Sacituzumab govitecan plus platinum-based chemotherapy mediates significant antitumor effects in triple-negative breast, urinary bladder, and small-cell lung carcinomas.

Sacituzumab govitecan (SG) is an antibody-drug conjugate composed of an anti-Trop-2-directed antibody conjugated with the topoisomerase I inhibitory drug, SN-38, via a proprietary hydrolysable linker. SG has received United States Food and Drug Administration (FDA) approval to treat metastatic triple-negative breast cancer (TNBC), unresectable locally advanced or metastatic hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer, and accelerated approval for metastatic urothelial cancer. We investigated the utility of combining SG with platinum-based chemotherapeutics in TNBC, urinary bladder carcinoma (UBC), and small-cell lung carcinoma (SCLC). SG plus carboplatin or cisplatin produced additive growth-inhibitory effects in vitro that trended towards synergy. Immunoblot analysis of cell lysates suggests perturbation of the cell-cycle and a shift towards pro-apoptotic signaling evidenced by an increased Bax to Bcl-2 ratio and down-regulation of two anti-apoptotic proteins, Mcl-1 and survivin. Significant antitumor effects were observed with SG plus carboplatin in mice bearing TNBC or SCLC tumors compared to all controls (P < 0.0062 and P < 0.0017, respectively) and with SG plus cisplatin in UBC and SCLC tumor-bearing animals (P < 0.0362 and P < 0.0001, respectively). These combinations were well tolerated by the animals. Combining SG with platinum-based chemotherapeutics demonstrates the benefit in these indications and warrants further clinical investigation.

Predictive biomarkers for sacituzumab govitecan efficacy in Trop-2-expressing triple-negative breast cancer.

Sacituzumab govitecan (SG) is an antibody-drug conjugate composed of a humanized anti-Trop-2 IgG antibody conjugated via a hydrolysable linker to SN-38, the topoisomerase I-inhibitory active component of irinotecan. We investigated whether Trop-2-expression and homologous recombination repair (HRR) of SN-38-mediated double-strand DNA (dsDNA) breaks play a role in the sensitivity of triple-negative breast cancer (TNBC) to SG. Activation of HRR pathways, as evidenced by Rad51 expression, was assessed in SG-sensitive cell lines with low and moderate Trop-2-expression (SK-MES-1 squamous cell lung carcinoma and HCC1806 TNBC, respectively), compared to a low Trop-2-expressing, less SG-sensitive TNBC cell line (MDA-MB-231). Further, two Trop-2-transfectants of MDA-MB-231, C13 and C39 (4- and 25-fold higher Trop-2, respectively), were treated in mice with SG to determine whether increasing Trop-2 expression improves SG efficacy. SG mediated >2-fold increase in Rad51 in MDA-MB-231 but had no effect in SK-MES-1 or HCC1806, resulting in lower levels of dsDNA breaks in MDA-MB-231. SG and saline produced similar effects in parental MDA-MB-231 tumor-bearing mice (median survival time (MST) = 21d and 19.5d, respectively). However, in mice bearing higher Trop-2-expressing C13 and C39 tumors after Trop-2 transfection, SG provided a significant survival benefit, even compared to irinotecan (MST = 97d vs. 35d for C13, and 81d vs. 28d for C39, respectively; P < 0.0007). These results suggest that SG could provide better clinical benefit than irinotecan in patients with HRR-proficient tumors expressing high levels of Trop-2, as well as to patients with HRR-deficient tumors expressing low/moderate levels of Trop-2.

Selective and Concentrated Accretion of SN-38 with a CEACAM5-Targeting Antibody-Drug Conjugate (ADC), Labetuzumab Govitecan (IMMU-130).

Labetuzumab govitecan (IMMU-130), an antibody-drug conjugate (ADC) with an average of 7.6 SN-38/IgG, was evaluated for its potential to enhance delivery of SN-38 to human colonic tumor xenografts. Mice bearing LS174T or GW-39 human colonic tumor xenografts were injected with irinotecan or IMMU-130 (SN-38 equivalents ∼500 or ∼16 µg, respectively). Serum and homogenates of tumors, liver, and small intestine were extracted, and SN-38, SN-38G (glucuronidated SN-38), and irinotecan concentrations determined by reversed-phase HPLC. Irinotecan cleared quickly from serum, with only 1% to 2% injected dose/mL after 5 minutes; overall, approximately 20% was converted to SN-38 and SN-38G. At 1 hour with IMMU-130, 45% to 63% injected dose/mL of the SN-38 was in the serum, with >90% bound to the ADC over 3 days, and with low levels of SN-38G. Total SN-38 levels decreased more quickly than the IgG, confirming a gradual SN-38 release from the ADC. AUC analysis found that SN-38 levels were approximately 11- and 16-fold higher in LS174T and GW-39 tumors, respectively, in IMMU-130-treated animals. This delivery advantage is amplified >30-fold when normalized to SN-38 equivalents injected for each product. Levels of SN-38 and SN-38G were appreciably lower in the liver and small intestinal contents in animals given IMMU-130. On the basis of the SN-38 equivalents administered, IMMU-130 potentially delivers >300-fold more SN-38 to CEA-producing tumors compared with irinotecan, while also reducing levels of SN-38 and SN-38G in normal tissues. These observations are consistent with preclinical and clinical data showing efficacy and improved safety. Mol Cancer Ther; 17(1); 196-203. ©2017 AACR.

IMMU-140, a Novel SN-38 Antibody-Drug Conjugate Targeting HLA-DR, Mediates Dual Cytotoxic Effects in Hematologic Cancers and Malignant Melanoma.

HLA-DR is a member of the MHC class II antigen family expressed on hematologic and solid tumors. Antibodies directed against HLA-DR have demonstrated some clinical success, but toxicities limited development. IMMU-140 is an anti-HLA-DR antibody-drug conjugate composed of the active metabolite of irinotecan, SN-38, conjugated to a humanized anti-HLA-DR IgG4 antibody (IMMU-114); the IgG4 naked antibody is devoid of immune functions. Our aim was to determine if SN-38, the metabolite of a drug not commonly used in hematopoietic cancers, would be effective and safe when targeted to HLA-DR-expressing tumors. IMMU-140 had dual-therapeutic mechanisms, as evidenced by its retention of nonoverlapping anti-HLA-DR nonclassical apoptotic signaling and classical apoptosis mediated by its SN-38 payload. In seven human disease models [acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), and melanoma], IMMU-140 provided significant therapeutic efficacy compared with controls, in vitro, in 3D spheroid models, and in vivo Except for MM and HL, IMMU-140 imparted significantly improved antitumor effects compared with parental IMMU-114. Even in intractable AML and ALL, where IMMU-114 only had modest antitumor effects, IMMU-140 therapy mediated >80% improvement in survival. Therapy was well tolerated, as demonstrated by no marked loss in body weight. Combined with doxorubicin, IMMU-140 produced significantly greater antitumor effects in HL than with monotherapy and without any added toxicity. The dual-therapeutic action of IMMU-140 resulted in promising therapeutic activity in a range of hematopoietic tumors and melanoma, and therefore warrants clinical development. Mol Cancer Ther; 17(1); 150-60. ©2017 AACR.

Combination Therapy with Bispecific Antibodies and PD-1 Blockade Enhances the Antitumor Potency of T Cells.

The DOCK-AND-LOCK (DNL) method is a platform technology that combines recombinant engineering and site-specific conjugation to create multispecific, multivalent antibodies of defined composition with retained bioactivity. We have applied DNL to generate a novel class of trivalent bispecific antibodies (bsAb), each comprising an anti-CD3 scFv covalently conjugated to a stabilized dimer of different antitumor Fabs. Here, we report the further characterization of two such constructs, (E1)-3s and (14)-3s, which activate T cells and target Trop-2- and CEACAM5-expressing cancer cells, respectively. (E1)-3s and (14)-3s, in the presence of human T cells, killed target cells grown as monolayers at subnanomolar concentrations, with a similar potency observed for drug-resistant cells. Antitumor efficacy was demonstrated for (E1)-3s coadministered with human peripheral blood mononuclear cells (PBMC) in NOD/SCID mice harboring xenografts of MDA-MB-231, a triple-negative breast cancer line constitutively expressing Trop-2 and PD-L1. Growth inhibition was observed following treatment with (E1)-3s or (14)-3s combined with human PBMC in 3D spheroids generated from target cell lines to mimic the in vivo behavior and microenvironment of these tumors. Moreover, addition of an antagonistic anti-PD-1 antibody increased cell death in 3D spheroids and extended survival of MDA-MB-231-bearing mice. These preclinical results emphasize the potential of combining T-cell-redirecting bsAbs with antagonists or agonists that mitigate T-cell inhibition within the tumor microenvironment to improve immunotherapy of solid cancers in patients. They also support the use of 3D spheroids as a predictive alternative to in vivo models for evaluating T-cell functions. Cancer Res; 77(19); 5384-94. ©2017 AACR.

Emergency percutaneous nephrostomy versus emergency percutaneous nephrolithotomy in patients with sepsis associated with large uretero-pelvic junction stone impaction: a randomized controlled trial

ABSTRACT Introduction A randomized trial was conducted prospectively to evaluate the efficacy, related complications, and convalescence of emergency percutaneous nephrolithotomy compared to percutaneous nephrostomy for decompression of the collecting system in cases of sepsis associated with large uretero-pelvic junction stone impaction. Materials and Methods The inclusion criteria included a WBC count of 10.000/mm3 or more and/or a temperature of 38°C or higher. Besides, all enrolled patients should maintain stable hemodynamic status and proper organ perfusions. A total of 113 patients with large, obstructive uretero-pelvic junction stones and clinical signs of sepsis completed the study protocol. Of those, 56 patients were placed in the emergency percutaneous nephrostomy group, while the other 57 patients were part of the percutaneous nephrolithotomy group. The primary end point was the time until normalization of white blood cells (WBC) at a count of 10.000/mm3 or less, and a temperature of 37.4°C or lower. The secondary end points included the comparison of analgesic consumption, length of stay, and related complications. Statistical analysis was performed using SPSS® version 14.0.1. The Mann-Whitney U test, chi-square test, and Fisher’s exact test were used as appropriate. Results The length of hospital stays (in days) was 10.09±3.43 for the emergency percutaneous nephrostomy group and 8.18±2.72 for the percutaneous nephrolithotomy group. This set of data noted a significant difference between groups. There was no difference between groups in regard to white blood cell count (in mm3), time to normalization of white blood cell count (in days), body temperature (in ºC), time to normalization of body temperature (in days), C-reactive proteins (in mg/dL), time taken for C-reactive proteins to decrease over 25% (in days), procalcitonin (in ng/mL), or complication rates. Conclusions This study confirms that emergency percutaneous nephrolithotomy may be as safe as early percutaneous nephrolithotomy in a selected low risk patients with sepsis-associated large, obstructive stone.

Emergency percutaneous nephrostomy versus emergency percutaneous nephrolithotomy in patients with sepsis associated with large uretero-pelvic junction stone impaction: a randomized controlled trial.

INTRODUCTION: A randomized trial was conducted prospectively to evaluate the efficacy, related complications, and convalescence of emergency percutaneous nephrolithotomy compared to percutaneous nephrostomy for decompression of the collecting system in cases of sepsis associated with large uretero-pelvic junction stone impaction. MATERIALS AND METHODS: The inclusion criteria included a WBC count of 10.000/mm3 or more and/or a temperature of 38°C or higher. Besides, all enrolled patients should maintain stable hemodynamic status and proper organ perfusions. A total of 113 patients with large, obstructive uretero-pelvic junction stones and clinical signs of sepsis completed the study protocol. Of those, 56 patients were placed in the emergency percutaneous nephrostomy group, while the other 57 patients were part of the percutaneous nephrolithotomy group. The primary end point was the time until normalization of white blood cells (WBC) at a count of 10.000/mm3 or less, and a temperature of 37.4°C or lower. The secondary end points included the comparison of analgesic consumption, length of stay, and related complications. Statistical analysis was performed using SPSS® version 14.0.1. The Mann-Whitney U test, chi-square test, and Fisher's exact test were used as appropriate. RESULTS: The length of hospital stays (in days) was 10.09±3.43 for the emergency percutaneous nephrostomy group and 8.18±2.72 for the percutaneous nephrolithotomy group. This set of data noted a significant difference between groups. There was no difference between groups in regard to white blood cell count (in mm3), time to normalization of white blood cell count (in days), body temperature (in ºC), time to normalization of body temperature (in days), C-reactive proteins (in mg/dL), time taken for C-reactive proteins to decrease over 25% (in days), procalcitonin (in ng/mL), or complication rates. CONCLUSIONS: This study confirms that emergency percutaneous nephrolithotomy may be as safe as early percutaneous nephrolithotomy in a selected low risk patients with sepsis-associated large, obstructive stone.

Efficacy of Silodosin in Expulsive Therapy for Distal Ureteral Stones: A Randomized Double-blinded Controlled Trial.

PURPOSE: To evaluate the efficacy of silodosin in the medical expulsive therapy for symptomatic distal ureteral stones. MATERIALS AND METHODS: This prospectively randomized controlled trial was carried out from May 2011 to December 2014. In all, 198 patients with radiopaque distal ureteral stones <10 mm in size were eligible: 61 patients in the control group and 62 patients in the silodosin group. The silodosin group received silodosin 8 mg daily, and the control group received lactose tablets. The primary outcome was the expulsion rate. The secondary outcomes the expulsion time, analgesic consumption, lower urinary tract symptoms, colic episodes, and adverse effects. Statistical analyses were performed using a Mann-Whitney U-test and chi-square test. RESULTS: The final analysis was conducted with 61 control and 62 silodosin patients as the denominator in each randomization arm. The average expulsion times were 6.31 ± 2.13 days for the silodosin group and 9.73 ± 2.76 days for the control group (P < .001). CONCLUSION: Treatment with silodosin proved to be safe and effective, as demonstrated by the increased stone expulsion rate, the reduced expulsion time, and the reduced analgesics consumption. .

Combining ABCG2 Inhibitors with IMMU-132, an Anti-Trop-2 Antibody Conjugate of SN-38, Overcomes Resistance to SN-38 in Breast and Gastric Cancers.

Sacituzumab govitecan (IMMU-132), an SN-38-conjugated antibody-drug conjugate, is showing promising therapeutic results in a phase I/II trial of patients with advanced Trop-2-expressing, metastatic, solid cancers. As members of the ATP-binding cassette (ABC) transporters confer chemotherapy resistance by active drug efflux, which is a frequent cause of treatment failure, we explored the use of known inhibitors of ABC transporters for improving the therapeutic efficacy of IMMU-132 by overcoming SN-38 resistance. Two human tumor cell lines made resistant to SN-38, MDA-MB-231-S120 (human breast cancer) and NCI-N87-S120 (human gastric cancer), were established by continuous exposure of the parental cells to stepwise increased concentrations of SN-38 and analyzed by flow cytometry for functional activities of ABCG2 and ABCB1, immunoblotting and qRT-PCR for the expression of ABCG2 at both protein and mRNA levels, and MTS assays for the potency of SN-38 alone or in combination with a modulator of ABC transporters. MDA-MB-231-S120 and NCI-N87-S120 displayed reduced sensitivity to SN-38 in vitro, with IC50 values approximately 50-fold higher than parental MDA-MB-231 and NCI-N87 cells. The increase in drug resistance of both S120 cell populations is associated with the expression of functional ABCG2, but not ABCB1. Importantly, treatment of both S120 sublines with known ABCG2 inhibitors (fumitremorgin C, Ko143, and YHO-13351) restored toxicity of SN-38, and the combination of YHO-13351 with IMMU-132 increased the median survival of mice bearing NCI-N87-S120 xenografts. These results provide a rationale for combination therapy of IMMU-132 and inhibitors of ABC transporters, such as YHO-13351. Mol Cancer Ther; 15(8); 1910-9. ©2016 AACR.

Percutaneous nephrostomy versus ureteroscopic management of sepsis associated with ureteral stone impaction: a randomized controlled trial.

A randomized trial was conducted prospectively to evaluate the efficacy, related complications, and convalescence of emergent retrograde ureteroscopic management, instead of percutaneous nephrostomy for decompression of the collecting system in cases of sepsis associated with ureteral stone obstruction. A total of 107 patients undergoing ureteroscopic stone manipulation for ureteral stones completed the study protocol, 53 patients in the percutaneous nephrostomy group, and 54 patients in the emergent retrograde ureteroscopic management group. The primary end point was the time to normalization of WBC of 10,000/mm or less and temperature of 37.4 °C or less. The second end point was the comparison analgesic consumption, length of stay, and related complications. Statistical analysis was performed using SPSS(®) version 14.0.1. The Mann-Whitney U test, Chi square test, and fisher's exact test was used as appropriate. The length of hospital stay (days) was 10.25 ± 3.53 and 8.24 ± 2.77 in the percutaneous nephrostomy group and emergent retrograde ureteroscopic management group, respectively, with significant difference (Table 2). However, patients in the emergent retrograde ureteroscopic management group had a significantly higher rate of s body temperature (°C). Meanwhile, the analgesic consumptions are 31.51 ± 11.16 and 40.00 ± 14.54 in the percutaneous nephrostomy group and emergent retrograde ureteroscopic management group, respectively, with significant difference. Our results show that it can be effectively and safely managed by retrograde ureteroscopic management combined with strong antibiotics in select clinical situations. Ureteroscopic management should no longer be considered a contraindication for the treatment of patients with sepsis associated with obstructing ureteral stones.

Enhanced Delivery of SN-38 to Human Tumor Xenografts with an Anti-Trop-2-SN-38 Antibody Conjugate (Sacituzumab Govitecan).

PURPOSE: This study examined the delivery of SN-38 to Trop-2-expressing tumors and assessed the constitutive products in the serum, liver, and small intestine in nude mice bearing human tumor xenografts (Capan-1 or NCI-N87) given a single injection of irinotecan (40 mg/kg; ∼ 0.8 mg/mouse, containing ∼ 460 µg SN-38 equivalents) or sacituzumab govitecan (IMMU-132), an antibody-drug conjugate composed of a humanized anti-Trop-2 IgG coupled site specifically with an average of 7.6 molecules of SN-38. EXPERIMENTAL DESIGN: At select times, tissues were extracted and concentrations of the products measured by reversed-phase high-performance liquid chromatography (HPLC). RESULTS: In serum, >98% irinotecan cleared within 5 minutes; peak levels of SN-38 and SN-38G (glucuronidated SN-38) were detected in equal amounts at this time, and no longer detected after 6 to 8 hours. IMMU-132 was detected in the serum over 3 days, and at each interval, ≥ 95% of total SN-38 was bound to the antibody. Intact IMMU-132 cleared with a half-life of 14 hours, which closely reflected the in vitro rate of SN-38 released from the conjugate in mouse serum (i.e., 17.5 hours), whereas the IgG portion of the conjugate cleared with a half-life of 67.1 hours. In vitro and in vivo studies disclosed IgG-bound SN-38 was protected from glucuronidation. Area under the curve (AUC) analysis indicated that IMMU-132 delivers 20-fold to as much as 136-fold more SN-38 to tumors than irinotecan, with tumor:blood ratios favoring IMMU-132 by 20- to 40-fold. Intestinal concentrations of SN-38/SN-38G also were 9-fold lower with IMMU-132. CONCLUSIONS: These studies confirm a superior SN-38 tumor delivery by IMMU-132 compared with irinotecan.

Sacituzumab Govitecan (IMMU-132), an Anti-Trop-2/SN-38 Antibody-Drug Conjugate: Characterization and Efficacy in Pancreatic, Gastric, and Other Cancers.

Sacituzumab govitecan (IMMU-132) is an antibody-drug conjugate (ADC) made from a humanized anti-Trop-2 monoclonal antibody (hRS7) conjugated with the active metabolite of irinotecan, SN-38. In addition to its further characterization, as the clinical utility of IMMU-132 expands to an ever-widening range of Trop-2-expressing solid tumor types, its efficacy in new disease models needs to be explored in a nonclinical setting. Unlike most ADCs that use ultratoxic drugs and stable linkers, IMMU-132 uses a moderately toxic drug with a moderately stable carbonate bond between SN-38 and the linker. Flow cytometry and immunohistochemistry disclosed that Trop-2 is expressed in a wide range of tumor types, including gastric, pancreatic, triple-negative breast (TNBC), colonic, prostate, and lung. While cell-binding experiments reveal no significant differences between IMMU-132 and parental hRS7 antibody, surface plasmon resonance analysis using a Trop-2 CM5 chip shows a significant binding advantage for IMMU-132 over hRS7. The conjugate retained binding to the neonatal receptor, but it lost greater than 60% of the antibody-dependent cell-mediated cytotoxicity activity compared to that of hRS7. Exposure of tumor cells to either free SN-38 or IMMU-132 demonstrated the same signaling pathways, with pJNK1/2 and p21(WAF1/Cip1) upregulation followed by cleavage of caspases 9, 7, and 3, ultimately leading to poly-ADP-ribose polymerase cleavage and double-stranded DNA breaks. Pharmacokinetics of the intact ADC in mice reveals a mean residence time (MRT) of 15.4 h, while the carrier hRS7 antibody cleared at a similar rate as that of the unconjugated antibody (MRT ∼ 300 h). IMMU-132 treatment of mice bearing human gastric cancer xenografts (17.5 mg/kg; twice weekly × 4 weeks) resulted in significant antitumor effects compared to that of mice treated with a nonspecific control. Clinically relevant dosing schemes of IMMU-132 administered either every other week, weekly, or twice weekly in mice bearing human pancreatic or gastric cancer xenografts demonstrate similar, significant antitumor effects in both models. Current Phase I/II clinical trials ( ClinicalTrials.gov , NCT01631552) confirm anticancer activity of IMMU-132 in cancers expressing Trop-2, including gastric and pancreatic cancer patients.

Identification of PAM4 (clivatuzumab)-reactive epitope on MUC5AC: a promising biomarker and therapeutic target for pancreatic cancer.

PAM4 is a monoclonal antibody showing high specificity for pancreatic ductal adenocarcinoma (PDAC). Humanized PAM4 labeled with 90Y in combination with low-dose gemcitabine has shown promising therapeutic activity, and is being evaluated in a phase III clinical trial. Prior efforts have suggested that PAM4 potentially reacts with MUC5AC, a secretory mucin expressed de novo in early pancreatic neoplasia and retained throughout disease progression. In present study, we provide further evidence validating MUC5AC as the PAM4 antigen, and locate PAM4-reactive epitope within the N-terminal cysteine-rich subdomain 2 (Cys2), thus differentiating PAM4 from most anti-MUC5AC antibodies known to-date. Specifically, we show (i) PAM4-antigen and MUC5AC were co-localized in multiple human cancer cell lines, including Capan-1, BxPC-3, and CFPAC-1; (ii) MUC5AC-specific siRNA prominently reduced the expression of both MUC5AC and PAM4-antigen in CFPAC-1 cells; (iii) PAM4 preferentially binds to the void-volume fractions from Sepharose-CL2B chromatography of Capan-1 culture supernatants, which were revealed by Western blot to display the ladder pattern characteristic of oligomeric MUC5AC; and (iv) the N-terminal Cys2 within several recombinant MUC5AC fragments is essential for binding to PAM4. These findings shed light on the mechanism of PAM4-based diagnosis and treatment for pancreatic cancer, and guide further exploration of its clinical utility.

Extensive crosslinking of CD22 by epratuzumab triggers BCR signaling and caspase-dependent apoptosis in human lymphoma cells.

Epratuzumab has demonstrated therapeutic activity in patients with non-Hodgkin lymphoma, acute lymphoblastic leukemia, systemic lupus erythematosus, and Sjögren's syndrome, but its mechanism of affecting normal and malignant B cells remains incompletely understood. We reported previously that epratuzumab displayed in vitro cytotoxicity to CD22-expressing Burkitt lymphoma cell lines (Daudi and Ramos) only when immobilized on plates or combined with a crosslinking antibody plus a suboptimal amount of anti-IgM (1 µg/mL). Herein, we show that, in the absence of additional anti-IgM ligation, extensive crosslinking of CD22 by plate-immobilized epratuzumab induced intracellular changes in Daudi cells similar to ligating B-cell antigen receptor with a sufficiently high amount of anti-IgM (10 µg/mL). Specifically, either treatment led to phosphorylation of CD22, CD79a and CD79b, along with their translocation to lipid rafts, both of which were essential for effecting caspase-dependent apoptosis. Moreover, such immobilization induced stabilization of F-actin, phosphorylation of Lyn, ERKs and JNKs, generation of reactive oxygen species (ROS), decrease in mitochondria membrane potential (Δψm), upregulation of pro-apoptotic Bax, and downregulation of anti-apoptotic Bcl-xl and Mcl-1. The physiological relevance of immobilized epratuzumab was implicated by noting that several of its in vitro effects, including apoptosis, drop in Δψm, and generation of ROS, could be observed with soluble epratuzumab in Daudi cells co-cultivated with human umbilical vein endothelial cells. These results suggest that the in vivo mechanism of non-ligand-blocking epratuzumab may, in part, involve the unmasking of CD22 to facilitate the trans-interaction of B cells with vascular endothelium.

In-vivo fusion of human cancer and hamster stromal cells permanently transduces and transcribes human DNA.

After demonstrating, with karyotyping, polymerase chain reaction (PCR) and fluorescence in-situ hybridization, the retention of certain human chromosomes and genes following the spontaneous fusion of human tumor and hamster cells in-vivo, it was postulated that cell fusion causes the horizontal transmission of malignancy and donor genes. Here, we analyzed gene expression profiles of 3 different hybrid tumors first generated in the hamster cheek pouch after human tumor grafting, and then propagated in hamsters and in cell cultures for years: two Hodgkin lymphomas (GW-532, GW-584) and a glioblastoma multiforme (GB-749). Based on the criteria of MAS 5.0 detection P-values ≤0.065 and at least a 2-fold greater signal expression value than a hamster melanoma control, we identified 3,759 probe sets (ranging from 1,040 to 1,303 in each transplant) from formalin-fixed, paraffin-embedded sections of the 3 hybrid tumors, which unambiguously mapped to 3,107 unique Entrez Gene IDs, representative of all human chromosomes; however, by karyology, one of the hybrid tumors (GB-749) had a total of 15 human chromosomes in its cells. Among the genes mapped, 39 probe sets, representing 33 unique Entrez Gene IDs, complied with the detection criteria in all hybrid tumor samples. Five of these 33 genes encode transcription factors that are known to regulate cell growth and differentiation; five encode cell adhesion- and transmigration-associated proteins that participate in oncogenesis and/or metastasis and invasion; and additional genes encode proteins involved in signaling pathways, regulation of apoptosis, DNA repair, and multidrug resistance. These findings were corroborated by PCR and reverse transcription PCR, showing the presence of human alphoid (α)-satellite DNA and the F11R transcripts in additional tumor transplant generations. We posit that in-vivo fusion discloses genes implicated in tumor progression, and gene families coding for the organoid phenotype. Thus, cancer cells can transduce adjacent stromal cells, with the resulting progeny having permanently transcribed genes with malignant and other gene functions of the donor DNA. Using heterospecific in-vivo cell fusion, genes encoding oncogenic and organogenic traits may be identified.

Redirected T-cell killing of solid cancers targeted with an anti-CD3/Trop-2-bispecific antibody is enhanced in combination with interferon-α.

Trop-2 has limited presence on normal tissues but is highly expressed in diverse epithelial cancers. (E1)-3s is a T-cell-redirecting trivalent bispecific antibody (bsAb), comprising an anti-CD3 scFv covalently linked to a stabilized dimer of a Trop-2-targeting Fab using Dock-and-Lock. We show for the first time that bsAb-mediated bidirectional trogocytosis occurs between target and T cells and involves immunologic synapses. We studied the effects of interferon-α (INFα) on (E1)-3s-mediated T-cell killing of human gastric and pancreatic cancer cell lines. T-cell activation, cytokine induction, and cytotoxicity were evaluated ex vivo using peripheral blood mononuclear cells (PBMC) or T cells with NCI-N87 gastric cancer as target cells. In vivo activity was assayed with NCI-N87 and Capan-1 (pancreatic) xenografts. In the presence of target cells and PBMCs, (E1)-3s did not cause excess cytokine production. When combined with (E1)-3s, peginterferonalfa-2a--which alone did not increase T-cell activation or raise cytokine levels over baseline--increased CD69 expression but did not significantly increase cytokine induction. (E1) 3s mediated a highly potent T-cell lysis of NCI-N87 target cells in vitro. Inclusion of peginterferonalfa-2a or a more potent form of INFα, 20*-2b, significantly potentiated the activity of (E1)-3s by more than 2.5- or 7-fold, respectively. In vivo, combining peginterferonalfa-2a with (E1)-3s delayed Capan-1 growth longer than each single agent. Similarly, combination therapy delayed tumor proliferation of NCI-N87 compared with (E1)-3s or peginterferonalfa-2a single-treatment groups. (E1)-3s effectively induced T-cell-mediated killing of Trop-2-expressing pancreatic and gastric cancers, which was enhanced with INFα.

Anti-CD22/CD20 Bispecific antibody with enhanced trogocytosis for treatment of Lupus.

The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenström's macroglobulinemia, Sjögren's syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. We reported recently that epratuzumab mediates Fc/FcR-dependent membrane transfer from B cells to effector cells via trogocytosis, resulting in a substantial reduction of multiple BCR modulators, including CD22, CD19, CD21, and CD79b, as well as key cell adhesion molecules, including CD44, CD62L, and ß7 integrin, on the surface of B cells in peripheral blood mononuclear cells obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and ß7-integrin, and with considerably less immunocompromising B-cell depletion that would result with anti-CD20 mAbs such as veltuzumab or rituximab, given either alone or in combination with epratuzumab. A CD22/CD19 bispecific hexavalent antibody, which exhibited enhanced trogocytosis of some antigens and minimal B-cell depletion, may also be therapeutically useful. The bispecific antibody is a candidate for improved treatment of lupus and other autoimmune diseases, offering advantages over administration of the two parental antibodies in combination.

Trop-2-targeting tetrakis-ranpirnase has potent antitumor activity against triple-negative breast cancer.

BACKGROUND: Ranpirnase (Rap) is an amphibian ribonuclease with reported antitumor activity, minimal toxicity, and negligible immunogenicity in clinical studies, but the unfavorable pharmacokinetics and suboptimal efficacy hampered its further clinical development. To improve the potential of Rap-based therapeutics, we have used the DOCK-AND-LOCK™ (DNL™) method to construct a class of novel IgG-Rap immunoRNases. In the present study, a pair of these constructs, (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2, comprising four copies of Rap linked to the CH3 and CK termini of hRS7 (humanized anti-Trop-2), respectively, were evaluated as potential therapeutics for triple-negative breast cancer (TNBC). METHODS: The DNL-based immunoRNases, (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2, were characterized and tested for biological activities in vitro on a panel of breast cancer cell lines and in vivo in a MDA-MB-468 xenograft model. RESULTS: (Rap)2-E1-(Rap)2 was highly purified (>95%), exhibited specific cell binding and rapid internalization in MDA-MB-468, a Trop-2-expressing TNBC line, and displayed potent in vitro cytotoxicity (EC50 ≤ 1 nM) against diverse breast cancer cell lines with moderate to high expression of Trop-2, including MDA-MB-468, BT-20, HCC1806, SKBR-3, and MCF-7. In comparison, structural counterparts of (Rap)2-E1-(Rap)2, generated by substituting hRS7 with selective non-Trop-2-binding antibodies, such as epratuzumab (anti-CD22), were at least 50-fold less potent than (Rap)2-E1-(Rap)2 in MDA-MB-468 and BT-20 cells, both lacking the expression of the cognate antigen. Moreover, (Rap)2-E1-(Rap)2 was less effective (EC50 > 50 nM) in MDA-MB-231 (low Trop-2) or HCC1395 (no Trop-2), and did not show any toxicity to human peripheral blood mononuclear cells. In a mouse TNBC model, a significant survival benefit was achieved with (Rap)2-E1*-(Rap)2 when given the maximal tolerated dose. CONCLUSIONS: A new class of immunoRNases was generated with enhanced potency for targeted therapy of cancer. The promising results from (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2 support their further investigation as a potential treatment option for TNBC and other Trop-2-expressing cancers.

Predictive patient-specific dosimetry and individualized dosing of pretargeted radioimmunotherapy in patients with advanced colorectal cancer.

PURPOSE: Pretargeted radioimmunotherapy (PRIT) with bispecific antibodies (bsMAb) and a radiolabeled peptide reduces the radiation dose to normal tissues. Here we report the accuracy of an (111)In-labeled pretherapy test dose for personalized dosing of (177)Lu-labeled IMP288 following pretargeting with the anti-CEA × anti-hapten bsMAb, TF2, in patients with metastatic colorectal cancer (CRC). METHODS: In 20 patients bone marrow absorbed doses (BMD) and doses to the kidneys were predicted based on blood samples and scintigrams acquired after (111)In-IMP288 injection for individualized dosing of PRIT with (177)Lu-IMP288. Different dose schedules were studied, varying the interval between the bsMAb and peptide administration (5 days vs. 1 day), increasing the bsMAb dose (75 mg vs. 150 mg), and lowering the peptide dose (100 µg vs. 25 µg). RESULTS: TF2 and (111)In/(177)Lu-IMP288 clearance was highly variable. A strong correlation was observed between peptide residence times and individual TF2 blood concentrations at the time of peptide injection (Spearman's ρ = 0.94, P < 0.0001). PRIT with 7.4 GBq (177)Lu-IMP288 resulted in low radiation doses to normal tissues (BMD <0.5 Gy, kidney dose <3 Gy). Predicted (177)Lu-IMP288 BMD were in good agreement with the actual measured doses (mean ± SD difference -0.0026 ± 0.028 mGy/MBq). Hematological toxicity was mild in most patients, with only two (10 %) having grade 3-4 thrombocytopenia. A correlation was found between platelet toxicity and BMD (Spearman's ρ = 0.58, P = 0.008). No nonhematological toxicity was observed. CONCLUSION: These results show that individual high activity doses in PRIT in patients with CEA-expressing CRC could be safely administered by predicting the radiation dose to red marrow and kidneys, based on dosimetric analysis of a test dose of TF2 and (111)In-IMP288.