Cathepsin B Processing Is Required for the In Vivo Efficacy of Albumin-Drug Conjugates.

Targeted drug delivery approaches that selectively and preferentially deliver therapeutic agents to specific tissues are of great interest for safer and more effective pharmaceutical treatments. We investigated whether cathepsin B cleavage of a valine-citrulline [VC(S)]-containing linker is required for the release of monomethyl auristatin E (MMAE) from albumin-drug conjugates. In this study, we used an engineered version of human serum albumin, Veltis High Binder II (HBII), which has enhanced binding to the neonatal Fc (fragment crystallizable) receptor (FcRn) to improve drug release upon binding and FcRn-mediated recycling. The linker-payload was conjugated to cysteine 34 of albumin using a carbonylacrylic (caa) reagent which produced homogeneous and plasma stable conjugates that retained FcRn binding. Two caa-linker-MMAE reagents were synthesized─one with a cleavable [VC(S)] linker and one with a noncleavable [VC(R)] linker─to question whether protease-mediated cleavage is needed for MMAE release. Our findings demonstrate that cathepsin B is required to achieve efficient and selective antitumor activity. The conjugates equipped with the cleavable [VC(S)] linker had potent antitumor activity in vivo facilitated by the release of free MMAE upon FcRn binding and internalization. In addition to the pronounced antitumor activity of the albumin conjugates in vivo, we also demonstrated their preferable tumor biodistribution and biocompatibility with no associated toxicity or side effects. These results suggest that the use of engineered albumins with high FcRn binding combined with protease cleavable linkers is an efficient strategy to target delivery of drugs to solid tumors.

Antibody-Drug Conjugate αEGFR-E-P125A Reduces Triple-negative Breast Cancer Vasculogenic Mimicry, Motility, and Metastasis through Inhibition of EGFR, Integrin, and FAK/STAT3 Signaling.

Primary tumor growth and metastasis in triple-negative breast cancer (TNBC) require supporting vasculature, which develop through a combination of endothelial angiogenesis and vasculogenic mimicry (VM), a process associated with aggressive metastatic behavior in which vascular-like structures are lined by tumor cells. We developed αEGFR-E-P125A, an antibody-endostatin fusion protein that delivers a dimeric, mutant endostatin (E-P125A) payload that inhibits TNBC angiogenesis and VM in vitro and in vivo. To characterize the mechanisms associated with induction and inhibition of VM, RNA sequencing (RNA-seq) of MDA-MB-231-4175 TNBC cells grown in a monolayer (two-dimensional) was compared with cells plated on Matrigel undergoing VM [three-dimensional (3D)]. We then compared RNA-seq between TNBC cells in 3D and cells in 3D with VM inhibited by αEGFR-E-P125A (EGFR-E-P125A). Gene set enrichment analysis demonstrated that VM induction activated the IL6-JAK-STAT3 and angiogenesis pathways, which were downregulated by αEGFR-E-P125A treatment.Correlative analysis of the phosphoproteome demonstrated decreased EGFR phosphorylation at Y1069, along with decreased phosphorylation of focal adhesion kinase Y397 and STAT3 Y705 sites downstream of α5ß1 integrin. Suppression of phosphorylation events downstream of EGFR and α5ß1 integrin demonstrated that αEGFR-E-P125A interferes with ligand-receptor activation, inhibits VM, and overcomes oncogenic signaling associated with EGFR and α5ß1 integrin cross-talk. In vivo, αEGFR-E-P125A treatment decreased primary tumor growth and VM, reduced lung metastasis, and confirmed the inhibition of signaling events observed in vitro. Simultaneous inhibition of EGFR and α5ß1 integrin signaling by αEGFR-E-P125A is a promising strategy for the inhibition of VM, tumor growth, motility, and metastasis in TNBC and other EGFR-overexpressing tumors. SIGNIFICANCE: αEGFR-E-P125A reduces VM, angiogenesis, tumor growth, and metastasis by inhibiting EGFR and α5ß1 integrin signaling, and is a promising therapeutic agent for TNBC treatment, used alone or in combination with chemotherapy.

An immunomodulatory antibody-drug conjugate targeting BDCA2 strongly suppresses plasmacytoid dendritic cell function and glucocorticoid responsive genes.

OBJECTIVES: Blood dendritic cell antigen 2 (BDCA2) is exclusively expressed on plasmacytoid dendritic cells (pDCs) whose uncontrolled production of type I IFN (IFN-I) is crucial in pathogenesis of SLE and other autoimmune diseases. Although anti-BDCA2 antibody therapy reduced disease activity in SLE patients, its clinical efficacy needs further improvement. We developed a novel glucocorticoid receptor agonist and used it as a payload to conjugate with an anti-BDCA2 antibody to form an BDCA2 antibody-drug conjugate (BDCA2-ADC). The activation of BDCA2-ADC was evaluated in vitro. METHODS: Inhibitory activity of BDCA2-ADC was evaluated in peripheral blood mononuclear cells or in purified pDCs under ex vivo toll-like receptor agonistic stimulation. The global gene regulation in purified pDCs was analysed by RNA-seq. The antigen-dependent payload delivery was measured by reporter assay. RESULTS: The BDCA2-ADC molecule causes total suppression of IFNα production and broader inhibition of inflammatory cytokine production compared with the parental antibody in human pDCs. Global gene expression analysis confirmed that the payload and antibody acted synergistically to regulate both type I IFN signature genes and glucocorticoid responsive genes in pDCs. CONCLUSION: Taken together, these data suggest dual mechanisms of BDCA2-ADC on pDCs and the potential for BDCA2-ADC to be the first ADC treatment for SLE in the world and a better treatment option than anti-BDCA2 antibody for SLE patients.

Generation and Characterization of Iduronidase-Cleavable ADCs.

A crucial design feature for the therapeutic success of antibody-drug conjugates (ADCs) is the linker that connects the antibody with the drug. Linkers must be stable in circulation and efficiently release the drug inside the target cell, thereby having a fundamental impact on ADC pharmacokinetics and efficacy. The variety of enzymatically cleavable linkers applied in ADCs is limited, and some are believed to be associated with unwanted side effects due to the expression of cleavage-mediating enzymes in nonmalignant cells. Based on a bioinformatic screen of lysosomal enzymes, we identified α-l-iduronidase (IduA) as an interesting candidate for ADC linker cleavage because of its low expression in normal tissues and its overexpression in several tumor types. In the present study, we report a novel IduA-cleavable ADC linker using exatecan and duocarmycin as payloads. We showed the functionality of our linker system in cleavage assays using recombinant IduA or cell lysates and compared it to established ADC linkers. Subsequently, we coupled iduronide-exatecan via interchain cysteines or iduronide-duocarmycin via microbial transglutaminase (mTG) to an anti-CEACAM5 (aCEA5) antibody. The generated iduronide-exatecan ADC showed high serum stability and similar target-dependent tumor cell killing in the subnanomolar range but reduced toxicity on nonmalignant cells compared to an analogous cathepsin B-activatable valine-citrulline-exatecan ADC. Finally, in vivo antitumor activity could be demonstrated for an IduA-cleavable duocarmycin ADC. The presented results emphasize the potential of iduronide linkers for ADC development and represent a tool for further balancing out tumor selectivity and safety.

The endosomal-lysosomal system in ADC design and cancer therapy.

INTRODUCTION: This discourse delves into the intricate connections between the endosomal-lysosomal system and antibody-drug conjugates (ADCs), shedding light on an essential yet less understood dimension of targeted therapy. While ADCs have revolutionized cancer treatment, resistance remains a formidable challenge, often involving diverse and overlapping mechanisms. AREAS COVERED: This discourse highlights the roles of various components within the endosomal machinery, including Rab proteins, in ADC resistance development. It also explores how the transferrin-transferrin receptor and epidermal growth factor-epidermal growth factor receptor complexes, known for their roles in recycling and degradation process, respectively, can offer valuable insights for ADC design. Selected strategies to enhance lysosomal targeting are discussed, and potentially offer solutions to improve ADC efficacy. EXPERT OPINION: By harnessing these different insights that connect ADCs with the endosomal-lysosomal system, the field may benefit to shape the next-generation of ADC design for increased efficacy and improved patient outcomes.

Targeting carcinoma-associated mesothelial cells with antibody-drug conjugates in ovarian carcinomatosis.

Ovarian carcinomatosis is characterized by the accumulation of carcinoma-associated mesothelial cells (CAMs) in the peritoneal stroma and mainly originates through a mesothelial-to-mesenchymal transition (MMT) process. MMT has been proposed as a therapeutic target for peritoneal metastasis. Most ovarian cancer (OC) patients present at diagnosis with peritoneal seeding, which makes tumor progression control difficult by MMT modulation. An alternative approach is to use antibody-drug conjugates (ADCs) targeted directly to attack CAMs. This strategy could represent the cornerstone of precision-based medicine for peritoneal carcinomatosis. Here, we performed complete transcriptome analyses of ascitic fluid-isolated CAMs in advanced OC patients with primary-, high-, and low-grade, serous subtypes and following neoadjuvant chemotherapy. Our findings suggest that both cancer biological aggressiveness and chemotherapy-induced tumor mass reduction reflect the MMT-associated changes that take place in the tumor surrounding microenvironment. Accordingly, MMT-related genes, including fibroblast activation protein (FAP), mannose receptor C type 2 (MRC2), interleukin-11 receptor alpha (IL11RA), myristoylated alanine-rich C-kinase substrate (MARCKS), and sulfatase-1 (SULF1), were identified as specific actionable targets in CAMs of OC patients, which is a crucial step in the de novo design of ADCs. These cell surface target receptors were also validated in peritoneal CAMs of colorectal cancer peritoneal implants, indicating that ADC-based treatment could extend to other abdominal tumors that show peritoneal colonization. As proof of concept, a FAP-targeted ADC reduced tumor growth in an OC xenograft mouse model with peritoneal metastasis-associated fibroblasts. In summary, we propose MMT as a potential source of ADC-based therapeutic targets for peritoneal carcinomatosis. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Trimming Crystallizable Fragment (Fc) Glycans Enables the Direct Enzymatic Transfer of Biomacromolecules to Antibodies as Therapeutics.

Glycoengineering has provided powerful tools to construct site-specific antibody conjugates. However, only small-molecule payloads can be directly transferred to native or engineered antibodies using existing glycoengineering strategies. Herein, we demonstrate that reducing the complexity of crystallizable fragment (Fc) glycans could dramatically boost the chemoenzymatic modification of immunoglobulin G (IgG) via an engineered fucosyltransferase. In this platform, antibodies with Fc glycans engineered to a simple N-acetyllactosamine (LacNAc) disaccharide are successfully conjugated to biomacromolecules, such as oligonucleotides and nanobodies, in a single step within hours. Accordingly, we synthesized an antibody-conjugate-based anti-human epidermal growth factor receptor 2 (HER2)/ cluster of differentiation 3 (CD3) bispecific antibody and used it to selectively destroy patient-derived cancer organoids by reactivating endogenous T lymphocyte cells (T cells) inside the organoid. Our results highlight that this platform is a general approach to construct antibody-biomacromolecule conjugates with translational values.

Targeting CD44 Variant 5 with an Antibody-Drug Conjugate Is an Effective Therapeutic Strategy for Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second most frequent type of primary liver cancer. ICC is among the deadliest malignancies, highlighting that novel treatments are urgently needed. Studies have shown that CD44 variant isoforms, rather than the CD44 standard isoform, are selectively expressed in ICC cells, providing an opportunity for the development of an antibody-drug conjugate (ADC)-based targeted therapeutic strategy. In this study, we observed the specific expression of CD44 variant 5 (CD44v5) in ICC tumors. CD44v5 protein was expressed on the surface of most ICC tumors (103 of 155). A CD44v5-targeted ADC, H1D8-DC (H1D8-drug conjugate), was developed that comprises a humanized anti-CD44v5 mAb conjugated to the microtubule inhibitor monomethyl auristatin E (MMAE) via a cleavable valine-citrulline-based linker. H1D8-DC exhibited efficient antigen binding and internalization in cells expressing CD44v5 on the cell surface. Because of the high expression of cathepsin B in ICC cells, the drug was preferentially released in cancer cells but not in normal cells, thus inducing potent cytotoxicity at picomolar concentrations. In vivo studies showed that H1D8-DC was effective against CD44v5-positive ICC cells and induced tumor regression in patient-derived xenograft models, whereas no significant adverse toxicities were observed. These data demonstrate that CD44v5 is a bona fide target in ICC and provide a rationale for the clinical investigation of a CD44v5-targeted ADC-based approach. SIGNIFICANCE: Elevated expression of CD44 variant 5 in intrahepatic cholangiocarcinoma confers a targetable vulnerability using the newly developed antibody-drug conjugate H1D8-DC, which induces potent growth suppressive effects without significant toxicity.

Development of 177Lu-Cetuximab-PAMAM dendrimeric nanosystem: a novel theranostic radioimmunoconjugate.

PURPOSE: Epidermal growth factor receptors (EGFRs) are overexpressed in a wide range of tumors and are attractive candidates to target in targeted therapies. This study aimed to introduce a novel radiolabeled compound, 177Lu-cetuximab-PAMAM G4, for the treatment of EGFR-expressing tumors. METHODS: In this study, the cetuximab mAb was bound to PAMAM G4 and labeled with 177Lu via DTPA-CHX chelator. The synthesized nanosystem was confirmed by different analyses such as DLS, FT-IR, TEM, and RT-LC. Cell viability of the radioimmunoconjugate was assessed over the EGFR-expressing cell line of SW480. The biodistribution of 177Lu-Cetuximab-PAMAMG4 was determined in different intervals after injection of the radiolabeled compound in normal and tumoral nude mice via scarification and SPECT images. RESULTS: The average size of PAMAM G4 and PAMAM-Cetuximab-DTPA-CHX nanoparticles were 2 and 70 nm, respectively. 177Lu-Cetuximab-PAMAMG4 was prepared with radiochemical purity of more than 98%. The survival rates of SW480 cells at 24, 48, and 72 h post-treatment with177Lu-Cetuximab-PAMAMG4 (500 nM) were 18%, 15%, and 14%, respectively. The biodistribution studies showed a significant accumulation of 177Lu-Cetuximab-PAMAM in the EGFR-expressing tumor. CONCLUSION: According to the results, this new agent can be considered as an efficient therapeutic complex for tumors expressing EGFR receptors.

Co-Clinical Study of [fam-] Trastuzumab Deruxtecan (DS8201a) in Patient-Derived Xenograft Models of Uterine Carcinosarcoma and Its Association with Clinical Efficacy.

PURPOSE: Uterine carcinosarcoma (UCS), a subtype of endometrial carcinoma, is a rare and aggressive cancer with a poor prognosis. High clinical efficacy of trastuzumab deruxtecan (T-DXd) in HER2-expressing UCS was recently reported in a phase II trial (STATICE trial). We performed a co-clinical study of T-DXd using patient-derived xenograft (PDX) models of participants in the STATICE trial. EXPERIMENTAL DESIGN: Tumor specimens were resected during primary surgery or biopsied at recurrence from patients with UCS and transplanted into immunodeficient mice. Seven UCS-PDXs from six patients were established and HER2, estrogen receptor (ER), and p53 expression in PDX and the original tumor was assessed. Drug efficacy tests were performed using six of the seven PDXs. Of the six UCS-PDXs tested, two were derived from patients enrolled in the STATICE trial. RESULTS: The histopathological characteristics of the six PDXs were well-conserved from the original tumors. HER2 expression was 1+ in all PDXs, and ER and p53 expression was almost similar to that in the original tumors. Remarkable tumor shrinkage after T-DXd administration was observed in four of the six PDXs (67%), comparable with the response rate (70%) of HER2 1+ patients in the STATICE trial. Two patients enrolled in the STATICE trial showed partial response as the best response, and the clinical effect was well-replicated with marked tumor shrinkage. CONCLUSIONS: We successfully performed a co-clinical study of T-DXd in HER2-expressing UCS, along with the STATICE trial. Our PDX models can predict clinical efficacy and serve as an effective preclinical evaluation platform.

In silico engineering a CD80 variant with increased affinity to CTLA-4 and decreased affinity to CD28 for optimized cancer immunotherapy.

CD80 or cluster of differentiation 80, also known as B7-1, is a member of the immunoglobulin super family, which binds to CTLA-4 and CD28 T cell receptors and induces inhibitory and inductive signals respectively. Although CTLA-4 and CD28 receptors belong to the same protein family, slight differences in their structures leads to CD80 having a higher binding affinity to CTLA-4 (-14.55 kcal/mol) compared with CD28(-12.51 kcal/mol). In this study, we constructed a variant of CD80 protein with increased binding affinity to CTLA-4 and decreased binding affinity to CD28. This variant has no signaling capability, and can act as a cap for these receptors to protect them from natural CD80 proteins existing in the body. The first step was the evolutionary and alanine scanning analysis of CD80 protein to determine conserved regions in this protein. Next, complex alanine scanning technique was employed to determine CD80 protein hotspots in CD80-CTLA-4 and CD80-CD28 protein complexes. This information was fed into a computational model developed in R for in silico mutagenesis and CD80 variant library construction. The 3D structures of variants were modeled using the Swiss model webserver. After modeling the 3D structures, HADDOCK server was employed to build all protein-protein complexes, which contain CTLA-4-CD80 variant complexes, Wild type CD80-CD28 complexes and CD28-CD80 variant complexes. Protein-protein binding free energy was determined using FoldX and the variant number 316 with mutations at 29, 31, 33 positions showed increased binding affinity to CTLA-4 (-21.43 kcal/mol) and decreased binding affinity to CD28 (- 9.54 kcal/mol). Finally, molecular dynamics (MD) simulations confirmed the stability of variant 316. In conclusion, we designed a new CD80 protein variant with potential immunotherapeutic applications.

Non-internalising antibody-drug conjugates.

Antibody-drug conjugates (ADCs) typically require internalisation into cancer cells to release their cytotoxic payload. However, this places stringent constraints on therapeutic development, requiring cancer targets that have high expression of internalising antigens and efficient intracellular processing. An alternative approach is emerging whereby the payloads can be released extracellularly from cleavable linkers upon binding to poorly-internalising antigens or other tumoral components. This removes the reliance on high antigen expression, avoids potentially inefficient internalisation, and can greatly expand the range of cancer targets to components of the extracellular tumour matrix. This review gives an overview of recent developments towards non-internalising ADCs, including emerging cancer-associated cell surface and extracellular proteins, cancer stromal targeting and the linking chemistry that enables extracellular payload release.

A Novel Family of Acid-Cleavable Linker Based on Cyclic Acetal Motifs for the Production of Antibody-Drug Conjugates with High Potency and Selectivity.

Cleavable linkers have become the subject of intense study in the field of chemical biology, particularly because of their applications in the construction of antibody-drug conjugates (ADC), where they facilitate lysosomal cleavage and liberation of drugs from their carrier protein. Due to lysosomes' acidic nature, acid-labile motifs have attracted much attention, leading to the development of hydrazone and carbonate linkers among several other entities. Continuing our efforts in designing new moieties, we present here a family of cyclic acetals that exhibit excellent plasma stability and acid lability, notably in lysosomes. Incorporated in ADC, they led to potent constructs with picomolar potency in vitro and similar in vivo efficacy as the commercially available ADC Kadcyla in mouse xenograft models.

An Enzymatically Cleavable Tripeptide Linker for Maximizing the Therapeutic Index of Antibody-Drug Conjugates.

Valine-citrulline is a protease-cleavable linker commonly used in many drug delivery systems, including antibody-drug conjugates (ADC) for cancer therapy. However, its suboptimal in vivo stability can cause various adverse effects such as neutropenia and hepatotoxicity, leading to dose delays or treatment discontinuation. Here, we report that glutamic acid-glycine-citrulline (EGCit) linkers have the potential to solve this clinical issue without compromising the ability of traceless drug release and ADC therapeutic efficacy. We demonstrate that our EGCit ADC resists neutrophil protease-mediated degradation and spares differentiating human neutrophils. Notably, our anti-HER2 ADC shows almost no sign of blood and liver toxicity in healthy mice at 80 mg kg-1. In contrast, at the same dose level, the FDA-approved anti-HER2 ADCs Kadcyla and Enhertu show increased levels of serum alanine aminotransferase and aspartate aminotransferase and morphologic changes in liver tissues. Our EGCit conjugates also exert greater antitumor efficacy in multiple xenograft tumor models compared with Kadcyla and Enhertu. This linker technology could substantially broaden the therapeutic windows of ADCs and other drug delivery agents, providing clinical options with improved efficacy and safety.

Discerning subsets of breast cancer with very low and absent HER2 protein expression.

Breast cancers are currently eligible for treatment with anti-HER2 therapies if they exhibit amplification of the gene ERBB2 and overexpression of its protein product HER2. Recently, breast cancers with low HER2 expression have shown response to novel anti-HER2 antibody-drug conjugates, and the lower end of "low-HER2" tumors has not yet been clinically delineated. The historically binary approach to HER2 scoring will need to evolve and reporting of HER2 status may require refinement to better stratify low-HER2 statuses. We performed a quality review of HER2 immunohistochemical (IHC) scoring of breast carcinomas with low HER2 expression (71 core biopsies and 51 excisions). We also investigated the feasibility of discerning cases with total lack of HER2 expression from those cases with "very low" HER2 expression that did not meet current criteria for a HER2(1+) score. Rescoring HER2 achieved substantial agreement when performed at 200×, and near-perfect agreement at 400× magnification. Examination under 400× magnification led to recognition of more cases with HER2 expression. Less than 10% of cases showed complete lack of HER2 protein expression by IHC. Cases with "very low" expression were readily identified, and such a category would be feasible to implement in pathologist workflow.

A recombinant scFv antibody-based fusion protein that targets EGFR associated with IMPDH2 downregulation and its drug conjugate show therapeutic efficacy against esophageal cancer.

The present study aimed to evaluate the anti-tumor efficacy of the epidermal growth factor receptor (EGFR)-targeting recombinant fusion protein Fv-LDP-D3 and its antibody-drug conjugate Fv-LDP-D3-AE against esophageal cancer. Fv-LDP-D3, consisting of the fragment variable (Fv) of an anti-EGFR antibody, the apoprotein of lidamycin (LDP), and the third domain of human serum albumin (D3), exhibited a high binding affinity for EGFR-overexpressing esophageal cancer cells, inhibited EGFR phosphorylation and down-regulated inosine monophosphate dehydrogenase type II (IMPDH2) expression. Fv-LDP-D3 was taken up by cancer cells through intensive macropinocytosis; it inhibited the proliferation and induced the apoptosis of esophageal cancer cells. In vivo imaging revealed that Fv-LDP-D3 displayed specific tumor-site accumulation and a long-lasting retention over a 26-day period. Furthermore, Fv-LDP-D3-AE, a pertinent antibody-drug conjugate prepared by integrating the enediyne chromophore of lidamycin into the Fv-LDP-D3 molecule, displayed highly potent cytotoxicity, inhibited migration and invasion, induced apoptosis and DNA damage, arrested cells at G2/M phase, and caused mitochondrial damage in esophageal cancer cells. More importantly, both of Fv-LDP-D3 and Fv-LDP-D3-AE markedly inhibited the growth of esophageal cancer xenografts in athymic mice at well tolerated doses. The present results indicate that Fv-LDP-D3, and Fv-LDP-D3-AE exert prominent antitumor efficacy associated with targeting EGFR, suggesting their potential as promising candidates for targeted therapy against esophageal cancer.

Impact of Drug Conjugation on Thermal and Metabolic Stabilities of Aglycosylated and N-Glycosylated Antibodies.

N-linked glycosylation is one of the most common and complex posttranslational modifications that govern the biological functions and physicochemical properties of therapeutic antibodies. We evaluated thermal and metabolic stabilities of antibody-drug conjugates (ADCs) with payloads attached to the C'E loop in the immunoglobulin G (IgG) Fc CH2 domain, comparing the glycosylated and aglycosylated Fc ADC variants. Our study revealed that introduction of small-molecule drugs into an aglycosylated antibody can compensate for thermal destabilization originating from structural distortions caused by elimination of N-linked glycans. Depending on the conjugation site, glycans had both positive and negative effects on plasma stability of ADCs. The findings highlight the importance of consideration for selection of conjugation site to achieve desirable physicochemical properties and plasma stability.

Antibody-drug conjugates targeting CD248 inhibits liver fibrosis through specific killing on myofibroblasts.

BACKGROUND: Chronic liver injury induces pathological repair, resulting in fibrosis, during which hepatic stellate cells (HSCs) are activated and transform into myofibroblasts. CD248 is mainly expressed on myofibroblasts and was considered as a promising target to treat fibrosis. The primary aim of this study was to generate a CD248 specific antibody-drug conjugate (ADC) and evaluate its therapeutic efficacy for liver fibrosis and its safety in vivo. METHODS: CD248 expression was examined in patients with liver cirrhosis and in mice with CCl4-induced liver fibrosis. The ADC IgG78-DM1, which targets CD248, was prepared and its bioactivity on activated primary HSCs was studied. The anti-fibrotic effects of IgG78-DM1 on liver fibrosis were evaluated in CCl4-induced mice. The reproductive safety and biosafety of IgG78-DM1 were also evaluated in vivo. RESULTS: CD248 expression was upregulated in patients with liver cirrhosis and in CCl4-induced mice, and was mainly expressed on alpha smooth muscle actin (α-SMA)+ myofibroblasts. IgG78-DM1 was successfully generated, which could effectively bind with and kill CD248+ activated HSCs in vitro and inhibit liver fibrosis in vivo. In addition, IgG78-DM1 was demonstrated to have qualified biosafety and reproductive safety in vivo. CONCLUSIONS: Our study demonstrated that CD248 could be an ideal target for myofibroblasts in liver fibrosis, and CD248-targeting IgG78-DM1 had excellent anti-fibrotic effects in mice with liver fibrosis. Our study provided a novel strategy to treat liver fibrosis and expanded the application of ADCs beyond tumors.

Sensitive LC-MS/MS quantification of unconjugated maytansinoid DM4 and its metabolite S-methyl-DM4 in human plasma.

Aim: To report the development and validation of an LC-MS/MS method for the simultaneous determination of unconjugated payload DM4 and its metabolite S-methyl-DM4 in human plasma. Methodology: A workflow of protein precipitation followed by reduction and solid phase extraction was employed to remove antibody-maytansinoid conjugates from plasma matrix, release DM4 from endogenous conjugates, and generate a clean sample extract for analysis, respectively. Sodium adduct species of both analytes were selected for multiple reaction monitoring to meet the assay sensitivity requirement in liquid chromatography with tandem mass spectrometry. Conclusion: The method was fully validated for a dynamic range of 0.100-50.0 ng/ml for both analytes along with desired stability and acceptable incurred sample reanalysis.

Switchable assembly and function of antibody complexes in vivo using a small molecule.

The antigen specificity and long serum half-life of monoclonal antibodies have made them a critical part of modern therapeutics. These properties have been coopted in a number of synthetic formats, such as antibody-drug conjugates, bispecific antibodies, or Fc-fusion proteins to generate novel biologic drug modalities. Historically, these new therapies have been generated by covalently linking multiple molecular moieties through chemical or genetic methods. This irreversible fusion of different components means that the function of the molecule is static, as determined by the structure. Here, we report the development of a technology for switchable assembly of functional antibody complexes using chemically induced dimerization domains. This approach enables control of the antibody's intended function in vivo by modulating the dose of a small molecule. We demonstrate this switchable assembly across three therapeutically relevant functionalities in vivo, including localization of a radionuclide-conjugated antibody to an antigen-positive tumor, extension of a cytokine's half-life, and activation of bispecific, T cell-engaging antibodies.