Prolonged Plasma Half-life Research Articles

Dextrin conjugation to colistin inhibits its toxicity, cellular uptake and acute kidney injury in vivo.

The acute kidney injury (AKI) and dose-limiting nephrotoxicity, which occurs in 20-60% of patients following systemic administration of colistin, represents a challenge in the effective treatment of multi-drug resistant Gram-negative infections. To reduce clinical toxicity of colistin and improve targeting to infected/inflamed tissues, we previously developed dextrin-colistin conjugates, whereby colistin is designed to be released by amylase-triggered degradation of dextrin in infected and inflamed tissues, after passive targeting by the enhanced permeability and retention effect. Whilst it was evident in vitro that polymer conjugation can reduce toxicity and prolong plasma half-life, without significant reduction in antimicrobial activity of colistin, it was unclear how dextrin conjugation would alter cellular uptake and localisation of colistin in renal tubular cells in vivo. We discovered that dextrin conjugation effectively reduced colistin's toxicity towards human kidney proximal tubular epithelial cells (HK-2) in vitro, which was mirrored by significantly less cellular uptake of Oregon Green (OG)-labelled dextrin-colistin conjugate, when compared to colistin. Using live-cell confocal imaging, we revealed localisation of both, free and dextrin-bound colistin in endolysosome compartments of HK-2 and NRK-52E cells. Using a murine AKI model, we demonstrated dextrin-colistin conjugation dramatically diminishes both proximal tubular injury and renal accumulation of colistin. These findings reveal new insight into the mechanism by which dextrin conjugation can overcome colistin's renal toxicity and show the potential of polymer conjugation to improve the side effect profile of nephrotoxic drugs.

ZGGS15, a Novel IgG4 Bispecific Monoclonal Antibody Targeting Both LAG-3 and Tigit, Demonstrates Outstanding Anti-Tumor Activity and Can be Used As Monotherapy or in Combination with Anti-PD-1 Antibodies for Cancer Immunotherapy

Background This study aims to evaluate the anti-tumor efficacy of ZGGS15, a novel IgG4 bispecific monoclonal antibody (BsAb) targeting TIGIT and LAG-3, for cancer immunotherapy. Methods: ZGGS15 was generated in-house with the standard recombinant BsAb technology. ZGGS15 proteins were purified with protein A affinity column chromatography and evaluated for purity by size-exclusion high-performance liquid chromatography. The ZGGS15 affinity was assessed using a biolayer interferometry (BLI) assay. The EC50s of ZGGS15 binding to TIGIT and LAG-3 were determined using the ELISA technique. The EC50s of ZGGS15 binding to REH, Hela, MCF-7, and HCC1954 cells were measured by flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities were also evaluated by flow cytometry. BALB/c-hPD-1/hLAG-3 and BALB/c-hPD-1/hTIGIT subcutaneously transplanted with CT26.WT tumor mouse models, and peripheral blood mononuclear cells (PBMC) -humanized NOG bearing A375 tumor mouse model were used for in vivo anti-tumor efficacy evaluation. Results ZGGS15 binds specifically to activated CD4 + and CD8 + T cells and human LAG-3 and TIGIT proteins expressed by CHO-K1 cells identically with a dose-dependent nanomolar affinity of 0.69 nM and 1.87 nM for human LAG-3 and TIGIT, respectively. In vitro kinetic analysis BLI results revealed ZGGS15 has high affinity for human LAG-3 and human TIGIT proteins, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 binds to human LAG-3 and human TIGIT simultaneously with similar affinity. Furthermore, ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50=0.24 nM). ZGGS15 induced better T cell activation than single anti-LAG3 or anti-TIGIT mAb agent or combined with different stimuli with or without cancer cells. When combined with nivolumab, ZGGS15 can further enhance the T cell activation in a dose-dependent manner. ADCC/CDC assay results showed that ZGGS15 binding to target cells does not induce ADCC or CDC. ZGGS15 interacts with the human neonatal Fc receptor in a pH-dependent manner, which can promote recirculation of ZGGS15 with a prolonged half-life in plasma. Besides, ZGGS15 interacts with human Fc-γ receptors and the human complement component, C1q, with low binding affinities. However, ZGGS15 does not induce obvious cytokine production from PBMCs, including IFN-γ, IL-2, IL-6, IL-10, and TNF-α. In vivo anti-tumor efficacy for ZGG15 in PBMC humanized NOG mice bearing A375 tumors showed that ZGGS15 had significant anti-tumor effects in the subcutaneous A375 xenograft model. ZGGS15 showed a better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agent and a synergetic effect when combined with nivolumab. On day 18 post-treatment, the ZSAB015 plus nivolumab treatment demonstrated a significantly tumor growth inhibition (TGI) of 95.80% ( p =0.001), compared with the vehicle group. In vivo efficacy tests in BALB/c-hPD-1/hLAG-3 mice subcutaneously transplanted with CT26.WT tumor model showed that both high and low doses of ZGGS15 could inhibit the growth of CT26.WT colon cancer. The combination of high-dose ZGGS15 plus nivolumab has better efficacy than high-dose ZGGS15 and is slightly better than nivolumab alone. The tumor volume inhibition rate (TGITV) for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% ( p < 0.001). During the experiment, the tumor-bearing mice tolerated it well, and no adverse reactions were observed. In vivo efficacy tests of ZGG15 in BALB/c-hPD-1/hTIGIT mice subcutaneously transplanted with CT26.WT tumor model showed that ZGGS15 at a low dose (1.5 mg/kg) demonstrated a significant tumor inhibitory effect, with the TGITV of 51.42% ( p < 0.01). The combination of ZGGS15 high-dose with nivolumab (5 mg/kg + 1 mg/kg) showed a better tumor inhibitory effect compared with ZGGS15 high-dose (5 mg/kg), with the TGITV of 81.15% and 41.54%, respectively ( p < 0.05). Conclusions ZGGS15 has excellent anti-tumor activity by inhibiting LAG-3 and TIGIT as a new IgG4 BsAb. ZGGS15 also exhibits potent anti-tumor efficacy in transgenic mouse models combined with nivolumab. Additionally, ZGGS15 does not elicit ADCC or CDC, therefore avoiding the cross-reaction of non-target binding. Finally, ZGGS15 does not induce cytokine production, mitigating any potential adverse effects of CRS.

A global comparative field study to evaluate the factor VIII activity of efanesoctocog alfa by one-stage clotting and chromogenic substrate assays at clinical haemostasis laboratories.

Structural and chemical modifications of factor VIII (FVIII) products may influence their behaviour in FVIII activity assays. Hence, it is important to assess the performance of FVIII products in these assays. Efanesoctocog alfa is a new class of FVIII replacement therapy designed to provide both high sustained factor activity levels and prolonged plasma half-life. Evaluate the accuracy of measuring efanesoctocog alfa FVIII activity in one-stage clotting assays (OSAs) and chromogenic substrate assays (CSAs). Human plasma with no detectable FVIII activity was spiked with efanesoctocog alfa or a full-length recombinant FVIII product comparator, octocog alfa, at nominal concentrations of 0.80 IU/mL, 0.20 IU/mL, or 0.05 IU/mL, based on labelled potency. Clinical haemostasis laboratories (N=35) tested blinded samples using in-house assays. Data from 51 OSAs (14 activated partial thromboplastin time [aPTT] reagents) and 42 CSAs (eight kits) were analyzed. Efanesoctocog alfa activity was reliably (±25% of nominal activity) measured across all concentrations using OSAs with Actin FSL and multiple other aPTT reagents. Under- and overestimation of FVIII activity occurred with some reagents. No specific trend was observed for any class of aPTT activators. A two- to three-fold overestimation was consistently observed using CSAs and the OSA with Actin FS as the aPTT reagent across evaluated concentrations. Under- or overestimation occurred with some specific OSAs and most CSAs, which has been previously observed with other modified FVIII replacement products. Efanesoctocog alfa FVIII activity was measured with acceptable accuracy and reliability using several OSA methods and commercial plasma standards.

Insertion of (Bioactive) Equatorial Ligands into Platinum(IV) Complexes.

Platinum(IV) prodrugs are highly interesting alternatives to platinum(II) anticancer therapeutics due to their increased tumor selectivity and reduced side effects. In contrast to the established theory, we recently observed that the equatorial ligand(s) of e.g. oxaliplatin(IV) complexes can be hydrolyzed with formation of [(DACH)Pt(OHeq )2 (OAcax )2 ]. In the work presented here, we investigated the reactivity and synthetic usability of this complex to be exploited as a precursor for the development of novel platinum(IV) complexes, not able to be synthesized by conventional protocols. Indeed, we could substitute the equatorial hydroxido ligand(s) e.g. by one or two monodentate biotin ligands (which would be oxidized under standard methods). The formed complexes turned out to be very stable with slow ligand release after reduction, ideal for long-circulating tumor-targeting strategies. Therefore, two platinum(IV) complexes with equatorial maleimides, capable of exploiting serum albumin as a natural nanocarrier, were synthesized as well. The complexes showed massively prolonged plasma half-life and distinctly improved anticancer activity in vivo compared to oxaliplatin. Taken together, the newly developed synthetic platform allows the simple and specific insertion of equatorial ligands into platinum(IV) complexes. This will enable the attachment of three different (bioactive) moieties generating targeted triple-action platinum(IV) prodrugs within one single platinum complex.

Biodistribution of Agmatine to Brain and Spinal Cord after Systemic Delivery.

Agmatine, an endogenous polyamine, has been shown to reduce chronic pain behaviors in animal models and in patients. This reduction is due to inhibition of the GluN2B subunit of the N-methyl-D-aspartate receptor (NMDAR) in the central nervous system (CNS). The mechanism of action requires central activity, but the extent to which agmatine crosses biologic barriers such as the blood-brain barrier (BBB) and intestinal epithelium is incompletely understood. Determination of agmatine distribution is limited by analytical protocols with low sensitivity and/or inefficient preparation. This study validated a novel bioanalytical protocol using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for quantification of agmatine in rat biologic matrices. These protocols were then used to determine the plasma pharmacokinetics of agmatine and the extent of distribution to the CNS. Precision and accuracy of the protocol met US Food and Drug Administration (FDA) standards in surrogate matrix as well as in corrected concentrations in appropriate matrices. The protocol also adequately withstood stability and dilution conditions. Upon application of this protocol to pharmacokinetic study, intravenous agmatine showed a half-life in plasma ranging between 18.9 and 14.9 minutes. Oral administration led to a prolonged plasma half-life (74.4-117 minutes), suggesting flip-flop kinetics, with bioavailability determined to be 29%-35%. Intravenous administration led to a rapid increase in agmatine concentration in brain but a delayed distribution and lower concentrations in spinal cord. However, half-life of agmatine in both tissues is substantially longer than in plasma. These data suggest that agmatine adequately crosses biologic barriers in rat and that brain and spinal cord pharmacokinetics can be functionally distinct. SIGNIFICANCE STATEMENT: Agmatine has been shown to be an effective nonopioid therapy for chronic pain, a significantly unmet medical necessity. Here, using a novel bioanalytical protocol for quantification of agmatine, we present the plasma pharmacokinetics and the first report of agmatine oral bioavailability as well as variable pharmacokinetics across different central nervous system tissues. These data provide a distributional rationale for the pharmacological effects of agmatine as well as new evidence for kinetic differences between brain and spinal cord.

Recent advances in self-assembled cyclic peptide-based smart nanostructures

Peptide chemistry has emerged as one of the growing fields of research. Peptide chemistry has positively impacted various areas, including biochemistry, medicine, hormonal therapy, drug delivery, food and the cosmetic industry, materials science, and nanotechnology, via the development of ways to change and imitate the shape and function of peptide structures. The structural changes of peptides and the employment of innovative synthetic techniques have left an indelible mark on a number of scientific disciplines. Numerous nanostructures based on simple and complicated peptides have been constructed so far; however, cyclic peptides have attracted a great deal of interest from the scientific community due to their wide range of applications and distinctive properties. These properties include self-assembly, morphogenesis, and charge distribution, among others. In addition, nanostructured cyclic peptides offer increased and effective performance due to their high stability, prolonged plasma half-life, membrane permeability, and efficient transport, among other attributes. Recent work indicates the manufacture of nanostructured cyclic peptides by chemical means. In this review, a brief investigation of the morphology of cyclic peptides was conducted. In addition, the therapeutic potential of these nanostructured cyclic peptides and the prognosis for a variety of potential applications are also discussed.

THE METABOLISM OF LUFOTRELVIR, A PRODRUG INVESTIGATED FOR THE TREATMENT OF SARS-COV-2, IN HUMANS FOLLOWING INTRAVENOUS ADMINISTRATION.

The metabolism of lufotrelvir, a novel phosphate prodrug of PF-00835231 for the treatment of COVID-19, was evaluated in healthy human volunteers and clinical trial participants with COVID-19 following intravenous infusion. The prodrug was completely converted to PF-00835231 which was subsequently cleared by hydrolysis, hydroxylation, ketoreduction, epimerization, renal clearance, and secretion into the feces. The main circulating metabolite was a hydrolysis product (M7) which was present at concentrations greater than PF-00835231, and this was consistent between healthy volunteers and participants with COVID-19. Upon administration of [14C]lufotrelvir, only 63% of the dose was obtained in excreta over 10 days and total drug-related material demonstrated a prolonged terminal phase half-life in plasma. A considerable portion of the labelled material was unextractable from fecal homogenate and plasma. The position of the carbon-14 atom in the labelled material was at a leucine carbonyl, and pronase digestion of the pellet derived from extraction of the fecal homogenate showed that [14C]leucine was released. Significance Statement Lufotrelvir is an experimental phosphate prodrug intravenous therapy investigated for the potential treatment of COVID-19 in a hospital setting. The overall metabolism of lufotrelvir was determined in human healthy volunteers and clinical trial participants with COVID-19. Conversion of the phosphate prodrug to the active drug PF-00835231 was complete and the subsequent metabolic clearance of the active drug was largely via amide bond hydrolysis. Substantial drug-related material was not recovered due to loss of the carbon-14 label to endogenous metabolism.

Nanocomplexes of Biodegradable Anticancer Macromolecules: Prolonged Plasma Half-life, Reduced Toxicity, and Increased Tumor Targeting.

Anticancer drug resistance is a large contributing factor to the global mortality rate of cancer patients. Anticancer macromolecules such as polymers were recently reported to overcome this issue. Anticancer macromolecules have unselective toxicity because they are highly positively charged. Herein, an anionic biodegradable polycarbonate carrier was synthesized and utilized to form nanocomplexes with an anticancer polycarbonate via self-assembly to neutralize its positive charges. Biotin was conjugated to the anionic carrier and served as a cancer cell-targeting moiety. The nanoparticles had sizes of <130nm with anticancer polymer loading levels of 38%-49%. Unlike the small molecular anticancer drug doxorubicin, the nanocomplexes effectively inhibited the growth of both drug-susceptible MCF7 and drug-resistant MCF7/ADR human breast cancer cell lines with low half maximal inhibitory concentration (IC50 ). The nanocomplexes increased the anticancer polymer's in vivo half-life from 1h to 6 - 8h, and rapidly killed BT474 human breast cancer cells primarily via an apoptotic mechanism. The nanocomplexes significantly increased the median lethal dose (LD50) and reduced the injection site toxicity of the anticancer polymer. They suppressed tumor growth by 32% - 56% without causing any damage to the liver and kidneys. These nanocomplexes may potentially be used for cancer treatment to overcome drug resistance. This article is protected by copyright. All rights reserved.

Abstract 2323: ZGGS18, a bispecific drug candidate of anti-VEGF and TGFbeta-trap, inhibiting tumor proliferation and improving tumor microenvironment with I/O therapy

Abstract ZGGS18 is a bifunctional fusion protein targeting both human vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β), which consist of a monoclonal antibody against VEGF and an engineered extracellular domain of TGF-β receptor II (TGF-βRII ECD). In vitro studies show ZGGS18 specifically and simultaneously binding towards various VEGFs and capturing different TGF-β isoforms by trap domains that play a synergistic role to block signaling of VEGF/TGF-β with their receptors. For instance, ZGGS18 inhibits human VEGF121 and 165 induced HUVEC proliferation; ZGGS18 also neutralizes TGF-β1 and -β3 related growth arrest of HT-2 cells. Further studies show that ZGGS18 does not induce ADCC- or CDC-dependent killing tumor cells expressing two soluble targets, suggesting its binding affinities towards Fc-gamma and complement C1q receptors without effector functions. These results support functional depletion of VEGF/TGF-β in tumor microenvironment (TME) by ZGGS18 essentially for its MOA to inhibit tumor growth. A dose-dependent in vivo anti-tumor efficacy of ZGGS18 (1, 3 and 15 mg/kg) has been found in PBMC humanized H460 lung cancer xenograft model. In addition, ZGGS18 has enhanced efficacies to kill tumor growth in MC38 xenograft models when it is combined with anti-PD-1 antibodies (keytruda), support the improvement of TME for I/O reagents by ZGGS18 in cooperation with its role in anti-proliferation and anti-angiogenesis. Toxicity and safety pharmacology studies with weekly repeat doses for four weeks in cynomolgus monkeys indicate that ZGGS18 is well tolerated over the dose range 13, 40 and 120 mg/kg without obviously toxic or adverse effects (NOAEL =120 mg/kg). PK/TK analyses indicate that ZGG18 has a prolonged half-life in plasma, attribute to its human neonatal Fc receptor (FcRn) binding and pH-dependent interaction for mAb recycling. The IND application of ZGGS18 has been approved by both FDA and Chinese NMPA for clinical trials in treatment of advanced tumors as monotherapy and/or in combined with PD-1/PD-L1 based I/O therapies. Citation Format: Ruifeng Liu, Alfonso Suarez, Tyler Liban, Bin Zhang, Tongcheng Dai, Margaret Karow, Jackie Sheng, Zelin Sheng, Binhua Lv, Bing Zhu. ZGGS18, a bispecific drug candidate of anti-VEGF and TGFbeta-trap, inhibiting tumor proliferation and improving tumor microenvironment with I/O therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2323.

Abstract 6313: IND-enabling studies for GrB-Fc-IT4: in vivo efficacy, pharmacokinetics, schedule optimization and maximum tolerated dose

Abstract Fn14 (fibroblast growth factor-inducible 14) is the only known receptor of TWEAK (tumor necrosis factor-related weak inducer of apoptosis). The binding of TWEAK to Fn14 activates several intracellular signal transduction cascades that lead to cell death, proliferation, migration, or survival depending on the cellular contexts. Several studies have shown that Fn14 is upregulated in many solid tumors when compared with healthy tissues. The activation of TWEAK/Fn14 signaling increases the proliferation, invasion, and migration of tumor cells. Moreover, angiogenesis, pro-inflammatory cytokine expression, and epithelial-mesenchymal transitions are promoted upon TWEAK/Fn14 activation. Therefore, Fn14 is considered a potential therapeutic target, and attempts to generate targeted therapeutics have achieved limited success in pre-clinical and clinical trials. We generated a completely human fusion protein, GrB-Fc-IT4, consisting of the IT4 scFv antibody targeting the Fn14 receptor and the serine protease granzyme B (GrB) as the cytotoxic payload. Comprehensive mechanism of action studies showed that GrB-Fc-IT4 effectively induced cell death in vitro against a broad selection of tumor types with high specificity. The intracellular pathway included rapid, irreversible activation of the caspase cascade and independent mitochondrial depolarization leading to intense apoptotic cellular damage. Pharmacokinetic studies in mice showed that GrB-Fc-IT4 cleared bi-exponentially from plasma with a rapid initial clearance (t ½α = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2β = 35 hours), similar to that of IgGs. Based on our PK study, we compared the in vivo antitumor efficacy of GrB-Fc-IT4 against A549 (NSCLC) tumors, using two different dosing schedules (QODx5 vs QWx5). Tumor efficacy showed a clear dose and schedule dependence. Mice bearing A549 tumors treated with 32 mg/kg/dose (QOWx5) exhibited complete tumor growth inhibition (7/10 tumors regressed) with 3/10 tumors showing no growth up to 50 days after the last dose. Thus, GrB-Fc-IT4 displays impressive in vitro and in vivo cytotoxic effects. Even though GrB-Fc-IT4 cross-reacts with the murine Fn14 antigen, maximum tolerated dose studies in mice have confirmed that GrB-Fc-IT4 is a safe product, as mice treated with the drug up to 500 mg/kg showed no evidence of toxicity (changes in respiration, rough hair coat, unusual behavior or hunched posture). Taking in consideration these results we estimate that the therapeutic index for this class of drug is higher than four. Our data suggest that GrB-Fc-IT4 is a novel antitumor agent with a unique mechanism of action compared to all other therapeutic agents. The exceptionally safe and effective profile of this drug makes it an ideal candidate for advancing to clinical trials. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Mihai Gagea, Michael G. Rosenblum. IND-enabling studies for GrB-Fc-IT4: in vivo efficacy, pharmacokinetics, schedule optimization and maximum tolerated dose [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6313.

B-Cell Targeting Anti-CD37 Humanized Antibodies Engineered for Potent Effector Functions and Extended Plasma Half-Life

Background: CD37 is a tetraspanin protein abundant on the surface of B-cells but absent in hematopoietic stem cells and plasma cells. Its expression pattern has attracted interest as a therapeutic target for B-cell malignancies including non-Hodgkin lymphomas (NHL), a heterogeneous disease with substantial unmet clinical need due to relapse and resistance to currently available treatments. During the last years, different approaches have been followed to exploit CD37 in targeting malignant B-cells, such as the generation of CAR-T cells and monoclonal antibodies. Of note, the anti-CD37 antibody-drug conjugates (ADC) naratuximab emtansine and the CD37-targeting radioimmunotherapy (RIT) Betalutin have shown efficacy in NHL patients during clinical testing. Here we report on the design of humanized anti-CD37 IgG1 antibodies (NNV029, NNV031 and NNV032) that are equipped with a unique Fc technology and N-glycan engineering for enhanced effector functions and extended plasma half-life. Methods: The humanized anti-CD37 candidates were characterized in vitro and in vivo and benchmarked against the anti-CD20 antibodies approved for NHL treatment, rituximab and obinutuzumab, and a recombinant version of the bi-paratopic anti-CD37 antibody DuoHexaBody-CD37, currently in Phase I clinical development. We assessed their Fcγ receptor binding properties and their ability to induce antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) against CD37-expressing NHL cell lines and patient-derived chronic lymphocytic leukemia (CLL) cells. Moreover, we evaluated their human FcRn binding and transport properties using the human endothelial recycling assay (HERA) followed by studies in human FcRn (Tg32) expressing mice. Lastly, we compared the therapeutic efficacy of the humanized antibodies versus obinutuzumab and recombinant DuoHexaBody-CD37 in a xenograft disseminated Daudi tumor model in Tg32 mice. Results: We revealed that the designed anti-CD37 IgG1 candidates have higher affinity towards FcγRIIa expressed on macrophages and that afucosylation enhanced FcγRIII engagement. Accordingly, using cell-based effector assays, potent ADCP and ADCC were measured. In NHL Burkitt's lymphoma cell lines, ADCP induction by the anti-CD37 humanized antibodies was higher than that displayed by obinutuzumab, rituximab and recombinant DuoHexaBody-CD37, while in DLBCL and MCL cell models it was in a similar range or slightly lower than that shown by the benchmarking antibodies. Moreover, the afucosylated version promoted very strong ADCC, which was superior to that of recombinant DuoHexaBody-CD37 and rituximab, but comparable to that of the afucosylated antibody obinutuzumab. In contrast, all the humanized anti-CD37 IgG1 candidates displayed very modest CDC activation. Fc-engineering for enhanced human FcRn binding was shown to increase human FcRn-mediated rescue from intracellular degradation, which translated into prolonged plasma half-life in Tg32 mice of the anti-CD37 IgG1 antibodies. Finally, all the designed anti-CD37 antibodies performed significantly better than vehicle control treatment in treating Daudi-induced disease in vivo, showing equal therapeutic effect as that of obinutuzumab and recombinant DuoHexaBody-CD37. Conclusions: Taken together, the humanized and Fc-engineered anti-CD37 antibodies show potent effector functions, favorable pharmacokinetic properties and in vivo therapeutic efficacy. As such, the designed CD37 antibodies should be attractive modalities in targeted treatment of B cell malignancies, such as NHL, including lymphomas non-responding to anti-CD20 treatments, but also potentially for B-cell driven autoimmune disorders.

NNV024, a Humanized Anti-CD37 Antibody with Enhanced ADCC and Extended Plasma Half-Life for the Treatment of B-Cell Malignancies

Background: There is a significant unmet clinical need for new therapeutic approaches for patients with B-cell non-Hodgkin lymphomas (B-NHL) who relapse or are refractory to anti-CD20 therapies. Moreover, certain B-cell malignancies do not express CD20. High expression of the CD37 antigen on malignant B-cells associated with limited or no expression on other hematopoietic cells makes CD37 a highly attractive alternative target for B-cell depleting therapies. We have therefore developed a humanized anti-CD37 monoclonal antibody (NNV024) engineered to exhibit a strong antibody-dependent cellular cytotoxicity (ADCC). Methods: Generation of NNV024 was performed by combining in silico CDR-grafting and structure-based back mutation. Candidates were then selected for their affinity to CD37 expressing cell line, Ramos. In silico immunogenicity prediction tools were integrated in the selection process to support the generation of a candidate with low immunogenicity potential (NNV023). Last, to increase ADCC, the selected candidate was afucosylated at the N-linked bi-antennary glycan at N297 of the Fc region (NNV024). The NNV024 was characterized in vitro and in vivo. Binding to human Fc gamma receptors (FcγRs), neonatal Fc receptor (FcRn), and complement component 1q (C1q) was confirmed by ELISA and SPR. ADCC and antibody-dependent cellular phagocytosis (ADCP) induction were measured using cell-based reporter assays with eleven cell lines derived from B-NHL subtypes such as Burkitt's lymphoma, diffuse large B-Cell lymphoma (DLBCL), and mantel cell lymphoma (MCL). All cell lines express both CD20 and CD37 on their cell surface. In addition, B cell chronic lymphocytic leukaemia (B-CLL) cells isolated from patient samples were used to test induction of ADCC, ADCP and complement-dependent cytotoxicity (CDC) in vitro. NNV024 plasma pharmacokinetics were assessed in Tg32 transgenic mice expressing human FcRn, which is considered a key regulator of IgG plasma half-life. A therapy study comparing NNV024 (n=10) and obinutuzumab (n=10) was performed using a disseminated tumor (Daudi) model in CB17-SCID mice (107 cells/mouse, i.v.). The anti-CD20 Abs rituximab and obinutuzumab as well as the anti-CD37 Ab, Duohexabody-CD37, were used for benchmarking. Results: As expected, the glyco-engineering increased affinity of NNV024 to FcγRIIIa (KD before afucosylation: 460.8 ng/mL; KD after: 104.1 ng/mL). Binding of Abs to FcγRIIIa activates immunological effector cells such as Natural Killer cells, macrophages, monocytes, and eosinophils. NNV024 demonstrated ADCC superior to that observed with obinutuzumab, rituximab, and Duohexabody-CD37. NNV024 exhibited a 2-fold prolonged plasma half-life in mice (8.9±0.8 days) compared to obinutuzumab (4.4±1.3 days) and Duohexabody-CD37 (4.1±1.2 days). Mice survival was enhanced up to ~ 40% when treated with NNV024 (10 µg/mouse - 70.5 days; 50 µg/mouse - 84 days) compared to obinutuzumab at a dose of 10 µg (70.5 days vs. 53 days) or 50 µg (84 days vs. 62 days). Conclusion: These encouraging preclinical results support further development of NNV024 as a potential therapeutic mAb candidate for the treatment of B-cell malignancies and B-cell driven autoimmune disorders.

Tumor-Targeting Polymer–Drug Conjugate for Liver Cancer Treatment In Vitro

Bufalin (buf) has poor solubility in aqueous solution, poor tumor targeting, and many non-specific toxic and side effects. The advantages of high-molecular-weight polymer conjugates are that they can improve the water solubility of buf, prolong plasma half-life, and reduce non-specific toxicity. A novel water-soluble polymer-drug conjugate with buf and fluorescein pendants was prepared by the combination of reversible addition-fragmentation transfer (RAFT) polymerization and click chemistry. Its anticancer performance and cellular uptake behavior against liver cancer were investigated in vitro. The polymer-buf conjugates exhibit controlled release and tumor-targeting capabilities, showing promise for clinical applications.

Evaluating the efficacy of prototype antiseizure drugs using a preclinical pharmacokinetic approach.

Pharmacokinetics (PK) of a drug drive its exposure, efficacy, and tolerability. A thorough preclinical PK assessment of antiseizure medications (ASMs) is therefore essential to evaluate the clinical potential. We tested protection against evoked seizures of prototype ASMs in conjunction with analysis of plasma and brain PK as a proof-of-principle study to enhance our understanding of drug efficacy and duration of action using rodent seizure models. In vivo seizure protection assays were performed in adult male CF-1 mice and Sprague Dawley rats. Clobazam (CLB), N-desmethyl CLB (NCLB), carbamazepine (CBZ), CBZ-10,11-epoxide (CBZE), sodium valproate (VPA), and levetiracetam (LEV) concentrations were quantified in plasma and brain using liquid chromatography-tandem mass spectrometry. Mean concentrations of each analyte were calculated and used to determine PK parameters via noncompartmental analysis in Phoenix WinNonLin. NCLB concentrations were approximately 10-fold greater than CLB in mice. The antiseizure profile of CLB was partially sustained by NCLB in mice. CLB concentrations were lower in rats than in mice. CBZE plasma exposures were approximately 70% of CBZ in both mice and rats, likely contributing to the antiseizure effect of CBZ. VPA showed a relatively short half-life in both mice and rats, which correlated with a sharp decline in efficacy. LEV had a prolonged brain and plasma half-life, associated with a prolonged duration of action in mice. The study demonstrates the utility of PK analyses for understanding the seizure protection time course in mice and rats. The data indicate that distinct PK profiles of ASMs between mice and rats likely drive differences in drug efficacy between rodent models.

Abstract 5216: Optimizing antitumor efficacy of granzyme B human fusion constructs targeting the Fn14 antigen in solid tumors. Pre IND pharmacokinetics, schedule, dose optimization and MTD studies

Abstract We previously demonstrated that a human fusion protein construct targeting the Fn14 receptor and containing the cytotoxic granzyme B (GrB) payload (GrB-Fc-IT4) displays impressive in vitro and in vivo cytotoxic effects. Comprehensive mechanism of action studies show that GrB-Fc-IT4 effectively induces cell death in vitro against a broad selection of tumor types with high specificity. The intracellular pathway includes irreversible activation of the caspase cascade and independent mitochondrial depolarization leading to an intense apoptotic cellular damage. This activation occurs quickly and efficiently. Pharmacokinetic studies in mice showed that GrB-Fc-IT4 was cleared bi-exponentially from plasma with a rapid initial clearance (t ½alpha = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2beta = 35 hours) similar to that of IgGs. Based on our pharmacokinetic study, we employed a QODX5 therapy schedule and demonstrated impressive antitumor activity against several tumor models including PDX-lung, A459 (NSCLC) and MDA-MB231(TNBC) tumor models. In the present study we compared the in vivo antitumor efficacy of GrB-Fc-IT4 against A549 (NSCLC) tumor cell line, using two different dosing schedules (QODx5 vs QWx5). There was a clear dose and schedule dependence and that treating mice bearing A549 tumors with 32mg/kg/dose using a QOWx5 schedule induced a complete tumor growth inhibition (7/10 tumors regressed) with 3/10 tumors showing no growth up to 50 days after the last dose. Preliminary results suggest that GrB-Fc-IT4 may reduce the formation of spontaneous A549 metastasis into lungs. Further studies are planned to examine the potential of the fusion construct to prevent spontaneous metastatic spread in lung tumor models. Although the fusion construct cross-reacts with the murine Fn14 antigen, maximum tolerated dose studies have confirmed that GrB-Fc-IT4 is a safe product, showing no evidence of toxicity (weight loss) in mice treated with up to 500mg/kg. This suggests that the therapeutic index (TI) for this class of agents is &amp;gt;4. Our data suggest that GrB-Fc-IT4 is a novel class of antitumor agents with a unique mechanism of action. This agent seems to be an exceptionally safe and effective drug and appears to be an ideal candidate for advancing to clinical trials. Research conducted, in part, by Clayton Foundation for Research. Citation Format: Ana Alvarez De Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Mihai Gagea, Michael G. Rosenblum. Optimizing antitumor efficacy of granzyme B human fusion constructs targeting the Fn14 antigen in solid tumors. Pre IND pharmacokinetics, schedule, dose optimization and MTD studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5216.

Fc-based Duokines: dual-acting costimulatory molecules comprising TNFSF ligands in the single-chain format fused to a heterodimerizing Fc (scDk-Fc)

ABSTRACT Targeting costimulatory receptors of the tumor necrosis factor superfamily (TNFSF) to activate T-cells and promote anti-tumor T-cell function have emerged as a promising strategy in cancer immunotherapy. Previous studies have shown that combining two different members of the TNFSF resulted in dual-acting costimulatory molecules with the ability to activate two different receptors either on the same cell or on different cell types. To achieve prolonged plasma half-life and extended drug disposition, we have developed novel dual-acting molecules by fusing single-chain ligands of the TNFSF to heterodimerizing Fc chains (scDuokine-Fc, scDk-Fc). Incorporating costimulatory ligands of the TNF superfamily into a scDk-Fc molecule resulted in enhanced T-cell proliferation translating in an increased anti-tumor activity in combination with a primary T-cell-activating bispecific antibody. Our data show that the scDk-Fc molecules are potent immune-stimulatory molecules that are able to enhance T-cell mediated anti-tumor responses.

Novel VHH-Based Tracers with Variable Plasma Half-Lives for Imaging of CAIX-Expressing Hypoxic Tumor Cells.

Hypoxic areas are present in the majority of solid tumors, and hypoxia is associated with resistance to therapies and poor outcomes. A transmembrane protein that is upregulated by tumor cells that have adapted to hypoxic conditions is carbonic anhydrase IX (CAIX). Therefore, noninvasive imaging of CAIX could be of prognostic value, and it could steer treatment strategies. The aim of this study was to compare variants of CAIX-binding VHH B9, with and without a C-terminal albumin-binding domain with varying affinity (ABDlow and ABDhigh), for SPECT imaging of CAIX expression. The binding affinity and internalization of the various B9-variants were analyzed using SK-RC-52 cells. Biodistribution studies were performed in mice with subcutaneous SCCNij153 human head and neck cancer xenografts. Tracer uptake was determined by ex vivo radioactivity counting and visualized by SPECT/CT imaging. Furthermore, autoradiography images of tumor sections were spatially correlated with CAIX immunohistochemistry. B9-variants demonstrated a similar moderate affinity for CAIX in vitro. Maximal tumor uptake and acceptable tumor-to-blood ratios were found in the SCCNij153 model at 4 h post injection for [111In]In-DTPA-B9 (0.51 ± 0.08%ID/g and 8.1 ± 0.85, respectively), 24 h post injection for [111In]In-DTPA-B9-ABDlow (2.39 ± 0.44%ID/g and 3.66 ± 0.81, respectively) and at 72 h post injection for [111In]In-DTPA-B9-ABDhigh (8.7 ± 1.34%ID/g and 2.43 ± 0.15, respectively). An excess of unlabeled monoclonal anti-CAIX antibody efficiently inhibited tumor uptake of [111In]In-DTPA-B9, while only a partial reduction of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh uptake was found. Immunohistochemistry and autoradiography images showed colocalization of all B9-variants with CAIX expression; however, [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh also accumulated in non-CAIX expressing regions. Tumor uptake of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh, but not of [111In]In-DTPA-B9, could be visualized with SPECT/CT imaging. In conclusion, [111In]In-DTPA-B9 has a high affinity to CAIX and shows specific targeting to CAIX in head and neck cancer xenografts. The addition of ABD prolonged plasma half-life, increased tumor uptake, and enabled SPECT/CT imaging. This uptake was, however, partly CAIX- independent, precluding the ABD-tracers for use in hypoxia quantification in this tumor type.

Development of improved SRC-3 inhibitors as breast cancer therapeutic agents.

Steroid receptor coactivators (SRCs) possess specific and distinct oncogenic roles in the initiation of cancer and in its progression to a more aggressive disease. These coactivators interact with nuclear receptors and other transcription factors to boost transcription of multiple genes, which potentiate cancer cell proliferation, migration, invasion, tumor angiogenesis and epithelial-mesenchymal transition (EMT). Targeting SRCs using small molecule inhibitors (SMIs) is a promising approach to control cancer progression and metastasis. By high-throughput screening analysis, we recently identified SI-2 as a potent SRC SMI. To develop therapeutic agents, SI-10 and SI-12, the SI-2 analogs are synthesized that incorporate the addition of F atoms to the SI-2 chemical structure. As a result, these analogs exhibit a significantly prolonged plasma half-life, minimal toxicity and improved hERG activity. Biological functional analysis showed that SI-10 and SI-12 treatment (5-50 nM) can significantly inhibit viability, migration and invasion of breast cancer cells in vitro and repress the growth of breast cancer PDX organoids. Treatment of mice with 10 mg/kg/day of either SI-10 or SI-12 was sufficient to repress the growth of xenograft tumors derived from MDA-MB-231 and LM2 cells. Furthermore, in spontaneous and experimental metastasis mouse models developed from MDA-MB-231 and LM2 cells, respectively, SI-10 and SI-12 effectively inhibited the progression of breast cancer lung metastasis. These results demonstrate that SI-10 and SI-12 are promising therapeutic agents and are specifically effective in blocking tumor metastasis, a key point in tumor progression to a more lethal state that results in patient mortality in the majority of cases.

Lipophilic Conjugates of Drugs: A Tool to Improve Drug Pharmacokinetic and Therapeutic Profiles.

Lipophilic conjugates (LCs) of small molecule drugs have been used widely in clinical and pre-clinical studies to achieve a number of pharmacokinetic and therapeutic benefits. For example, lipophilic derivatives of drugs are employed in several long acting injectable products to provide sustained drug exposure for hormone replacement therapy and to treat conditions such as neuropsychiatric diseases. LCs can also be used to modulate drug metabolism, and to enhance drug permeation across membranes, either by increasing lipophilicity to enhance passive diffusion or by increasing protein-mediated active transport. Furthermore, such conjugation strategies have been employed to promote drug association with endogenous macromolecular carriers (e.g. albumin and lipoproteins), and this in turn results in altered drug distribution and pharmacokinetic profiles, where the changes can be 'general' (e.g. prolonged plasma half-life) or 'specific' (e.g. enhanced delivery to specific tissues in parallel with the macromolecular carriers). Another utility of LCs is to enhance the encapsulation of drugs within engineered nanoscale drug delivery systems, in order to best take advantage of the targeting and pharmacokinetic benefits of nanomedicines. The current review provides a summary of the mechanisms by which lipophilic conjugates, including in combination with delivery vehicles, can be used to control drug delivery, distribution and therapeutic profiles. The article is structured into sections which highlight a specific benefit of LCs and then demonstrate this benefit with case studies. The review attempts to provide a toolbox to assist researchers to design and optimise drug candidates, including consideration of drug-formulation compatibility.

Surfactant Assisted Rapid-Release Liposomal Strategies Enhance the Antitumor Efficiency of Bufalin Derivative and Reduce Cardiotoxicity.

BackgroundBF211, a derivative of bufalin (BF), shows significantly improved solubility and potent antitumor efficiency compared to BF. Unfortunately, the unwanted toxicity such as cardiotoxicity caused by unspecific distribution has hindered its clinical use.MethodsPEGylated BF211 liposomes (BF211@Lipo) were designed and optimizely prepared based on the pre-prescription research. In vitro and in vivo cardiotoxicity was evaluated. In vivo pharmacokinetics and biodistribution of BF211@Lipo were investigated. In vivo antitumor activity and toxicity were evaluated in HepG2 cell xenograft models. The rapid-release triggered by Poloxamer 188 (P188) was assessed in vitro and in vivo.ResultsThe optimized BF211@Lipo displayed a spherical morphology with a size of (164.6 ± 10.3) nm and a high encapsulation efficiency of (93.24 ± 2.15) %. The in vivo concentration–time curves of BF211 loaded in liposomes showed a prolonged half-life in plasma and increased tumor accumulation. No obvious abnormality in electrocardiograms was observed in guinea pigs even at 9 mg/kg. Moreover, to improve the efficient release of BF211@Lipo, a surfactant-assisted rapid-release strategy was developed, and the release-promoting mechanism was revealed by the fluorescence resonance energy transfer (FRET) and fluorescence nanoparticle tracking analysis (fl-NTA) technology. Sequential injection of BF211@Lipo and P188 could ignite the “cold” liposomes locally in tumor regions, facilitating the burst release of BF211 and enhancing the therapeutic index.ConclusionOur progressive efforts that begin with preparation technology and dosage regimen enable BF211 to like a drug, providing a promising nano platform to deliver the cardiac glycosides and alleviate the side effects by decreasing unspecific biodistribution.