Here, we show that SLC46A3 dC, which localizes to the plasma membrane owing to lacking a lysosomal-sorting motif, has a great ability to transport 5-carboxyfluorescein (5-CF), a fluorescent probe, in a pH-dependent manner. 5-CF uptake mediated by SLC46A3 was significantly inhibited by compounds reported to be SLC46A3 substrates/inhibitors and competitively inhibited by estrone 3-sulfate, a typical SLC46A3 substrate. The inhibition assays followed by uptake studies revealed that SG3199, a pyrrolobenzodiazepine dimer, which has been used as an ADC payload, is a substrate of SLC46A3. Accordingly, the fluorescence-based assay system for the SLC46A3 function using 5-CF can provide a valuable tool to evaluate the interaction of drugs/drug candidates with SLC46A3.

Background: Trastuzumab-emtansine (T-DM1, commercial name: Kadcyla) is well-known antibody-drug conjugate (ADC) and was first approved for human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. This molecular format consisting of trastuzumab and maytansinoid payload (emtansine) is very simple, however, T-DM1 has wide heterogeneity due to non-specific conjugation, lowering its therapeutic index (TI)., Methods: To overcome this issue during the chemical modification of the random conjugation approach to generate T-DM1, we developed a novel chemical conjugation technology termed "AJICAP®" for modification of antibodies in site-specific manner by IgG Fc-affinity peptide based reagents., Results: In this study, we compared site-specific maytansinoid-based ADCs synthesized by AJICAP and T-DM1 in rat safety studies. The results indicated an increase in the maximum tolerated dose, demonstrating an expansion of the AJICAP-ADC therapeutic index compared with that of commercially available T-DM1. Gram scale preparation of this AJICAP-ADC and the initial stability study are also described., Conclusions: Trastuzumab-AJICAP-maytansinoid produced by this unique chemical conjugation methodology showed higher stability and tolerability than commercially available T-DM1., Competing Interests: The authors declare no conflict of interest. YM and BAM are serving as the Guest editors of this journal. We declare that YM and BAM had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to GP., (© 2022 The Author(s). Published by IMR Press.)

Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate (ADC), combining a humanized monoclonal antibody (IgG1) targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and a potent cytotoxic maytansinoid derivative, DM4, inhibiting microtubule assembly. SAR408701 is currently in clinical development for the treatment of advanced solid tumors expressing CEACAM5. It is administered intravenously as a conjugated antibody with an average Drug Antibody Ratio (DAR) of 3.8. During SAR408701 clinical development, four entities were measured in plasma: conjugated antibody (SAR408701), naked antibody (NAB), DM4 and its methylated metabolite (MeDM4), both being active. Average DAR and proportions of individual DAR species were also assessed in a subset of patients. An integrated and semi-mechanistic population pharmacokinetic model describing the time-course of all entities in plasma and DAR measurements has been developed. All DAR moieties were assumed to share the same drug disposition parameters, excepted for clearance which differed for DAR0 (i.e. NAB entity). The conversion of higher DAR to lower DAR resulted in a DAR-dependent ADC deconjugation and was represented as an irreversible first-order process. Each conjugated antibody was assumed to contribute to DM4 formation. All data were fitted simultaneously and the model developed was successful in describing the pharmacokinetic profile of each entity. Such a structural model could be translated to other ADCs and gives insight of mechanistic processes governing ADC disposition. This framework will further be expanded to evaluate covariates impact on SAR408701 pharmacokinetics and its derivatives, and thus can help identifying sources of pharmacokinetic variability and potential efficacy and safety pharmacokinetic drivers., (© 2022. The Author(s).)

Maytansinoids, the chemical derivatives of Maytansine, are commonly used as potent cytotoxic payloads in antibody-drug conjugates (ADC). Structure-activity-relationship studies had identified the C3 ester side chain as a critical element for antitumor activity of maytansinoids. The maytansinoids bearing the methyl group at C3 position with D configuration were about 100 to 400-fold less cytotoxic than their corresponding L-epimers toward various cell lines. The detailed mechanism of how chirality affects the anticancer activity remains elusive. In this study, we determined the high-resolution crystal structure of tubulin in complex with maytansinol, L-DM1-SMe and D-DM1-SMe. And we found the carbonyl oxygen atom of the ester moiety and the tail thiomethyl group at C3 side chain of L-DM1-SMe form strong intramolecular interaction with the hydroxyl at position 9 and the benzene ring, respectively, fixing the bioactive conformation and enhancing the binding affinity. Additionally, ligand-based and structure-based virtually screening methods were used to screen the commercially macrocyclic compounds library, and 15 macrocyclic structures were picketed out as putatively new maytansine-site inhibitors. Our study provides a possible strategy for the rational discovery of next-generation maytansine site inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The stability and exposure of toxin-related catabolites in system circulation contributes to the evaluation of the stability, targeted delivery and off-target toxicity for antibody-drug conjugates (ADC) at different stages during drug development. In this study, simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for determination catabolites of Mertansine (DM1), MCC-DM1 and Lys-MCC-DM1 in cynomolgus serum have been developed. The serum samples are processed by protein precipitation. The LC-MS/MS methods are applied on a Phenomenex C8 column (50 × 2.0 mm, 5 μm) with gradient elution with water-formic acid 0.1% (A) and acetonitrile-formic acid 0.1% (B) at a flow rate of 0.5 mL/min. The analytical run time is only 4.0 min and the calibration ranges of the standard curve are 0.500-200 ng/mL for DM1, 1.00-500 ng/mL for MCC-DM1 and 2.00-1000 ng/mL for Lys-MCC-DM1. Intra- and inter-day precision of low, middle and high quality controls was <15%, and accuracy was 99.2-110.9%. The methods were successfully applied to evaluate three catabolites of novel ADCs with N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate linker in vitro and in vivo studies., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/β-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/β-tubulin-GB1 complex., Competing Interests: Competing interest statement: The University of Florida has filed a patent application relating to the content of this article.

Signaling through the CD19-CD81 co-receptor complex, in combination with the B cell receptor, is a critical determinant of B cell development and activation. It is unknown how CD81 engages CD19 to enable co-receptor function. Here, we report a 3.8-angstrom structure of the CD19-CD81 complex bound to a therapeutic antigen-binding fragment, determined by cryo-electron microscopy (cryo-EM). The structure includes both the extracellular domains and the transmembrane helices of the complex, revealing a contact interface between the ectodomains that drives complex formation. Upon binding to CD19, CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface and reorganizes its transmembrane helices to occlude a cholesterol binding pocket present in the apoprotein. Our data reveal the structural basis for CD19-CD81 complex assembly, providing a foundation for rational design of therapies for B cell dysfunction., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Purpose: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a glycoprotein that has limited expression in normal adult tissues, but is overexpressed in carcinomas of the gastrointestinal tract, the genitourinary and respiratory systems, and breast cancer. As such, CEACAM5 is an attractive target for antibody-based therapies designed to selectively deliver cytotoxic drugs to certain epithelial tumors. Here, we describe preclinical data for a novel antibody-drug conjugate (ADC), SAR408701, which consists of an anti-CEACAM5 antibody (SAR408377) coupled to a maytansinoid agent DM4 via a cleavable linker., Experimental Design: The specificity and binding affinity of SAR408701 to human and cynomolgus monkey CEACAM5 were tested in vitro . The cytotoxic activity of SAR408701 was assessed in CEACAM5-expressing tumor cell lines and using patient-derived xenograft mouse models of CEACAM5-positive tumors. Pharmacokinetic-pharmacodynamic and pharmacokinetic-efficacy relationships were established. SAR408701 toxicity was evaluated in cynomolgus monkey., Results: SAR408701 bound selectively to human and cynomolgus monkey CEACAM5 with similar apparent Kd values (0.017 nmol/L and 0.024 nmol/L, respectively). Both in vitro and in vivo evaluations showed that SAR408701 has cytotoxic activity, leading to in vivo efficacy in single and repeated dosing. Single doses of SAR408701 induced significant increases in the tumor expression of phosphorylated histone H3, confirming the tubulin-targeting mechanism of action. The overall toxicity profile of SAR408701 in cynomolgus monkey was similar to that observed after intravenous administration of DM4 alone., Conclusions: On the basis of these preclinical data, the ADC SAR408701 is a promising candidate for development as a potential treatment for patients with CEACAM5-positive tumors., (©2020 American Association for Cancer Research.)

ADCs based on the natural product maytansine have been successfully employed clinically. In a previous report, ADCs based on hydrophilic non-cell permeable maytansinoids was presented. The authors in this report further explore the maytansine scaffold to develop tubulin inhibitors capable of cell permeation. The research resulted in amino-benzoyl-maytansinoid payloads that were further elaborated with linkers for conjugating to antibodies. This approach was applied to MUC16 tumor targeting antibodies for ovarian cancers. A positive control ADC was evaluated alongside the amino-benzoyl-maytansinoid ADC and the efficacy observed was equivalent while the isotype control ADCs had no effect., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A multiblocked mutant strain (ΔAHBA and Δasm12, asm21) of Actinosynnema pretiosum, the producer of the highly toxic maytansinoid ansamitocin, has been used for the mutasynthetic production of new proansamitocin derivatives. The use of mutant strains that are blocked in the biosynthesis of an early building block as well as in the expression of two tailoring enzymes broadens the scope of chemo-biosynthetic access to new maytansinoids. Remarkably, a ring-expanded macrolactone derived from ansamitocin was created for the first time., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Trastuzumab and the related ADC, ado-trastuzumab emtansine (T-DM1), both target HER2-overexpressing cells. Together, these drugs have treatment indications in both early-stage and metastatic settings for HER2 + breast cancer. T-DM1 retains the antibody functionalities of trastuzumab and adds the potency of a cytotoxic maytansine payload. Interestingly, in the clinic, T-DM1 cannot always replace the use of trastuzumab plus chemotherapy administered together as single agents. We hypothesize that this failure may be due, in part, to the limited systemic exposure achieved by T-DM1 relative to trastuzumab because of toxicity-related dosing constraints on the ADC. We have developed a trastuzumab-based ADC site specifically conjugated to maytansine through a noncleavable linker. This construct, termed CAT-01-106, has a drug-to-antibody ratio (DAR) of 1.8, approximately half the average DAR of T-DM1, which comprises a mixture of antibodies variously conjugated with DARs ranging from 0 to 8. The high DAR species present in T-DM1 contribute to its toxicity and limit its clinical dose. CAT-01-106 showed superior in vivo efficacy compared with T-DM1 at equal payload dosing and was equally or better tolerated compared with T-DM1 at equal payload dosing up to 120 mg/kg in Sprague-Dawley rats and 60 mg/kg in cynomolgus monkeys. CAT-01-106 also showed improved pharmacokinetics in rats relative to T-DM1, with 40% higher ADC exposure levels. Together, the data suggest that CAT-01-106 may be sufficiently tolerable to enable clinical dosing at trastuzumab-equivalent exposure levels, combining the functions of both the antibody and the payload in one drug and potentially improving patient outcomes., (©2020 American Association for Cancer Research.)

Linker design is crucial to the success of antibody-drug conjugates (ADCs). In this work, we developed a modular linker format for attaching molecular cargos to antibodies based on strand pairing between complementary oligonucleotides. We prepared antibody-oligonucleotide conjugates (AOCs) by attaching 18-mer oligonucleotides to an anti-HER2 antibody through thiol-maleimide chemistry, a method generally applicable to any immunoglobulin with interchain disulfide bridges. The hybridization of drug-bearing complementary oligonucleotides to our AOCs was rapid, stoichiometric, and sequence-specific. AOCs loaded with cytotoxic payloads were able to selectively target HER2-overexpressing cell lines such as SK-BR-3 and N87, with in vitro potencies similar to that of the marketed ADC Kadcyla (T-DM1). Our results demonstrated the potential of utilizing AOCs as a highly versatile and modular platform, where a panel of well-characterized AOCs bearing DNA, RNA, or various nucleic acid analogs, such as peptide nucleic acids, could be easily paired with any cargo of choice for a wide range of diagnostic or therapeutic applications.

The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single-site labeling of a high-frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent-accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys-selective electrophile connected by a spacer. Consequently, it enables the irreversible single-site labeling of a Lys residue independent of its place in the reactivity order. The user-friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site-selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe-tagged proteins. Besides, the methodology provides access to antibody-drug conjugate (ADC), which exhibits highly selective anti-proliferative activity towards HER-2 expressing SKBR-3 breast cancer cells., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Antibody-Drug Conjugates (ADCs) developed as a targeted treatment approach to deliver toxins directly to cancer cells are one of the fastest growing classes of oncology therapeutics, with eight ADCs and two immunotoxins approved for clinical use. However, selection of an optimum target and payload combination, to achieve maximal therapeutic efficacy without excessive toxicity, presents a significant challenge. We have developed a platform to facilitate rapid and cost-effective screening of antibody and toxin combinations for activity and safety, based on streptavidin-biotin conjugation. For antibody selection, we evaluated internalization by target cells using streptavidin-linked antibodies conjugated to biotinylated saporin, a toxin unable to cross cell membranes. For payload selection, we biotinylated toxins and conjugated them to antibodies linked to streptavidin to evaluate antitumour activity and pre-clinical safety. As proof of principle, we compared trastuzumab conjugated to emtansine via streptavidin-biotin (Trastuzumab-SB-DM1) to the clinically approved trastuzumab emtansine (T-DM1). We showed comparable potency in reduction of breast cancer cell survival in vitro and in growth restriction of orthotopic breast cancer xenografts in vivo. Our findings indicate efficient generation of functionally active ADCs. This approach can facilitate the study of antibody and payload combinations for selection of promising candidates for future ADC development.

Antibody-Drug Conjugates (ADCs) consist of antibodies attached to cytotoxic small molecules or biological agents (i.e., payloads) through chemical linkers which may be cleavable or non-cleavable. The development of new ADCs is challenging, particularly the process of attaching the linker-payload construct to the antibody (i.e., the conjugation process). One of the major problems associated with conjugation is high hydrophobicity of the payload which can lead to low yields of the ADC through aggregation and/or lower than desired Drug-Antibody Ratios (DARs). We report here a UPLC-based assay that can be used to study the physicochemical properties of ADC payloads at an early stage of development, and to provide information on whether the hydrophilic-hydrophobic balance is suitable for conjugation or further physicochemical optimization is required. The assay is relatively simple to establish and should be of use to those working in the ADC area., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: IP has share options and PJMJ, KMR and DET have founding stock in Femtogenix Ltd, the company supplying one of the experimental ADC payloads evaluated in this study. IP and PJMJ are fully employed by Femtogenix Ltd, and KMR and DET have consultancies with the company. PJMJ, KMR and DET are named inventors on patents filed and owned by the company., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The major drawbacks of the cytotoxin like DM1 are the off-target effects. Here, the targeting nanovesicles were developed by synthesizing tocopherol-SS-DM1 and conjugating a pH low insertion peptide (pHLIP) to PEGylated phospholipids, in which tocopherol-SS-DM1 improves the drug loading and is glutathione responsive in the cytoplasm, meanwhile, the pH insertion peptide targets the acidic microenvironment of cancer cells. Besides, these nanovesicles can accumulate at the endoplasmic reticulum and show increased cancer therapeutic effects both in vitro and in vivo. These targeting nanovesicles provide a novel formulation for subcellular organelle targeting, a platform for precisely delivery of cytotoxic DM1 to cancer cells, and an alternative strategy for antibody-drug conjugates (ADCs)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using time-of-flight (ToF) analyzers, due to their routine high m / z transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance mass spectrometer (15 T FT-ICR MS) is more than capable of analyzing a wide range of ions in the high m / z scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic cesium iodide salt clusters; a humanized IgG1 k monoclonal antibody (mAb; 148.2 kDa); an IgG1-mertansine drug conjugate (148.5 kDa, drug-to-antibody ratio; DAR 2.26); an IgG1-siRNA conjugate (159.1 kDa; ribonucleic acid to antibody ratio; RAR 1); the membrane protein aquaporin-Z (97.2 kDa) liberated from a C8E4 detergent micelle; the empty MSP1D1-nanodisc (142.5 kDa) and the tetradecameric chaperone protein complex GroEL (806.2 kDa; GroEL dimer at 1.6 MDa). We also investigate different regions of the FT-ICR MS that impact ion transmission and desolvation. Finally, we demonstrate how the transmission of these species and resultant spectra are highly consistent with those previously generated on both quadrupole-ToF (Q-ToF) and Orbitrap instrumentation. This report serves as an impactful example of how FT-ICR mass analyzers are competitive to Q-ToFs and Orbitraps for high mass detection at high m / z .

Covering: 2014-2019We review recent progress on natural products that target cytoskeletal components, including microtubules, actin, intermediate filaments, and septins and highlight their demonstrated and potential utility in the treatment of human disease. The anticancer efficacy of microtubule targeted agents identified from plants, microbes, and marine organisms is well documented. We highlight new microtubule targeted agents currently in clinical evaluations for the treatment of drug resistant cancers and the accumulating evidence that the anticancer efficacy of these agents is not solely due to their antimitotic effects. Indeed, the effects of microtubule targeted agents on interphase microtubules are leading to their potential for more mechanistically guided use in cancers as well as neurological disease. The discussion of these agents as more targeted drugs also prompts a reevaluation of our thinking about natural products that target other components of the cytoskeleton. For instance, actin active natural products are largely considered chemical probes and non-selective toxins. However, studies utilizing these probes have uncovered aspects of actin biology that can be more specifically targeted to potentially treat cancer, neurological disorders, and infectious disease. Compounds that target intermediate filaments and septins are understudied, but their continued discovery and mechanistic evaluations have implications for numerous therapeutic indications.

Ansamitocin P-3 (AP-3) is an important antitumor agent. The antitumor activity of AP-3 is a result of its affinity towards β-tubulin in eukaryotic cells. In this study, in order to improve AP-3 production, the reason for severe growth inhibition of the AP-3 producing strain Actinosynnema pretiosum WXR-24 under high concentrations of exogenous AP-3 was investigated. The cell division protein FtsZ, which is the analogue of β-tubulin in bacteria, was discovered to be the AP-3 target through structural comparison followed by a SPR biosensor assay. AP-3 was trapped into a less hydrophilic groove near the GTPase pocket on FtsZ by hydrogen bounding and hydrophobic interactions, as revealed by docking analysis. After overexpression of the APASM&#95;5716 gene coding for FtsZ in WXR-30, the resistance to AP-3 was significantly improved. Moreover, AP-3 yield was increased from 250.66 mg/L to 327.37 mg/L. After increasing the concentration of supplemented yeast extract, the final yield of AP-3 reached 371.16 mg/L. In summary, we demonstrate that the cell division protein FtsZ is newly identified as the bacterial target of AP-3, and improving resistance is an effective strategy to enhance AP-3 production.

Radiotherapy remains a major treatment modality for cancer types such as non-small cell lung carcinoma (or NSCLC). To enhance treatment efficacy at a given radiation dose, radiosensitizers are often used during radiotherapy. Herein, we report a nanoparticle agent that can selectively sensitize cancer cells to radiotherapy. Specifically, we nitrosylated maytansinoid DM1 and then loaded the resulting prodrug, DM1-NO, onto poly(lactide- co -glycolic)- block -poly(ethylene glycol) (PLGA- b -PEG) nanoparticles. The toxicity of DM1 is suppressed by nanoparticle encapsulation and nitrosylation, allowing the drug to be delivered to tumors through the enhanced permeability and retention effect. Under irradiation to tumors, the oxidative stress is elevated, leading to the cleavage of the S-N bond and the release of DM1 and nitric oxide (NO). DM1 inhibits microtubule polymerization and enriches cells at the G2/M phase, which is more radiosensitive. NO under irradiation forms highly toxic radicals such as peroxynitrites, which also contribute to tumor suppression. The two components work synergistically to enhance radiotherapy outcomes, which was confirmed in vitro by clonogenic assays and in vivo with H1299 tumor-bearing mice. Our studies suggest the great promise of DM1-NO PLGA nanoparticles in enhancing radiotherapy against NSCLC and potentially other tumor types.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma with a 95% mortality rate with no improvement to treatment in decades, and new therapies are desperately needed. PEN-221 is a miniaturized peptide-drug conjugate (∼2 kDa) designed to target SCLC via a Somatostatin Receptor 2 (SSTR2)-targeting ligand and to overcome the high proliferation rate characteristic of this disease by using the potent cytotoxic payload, DM1. SSTR2 is an ideal target for a drug conjugate, as it is overexpressed in SCLC with limited normal tissue expression. In vitro , PEN-221 treatment of SSTR2-positive cells resulted in PEN-221 internalization and receptor-dependent inhibition of cellular proliferation. In vivo , PEN-221 exhibited rapid accumulation in SSTR2-positive SCLC xenograft tumors with quick clearance from plasma. Tumor accumulation was sustained, resulting in durable pharmacodynamic changes throughout the tumor, as evidenced by increases in the mitotic marker of G 2 -M arrest, phosphohistone H3, and increases in the apoptotic marker, cleaved caspase-3. PEN-221 treatment resulted in significant antitumor activity, including complete regressions in SSTR2-positive SCLC xenograft mouse models. Treatment was effective using a variety of dosing schedules and at doses below the MTD, suggesting flexibility of dosing schedule and potential for a large therapeutic window in the clinic. The unique attributes of the miniaturized drug conjugate allowed for deep tumor penetration and limited plasma exposure that may enable long-term dosing, resulting in durable tumor control. Collectively, these data suggest potential for antitumor activity of PEN-221 in patients with SSTR2-positive SCLC., (©2019 American Association for Cancer Research.)

Folate receptor α (FRα) is a well-studied tumor biomarker highly expressed in many epithelial tumors such as breast, ovarian, and lung cancers. Mirvetuximab soravtansine (IMGN853) is the antibody-drug conjugate of FRα-binding humanized monoclonal antibody M9346A and cytotoxic maytansinoid drug DM4. IMGN853 is currently being evaluated in multiple clinical trials, in which the immunohistochemical evaluation of an archival tumor or biopsy specimen is used for patient screening. However, limited tissue collection may lead to inaccurate diagnosis due to tumor heterogeneity. Herein, we developed a zirconium-89 ( 89 Zr)-radiolabeled M9346A ( 89 Zr-M9346A) as an immuno-positron emission tomography (immuno-PET) radiotracer to evaluate FRα expression in triple-negative breast cancer (TNBC) patients, providing a novel means to guide intervention with therapeutic IMGN853. In this study, we verified the binding specificity and immunoreactivity of 89 Zr-M9346A by in vitro studies in FRα high cells (HeLa) and FRα low cells (OVCAR-3). In vivo PET/computed tomography (PET/CT) imaging in HeLa xenografts and TNBC patient-derived xenograft (PDX) mouse models with various levels of FRα expression demonstrated its targeting specificity and sensitivity. Following PET imaging, the treatment efficiencies of IMGN853, pemetrexed, IMGN853 + pemetrexed, paclitaxel, and saline were assessed in FRα high and FRα low TNBC PDX models. The correlation between 89 Zr-M9346A tumor uptake and treatment response using IMGN853 in FRα high TNBC PDX model suggested the potential of 89 Zr-M9346A PET as a noninvasive tool to prescreen patients based on the in vivo PET imaging for IMGN853-targeted treatment.

CD205 is a type I transmembrane glycoprotein and is a member of the C-type lectin receptor family. Analysis by mass spectrometry revealed that CD205 was robustly expressed and highly prevalent in a variety of solid malignancies from different histotypes. IHC confirmed the increased expression of CD205 in pancreatic, bladder, and triple-negative breast cancer (TNBC) compared with that in the corresponding normal tissues. Using immunofluorescence microscopy, rapid internalization of the CD205 antigen was observed. These results supported the development of MEN1309/OBT076, a fully humanized CD205-targeting mAb conjugated to DM4, a potent maytansinoid derivate, via a cleavable N-succinimidyl-4-(2-pyridyldithio) butanoate linker. MEN1309/OBT076 was characterized in vitro for target binding affinity, mechanism of action, and cytotoxic activity against a panel of cancer cell lines. MEN1309/OBT076 displayed selective and potent cytotoxic effects against tumor cells exhibiting strong and low to moderate CD205 expression. In vivo , MEN1309/OBT076 showed potent antitumor activity resulting in durable responses and complete tumor regressions in many TNBC, pancreatic, and bladder cancer cell line-derived and patient-derived xenograft models, independent of antigen expression levels. Finally, the pharmacokinetics and pharmacodynamic profile of MEN1309/OBT076 was characterized in pancreatic tumor-bearing mice, demonstrating that the serum level of antibody-drug conjugate (ADC) achieved through dosing was consistent with the kinetics of its antitumor activity. Overall, our data demonstrate that MEN1309/OBT076 is a novel and selective ADC with potent activity against CD205-positive tumors. These data supported the clinical development of MEN1309/OBT076, and further evaluation of this ADC is currently ongoing in the first-in-human SHUTTLE clinical trial., (©2019 American Association for Cancer Research.)

Antibody-drug conjugates (ADC) are a novel way to deliver potent cytotoxic compounds to cells expressing a specific antigen. Four ADC targeting CD19, including SAR3419 (coltuximab ravtansine), have entered clinical development. Here, we present huB4-DGN462, a novel ADC based on the SAR3419 anti-CD19 antibody linked via sulfo-SPDB to the potent DNA-alkylating agent DGN462. huB4-DGN462 had improved in vitro anti-proliferative and cytotoxic activity compared to SAR3419 across multiple B-cell lymphoma and human acute lymphoblastic leukemia cell lines. In vivo experiments using lymphoma xenografts models confirmed the in vitro data. The response of B-cell lymphoma lines to huB4-DGN462 was not correlated with CD19 expression, the presence of BCL2 or MYC translocations, TP53 inactivation or lymphoma histology. In conclusion, huB4-DGN462 is an attractive candidate for clinical investigation in patients with B-cell malignancies., (Copyright© 2019 Ferrata Storti Foundation.)

The tumor stromal microenvironments (TSM) including stromal cells and extracellular matrix (ECM) form an abominable barrier hampering nanoparticles accessibility to cancer cells, significantly compromising their antitumor effects. Herein, we report a bioinspired lipoprotein (bLP) that can induce efficient photothermia to remodel TSM and improve second bLP accessibility to cancer cells for antitumor therapy. The multiple stromal cells and ECM components in TSM are remarkably disrupted by bLP-mediated photothermal effects, which cause a 4.27-fold enhancement of second bLP accumulation in tumor, deep penetration in whole tumor mass and 27.0-fold increase of accessibility to cancer cells. Of note, this bLP-mediated TSM-remodeling to enhance cancer cell accessibility (TECA) strategy produces an eminent suppression of tumor growth and results in a 97.4% inhibition of lung metastasis, which is superior to the counterpart liposomes. The bLP-mediated TECA strategy provides deeper insights into enhancing nanoparticle accessibility to cancer cells for antitumor therapy.

To develop a highly efficient strategy against tumors, here, a nanocombination (PDC/P@HCuS) was designed and constructed to actualize chemo-phototherapy with the assistance of fluorescence (FL) and photoacoustic (PA) images. First, a type of organic-inorganic hybrid nanosystem (P@HCuS) was engineered by coupling the fluorescence-labeled amphiphilic fPEDC copolymer on the surface of hollow mesoporous copper sulfide nanoparticle (HCuS), in which HCuS was used as a photothermal and PA agent; fPEDC as a stabilizer, chromophore, and redox/pH-sensitive gatekeeper; and both of them as drug carriers. Then, a peptide-drug conjugate (cRGD-SMCC-DM1, PDC), as a molecular targeted maytansinoid, was loaded inside of P@HCuS to form PDC/P@HCuS. Next, the PDC/P@HCuS was investigated carefully with or without near-infrared (NIR) laser irradiation. In vitro, the nanocombination exhibited stimuli-responsive drug release, obvious cellular uptake, strong cytotoxicity to tumor cells, significant impact on cell cycle, and cytoskeleton and cellular proteomics as well as evident permeability into the tumor sphere, most of which could be boosted by NIR laser irradiation. In vivo, the nanocombinaiton exerted good FL/PA imaging features and photothermal efficiency, achieved the best antitumor efficacy in the presence of NIR laser irradiation, and showed excellent biosafety. Together, it demonstrated that the PDC/P@HCuS, representing a chemo-phototherapy based on a nanocombination associated with peptide-drug conjugate, could achieve the highly efficient antitumor effect.

Somatostatin receptor 2 (SSTR2) is frequently overexpressed on several types of solid tumors, including neuroendocrine tumors and small-cell lung cancer. Peptide agonists of SSTR2 are rapidly internalized upon binding to the receptor and linking a toxic payload to an SSTR2 agonist is a potential method to kill SSTR2-expressing tumor cells. Herein, we describe our efforts towards an efficacious SSTR2-targeting cytotoxic conjugate; examination of different SSTR2-targeting ligands, conjugation sites, and payloads led to the discovery of 22 (PEN-221), a conjugate consisting of microtubule-targeting agent DM1 linked to the C-terminal side chain of Tyr 3 -octreotate. PEN-221 demonstrates in vitro activity which is both potent (IC 50 = 10 nM) and receptor-dependent (IC 50 shifts 90-fold upon receptor blockade). PEN-221 targets high levels of DM1 to SSTR2-expressing xenograft tumors, which has led to tumor regressions in several SSTR2-expressing xenograft mouse models. The safety and efficacy of PEN-221 is currently under evaluation in human clinical trials.

Maytansine and its related analogues are a class of highly potent anti-proliferation agents that have failed to be exploited as clinical drugs for human therapy due to unacceptable systemic toxicity. Here, we delineate a novel strategy that combines rational drug conjugation with subsequent nanoparticle assembly to systemically deliver this highly potent and toxic drug. To demonstrate this concept, we covalently coupled the thiolated maytansine derivative, the DM1 agent, to amphiphilic block co-polymers, polyethylene glycol (PEG)-block-polylactide (PLA), in varying molecular weights to generate two prodrug constructs (i.e., PEG 2K -PLA 2K -DM1 and PEG 2K -PLA 4K -DM1) via the maleimide-thiol reaction. The resulting two constructs are amenable to self-assembly in aqueous solutions and are systemically injectable for preclinical studies. In vivo evaluations indicate that PEG-PLA-DM1 conjugate-assembled nanoparticles (NPs) display substantially reduced drug toxicity compared to the free drug forms and NPs that physically encapsulate DM1. Furthermore, following systemic administration, these nanodrugs produced superior therapeutic efficacy over free DM1 in a colon tumor xenograft-bearing mouse model. Therefore, this study provides evidence that the conjugation of toxic drugs to assembling copolymers enables the alleviation of cancer drug toxicity and effective delivery of anticancer drugs. Thus, this DM1-formulated platform represents a new generation of nanotherapeutics that are available for further clinical evaluation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Daratumumab, an FDA approved antibody drug, displays specific targeting ability to abnormal white blood cells overexpressing CD38 and provides efficacious therapy for multiple myeloma. Here, in order to achieve enhanced remission of multiple myeloma, we designed Dara-DM4, antibody drug conjugates (ADCs) by conjugating Daratumumab and DM4 via a disulfide linker. Dara-DM4 showed significantly higher cellular uptake and inhibitory efficacy on MM1S cells that overexpressing CD38 with an IC 50 of 0.88 µg/mL post 72 hr treatment. These results support a promising ADCs strategy for multiple myeloma treatment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cancer stem-like cells (CSCs) are proposed to be responsible for tumor metastasis, resistance and relapse after therapy, but are unable to be eliminated by many current therapies. Herein, we report that the apoferritin nanocages loading cytotoxic mertansine (M-AFN) can significantly improve their uptake in CSCs-enriched tumorspheres and effectively eradicate CSCs in tumorspheres for anticancer therapy. M-AFN were uniformly nanocage structures with the mean diameter of 11.26 ± 2.58 nm and the loading capacity of 0.62%. In the CSCs-enriched tumorsphere model, M-AFN could be preferentially internalized by tumorsphere cells and the average half-inhibitory concentration (IC 50 ) of M-AFN was obviously reduced by 5.46-fold when comparing to the parent 4T1 breast cancer cells. Moreover, both the already existing tumorspheres and the formation of secondary tumorspheres were drastically disrupted by M-AFN, but barely impacted by mertansine alone. The flow cytometer analysis showed the CSCs fractions in tumorspheres were considerably reduced by the M-AFN treatment. Therefore, the apoferritin nanocages represent an encouraging nanoplatform to eradicate CSCs for effective anticancer therapy., (Copyright © 2018. Published by Elsevier B.V.)

Purpose: Antibody-drug conjugates (ADC) utilizing noncleavable linker drugs have been approved for clinical use, and several are in development targeting solid and hematologic malignancies including multiple myeloma. Currently, there are no reliable biomarkers of activity for these ADCs other than presence of the targeted antigen. We observed that certain cell lines are innately resistant to such ADCs, and sought to uncover the underlying mechanism of resistance., Experimental Design: The expression of 43 lysosomal membrane target genes was evaluated in cell lines resistant to ADCs bearing the noncleavable linker, pyrrolobenzodiazepine payload SG3376, in vitro . The functional relevance of SLC46A3, a lysosomal transporter of noncleavable ADC catabolites whose expression uniquely correlated with SG3376 resistance, was assessed using EPHA2-, HER2-, and BCMA-targeted ADCs and isogenic cells overexpressing or genetically inactivated for SLC46A3 . SLC46A3 expression was also examined in patient-derived xenograft and in vitro models of acquired T-DM1 resistance and multiple myeloma bone marrow samples by RT-PCR., Results: Loss of SLC46A3 expression was found to be a mechanism of innate and acquired resistance to ADCs bearing DM1 and SG3376. Sensitivity was restored in refractory lines upon introduction of SLC46A3 , suggesting that expression of SLC46A3 may be more predictive of activity than target antigen levels alone. Interrogation of primary multiple myeloma samples indicated a range of SLC46A3 expression, including samples with undetectable levels like multiple myeloma cell lines resistant to BCMA-targeting DM1 and SG3376 ADCs., Conclusions: Our findings support SLC46A3 as a potential patient selection biomarker with immediate relevance to clinical trials involving these ADCs., (©2018 American Association for Cancer Research.)

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are highly potent biopharmaceuticals designed for targeted cancer therapies. mAbs and ADCs can undergo modifications during production and storage which may affect binding to target receptors, potentially altering drug efficacy. In this work, liquid chromatography was coupled online to surface plasmon resonance (LC-SPR) to allow label-free affinity evaluation of mAb and ADC sample constituents (size and charge variants), under near-native conditions. Trastuzumab and its ADC trastuzumab emtansine (T-DM1) were used as a test sample and were analyzed by aqueous size-exclusion chromatography (SEC)-SPR before and after exposure to aggregate-inducing conditions. SEC-SPR allowed separation of the formed aggregates and measurement of their affinity towards the ligand-binding domain of the human epidermal growth factor receptor 2 (HER2) receptor immobilized on the surface of the SPR sensor chip. The monomer and aggregates of the mAb and ADC were shown to have similar antigen affinity. Conjugation of drugs to trastuzumab appeared to accelerate the aggregate formation. In addition, cation-exchange chromatography (CEX) was coupled to SPR enabling monitoring the maximum ligand-analyte binding capacity (R max ) of individual charge variants present in mAbs. Deamidated species and lysine variants in trastuzumab sample were separated but did not show different binding affinities to the immobilized HER2-binding domain. In order to allow protein variant assignment, parallel MS detection was added to the LC-SPR setup using a column effluent split. The feasibility of the LC-MS/SPR system was demonstrated by analysis of trastuzumab and T-DM1 providing information on antibody glycoforms and/or determination of the drug-to-antibody ratio (DAR), while simultaneously monitoring binding of eluting species to HER2. Graphical abstract ᅟ.

Specific drug delivery to metastatic tumors remains a great challenge for antimetastasis therapy. We herein report a bioengineered macrophage-based delivery system (LD-MDS) that can be preferentially delivered to lung metastases and intelligently transformed into nanovesicles and secondary nanovesicles for antimetastasis therapy. LD-MDS was prepared by anchoring a legumain-specific propeptide of melittin (legM) and cytotoxic soravtansine (DM4) prodrug onto the membrane of living macrophages. LD-MDS is responsively activated by legumain protease and converted into DM4-loaded exosome-like nanovesicles (DENs), facilitating efficient internalization by metastatic 4T1 cancer cells and considerable cell death. Afterward, the damaged 4T1 cells can release secondary nanovesicles and free drug molecules to destroy neighboring cancer cells. In vivo, LD-MDS displays superior targeting efficiency for lung metastatic lesions with diameters less than 100 μm and remarkably inhibits lung metastasis. This study provides a new opportunity to explore endogenous macrophages as living drug delivery vehicles with controlled drug release to target metastatic lung tumors.

Our aim was to synthesize 89 Zr-labeled trastuzumab-emtansine ( 89 Zr-DFO-T-DM1) to probe the delivery of trastuzumab-emtansine (T-DM1) to HER2-positive breast cancer (BC) by positron emission tomography (PET). We further aimed to compare the tumor and normal tissue uptake of 89 Zr-DFO-T-DM1 with 89 Zr-DFO-trastuzumab. T-DM1 was modified with 3.0 ± 0.2 desferrioxamine (DFO) chelators for complexing 89 Zr by reaction with a 14-fold molar excess of p-NCS-Bz-DFO. The number of DFO chelators per T-DM1 molecule was quantified spectrophotometrically at 430 nm after the reaction with FeCl 3 . SDS-PAGE and SE-HPLC demonstrated a pure and homogeneous immunoconjugate. DFO-T-DM1 and DFO-trastuzumab were labeled to high efficiency (>97%) with 89 Zr at a specific activity of 0.55 MBq/μg in a 2 M Na 2 CO 3 /0.5 M HEPES buffer, pH 7.0, at RT for 60-90 min. The labeling efficiency was measured by instant thin layer-silica gel chromatography (ITLC-SG) and SE-HPLC. HER2 immunoreactivity was measured in a saturation binding assay using SK-BR-3 human BC cells. 89 Zr-DFO-T-DM1 exhibited high affinity HER2 binding ( K d = 3.7 ± 0.4 nM) that was not significantly different than 89 Zr-DFO-trastuzumab (4.4 ± 0.5 nM; P = 0.06). The optimal time for tumor imaging with 89 Zr-DFO-T-DM1 was 96 h post-injection in NOD-scid mice with s.c. HER2 overexpressing (HER2 3+) BT-474 human BC xenografts. Tumor uptake was dependent on the level of HER2 expression in mice with s.c. BT-474 (HER2 3+), MDA-MB-231/H2N (HER2 2+), MDA-MB-231 (HER2 0-1+), or MDA-MB-468 (HER2 0) human BC xenografts injected with 89 Zr-DFO-T-DM1 (10 μg, 5.2 MBq). All tumors were visualized by microPET/CT, but the tumor intensity was greatest for BT-474 and MDA-MB-231/H2N xenografts. The tumor uptake of 89 Zr-DFO-T-DM1 was 4.1-fold significantly higher than 89 Zr-DFO-trastuzumab in mice with s.c. BT-474 (HER2 3+) xenografts (43.5 ± 4.3%ID/g vs 10.6 ± 5.4%ID/g, respectively; P < 0.001). Tumor uptake of 89 Zr-DFO-T-DM1 in MDA-MB-231/H2N xenografts (HER2 2+) was 3.7-fold significantly higher than 89 Zr-DFO-trastuzumab (10.1 ± 3.6%ID/g vs 2.7 ± 0.5%ID/g; P < 0.001). The higher tumor uptake of 89 Zr-DFO-T-DM1 compared to 89 Zr-DFO-trastuzumab was not due to a higher HER2 binding affinity or to differences in the residence time in the blood or tumor size. We conclude that 89 Zr-DFO-T-DM1 is a useful probe to assess the delivery of T-DM1 to HER2-positive BC. PET with 89 Zr-DFO-trastuzumab has been studied clinically to predict response to T-DM1, but our results suggest that 89 Zr-DFO-T-DM1 may be more accurate due to the differences in the tumor uptake observed in the preclinical BC xenograft mouse models.

The commercially available antibody-drug conjugate (ADC) product, Kadcyla ® is synthesized using a 2-step reaction, wherein the linker is conjugated to native lysines on the mAb in step 1, followed by drug conjugation to the linker-modified antibody in step 2. In our study, we synthesized a lysine-conjugated ADC (Syn-ADC) on the same trastuzumab scaffold as Kadcyla ® using a 1-step reaction. Mass spectrometry of both products revealed a subpopulation of Kadcyla ® containing free linkers conjugated to the mAb, but not conjugated to the drug, which were absent in the 1-step reaction ADC product. Differential scanning calorimetry thermograms showed that the drug and linker conjugation significantly reduced the thermal stability and energies of activation for the denaturation of the C H 2 domain of the ADCs. The heating induced aggregation events started as early as ∼57°C and ∼45°C for Kadcyla ® and Syn-ADC, respectively, compared with 71°C for Herceptin ® . The colloidal stability measurements clearly showed that the hydrophobic drug payload on ADCs significantly reduced the repulsive interprotein interactions when compared to the unconjugated antibody under formulation buffer conditions (pH 6.0). Attaching hydrophobic drug and linker moieties onto the antibody lowered the thermal and colloidal stabilities and increased the aggregation propensity of the ADCs., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Aim: Compared with small molecules, LC-MS quantitation of larger biotherapeutic proteins such as antibodies and antibody-drug conjugates at the intact level presents many challenges in both LC and MS due to their higher molecular weight, bigger size, structural complexity and heterogeneity., Results & Conclusion: In this study, quantitation of an intact lysine-linked antibody-drug conjugate, trastuzumab emtansine is presented. Trastuzumab emtansine was extracted from rat plasma using bead-based immunoaffinity capture; after elution from the beads, it was directly analyzed on a LC-HRMS system. Quantitation using both extracted ion chromatogram and deconvoluted mass peaks was evaluated. A limit of quantitation was approximately 20 ng on column with a linear dynamic range from 5 to 100 μg/ml. In addition, the reproducibility and distribution of the drug-to-antibody ratio at different trastuzumab emtansine concentrations were discussed.

Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Grade 3 endometrioid and uterine serous carcinomas (USC) account for the vast majority of endometrial cancer deaths. The purpose of this study was to determine folic acid receptor alpha (FRα) expression in these biologically aggressive (type II) endometrial cancers and evaluate FRα as a targetable receptor for IMGN853 (mirvetuximab soravtansine). The expression of FRα was evaluated by immunohistochemistry (IHC) and flow cytometry in 90 endometrioid and USC samples. The in vitro cytotoxic activity and bystander effect were studied in primary uterine cancer cell lines expressing differential levels of FRα. In vivo antitumor efficacy of IMGN853 was evaluated in xenograft/patient-derived xenograft (PDX) models. Semiquantitative IHC analysis indicated that 41% of the USC patients overexpress FRα. Further, overexpression of FRα (i.e., 2+) was detected via flow cytometry in 22% (2/9) of primary endometrioid and in 27% (3/11) of primary USC cell lines. Increased cytotoxicity was seen with IMGN853 treatment compared with control in 2+ expressing uterine tumor cell lines. In contrast, tumor cell lines with low FRα showed no difference when exposed to IMGN853 versus control. IMGN853 induced bystander killing of FRα = 0 tumor cells. In an endometrioid xenograft model (END(K)265), harboring 2+ FRα, IMGN853 treatment showed complete resolution of tumors ( P < 0.001). Treatment with IMGN853 in the USC PDX model (BIO(K)1), expressing 2+ FRα, induced twofold increase in median survival ( P < 0.001). IMGN853 shows impressive antitumor activity in biologically aggressive FRα 2+ uterine cancers. These preclinical data suggest that patients with chemotherapy resistant/recurrent endometrial cancer overexpressing FRα may benefit from this treatment. Mol Cancer Ther; 17(5); 1003-11. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Trastuzumab emtansine is an antibody-drug conjugate comprised of the anti-HER2 monoclonal antibody trastuzumab linked to DM1 (emtansine), a potent cytotoxic maytansinoid derivative, by a stable linker. This structure results in improved tumor-directed cytotoxicity in HER2+ breast cancer with reduced systemic toxicities, particularly the cardiac toxicities associated with single agent trastuzumab. Phase III trials have demonstrated improved progression-free and overall survival in heavily pretreated patients with advanced HER2+ breast cancer, with an acceptable toxicity profile. However, its role in first-line treatment is less clear. Ongoing studies continue to evaluate its role in neoadjuvant and adjuvant management of HER2+ breast cancer.

Antibody-drug conjugates (ADC) represent an emerging, novel class of biopharmaceuticals. The heterogeneity originating from the sophisticated structure requires orthogonal analytical techniques for quality and stability assessment of ADC to ensure safety and efficacy. In this study, the stability of Trastuzumab (recombinant humanized IgG1 mAb, targeting HER2 receptor) and its ADC with DM1 (anti-tubulin anticancer drug), Trastuzumab emtansine (T-DM1) were studied. SE-HPLC was used to monitor formation of aggregates and/or fragments of the monoclonal antibodies (mAb). Correlation with the results of reducing and non-reducing sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS) were performed to interpret the obtained results. RP-HPLC was used for assessment of the stability of DM1 in ADC while spectrophotometry was employed to determine drug antibody ratio (DAR) . The studied drugs were subjected to several stress conditions including pH, temperature, mechanical agitation and repeated freeze and thaw to generate possible degradation products and ensure suitability of the assay protocol. The degradation pattern and extent were demonstrated under the indicated stress conditions. The correlation between the results of SE-HPLC and those of SDS-PAGE and DLS ensured the validity of the orthogonal assay protocol and indicated aggregates that were not detected using SE-HPLC. Results showed clearly that T-DM1 is relatively less stable than its parent mAb. This was attributed to the presence of the drug-linker part that is attached to the mAb. RP-HPLC showed that the cytotoxic drug moiety is liable for degradation under the studied conditions resulting in alteration of DAR as well as formation of degradation products. This confirmed the need for more robust coupling chemistries for production of safe and effective ADC and highlighted the importance of orthogonal testing protocols for quality assessment. The assay protocol should be applicable for quality and stability assessment of various ADC., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The selective destruction of tumour cells while sparing healthy tissues is one of the main challenges in cancer therapy. Antibody-drug conjugates (ADCs) are arguably the most rapidly expanding class of targeted cancer therapies. Efficient drug conjugation and release technologies are essential for the development of these new therapeutic agents. In response to the ever-increasing demand for efficient drug release systems, we have developed a new class of β-galactosidase-cleavable linkers for ADCs. Within this framework, novel payloads comprising a galactoside linker, the monomethyl auristatin E (MMAE) and cysteine-reactive groups were synthesized, conjugated with trastuzumab and evaluated both in vitro and in vivo. The ADCs with galactoside linkers demonstrated superior therapeutic efficacy in mice compared to the marketed trastuzumab emtansine used for the treatment of breast cancer., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Background: The aim of this review was to systematically evaluate the safety and efficacy of the addition of pertuzumab to trastuzumab emtansine (T-DM1) ± taxane in patients with human epidermal growth factor receptor 2 (HER2)-positive, locally advanced breast cancer (LABC) or metastatic breast cancer (MBC)., Materials and Methods: Several databases were searched for relevant clinical trials. The study characteristics, details of adverse events (AEs) and details of treatment efficacy were extracted for analysis., Results: Six studies with 996 patients were included. Common AEs of T-DM1 + pertuzumab ± taxane included fatigue, diarrhea, nausea, epistaxis, peripheral neuropathy, increased aspartate transaminase (AST), increased alanine transaminase (ALT) and thrombocytopenia. Major grade ≥3 AEs of T-DM1 + pertuzumab ± taxane included thrombocytopenia, neutropenia, fatigue, increased ALT, anemia and peripheral neuropathy. The addition of pertuzumab to T-DM1 ± taxane led to higher risks of diarrhea (especially grade ≥3 diarrhea), rash and vomiting, and decreased risks of thrombocytopenia and grade ≥3 increased AST. The relative risks of the addition of pertuzumab to T-DM1 ± taxane for objective response (1.068, 95% CI 0.945-1.207) and clinical benefit (1.038, 95% CI 0.974-1.106) were not statistically significant., Conclusion: Common AEs should be carefully monitored in HER2-positive LABC or MBC patients treated with T-DM1 + pertuzumab ± taxane. The addition of pertuzumab to T-DM1 ± taxane showed noninferior, but not superior, objective response rate and clinical benefit rate. However, more studies are needed to further verify these findings., Competing Interests: Disclosure The authors report no conflicts of interest in this work.