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The cell-surface receptor tyrosine kinase c-mesenchymal-epithelial transition factor (c-Met) is overexpressed in a wide range of solid tumors, making it an appropriate target antigen for the development of anticancer therapeutics. Various antitumor c-Met-targeting therapies (including monoclonal antibodies [mAbs] and tyrosine kinases) have been developed for the treatment of c-Met-overexpressing tumors, most of which have so far failed to enter the clinic because of their efficacy and complications. Antibody–drug conjugates (ADCs), a new emerging class of cancer therapeutic agents that harness the target specificity of mAbs to deliver highly potent small molecules to the tumor with the minimal damage to normal cells, could be an attractive therapeutic approach to circumvent these limitations in patients with c-Met-overexpressing tumors. Of great note, there are currently nine c-Met-targeting ADCs being examined in different phases of clinical studies as well as eight preclinical studies for treating various solid tumors. The purpose of this study is to present a broad overview of clinical- and preclinical-stage c-Met-targeting ADCs. [ABSTRACT FROM AUTHOR]

HIV-infected cells persisting in the face of suppressive antiretroviral therapy are the barrier to curing infection. Cytotoxic immunoconjugates targeted to HIV antigens on the cell surface may clear these cells. We showed efficacy in mouse and macaque models using immunotoxins, but immunogenicity blunted the effect. As an alternative, we propose antibody drug conjugates (ADCs), as used in cancer immunotherapy. In cancer, the target is a dividing cell, whereas it may not be in HIV. We screened cytotoxic drugs on human primary cells and cell lines. An anthracycline derivative, PNU-159682 (PNU), was highly cytotoxic to both proliferating and resting cells. Human anti-gp41 mAb 7B2 was conjugated to ricin A chain or PNU. The conjugates were tested in vitro for cytotoxic efficacy and anti-viral effect, and in vivo for tolerability. The specificity of killing for both conjugates was demonstrated on Env+ and Env- cells. The toxin conjugate was more potent and killed more rapidly, but 7B2-PNU was effective at levels achievable in patients. The ricin conjugate was well tolerated in mice; 7B2-PNU was toxic when administered intraperitoneally but was tolerated intravenously. We have produced an ADC with potential to target the persistent HIV reservoir in both dividing and non-dividing cells while avoiding immunogenicity. Cytotoxic anti-HIV immunoconjugates may have greatest utility as part of an "activate and purge" regimen, involving viral activation in the reservoir. This is a unique comparison of an immunotoxin and ADC targeted by the same antibody and tested in the same systems. [ABSTRACT FROM AUTHOR]

We are developing cytotoxic immunoconjugates (CICs) targeting the envelope protein (Env) of the Human Immunodeficiency Virus, type 1 (HIV) to purge the persistent reservoirs of viral infection. We have previously studied the ability of multiple monoclonal antibodies (mAbs) to deliver CICs to an HIV-infected cell. We have found that CICs targeted to the membrane-spanning gp41 domain of Env are most efficacious, in part because their killing is enhanced in the presence of soluble CD4. The ability of a mAb to deliver a CIC does not correlate with its ability to neutralize nor mediate Ab-dependent cellular cytotoxicity. In the current study, we seek to define the most effective anti-gp41 mAbs for delivering CICs to HIV-infected cells. To do this, we have evaluated a panel of human anti-gp41 mAbs for their ability to bind and kill two different Env-expressing cell lines: persistently infected H9/NL4-3 and constitutively transfected HEK293/92UG. We measured the binding and cytotoxicity of each mAb in the presence and absence of soluble CD4. We found that mAbs to the immunodominant helix-loop-helix region (ID-loop) of gp41 are most effective, whereas neutralizing mAbs to the fusion peptide, gp120/gp41 interface, and the membrane proximal external region (MPER) are relatively ineffective at delivering CICs. There was only a weak correlation between antigen exposure and killing activity. The results show that the ability to deliver an effective IC and neutralization are distinct functions of mAbs. [ABSTRACT FROM AUTHOR]

Prostate-specific membrane antigen (PSMA)-directed antibody drug conjugates (ADCs) [Display omitted] Prostate-specific membrane antigen (PSMA) is a type II membrane glycoprotein overexpressed in a variety of tumors, especially in nearly all prostate cancers, which makes it a potentially attractive antigen for targeted cancer therapies. More importantly, PSMA, due to no shedding into circulation and efficient internalization after antibody binding, becomes a potential target for antibody-drug conjugates (ADCs), a valid and emerging paradigm of cancer treatment. Four and eight PSMA-directed ADCs have been or are currently being investigated in clinical trials (three of which failed to confirm the promising results while one is currently being evaluated in an ongoing clinical study) and preclinical studies, respectively, for the treatment of PSMA-positive solid tumors, especially prostate cancer. The present study aims to completely review clinical- and preclinical-stage PSMA-directed ADCs. [ABSTRACT FROM AUTHOR]

Antibody-drug conjugates (ADCs) are revolutionizing metastatic breast cancer treatment, resulting in a better prognosis and a higher safety profile than chemotherapy. Nevertheless, treatment-related adverse events (TRAE) have been extensively documented. We searched five databases for articles published up to December 2023 and conducted a meta-analysis on 23 clinical trials to estimate TRAE prevalence related to currently approved ADCs. The prevalence of the most common TRAEs ranged from 12 % to 33 %, depending on the ADC type and study design. Gastrointestinal disorders were highly prevalent during Trastuzumab Deruxtecan, general disorders were extremely common during Trastuzumab Emtansine, and blood system disorders and gastrointestinal disorders were the most prevalent during Sacituzumab Govitecan. This study provides an estimate of ADC-related TRAEs for each treatment based on study design. Despite each ADC having specific toxicities, gastrointestinal symptoms were highly prevalent in all treatments. This study lays the groundwork for developing personalized risk-stratified care pathways. [Display omitted] • ADC (antibody-drug conjugates)-related adverse events have been widely reported. • The three currently approved ADCs have distinct toxicity profiles. • Trastuzumab deruxtecan has a higher symptom burden than trastuzumab emtansine. • Sacituzumab govitecan has a higher symptom burden than trastuzumab emtansine. • Gastrointestinal disorders were highly prevalent in all three treatments. [ABSTRACT FROM AUTHOR]

Nectin-4 (Nectin cell adhesion molecule 4), a type I transmembrane cell adhesion protein, was demonstrated to be overexpressed in a variety of tumors, making it an attractive antigen for targeted therapies such as antibody-drug conjugates (ADCs). Of great note, the US Food and Drug Administration (FDA)-approval of the first Nectin-4-directed ADC, enfortumab vedotin (EV), in urothelial cancer (UC) not only introduced Nectin-4 as a clinically validated and reliable target antigen but also confirmed the evolving role of Nectin-4-directed ADCs as novel and promising cancer therapeutics. In addition to EV, there have been or are currently being seven and eleven Nectin-4-directed ADCs, respectively, in various stages of clinical trials and preclinical development, offering a promising future for the treatment of Nectin-4-positive cancer patients. This study reviewed clinical- and preclinical-stage Nectin-4-directed ADCs. [Display omitted] [ABSTRACT FROM AUTHOR]

We are developing cytotoxic immunoconjugates (CICs) targeting the envelope protein (Env) of the Human Immunodeficiency Virus, type 1 (HIV) to purge the persistent reservoirs of viral infection. We have previously studied the ability of multiple monoclonal antibodies (mAbs) to deliver CICs to an HIV-infected cell. We have found that CICs targeted to the membrane-spanning gp41 domain of Env are most efficacious, in part because their killing is enhanced in the presence of soluble CD4. The ability of a mAb to deliver a CIC does not correlate with its ability to neutralize nor mediate Ab-dependent cellular cytotoxicity. In the current study, we seek to define the most effective anti-gp41 mAbs for delivering CICs to HIV-infected cells. To do this, we have evaluated a panel of human anti-gp41 mAbs for their ability to bind and kill two different Env-expressing cell lines: persistently infected H9/NL4-3 and constitutively transfected HEK293/92UG. We measured the binding and cytotoxicity of each mAb in the presence and absence of soluble CD4. We found that mAbs to the immunodominant helix-loop-helix region (ID-loop) of gp41 are most effective, whereas neutralizing mAbs to the fusion peptide, gp120/gp41 interface, and the membrane proximal external region (MPER) are relatively ineffective at delivering CICs. There was only a weak correlation between antigen exposure and killing activity. The results show that the ability to deliver an effective IC and neutralization are distinct functions of mAbs.

Background: Outcomes for patients with relapsed/refractory acute myeloid leukemia (R/R AML) remain poor. Novel therapies specifically targeting AML are of high interest. Brentuximab vedotin (BV) is an antibody-drug conjugate that is specific for human CD30. In this phase 1 dose escalation study, the authors evaluated the safety of BV combined with mitoxantrone, etoposide, and cytarabine (MEC) re-induction chemotherapy for patients with CD30-expressing R/R AML.Methods: Using a standard dose escalation design, the authors evaluated 3 dose levels of BV (0.9 mg/kg, 1.2 mg/kg, and 1.8 mg/kg) administered once on day 1 followed by MEC on days 3 through 7.Results: There were no dose-limiting toxicities noted and the maximum tolerated dose was not reached. The recommended phase 2 dose of BV was determined to be 1.8 mg/kg when combined with MEC. The side effect profile was similar to that expected from MEC chemotherapy alone, with the most common grade ≥3 toxicities being febrile neutropenia, thrombocytopenia, and anemia (toxicities were graded using the National Cancer Institute Common Terminology Criteria for Adverse Events [version 4.0]). Among the 22 patients enrolled on the trial, the composite response rate was 36%, with a composite response rate of 42% noted among those who received the highest dose of BV. The median overall survival was 9.5 months, with a median disease-free survival of 6.8 months observed among responders. Approximately 55% of patients were able to proceed with either allogeneic hematopoietic stem cell transplantation or donor lymphocyte infusion.Conclusions: The combination of BV with MEC was found to be safe in patients with CD30-expressing R/R AML and warrants further study comparing this combination with the use of MEC alone in this population (ClinicalTrials.gov identifier NCT01830777).Lay Summary: The outcomes for patients with relapsed/refractory acute myeloid leukemia (R/R AML) are exceptionally poor. New and emerging treatment combinations are actively being studied in an effort to improve outcomes. The authors examined the combination of brentuximab vedotin, an antibody product that recognizes a marker called CD30, with mitoxantrone, etoposide, and cytarabine (MEC), a common chemotherapy regimen, in patients with R/R AML that expressed the CD30 marker. The authors found that the combination was safe and well tolerated. Future studies comparing this new combination with the use of MEC alone can help to inform its effectiveness for this patient population. [ABSTRACT FROM AUTHOR]

Immunotoxins (ITs) are attractive anticancer modalities aimed at cancer-specific delivery of highly potent cytotoxic protein toxins. An IT consists of a targeting domain (an antibody, cytokine, or another cell-binding protein) chemically conjugated or recombinantly fused to a highly cytotoxic payload (a bacterial and plant toxin or human cytotoxic protein). The mode of action of ITs is killing designated cancer cells through the effector function of toxins in the cytosol after cellular internalization via the targeted cell-specific receptor-mediated endocytosis. Although numerous ITs of diverse structures have been tested in the past decades, only 3 ITs—denileukin diftitox, tagraxofusp, and moxetumomab pasudotox—have been clinically approved for treating hematological cancers. No ITs against solid tumors have been approved for clinical use. In this review, we discuss critical research and development issues associated with ITs that limit their clinical success as well as strategies to overcome these obstacles. The issues include off-target and on-target toxicities, immunogenicity, human cytotoxic proteins, antigen target selection, cytosolic delivery efficacy, solid-tumor targeting, and developability. To realize the therapeutic promise of ITs, novel strategies for safe and effective cytosolic delivery into designated tumors, including solid tumors, are urgently needed. [ABSTRACT FROM AUTHOR]

According to a 2020 World Health Organization report (Globocan 2020), cancer was a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. The aim of anticancer therapy is to specifically inhibit the growth of cancer cells while sparing normal dividing cells. Conventional chemotherapy, radiotherapy and surgical treatments have often been plagued by the frequency and severity of side effects as well as severe patient discomfort. Cancer targeting by drug delivery systems, owing to their selective targeting, efficacy, biocompatibility and high drug payload, provides an attractive alternative treatment; however, there are technical, therapeutic, manufacturing and clinical barriers that limit their use. This article provides a brief review of the challenges of conventional anticancer therapies and anticancer drug targeting with a special focus on liposomal drug delivery systems.

Modifying biological agents with polymers such as polyethylene glycol (PEG) has demonstrated clinical benefits; however, post-market surveillance of PEGylated derivatives has revealed PEG-associated toxicity issues, prompting the search for alternatives. We explore how conjugating a poly- l -glutamic acid (PGA) to an anti-insulin growth factor 1 receptor antibody (AVE1642) modulates the bio-nano interface and anti-tumor activity in preclinical prostate cancer models. Native and PGA-modified AVE1642 display similar anti-tumor activity in vitro ; however, AVE1642 prompts IGF-1R internalization while PGA conjugation prompts higher affinity IGF-1R binding, thereby inhibiting IGF-1R internalization and altering cell trafficking. AVE1642 attenuates phosphoinositide 3-kinase signaling, while PGA-AVE1642 inhibits phosphoinositide 3-kinase and mitogen-activated protein kinase signaling. PGA conjugation also enhances AVE1642's anti-tumor activity in an orthotopic prostate cancer mouse model, while PGA-AVE1642 induces more significant suppression of cancer cell proliferation/angiogenesis than AVE1642. These findings demonstrate that PGA conjugation modulates an antibody's bio-nano interface, mechanism of action, and therapeutic activity. Poly- l -glutamic acid (PGA) conjugation alters the biological properties of a human monoclonal antibody against IGFR (AVE1642), increasing its antitumoral activity due to the capability to alter the bio-nano interaction between PGA-AVE1642 conjugate and IGF-1R in aggressive prostate cancer. These differential bio-nano interactions result in differential effects on critical cellular signaling pathways and the tumor microenvironment. [Display omitted] [ABSTRACT FROM AUTHOR]

Iron is essential for cell growth and is imported into cells in part through the action of transferrin (Tf), a protein that binds its receptor (TfR1 or CD71) on the surface of a cell, and then releases iron into endosomes. TfR1 is a single pass type-II transmembrane protein expressed at basal levels in most tissues. High expression of TfR1 is typically associated with rapidly proliferating cells, including various types of cancer. TfR1 is targeted by experimental therapeutics for several reasons: its cell surface accessibility, constitutive endocytosis into cells, essential role in cell growth and proliferation, and its overexpression by cancer cells. Among the therapeutic agents used to target TfR1, antibodies stand out due to their remarkable specificity and affinity. Clinical trials are being conducted to evaluate the safety and efficacy of agents targeting TfR1 in cancer patients with promising results. These observations suggest that therapies targeting TfR1 as direct therapeutics or delivery conduits remain an attractive alternative for the treatment of cancers that overexpress the receptor.

Human EGF Receptor 2 (HER2) is an important oncogene driving aggressive metastatic growth in up to 20% of breast cancer tumors. At the same time, it presents a target for passive immunotherapy such as trastuzumab (TZM). Although TZM has been widely used clinically since 1998, not all eligible patients benefit from this therapy due to primary and acquired drug resistance as well as potentially lack of drug exposure. Hence, it is critical to directly quantify TZM–HER2 binding dynamics, also known as cellular target engagement, in undisturbed tumor environments in live, intact tumor xenograft models. Herein, we report the direct measurement of TZM–HER2 binding in HER2-positive human breast cancer cells and tumor xenografts using fluorescence lifetime Forster Resonance Energy Transfer (FLI-FRET) via near-infrared (NIR) microscopy (FLIM-FRET) as well as macroscopy (MFLI-FRET) approaches. By sensing the reduction of fluorescence lifetime of donor-labeled TZM in the presence of acceptor-labeled TZM, we successfully quantified the fraction of HER2-bound and internalized TZM immunoconjugate both in cell culture and tumor xenografts in live animals. Ex vivo immunohistological analysis of tumors confirmed the binding and internalization of TZM–HER2 complex in breast cancer cells. Thus, FLI-FRET imaging presents a powerful analytical tool to monitor and quantify cellular target engagement and subsequent intracellular drug delivery in live HER2-positive tumor xenografts.

Breast cancer is a heterogeneous group of malignancies with a spectrum of molecular subtypes, pathologies and outcomes that together comprise the most common non-cutaneous cancer in women. Currently, over 80% of breast cancer patients are diagnosed at relatively early stages of disease where there are encouraging data on outcomes and long term survival. However, there is currently no curative option for those patients with metastatic disease and there is a substantial medical need to identify effective and safe treatment options for these patients. One approach to improve cancer therapy is by designing therapeutics directed against targets with differential levels of expression on malignant versus normal cells with the goal of improving tumor selectivity and reducing damage to normal tissues. Antibody drug conjugates (ADCs) are a rapidly evolving therapeutic class that exploits the target-selectivity of monoclonal antibodies (MAbs) to deliver cytotoxic drugs to antigen-expressing cells (Lambert & Morris, 2017; Senter, 2009; Thomas, Teicher, & Hassan, 2016; Trail, 2013). The regulatory approval of ADCs for both hematologic malignancies (brentuximab vedotin) (Younes et al., 2010) and solid tumors (ado-trastuzumab emtansine) ( Amiri-Kordestani et al., 2014 ; Verma et al., 2012) clearly demonstrates the clinical potential of ADCs. This review will focus on targets under consideration for breast cancer directed ADCs and on the technology modifications being considered to improve ADC efficacy and safety. [ABSTRACT FROM AUTHOR]

Vaccination is a reliable method of prophylaxis and a crucial measure for public health. However, the majority of vaccines cannot be administered orally due to their degradation in the harsh gut environment or inability to cross the GI tract. In this study, we report the first proof-of-concept study of orally producible chemically programmed antibodies via specific conjugation of adaptor ligands to endogenous antibodies, in vivo . Pre-immuniztion with 2,4-dinitrophenyl (DNP), or the reactive hapten, 1,3-diketone (DK), or a novel reactive hapten, vinyl sulfone (VS) in mice, followed by oral administration of adaptor ligands composed of the hapten and biotin to the pre-immunized mice resulted in successful in vivo formation of the biotin-hapten-antibody complexes within 2 h. Pharmacokinetic evaluations revealed that apparent serum concentrations of programmed antibodies were up to 144 nM and that the serum half-lives reached up to 34.4 h. These findings show promise for the future development of orally bioavailable drug-hapten-antibody complexes as a strategy to quickly and easily modulate immune targets for aggressive pathogens as well as cancer. [ABSTRACT FROM AUTHOR]

Background The antibody-drug conjugate PF-06263507 targets the cell-surface, tumor-associated antigen 5T4 and consists of a humanized IgG1 conjugated to the microtubule-disrupting agent monomethylauristatin-F by a non-cleavable maleimidocaproyl linker. In this first-in-human, dose-finding trial (NCT01891669), we evaluated safety, pharmacokinetics, and preliminary antitumor activity of PF-06263507 in pretreated patients with advanced solid tumors, unselected for 5T4 expression. starting at 0.05 mg/kg, with 25, 56, and 95% dose increments, depending on observed dose-limiting toxicities (DLTs), applying a modified continual reassessment method. Results Twenty-six patients received PF-06263507 at 0.05 to 6.5 mg/kg. The first DLT, grade 3 photophobia, occurred at 4.34 mg/kg and two additional DLTs, grade 2 keratitis and grade 1 limbal stem cell deficiency (> 2-week dosing delay), at 6.5 mg/kg. The most common adverse events (AEs) were fatigue (38.5%), photophobia (26.9%), and decreased appetite, dry eye, nausea, and thrombocytopenia (23.1% each). No treatment-related grade 4-5 AEs were reported. Systemic exposure of PF-06263507 increased in a dose-related manner. At the maximum tolerated dose (MTD, 4.34 mg/kg), mean terminal half-life for PF-06263507 and unconjugated payload were ~6 and 3 days, respectively. Payload serum concentrations were substantially lower compared with PF-06263507. No objective responses were observed. Conclusions The MTD and recommended phase II dose were determined to be 4.34 mg/kg. Ocular toxicities accounted for the DLTs observed, as previously reported with monomethylauristatin-F payloads. Further studies are warranted to investigate clinical activity of this agent in patients with 5T4-expressing tumors. Trial registration ID: NCT01891669 [ABSTRACT FROM AUTHOR]

The conjugation of toxins, dyes, peptides, or proteins to monoclonal antibodies is often performed via free thiol groups generated by either partial reduction methods or engineering free cysteine residues into the antibody sequence. Antibodies from the rabbitOryctolagus cuniculushave an additional intrachain disulfide bond, whereby the light chain variable kappa domain is bridged to the constant kappa region between cysteine residues at positions 80 and 171, respectively. Chimerization of rabbit antibodies with human constant domains allows for the generation of a free thiol group at the light chain position 80 (C80) that can be used for site-specific conjugation. An efficient process for the purification and simultaneous removal of cysteinylation at the C80 site was developed. The unpaired C80 was shown to be efficiently conjugated using several different maleimido-based ligands. REsidue SPEcific Conjugation Technology (RESPECT) antibody-drug conjugates prepared using rabbit-human chimeric anti-human mesothelin rabbit antibodies and maleimido-PEG2-auristatin conjugated to C80 were shown to be highly potent and specificin vitroand effectivein vivoin reduction of tumor growth in a highly aggressive mesothelin-expressing xenograft tumor model. [ABSTRACT FROM PUBLISHER]

The primary treatment for malignant tumors remains to be surgical removal of the diseased tissue. The presence or absence of residual diseased tissue at the tumor margin is the strongest predictor of postoperative prognosis and recurrence. Accordingly, reliance on the ability of surgeons to visually distinguish diseased tissue from healthy tissue unambiguously in real time is crucial. Near infrared-I (NIR I) fluorescence-emitting targeting biomolecular constructs such as anticancer antibody-fluorophore conjugates, namely cetuximab-IRDye® 800CW (CTB-IRDye® 800CW), are FDA-approved for clinical trial usage in the fluorescence-guided resection of diseased tissue due to affording improved direct visualization of tumor tissue when compared to the use of either the unaided eye under standard white light illumination (WLI) surgical techniques or non-targeting fluorophores. Unfortunately, though helpful, CTB-IRDye® 800CW affords limited (i) identification of diseased tissue and (ii) tumor margin delineation, because the immunoconjugate generates suboptimal tumor-to-background ratios (TBRs) as a result of its spectral/photophysical profiles poorly aligning with the fixed optical windows of pre−/clinical setups. As such, CTB-IRDye® 800CW is more prone to affording incomplete resection compared to if TBRs were higher due to otherwise. To aid in accurately identifying deep-seated diseased tissue, photoacoustic (PA) tomography has been implemented alongside CTB-IRDye® 800CW to achieve PA signals that could result in higher TBRs. However, in clinical trial practice, using IRDye® 800CW for PA imaging also yields subpar TBRs due to it affording low PA signals. To overcome such limitations, we developed NIRDye 812 , a structurally-modified topological equivalent of IRDye® 800CW, to confer it the capability to yield both higher TBRs and superior PA signal than that of the equivalent CTB-conjugate and fluorophore IRDye® 800CW itself, respectively. To do so, we substituted the oxygen atom at its meso -position with a sulfur atom. CTB- NIRDye 812 demonstrated a red-shifted absorption wavelength at 796 nm and a peak NIR-I fluorescence emission wavelength at 820 nm, which better dovetails with the fixed windows of preinstalled fixed emission filters within commercial pre−/clinical NIR-I fluorescence imaging instruments. Overall, CTB- NIRDye 812 provided a ∼ 2-fold increase in TBRs compared to those of CTB-IRDye® 800CW in vivo. Also, NIRDye 812 displayed an ∼60% higher PA signal than that of IRDye® 800CW. Collectively, we achieved our goal of improving upon the spectral/photophysical and PA properties of IRDye® 800CW via introducing a subtle modification to its electronic core such that its CTB immunoconjugate could potentially allow for fast track or breakthrough designation by the FDA due to its near-identical structure displaying considerably improved efficacy. [Display omitted] • NIRDye 812 is a topological equivalent of IRDye 800 with superior spectrophysics. • Antibody-conjugate cetuximab- NIRDye 812 affords higher TBRs than cetuximab-IRDye 800. • NIRDye 812 exhibits ∼2-fold increase in photoacoustic signal compared to IRDye 800. [ABSTRACT FROM AUTHOR]

Monoclonal antibody (MAb) based therapies have achieved considerable success in oncology, primarily when used in combination with cytotoxic drugs. Antibody drug conjugates (ADCs) are a class of therapeutics that harness the antigen-selectivity of MAbs to deliver highly potent cytotoxic drugs to antigen-expressing tumor cells. The use of MAb directed delivery can confer a therapeutic index to highly potent cytotoxic drugs, increasing both the efficacy and safety of therapy. Although simple in concept, to achieve the design goal of improved therapeutic efficacy and reduced toxicity, each of the components of an ADC; the MAb, linker and drug need to considered in the context of the targeted antigen, the selectivity of antigen expression and the biology of the tumor type on which the target antigen is expressed. The characteristics of targets, MAbs, linkers and drugs being used in ADC design are discussed.

Iron is essential for cell growth and is imported into cells in part through the action of transferrin (Tf), a protein that binds its receptor (TfR1 or CD71) on the surface of a cell, and then releases iron into endosomes. TfR1 is a single pass type-II transmembrane protein expressed at basal levels in most tissues. High expression of TfR1 is typically associated with rapidly proliferating cells, including various types of cancer. TfR1 is targeted by experimental therapeutics for several reasons: its cell surface accessibility, constitutive endocytosis into cells, essential role in cell growth and proliferation, and its overexpression by cancer cells. Among the therapeutic agents used to target TfR1, antibodies stand out due to their remarkable specificity and affinity. Clinical trials are being conducted to evaluate the safety and efficacy of agents targeting TfR1 in cancer patients with promising results. These observations suggest that therapies targeting TfR1 as direct therapeutics or delivery conduits remain an attractive alternative for the treatment of cancers that overexpress the receptor. [ABSTRACT FROM AUTHOR]

CpG oligodeoxynucleotides (CpG) potently activate the immune system by mimicking microbial DNA. Conjugation of CpG to chTNT-3, an antibody targeting the necrotic centers of tumors, enabled CpG to accumulate in tumors after systemic delivery, where it can activate the immune system in the presence of tumor antigens. CpG chemically conjugated to chTNT-3 (chTNT-3/CpG) were compared to free CpG in their ability to stimulate the immune system in vitro and reduce tumor burden in vivo. In subcutaneous Colon 26 adenocarcinoma and B16-F10 melanoma models in BALB/c and C57BL/6 mice, respectively, chTNT-3/CpG, free CpG, or several different control constructs were administered systemically. Intraperitoneal injections of chTNT-3/CpG delayed tumor growth and improved survival and were comparable to intratumorally administered CpG. Compared to saline-treated mice, chTNT-3/CpG-treated mice had smaller average tumor volumes by as much as 72 % in Colon 26-bearing mice and 79 % in B16-bearing mice. Systemically delivered free CpG and CpG conjugated to an isotype control antibody did not reduce tumor burden or improve survival. In this study, chTNT-3/CpG retained immunostimulatory activity of the CpG moiety and enabled delivery to tumors. Because systemically administered CpG rapidly clear the body and do not accumulate into tumors, chTNT-3/CpG provide a solution to the limitations observed in preclinical and clinical trials. [ABSTRACT FROM AUTHOR]

Antibody drug conjugates (ADCs) are an emerging class of chemotherapeutic cancer treatment agents that combine the targeting specificity of antibodies with the efficient cell-killing potential of cytotoxic drugs. Unlike their protein and small-molecule therapeutic counterparts, the stability and degradation properties of ADCs are relatively unknown. Theoretically, ADC stability could be governed by properties and processes stemming from both the antibody and the linker-toxin chemistry. Recently, systematic studies of intrinsic ADC molecule stability have been presented in the primary literature. As there are burgeoning industrial and academic efforts aimed at developing optimized conjugation chemistries and antibody engineering approaches for next-generation ADCs, it is important to capture the current state of understanding of ADC stability. In this minireview, we discuss aspects of physical and chemical stability of ADCs gathered from a survey of the literature and illustrate how investigations into stability enable the development of more effective ADC molecules for the future. [ABSTRACT FROM AUTHOR]

Background: Alpha (α)-amidation of peptides is a mechanism required for the conversion of prohormones into functional peptide sequences that display biological activities, receptor recognition and signal transduction on target cells. Alpha (α)-amidation occurs in almost all species and amino acids identified in nature. C-terminal valine amide neuropeptides constitute the smallest group of functional peptide compounds identified in neurosecretory structures in vertebrate and invertebrate species.Methods: The α-amidated isoform of valine residue (Val-CONH2) was conjugated to KLH-protein carrier and used to immunize mice. Hyperimmune animals displaying high titers of valine amide antisera were used to generate stable hybridoma-secreting mAbs. Three productive hybridoma (P15A4, P17C11, and P18C5) were tested against peptides antigens containing both the C-terminal α-amidated (-CONH2) and free α-carboxylic acid (-COO(-)) isovariant of the valine residue.Results: P18C5 mAb displayed the highest specificity and selectivity against C-terminal valine amidated peptide antigens in different immunoassays. P18C5 mAb-immunoreactivity exhibited a wide distribution along the neuroaxis of the rat brain, particularly in brain areas that did not cross-match with the neuronal distribution of known valine amide neuropeptides (α-MSH, adrenorphin, secretin, UCN1-2). These brain regions varied in the relative amount of putative novel valine amide peptide immunoreactive material (nmol/μg protein) estimated through a fmol-sensitive solid-phase radioimmunoassay (RIA) raised for P18C5 mAb.Conclusions: Our results demonstrate the versatility of a single mAb able to differentiate between two structural subdomains of a single amino acid. This mAb offers a wide spectrum of potential applications in research and medicine, whose uses may extend from a biological reagent (used to detect valine amidated peptide substances in fluids and tissues) to a detoxifying reagent (used to neutralize exogenous toxic amide peptide compounds) or as a specific immunoreagent in immunotherapy settings (used to reduce tumor growth and tumorigenesis) among many others. [ABSTRACT FROM AUTHOR]

Antibody-drug conjugates (ADCs) are a new class of therapeutic agents that combine the targeting ability of monoclonal antibodies (mAbs) with small molecule drugs. The combination of a mAb targeting a cancer-specific antigen with a cytotoxin has tremendous promise as a new type of targeted cancer therapy. Two ADCs have been approved and many more are in clinical development, suggesting that this new class of drugs is coming to the forefront. Because of their unique nature as biologic-small drug hybrids, ADCs are challenging to develop, from both the scientific and regulatory perspectives. This review discusses both these aspects in current practice, and surveys the current state of the art of ADC drug development. [ABSTRACT FROM AUTHOR]

Biotherapeutics are attractive anti-cancer agents due to their high specificity and limited toxicity compared to conventional small molecules. Antibodies are widely used in cancer therapy, either directly or conjugated to a cytotoxic payload. Peptide therapies, though not as prevalent, have been utilized in hormonal therapy and imaging. The limitations associated with unmodified forms of both types of biotherapeutics have led to the design and development of novel structures, which incorporate key features and structures that have improved the molecules' abilities to bind to tumor targets, avoid degradation, and exhibit favorable pharmacokinetics. In this review, we highlight the current status of monoclonal antibodies and peptides, and provide a perspective on the future of biotherapeutics using novel constructs. [ABSTRACT FROM AUTHOR]

Immunoconjugates that deliver cytotoxic payloads to cancer cells represent a promising class of therapeutic agents which are intensively investigated in various clinical applications. Prerequisites for the generation of effective immunoconjugates are antibodies which efficiently deliver the respective cytotoxic payload. To facilitate the selection of human or mouse antibodies that display favorable characteristics as immunotoxins, we developed a novel Pseudomonas exotoxin A (ETA)-based screening protein. The α-Fc-ETA′ consists of a multispecies-specific Fc-binding domain antibody genetically fused to a truncated ETA version (ETA′). α-Fc-ETA′ non-covalently bound to human and mouse antibodies but did not form immune complexes with bovine immunoglobulins. In combination with antibodies harboring human or mouse Fc domains α-Fc-ETA′ inhibited proliferation of antigen-expressing tumor cells. The cytotoxic effects were strictly antibody dependent and were observed with low α-Fc-ETA′ concentrations. Mouse antibodies directed against CD7 and CD317/HM1.24 that previously had been used for the generation of functional recombinant immunotoxins, also showed activity in combination with α-Fc-ETA′ by inhibiting growth of antigen-positive myeloma and leukemia cell lines. In contrast, α-kappa-ETA′, a similarly designed human kappa light chain-specific fusion protein, was only specifically active in combination with antibodies containing a human kappa light chain. Thus, the novel α-Fc-ETA′ fusion protein is broadly applicable in screening antibodies and Fc-containing antibody derivatives from different species to select for candidates with favorable characteristics for immunotoxin development. [ABSTRACT FROM AUTHOR]

Astatine-211 is possibly the most promising radionuclide for targeted α-particle therapy when it comes to the treatment of occult disseminated cancer. Preclinical research has proven effective, and patient studies have been initiated based on these results. However, a lack of production capacity and the complex radiochemistry of 211At are major obstacles for research and prospective clinical applications. In the present study, astatination of immunoconjugates, already prepared well in advance before radiolabeling, was performed to investigate the possibility of formulating a kit-like reagent for the production of 211At radiopharmaceuticals. The shelf-life of ɛ -lysyl-3-(trimethylstannyl)benzamide immunoconjugates was evaluated, that is, the effect of different storage times on the quality of the immunoconjugates. The quality being referred to is the capacity to maintain a good radiochemical yield and good cell-binding property after labeling with 211At. The stability of the conjugates was found to be pH dependent with high stability at pH≥7 and less stability at pH≤5.5. The immunoconjugates (based on trastuzumab) could be kept for more than 3 months in a phosphate buffered saline solution (pH 7.4) at 4°C before labeling, without compromising the quality of the labeled product. The conjugates are also unaffected by storage at −20°C. Conjugates with a good shelf-life compatible with distant shipping as well as improved radiochemistry are important steps to facilitate further clinical progress with 211At. [ABSTRACT FROM AUTHOR]

Sperm protein 17 (Sp17) is selectively overexpressed in several human malignancies including ovarian carcinoma, but is absent or expressed at low levels in most normal tissues. Previous work from our group characterized an anti-Sp17 monoclonal antibody (clone 3C12) and showed that it specifically targeted tumor cells. In this report, we investigated whether a novel immunoconjugate containing 3C12 linked to the chemotherapeutic agent doxorubicin [(DOX) Adriamycin] had antitumor activity against ovarian cancer cell lines and tumor models. DOX was conjugated to 3C12 using a linker, and the specificity of 3C12-DOX was examined in Sp17-positive SKOV3 and Sp17-negative COC2 ovarian cancer cells using cell-based ELISA and internalization assays. The cytotoxicity of 3C12-DOX was assessed with the MTT assay, and its therapeutic effectiveness was evaluated in immunodeficient mice bearing SKOV3 cells. In vitro, the 3C12-DOX immunoconjugate specifically bound to and was internalized by Sp17-positive SKOV3 cells but did not bind to Sp17-negative cells. Treatment with 3C12-DOX (0.001 to 10 μg/mL) decreased the viability of SKOV3 cells in a Sp17-specific manner. In vivo, 3C12-DOX (3 mg/kg) induced the regression of established SKOV3 xenograft tumors in BALB/c mice compared with control treatment. The antitumor effects of 3C12-DOX were significantly associated with the induction of apoptosis in tumor cells. In addition, 3C12-DOX showed no observable adverse effects or toxicity when compared with DOX alone in mice bearing ovarian tumor xenografts. Our findings suggest that 3C12-DOX may be a potential antibody-drug conjugate for clinical development. [ABSTRACT FROM AUTHOR]

Abstract: Screening for expression of the high affinity receptor for IgE by reverse transcriptase PCR, revealed that almost all canine mast cell tumors expressed FcɛRIα mRNA, supporting the rationale for developing anti-neoplastic treatments based on molecules that could target this receptor. Use of cytotoxic cytokines to trigger an apoptotic signal is one strategy for inducing cell death in malignant mast cells. The coding sequences for canine IgE and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were identified through genome analyses. Selected regions of the coding sequences for these genes were cloned and compared to the predicted genome sequences. The Fc region of canine IgE, death domain of canine TRAIL and an IgE Fc: TRAIL fusion construct were generated and epitope-tagged proteins expressed, using a eukaryotic expression system. Specific binding of recombinant canine IgE Fc-containing proteins to recombinant human FcɛRIα and to a canine mast cell tumor line expressing FcɛRIα (C2), but not one failing to express FcɛRIα (MCLA), was demonstrated. Specific binding of the IgE: TRAIL fusion protein was not abrogated by the TRAIL moiety. These results are proof of principle that canine IgE targeting to FcɛRIα can be used as a platform for selective delivery of therapies to FcɛRIα-expressing cells, potentially enhancing their therapeutic index and efficacy. [Copyright &y& Elsevier]

Current cancer management strategies fail to adequately treat malignancies with multivariable dose-restricting factors such as systemic toxicity and multi-drug resistance limiting therapeutic benefit, quality of life and complete long-term remission rates. The targeted delivery of a therapeutic compound aims to enhance its circulation and cellular uptake, decrease systemic toxicity and improve therapeutic benefit with disease specificity. The transferrin peptide, its receptor and their biological significance, has been widely characterised and vastly relevant when applied to targeting strategies. Utilising knowledge about the physiological function of the transferrin-transferrin receptor complex and the efficiency of its receptor-mediated endocytosis provides rationale to continue the development of transferrin-targeted anticancer modalities. Furthermore, multiple studies report an upregulation in expression of the transferrin receptor on metastatic and drug resistant tumours, highlighting its selectivity to cancer. Due to the increased expression of the transferrin receptor in brain glioma, the successful delivery of anticancer compounds to the tumour site and the ability to cross the blood brain barrier has shown to be an important discovery. Its significance in the development of cancer-specific therapies is shown to be important by direct conjugation and immunotoxin studies which use transferrin and anti-transferrin receptor antibodies as the targeting moiety. Such conjugates have demonstrated enhanced cellular uptake via transferrin-mediated mechanisms and increased selective cytotoxicity in a number of cancer cell lines and tumour xenograft animal models. In addition, incubation of chemotherapy-insensitive cancer cells with transferrin-targeted conjugates in vitro has resulted in a reversal of their drug resistance. Transferrin immunotoxins have also shown similar promise, with a diphtheria toxin mutant covalently bound to transferrin (Tf-CRM107) currently involved in human clinical trials for the treatment of glioblastoma. Despite this, the inability to translate preliminary research into a clinical setting has compelled research into novel targeting strategies including the use of nanoparticulate theory in the design of drug delivery systems. The main objective of this review is to evaluate the importance of the transferrin-transferrin receptor complex as a target for cancer therapy through extensive knowledge of both the physiological and pathological interactions between the complex and different cell types. In addition, this review serves as a summary to date of direct conjugation and immunotoxin studies, with an emphasis on transferrin as an important targeting moiety in the directed delivery of anticancer therapeutic compounds. [ABSTRACT FROM AUTHOR]

An important step in cancer drug research and development is to generate molecules and agents with better outcomes for inhibition of cancer progression. To date, classic cytotoxic chemotherapy is one of the most frequent methods of treatment. Cytotoxic agents currently used frequently have severe side effects. Thus, clinicians are forced to reduce the recommended dosage, or even avoid the extreme usage of these agents because they are nonselective and are distributed to healthy cells as well as cancer cells. Antibody drug conjugates (ADCs) are a new generation of drugs that consist of three parts: a monoclonal antibody (monoclonal antibodies), a small molecule, and a linker between the two. The use of this structure in research pipelines and recently in clinical practice is likely to achieve more precise and selective distribution and delivery of the cytotoxic agents to cancer cells. This review analyzes the structure of ADCs and the advantages of targeted chemotherapeutics. Possible methods of improvement are also discussed. [ABSTRACT FROM AUTHOR]