Porphyrin photosensitizer molecules as effective medicine candidates for photodynamic therapy: electronic structure information aided design.

Traditional photosensitizers (PS) in photodynamic therapy (PDT) have restricted tissue penetrability of light and a lack of selectivity for tumor cells, which diminishes the efficiency of PDT. Our aim is to effectively screen porphyrin-based PS medication through computational simulations of large-scale design and screening of PDT candidates via a precise description of the state of the light-stimulated PS molecule. Perylene-diimide (PDI) shows an absorption band in the near-infrared region (NIR) and a great photostability. Meanwhile, the insertion of metal can enhance tumor targeting. Therefore, on the basis of the original porphyrin PS segments, a series of metalloporphyrin combined with PDI and additional allosteric Zn-porphyrin-PDI systems were designed and investigated. Geometrical structures, frontier molecular orbitals, ultraviolet-visible (UV-vis) absorption spectra, adiabatic electron affinities (AEA), especially the triplet excited states and spin-orbit coupling matrix elements (SOCME) of these expanded D-A porphyrin were studied in detail using the density functional theory (DFT) and time-dependent density functional theory (TDDFT) methods. PS candidates, conforming type I or II mechanism for PDT, have been researched carefully by molecular docking which targeted Factor-related apoptosis (Fas)/Fas ligand (Fasl) mediated signaling pathway. It was found that porphyrin-PDI, Fe2-porphyrin-PDI, Zn-porphyrin-PDI, Mg-porphyrin-PDI, Zn-porphyrin combined with PDI through single bond (compound 1), and two acetylenic bonds (compound 2) in this work would be proposed as potential PS candidates for PDT process. This study was expected to provide PS candidates for the development of novel medicines in PDT.

Optimized combination of zero-valent iron and oxygen-releasing biochar as cathodes of microbial fuel cells to enhance copper migration in sediment.

Membrane-less single-medium sediment microbial fuel cells (single-SMFC) can remove Cu2+ from sediment through electromigration. However, the high mass transfer resistance of the sediment and amount of oxygen at the cathode of the SMFC limit its Cu2+ removal ability. Therefore, this study used an oxygen-releasing bead (ORB) for slow oxygen release to increase oxygen at the SMFC cathode and improve the mass transfer property of the sediment. Resultantly, the copper removal efficiency of SMFC increased significantly. Response surface methodology was used to optimize the nano zero-valent iron (nZVI)-modified biochar as the catalyst to enhance the ability of the modified ORB (ORBm) to remove Cu2+ and slow release of O2. The maximum Cu2+ removal (95 %) and the slowest O2 release rate (0.41 mg O2/d·g ORBm) were obtained when the CaO2 content and ratio of nZVI-modified biochar to unmodified biochar were 0.99 g and 4.95, respectively. When the optimized ORBm was placed at the single-SMFC cathode, the voltage output and copper removal increased by 4.6 and 2.1 times, respectively, compared with the system without ORBm. This shows that the ORBm can improve the migration of Cu2+ in the sediment, providing a promising remediation method for Cu-contaminated sediments.

A Cell Surface-Binding Antibody Atlas Nominates a MUC18-Directed Antibody-Drug Conjugate for Targeting Melanoma.

Recent advances in targeted therapy and immunotherapy have substantially improved the treatment of melanoma. However, therapeutic strategies are still needed for unresponsive or treatment-relapsed patients with melanoma. To discover antibody-drug conjugate (ADC)-tractable cell surface targets for melanoma, we developed an atlas of melanoma cell surface-binding antibodies (pAb) using a proteome-scale antibody array platform. Target identification of pAbs led to development of melanoma cell killing ADCs against LGR6, TRPM1, ASAP1, and MUC18, among others. MUC18 was overexpressed in both tumor cells and tumor-infiltrating blood vessels across major melanoma subtypes, making it a potential dual-compartment and universal melanoma therapeutic target. AMT-253, an MUC18-directed ADC based on topoisomerase I inhibitor exatecan and a self-immolative T moiety, had a higher therapeutic index compared with its microtubule inhibitor-based counterpart and favorable pharmacokinetics and tolerability in monkeys. AMT-253 exhibited MUC18-specific cytotoxicity through DNA damage and apoptosis and a strong bystander killing effect, leading to potent antitumor activities against melanoma cell line and patient-derived xenograft models. Tumor vasculature targeting by a mouse MUC18-specific antibody-T1000-exatecan conjugate inhibited tumor growth in human melanoma xenografts. Combination therapy of AMT-253 with an antiangiogenic agent generated higher efficacy than single agent in a mucosal melanoma model. Beyond melanoma, AMT-253 was also efficacious in a wide range of MUC18-expressing solid tumors. Efficient target/antibody discovery in combination with the T moiety-exatecan linker-payload exemplified here may facilitate discovery of new ADC to improve cancer treatment. SIGNIFICANCE: Discovery of melanoma-targeting antibodies using a proteome-scale array and use of a cutting-edge linker-payload system led to development of a MUC18-targeting antibody-exatecan conjugate with clinical potential for treating major melanoma subtypes.

Antibody-Exatecan Conjugates with a Novel Self-immolative Moiety Overcome Resistance in Colon and Lung Cancer.

Antibody-drug conjugates (ADC) using DNA topoisomerase I inhibitor DXd/SN-38 have transformed cancer treatment, yet more effective ADCs are needed for overcoming resistance. We have designed an ADC class using a novel self-immolative T moiety for traceless conjugation and release of exatecan, a more potent topoisomerase I inhibitor with less sensitivity to multidrug resistance (MDR). Characterized by enhanced therapeutic indices, higher stability, and improved intratumoral pharmacodynamic response, antibody-T moiety-exatecan conjugates targeting HER2, HER3, and TROP2 overcome the intrinsic or treatment resistance of equivalent DXd/SN-38 ADCs in low-target-expression, large, and MDR+ tumors. T moiety-exatecan ADCs display durable antitumor activity in patient-derived xenograft and organoid models representative of unmet clinical needs, including EGFR ex19del/T790M/C797S triple-mutation lung cancer and BRAF/KRAS-TP53 double-mutant colon cancer, and show synergy with PARP/ATR inhibitor and anti-PD-1 treatment. High tolerability of the T moiety-exatecan ADC class in nonhuman primates supports its potential to expand the responding patient population and tumor types beyond current ADCs. SIGNIFICANCE: ADCs combining a novel self-immolative moiety and topoisomerase I inhibitor exatecan as payload show deep and durable response in low-target-expressing and MDR+ tumors resistant to DXd/SN-38 ADCs without increasing toxicity. This new class of ADCs has the potential to benefit an additional patient population beyond current options. See related commentary by Gupta et al., p. 817. This article is highlighted in the In This Issue feature, p. 799.

Performance of bioelectrochemical systems in treating exhaust gas with power generation: Effects of shock-load, shut-down episodes and microbial community.

A diffusive packed anode-bioelectrochemical (Dpa-Bes) system was constructed by feeding waste gas from the cathode to the anode tank in DPa-Bes through a proton exchange membrane (PEM). The high removal of oxygen by the PEM and the effective combination of the two packing materials reduced the electron loss and enhanced the proton transfer capacity, promoting the removal of acetone from the exhaust gas and increasing the output power. The maximum acetone removal efficiency of the modified Dpa-Bes reached ∼99 % after seven days of closed-circuit operation, with a 3.2-fold increase in maximum power density and a 2.27-fold increase in closed-circuit voltage relative to those of the unmodified Dpa-Bes. When the acetone concentration was 2400 ppm, the removal efficiency was 73.22 % and the elimination capacity was at its highest value of 290.21 g/m3/h. Microbial analysis revealed that the conductive filter contained abundant facultative and anaerobic bacteria, whereas the non-conductive filter was rich in aerobic bacteria. The abundance of anaerobic and facultative microorganisms in Dpa-Bes was much higher than in the unmodified Dpa-Bes, and the dominant bacteria were Flavobacterium and Ferruginibacter.

Performance of a packed-bed anode bio-electrochemical reactor for power generation and for removal of gaseous acetone.

The packed anode bioelectrochemical system (Pa-BES) developed in this study is a type of BES that introduces waste gas into a cathode and then into an anode, thereby providing the cathode with sufficient oxygen and reducing the amount of oxygen to the anode to promote the output of electricity. When the empty-bed residence time was 45 s and the liquid flowrate was 35 mL/s, the system achieved optimal performance. Under these conditions, removal efficiency, mineralization efficiency, voltage output, and power density were 93.86%, 93.37%, 296.3 mV, and 321.12 mW/m3, respectively. The acetone in the waste gas was almost completely converted into carbon dioxide, indicating that Pa-BES can effectively remove acetone and has the potential to be used in practical situations. A cyclic voltammetry analysis revealed that the packings exhibited clear redox peaks, indicating that the Pa-BES has outstanding biodegradation and power generation abilities. Through microbial community dynamics, numerous organics degraders, electrochemically active bacteria, nitrifying and denitrifying bacteria were found, and the spatial distribution of these microbes were identified. Among them, Xanthobacter, Bryobacter, Mycobacteriums and Terrimonawas were able to decompose acetone or other organic substances, with Xanthobacter dominating. Bacterium_OLB10 and Ferruginibacter are the electrochemically active bacteria in Pa-BES, while Ferruginibacter is the most abundant in the main anode, which is responsible for electron collection and transfer.

Gaseous isopropanol removal in a microbial fuel cell with deoxidizing anode: Performance, anode characteristics and microbial community.

A deoxidizing packing material (DPM) with an encapsulated deoxidizing agent (DA) was developed to construct the packed anodes of a trickle-bed microbial fuel cell (TB-MFC) for treating waste gas. The encapsulated DA can consume O2 in waste gas and increase the voltage output and power density (PD) of the constructed TB-MFC. The DPM effectively enables the circulating water in TB-MFC for maintaining a low level of dissolved oxygen for 80 h. The results revealed that when the concentration of isopropanol (IPA) in waste gas was 0.74 g/m3, the TB-MFC (DPM with DA) exhibited an IPA removal efficiency (RE) of up to 99.7%. When DPM with DA was used as the packing material of the TB-MFC (486.6 mW/m3), the PD was 2.54 times that obtained when using coke as the packing material (191.6 mW/m3). The next-generation sequencing results demonstrated that because the oxygen content of the MFC anode chamber decreased over time in the TB-MFC, the richness of anaerobic electrogens (Pseudoxanthomonas, Flavobacterium, and Ferruginibacter) in the packing materials was increased. These electrogens mainly attached to the DPM, and IPA-degraders appeared in the circulating water of the TB-MFC. This enabled the TB-MFC to simultaneously achieve a high voltage output and IPA RE.

Waste expanded polystyrene modified with H2SO4/biodegradable chelating agent for reuse: As a highly efficient adsorbent to remove fluoroquinolone antibiotic from water.

Untreated wastewater containing fluoroquinolone antibiotics poses serious hazards to aquatic species and human health; therefore, treatment of waste expanded polystyrene (EPS) is a crucial environmental matter. In this study, waste EPS was modified with a H2SO4/biodegradable chelating agent, [S,S]-ethylenediamine-N,N'-disuccinic acid (EDDS), and used for highly efficient adsorption of the fluoroquinolone antibiotic ciprofloxacin. When ciprofloxacin of 25 mg/L was used, the H2SO4-modified EPS (EPSH2SO4) adsorbed 60.5% of the ciprofloxacin. During sulfonation, adding a low dose of EDDS markedly improved the adsorption ability of EPSH2SO4+EDDS. The optimal modification conditions were 95% H2SO4, 0.002 M EDDS, 80 °C, and 40 min. The increased adsorbent doses enhanced the adsorption. Approximately 0.2 g/L of EPSH2SO4+EDDS could effectively adsorb 97.8% of the ciprofloxacin (554.3 mg/g) within 30 min. Solution pH0 greatly influenced the adsorption, and the most suitable pH0 was 6. The Langmuir isotherm accurately described the adsorption behaviors of both EPSH2SO4 and EPSH2SO4+EDDS (R2 = 0.997-0.998). The adsorption ability of EPSH2SO4+EDDS (qmax = 1250 mg/g) was 32 times higher than that of EPSH2SO4 (qmax = 38.6 mg/g). A total of 1 M HCl effectively regenerated the exhausted adsorbent. The optimal solid/liquid ratio and time were 0.08 g/20 mL and 60 min, respectively. The regenerated EPSH2SO4+EDDS maintained a high adsorption ability (87.2%) after 10 regeneration cycles. The results thus indicate that the EPSH2SO4+EDDS adsorption-regeneration process is a potential approach to remove ciprofloxacin from water.

Enhancement of isopropanol removal with concomitant power generation by microbial fuel cells: Optimization of deoxidizing composite anodes using response surface methodology.

This study used a response surface method to develop a deoxidizing anode, which was introduced into microbial fuel cells (MFCs) to treat isopropanol (IPA) wastewater and waste gas. By embedding a deoxidizing agent (DA) into the anode of MFCs, a hypoxic environment can be created to enable anaerobic electrogens to be effectively attached to the anode surface and grow. Consequently, MFC power generation performance can be enhanced. The optimal coke and conductive carbon black ratio of an anode and percentage of DA added were 3.61 g/g and 3.15 %, respectively. The research design concurrently achieved the maximum deoxygenation efficiency (0.86 mg O2/bead), minimum disintegration ratio (3.51 %), and minimum resistance (30.2 Ω). The regression model had high prediction power (R2 > 0.93) for anode performance. As determined through multi-objective optimization, the results highly satisfied the target expectation (desirability = 0.82). The optimized deoxidizing anode was filled into an air-cathode MFC, which had a higher IPA removal efficiency (1.15-fold) and voltage output (1.24-fold) than an MFC filled with coke. The results for the trickling-bed MFC filled with a deoxidizing anode revealed that when the inlet concentration was 0.74 g/m3, the voltage output and power density were highest at 416.3 mV and 486.6 mW/m3, respectively. The deoxidizing anode developed has the potential to increase the MFC voltage output and the pollutant removal.

Improving the performance of biotrickling filter microbial fuel cells in treating exhaust gas by adjusting the oxygen content of the anode tank.

A biotrickling filter (BTF) was combined with a microbial fuel cell (MFC) to remove ethyl acetate from exhaust gas while generating electricity in the process. The results indicated that the use of carbide porous ceramic rings (CPCR) as auxiliary anodes produced more biomass and exhibited a high average removal efficiency (98%), making it a superior microorganism growth carrier compared with carbon coke. When CPCR was used as the cathode in the BTF-MFC, the maximum power density (PD) was 5.64-14.8% of that achieved when carbon cloth was used as the cathode, revealing that CPCR is not a suitable cathode. The maximum elimination capacity (EC) and output voltage of the two-stage BTF-MFC (tBTF-MFC) were only 69.4% and 68.4% of those of the single-stage BTF-MFC (sBTF-MFC), presumably because of voltage reversal. Although the output voltage and EC in the tBTF-MFC were less than those in the sBTF-MFC, the follow-up field application involves stacking multiple small MFCs to remove high-concentration pollutants and generate a high power output. Additionally, continuously adding sodium sulfite decreased the average dissolved oxygen; generated an averaged closed-circuit voltage of 477 mV; and produced a maximum PD of 71.7 mW/m3. These findings demonstrated that the aforementioned method can effectively improve the problem of oxygen and MFC anodes competing for electrons, thus delivering a method that enhances MFC performance through controlling the amount of oxygen in practical applications.

α-Linolenic acid inhibits the migration of human triple-negative breast cancer cells by attenuating Twist1 expression and suppressing Twist1-mediated epithelial-mesenchymal transition.

α-Linolenic acid (ALA), an essential fatty acid, has anticancer activity in breast cancer, but the mechanism of its effects in triple-negative breast cancer (TNBC) remains unclear. We investigated the effect of ALA on Twist1, which is required to initiate epithelial-mesenchymal transition (EMT) and promotes tumor metastasis, and Twist1-mediated migration in MDA-MB231, MDA-MB468 and Hs578T cells. Twist1 protein was constitutively expressed in these TNBC cells, particularly MDA-MB-231 cells. Treatment with 100 µM ALA and Twist1 siRNA markedly decreased the Twist1 protein level and cell migration. Moreover, ALA transiently attenuated the nuclear accumulation of STAT3α as well as Twist1 mRNA expression. Treatment with ALA significantly attenuated the phosphorylation of JNK, ERK and Akt and decreased the phosphorylation of Twist1 at serine 68 in MDA-MB-231 cells. ALA accelerated Twist1 degradation in the presence of cycloheximide, whereas the ubiquitination and degradation of Twist1 by ALA was suppressed by MG-132. Pretreatment with ALA mimicked Twist1 siRNA, increased the protein expression of epithelial markers such as E-cadherin, and decreased the protein expression of mesenchymal markers including Twist1, Snail2, N-cadherin, vimentin, and fibronectin. Our findings suggest that ALA can be used not only to abolish EMT but also to suppress Twist1-mediated migration in TNBC cells.

Author Correction: Targeting CLDN18.2 by CD3 Bispecific and ADC Modalities for the Treatments of Gastric and Pancreatic Cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeting CLDN18.2 by CD3 Bispecific and ADC Modalities for the Treatments of Gastric and Pancreatic Cancer.

Human CLDN18.2 is highly expressed in a significant proportion of gastric and pancreatic adenocarcinomas, while normal tissue expression is limited to the epithelium of the stomach. The restricted expression makes it a potential drug target for the treatment of gastric and pancreatic adenocarcinoma, as evidenced by efforts to target CLDN18.2 via naked antibody and CAR-T modalities. Herein we describe CLDN18.2-targeting via a CD3-bispecific and an antibody drug conjugate and the characterization of these potential therapeutic molecules in efficacy and preliminary toxicity studies. Anti-hCLDN18.2 ADC, CD3-bispecific and diabody, targeting a protein sequence conserved in rat, mouse and monkey, exhibited in vitro cytotoxicity in BxPC3/hCLDN18.2 (IC50 = 1.52, 2.03, and 0.86 nM) and KATO-III/hCLDN18.2 (IC50 = 1.60, 0.71, and 0.07 nM) respectively and inhibited tumor growth of pancreatic and gastric patient-derived xenograft tumors. In a rat exploratory toxicity study, the ADC was tolerated up to 10 mg/kg. In a preliminary assessment of tolerability, the anti-CLDN18.2 diabody (0.34 mg/kg) did not produce obvious signs of toxicity in the stomach of NSG mice 4 weeks after dosing. Taken together, our data indicate that targeting CLDN18.2 with an ADC or bispecific modality could be a valid therapeutic approach for the treatment of gastric and pancreatic cancer.

Optimal design, anti-tumour efficacy and tolerability of anti-CXCR4 antibody drug conjugates.

Antibody-drug conjugates (ADCs) are promising therapies for haematological cancers. Historically, their therapeutic benefit is due to ADC targeting of lineage-restricted antigens. The C-X-C motif chemokine receptor 4 (CXCR4) is attractive for targeted therapy of haematological cancers, given its expression in multiple tumour types and role in cancer "homing" to bone marrow. However, CXCR4 is also expressed in haematopoietic cells and other normal tissues, raising safety challenges to the development of anti-CXCR4 ADCs for cancer treatment. Here, we designed the first anti-CXCR4 ADC with favourable therapeutic index, effective in xenografts of haematopoietic cancers resistant to standard of care and anti-CXCR4 antibodies. We screened multiple ADC configurations, by varying type of linker-payload, drug-to-antibody ratio (DAR), affinity and Fc format. The optimal ADC bears a non-cleavable linker, auristatin as payload at DAR = 4 and a low affinity antibody with effector-reduced Fc. Contrary to other drugs targeting CXCR4, anti-CXCR4 ADCs effectively eliminated cancer cells as monotherapy, while minimizing leucocytosis. The optimal ADC selectively eliminated CXCR4+ cancer cells in solid tumours, but showed limited toxicity to normal CXCR4+ tissues, sparing haematopoietic stem cells and progenitors. Our work provides proof-of-concept that through empirical ADC design, it is possible to target proteins with broad normal tissue expression.

Bioremediation capability evaluation of benzene and sulfolane contaminated groundwater: Determination of bioremediation parameters.

Benzene and sulfolane are commonly used but hazardous chemicals in the petrochemical industry and their leakage and inappropriate disposal certainly causes serious soil and groundwater contamination. In this research, the bioremediation potential of groundwater contaminated with benzene and sulfolane was evaluated, and the operating parameters for bioremediation were established through laboratory batch experiments. Among the various bacterial consortia, the bacterial population of monitoring well c (MWc) contained the highest sulfolane and benzene removal efficiencies. When the dissolved oxygen (DO) level was >1 mg L-1, the bacterial population of MWc showed excellent removal efficiencies toward high and low concentrations of benzene and sulfolane. The C:N:P ratio of 100:10:1 in media facilitated sulfolane and benzene biodegradation, and the degradation time was greatly reduced. Adding additional phosphate into real groundwater could slightly increase benzene removal efficiency. Trace elements only slightly enhanced benzene degradation. On the contrary, additional phosphate and trace elements supplementary did not enhance sulfolane degradation. However, sulfolane removal efficiency could be significantly improved through bioaugmentation of specific sulfolane degrading bacterium and 100% sulfolane removal efficiency was achieved.

RN765C, a low affinity EGFR antibody drug conjugate with potent anti-tumor activity in preclinical solid tumor models.

Epidermal growth factor receptor (EGFR) is a clinically validated target and often overexpressed in some solid tumors. Both EGFR tyrosine kinase inhibitors and ligand-blocking antibodies have been approved for treatment of NSCLC, head and neck cancers and colorectal cancers. However, clinical response is limited and often accompanied by significant toxicities due to normal tissue expression. To improve the effectiveness of targeting EGFR while minimizing the toxicities on normal tissues, we developed a low-affinity anti-EGFR antibody drug conjugate (ADC), RN765C. Potent in vitro cytotoxicity of RN765C, with nanomolar to subnanomolar EC50, was observed on a panel of cancer cell lines expressing moderate to high level of EGFR. In contrast, RN765C was less effective in killing normal human keratinocytes, presumably due to its lower receptor expression. Mechanistically, RN765C has multiple modes of action: inducing payload mediated mitotic arrest and cell death, blocking EGFR pathway signal and mediating antibody dependent cell cytotoxicity. In preclinical studies, a single dose of RN765C at 1.5-3 mg/kg was generally sufficient to induce tumor regression in multiple cell line and patient-derived xenograft models, including those that are resistant to EGFR-directed tyrosine kinase inhibitors. Our data support further investigation of RN765C in the clinic to treat EGFR expressing solid tumors.

A mouse model for evaluation of efficacy and concomitant toxicity of anti-human CXCR4 therapeutics.

The development of therapeutic monoclonal antibodies through mouse immunization often originates drug candidates that are not cross-reactive to the mouse ortholog. In such cases, and particularly in oncology, drug efficacy studies are performed on human tumor xenografts or with "surrogate" anti-mouse ortholog antibodies if targeting tumor host cells. Safety assessment of drug candidate(s) is performed at a later development stage in healthy non-human primates. While the latter remains necessary before a drug advances into human subjects, it precludes evaluation of safety in disease conditions and drug de-risking during early development. Therefore, mouse models that allow concomitant evaluation of drug efficacy and safety are highly desirable. The C-X-C motif chemokine receptor 4 (CXCR4) is an attractive target for tumor-targeted and immuno-oncology therapeutics, with multiple mouse immunization-derived antibodies undergoing clinical trials. Given the pleiotropic role of CXCR4 in cancer biology, we anticipate continuous interest in this target, particularly in the testing of therapeutic combinations for immuno-oncology. Here, we describe the generation and validation of the first mouse knock-in of the whole coding region of human CXCR4. Homozygous human CXCR4 knock-in (hereafter designated as HuCXCR4KI) mice were viable and outwardly healthy, reproduced normally and nursed their young. The expression pattern of human CXCR4 in this model was similar to that of CXCR4 expression in normal human tissues. The human CXCR4 knock-in gene was expressed as a biologically active protein, thereby allowing normal animal development and adequate"homing" of leukocytes to the bone marrow. To further validate our model, we used an in vivo functional assay of leukocyte mobilization from bone marrow to peripheral blood by blocking CXCR4 signaling. Both an anti-human CXCR4 -specific blocking antibody and the small molecule CXCR4 inhibitor AMD3100 induced increased leukocyte counts in peripheral blood, whereas an anti-mouse CXCR4 -specific blocking antibody had no effect. This new mouse model is useful to evaluate efficacy and safety of anti-human CXCR4 -specific drugs as single agents or in combination therapies, particularly in the oncology, immuno-oncology, wound healing and chronic inflammation therapeutic areas.

Targeting primary acute myeloid leukemia with a new CXCR4 antagonist IgG1 antibody (PF-06747143).

The chemokine receptor CXCR4 mediates cell anchorage in the bone marrow (BM) microenvironment and is overexpressed in 25-30% of patients with acute myeloid leukemia (AML). Here we have shown that a new CXCR4 receptor antagonist IgG1 antibody (PF-06747143) binds strongly to AML cell lines and to AML primary cells inhibiting their chemotaxis in response to CXCL12. PF-06747143 also induced cytotoxicity in AML cells via Fc-effector function. To characterize the effects of PF-06747143 on leukemia progression, we used two different patient-derived xenograft (PDX) models: Patient 17CXCR4-low and P15CXCR4-high models, characterized by relatively low and high CXCR4 expression, respectively. Weekly administration of PF-06747143 to leukemic mice significantly reduced leukemia development in both models. Secondary transplantation of BM cells from PF-06747143-treated or IgG1 control-treated animals showed that leukemic progenitors were also targeted by PF-06747143. Administration of a single dose of PF-06747143 to PDX models induced rapid malignant cell mobilization into the peripheral blood (PB). These findings support evaluation of this antibody in AML therapy, with particular appeal to patients resistant to chemotherapy and to unfit patients, unable to tolerate intensive chemotherapy.

Targeting the CXCR4 pathway using a novel anti-CXCR4 IgG1 antibody (PF-06747143) in chronic lymphocytic leukemia.

BACKGROUND: The CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL)-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL. METHODS: Patient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model. RESULTS: PF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD). PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity activity (CDC). PF-06747143 had significant combinatorial effect with standard of care (SOC) agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A), ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy, and in combination with bendamustine. CONCLUSIONS: We show evidence that PF-06747143 has biological activity in CLL primary cells, supporting a rationale for evaluation of PF-06747143 for the treatment of CLL patients.

Improved Lysosomal Trafficking Can Modulate the Potency of Antibody Drug Conjugates.

Antibody drug conjugates (ADCs) provide an efficacious and relatively safe means by which chemotherapeutic agents can be specifically targeted to cancer cells. In addition to the selection of antibody targets, ADCs offer a modular design that allows selection of ADC characteristics through the choice of linker chemistries, toxins, and conjugation sites. Many studies have indicated that release of toxins bound to antibodies via noncleavable linker chemistries relies on the internalization and intracellular trafficking of the ADC. While this can make noncleavable ADCs more stable in the serum, it can also result in lower efficacy when their respective targets are not internalized efficiently or are recycled back to the cell surface following internalization. Here, we show that a lysosomally targeted ADC against the protein APLP2 mediates cell killing, both in vitro and in vivo, more effectively than an ADC against Trop2, a protein with less efficient lysosomal targeting. We also engineered a bispecific ADC with one arm targeting HER2 for the purpose of directing the ADC to tumors, and the other arm targeting APLP2, whose purpose is to direct the ADC to lysosomes for toxin release. This proof-of-concept bispecific ADC demonstrates that this technology can be used to shift the intracellular trafficking of a constitutively recycled target by directing one arm of the antibody against a lysosomally delivered protein. Our data also show limitations of this approach and potential future directions for development.