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Molecular structure of fumitremorgin C

Functional activity of ABCB1 shown for HCT15, but not for NCI-N87 or NCI-N87-S120

Relative mRNA levels of ABCG2 determined by RT-qPCR

Molecular Structure of IMMU-132

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.

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.

Background: In current clinical trials (ClinicalTrials.gov, NCT01631552), triple-negative breast cancer (TNBC) patients treated with IMMU-132, which is composed of the active metabolite of irinotecan, SN-38, conjugated to an anti-Trop-2 antibody, shows manageable toxicity and very encouraging responses in relapsed/refractory cases. Synthetic lethality is a concept in which a cell harboring one out of two possible gene or protein defects is viable, while a cell containing both defects is nonviable. BRCA1/2 mutations are linked to deficiencies in DNA repair and are associated with TNBC. Other repair mechanisms involve poly(adenosine diphosphoribose) polymerase (PARP), which can be used by cancer cells to overcome loss of BRACA1/2. Treatment of TNBC cells with either IMMU-132 or paclitaxel results in cleavage and deactivation of PARP, whereas the small molecule olaparib directly inhibits PARP. Therefore, the rationale of combining IMMU-132 with either paclitaxel or olaparib to effectively knock-out PARP activity was investigated in TNBC xenografts to ascertain if these combinations will result in synthetic lethality. Methods: Mice bearing human TNBC xenografts (MDA-MB-468 or HCC1806) were treated with 15 mg/kg paclitaxel weekly for 5 weeks. IMMU-132 was administered either at 10 mg/kg or 12.5 mg/kg on days 1, 8, 22, and 29. In vitro, various human TNBC cell lines were incubated with either a constant amount of IMMU-132 in combination with various amounts of olaparib or constant olaparib with varying amounts of IMMU-132. A combination index number was calculated to determine whether the interaction was synergistic, additive, or antagonistic. Mice bearing TNBC tumors were treated with olaparib (50 mg/kg, qdx5d, for 4 wks), or IMMU-132 (10 mg/kg, 2xwkly x 4 wks), or the combination of both. Results: Mice bearing MDA-MB-468 tumors treated with the combination of IMMU-132 and paclitaxel exhibited superior anti-tumor effects with >11-fold shrinkage of tumors in comparison to 1.4-fold shrinkage in the IMMU-132 group alone (P=0.0003) or 11.4-fold increase in tumor size in those mice treated with paclitaxel alone (P Conclusions: Targeting the PARP DNA repair pathway in BRCA1/2 mutant TNBC tumors by combining IMMU-132 therapy with either paclitaxel or olaparib achieved synthetic lethality in this disease model with no observable toxicity. These data provide the rationale for the clinical evaluation of IMMU-132 in combination with other chemotherapeutics that likewise target DNA-repair mechanisms in patients with TNBC. Citation Format: Goldenberg DM, Cardillo TM, Govindan SV, Zalath M, Arrojo R, Sharkey RM. Synthetic lethality in TNBC mediated by an anti-Trop-2 antibody-drug conjugate, sacituzumab govitecan (IMMU-132), when combined with paclitaxel or the PARP inhibitor, olaparib. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-15-02.

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.

Purpose: The humanized anti-CD74 monoclonal antibody, milatuzumab, is in clinical evaluation for the therapy of multiple myeloma (MM). The ability of milatuzumab to increase the efficacy of bortezomib, doxorubicin, and dexamethasone was examined in three human CD74+ MM cell lines, CAG, KMS11, KMS12-PE, and one CD74-MM cell line, OPM-2.Experimental Design: Activity of milatuzumab as a monotherapy and combined with the drugs was evaluated by studying in vitro cytotoxicity, signaling and apoptotic pathways, and in vivo therapeutic activity in severe combined immunodeficient (SCID) mouse models of MM.Results: Given as a monotherapy, cross-linked milatuzumab, but not milatuzumab alone, yielded significant antiproliferative effects in CD74+ cells. The combination of cross-linked milatuzumab with bortezomib, doxorubicin, or dexamethasone caused more growth inhibition than either cross-linked milatuzumab or drug alone, producing significant reductions in the IC50 of the drugs when combined. Efficacy of combined treatments was accompanied by increased levels of apoptosis measured by increases of activated caspase-3 and hypodiploid DNA. Both milatuzumab and bortezomib affect the nuclear factor-κB pathway in CAG MM cells. In CAG- or KMS11-SCID xenograft models of disseminated MM, milatuzumab more than doubled median survival time, compared with up to a 33% increase in median survival with bortezomib but no significant benefit with doxorubicin. Moreover, combining milatuzumab and bortezomib increased survival significantly compared with either treatment alone.Conclusions: The therapeutic efficacies of bortezomib, doxorubicin, and dexamethasone are enhanced in MM cell lines when given in combination with milatuzumab, suggesting testing these combinations clinically.

BACKGROUND: IMMU-130 is an antibody-drug conjugate (ADC) undergoing clinical investigation in patients with metastatic colorectal cancer (ClinicalTrials.gov, NCT01605318). It is composed of a humanized anti-CEACAM5 IgG conjugated via a cleavable linker to SN-38, a topoisomerase-I inhibitor and active form of irinotecan. We investigated the potential advantage of IMMU-130 versus irinotecan for SN-38 delivery in nude mice bearing CEA-expressing human colonic tumor xenografts (LS174T or GW-39). METHODS: Mice were injected with irinotecan (~ 900 µg; SN-38 equivalents = ~500 µg) or 1.0 mg of IMMU-130 (16 µg SN-38 equivalents). Irinotecan-treated animals were necropsied 5 min, 1, 2, 6-8 h post-injection, while IMMU-130-treated animals were evaluated at 1, 6, 24, 48-72 h. Serum and homogenates of tumors, liver, and small intestinal contents were extracted, and SN-38, SN-38G, and irinotecan concentrations were determined by reversed-phase HPLC. For IMMU-130-treated specimens, SN-38 concentrations were assessed in the extracted samples (Free SN-38), as well as in acid-hydrolyzed samples to determine Total SN-38 (Free + bound). IgG was measured by ELISA. RESULTS: Irinotecan cleared quickly from serum, with [SN-38]averaging ~900 ng/mL to 200 ng/mL from 5 min to 6 h. SN-38G and SN-38 levels were similar. With IMMU-130, Free SN-38 was detected in serum over the entire monitoring period, but levels were only a small fraction of the Total SN-38 (~10%). Importantly, Free SN-38G was very low, being detected only within the first 6 h. Total SN-38 levels dropped more quickly than the IgG, confirming in vitro studies showing gradual SN-38 release from the ADC. In tumors, for irinotecan-treated animals, SN-38 peaked at 5 min, representing ≤0.2%/g of the SN-38 equivalent given. In IMMU-130-treated animals, no Free SN-38 was detected in tumors, but levels of Total SN-38 peaked at 6 h, with ~5%/g of the injected SN-38 dose present at that time, and were sustained longer than SN-38 delivered by irinotecan. Area under the curve analysis found SN-38 levels were ~10- and 17-fold higher in LS174T and GW-39 tumors, respectively, from IMMU-130-dosed versus irinotecan-dosed animals. This delivery advantage is amplified > 30-fold when normalized to SN-38 equivalents injected for each product, illustrating the improved bioavailability with IMMU-130-targeted SN-38. Levels of SN-38 and SN-38G were appreciably lower in the liver and small intestinal contents, which likely explains the lower incidence of severe diarrhea reported in patients given IMMU-130. CONCLUSION: IMMU-130 delivers >300-fold more SN-38 to CEA-producing tumors compared to irinotecan, while also reducing levels of potentially harmful SN-38 and SN-38G in normal tissues. These observations are consistent with preclinical data showing improved efficacy and safety. Citation Format: Thomas M. Cardillo, Robert M. Sharkey, Serengulam V. Govindan, Jennifer Donnel, Maria Zalath, David M. Goldenberg. Superior SN-38 pharmacodynamic and tumor-accretion profiles of labetuzumab govitecan (IMMU-130) versus irinotecan in experimental human colonic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4081. doi:10.1158/1538-7445.AM2017-4081

Introduction: Relapsed AML, ALL, and MM continue to be a therapy challenge. IMMU-114 (hL243) is a humanized anti-HLA-DR IgG4 monoclonal antibody engineered to lack effector-cell functions, but retains binding and a broad range of antitumor effects in diverse hematological neoplasms (Stein et al., Blood. 2010;115:5180-90). When given subcutaneously, it has encouraging efficacy in an initial Phase I clinical trial in relapsed or refractory NHL and CLL, with a good safety profile (ClinicalTrials.gov, NCT01728207). In vitro, AML has proven to be resistant to the antitumor effects of IMMU-114, despite high expression levels of HLA-DR. Likewise, in several different human ALL and MM cell lines, IMMU-114 has demonstrated a range of antitumor effects from a low of 9% to a high of 69%. In an effort to improve the antitumor activity of IMMU-114, an antibody-drug conjugate (ADC) was made in which IMMU-114 was conjugated with the active metabolite of irinotecan, SN-38. Another ADC utilizing SN-38 (sacituzumab govitecan) being studied in solid tumors has been well tolerated, with clinically significant objective responses in patients given multiple cycles over >6 months, with manageable neutropenia being the major toxicity. Thus, our goal was to determine if SN-38, a drug not commonly used in hematopoietic cancers, would prove to be an effective and safe therapeutic when targeted with the IMMU-114 antibody. In this current work, the in vivo activity of hL243-SN-38 versus parental IMMU-114 is examined in human AML, ALL, and MM xenografts. Methods : Conjugation of SN-38 to hL243 IgG4 resulted in a drug-to-antibody-ratio range of 6.1 to 6.6. For AML and MM disease models, NSG/SCID and C.B.-17 SCID mice received 2 Gy irradiation 24 h prior to an i.v. injection of MOLM-14 (2x106) or CAG cells (1x107), respectively. ALL was established in C.B-17 SCID mice injected with MN-60 cells (1x107). All therapies began 5 days post-tumor-cell injection. Test agents, including a non-targeting anti-CEA-SN-38 ADC, were administered as 500-mg injections twice-weekly for 4 wks. Animals were sacrificed at disease progression, characterized by the onset of hind-limb paralysis or loss of more than 15% body weight. Results: In experimental AML, saline control and IMMU-114 treated mice succumbed to disease progression quickly, with a median survival time (MST) of only 14 and 15 days, respectively. Conversely, mice treated with hL243-SN-38 had a greater than 1.5-fold increase in survival (MST=37 d, P=0.0031). Further, hL243-SN-38 therapy provided a significant survival benefit when compared to anti-CEA-SN-38 control (MST=21 d, P=0.0031). In mice bearing ALL xenografts, IMMU-114 provided a >60% improvement in survival compared to saline control (MST = 37 d vs. 22.5 d, respectively; P30% improvement in survival when compared to IMMU-114 therapy (MST=94.5 d, P=0.1313). In all three experiments, therapy with hL243-SN-38 was well tolerated, as evidenced by no significant loss in body weight. Conclusions: Therapy with the hL243-SN-38 ADC proved to be superior to IMMU-114 (which is active clinically in NHL and CLL) in both AML and ALL xenografts and beneficial in MM. Most importantly, in IMMU-114-refractive AML, hL243-SN-38 demonstrated a significant antitumor effect without any undue toxicity. This new ADC is a candidate for clinical evaluation in these intractable malignancies. Disclosures Goldenberg: Immunomedics: Employment, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Sharkey:Immunomedics, Inc.: Consultancy, Employment, Equity Ownership. Govindan:Immunomedics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zalath:Immunomedics, Inc.: Employment, Equity Ownership. Mostafa:Immunomedics, Inc.: Employment, Equity Ownership. Cardillo:Immunomedics, Inc.: Employment, Equity Ownership.

Purpose: IMMU-114 is a humanized anti-HLA-DR IgG4 monoclonal antibody currently under investigation for non-Hodgkin's lymphoma and CLL (ClinicalTrials.gov, NCT01728207). This study was undertaken to continue preclinical evaluations in CLL and ALL models, comparing IMMU-114 efficacy to anti-CD20 or doxorubicin therapy, respectively, as well in combination with Bruton's tyrosine kinase (Btk) or phosphoinositide-3-kinase (PI3K) inhibitors in CLL. Procedures: The human CLL cell line, JVM-3, was grown s.c. in SCID mice. Once tumors reached ∼0.2 cm3, they were divided into treatment groups of either IMMU-114 or rituximab (200, 100, or 50 μg, twice weekly for 4 weeks). Study survival endpoint was tumor progression to >1.0 cm3. In vitro, JMV-3 was treated with various concentrations of either a Btk inhibitor (ibrutinib) or PI3K inhibitor (idelalisib) in the presence of a constant amount of IMMU-114. IC50-values were determined, data were normalized, and isobolograms generated for each inhibitor to determine overall effect. For ALL, MN-60 cells were injected i.v. into SCID mice. After 5 days, animals received IMMU-114 (50 or 25 μg, 2 x weekly for 4 weeks) or doxorubicin (3×20 μg qdx3d induction phase, followed by a 60μg bolus injection maintenance phase on week 3). Disease progression was declared upon the onset of hind-limb paralysis. Results: Mice with JVM-3 tumors had a median survival time (MST) of 14 days for saline controls, while therapy with rituximab significantly improved survival (P39 days, P Conclusions: In a preclinical model of human CLL, IMMU-114 was superior to anti-CD20 therapy using rituximab, and had an additive effect when combined with Btk or PI3K inhibitors. IMMU-114 also achieved a significant survival benefit in the doxorubicin-refractive MN-60 ALL model. These data demonstrate IMMU-114's overall activity in diverse hematopoietic cancers and show the need for continued clinical and preclinical evaluation. Citation Format: Thomas M. Cardillo, Maria Zalath, Roberto Arrojo, Robert M. Sharkey, David M. Goldenberg. Superior anti-tumor effects of an anti-HLA-DR IgG4 antibody, IMMU-114, in chronic and acute lymphocytic leukemia (CLL and ALL): Comparison to anti-CD20 therapy, chemotherapy, or combined with kinase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 587.

Purpose: In current clinical trials (ClinicalTrials.gov, NCT01631552), TNBC patients treated with IMMU-132, which is composed of the active metabolite of irinotecan, SN-38, conjugated to an anti-Trop-2 antibody (drug:Ab ratio = 7.6), shows manageable toxicity and encouraging responses in relapsed/refractory cases. Preclinical studies were performed to determine the utility of combinations of IMMU-132 with either a poly(adenosine diphosphoribose) polymerase (PARP) inhibitor (olaparib) or microtubule inhibitors (paclitaxel or eribulin mesylate) in mice bearing BRCA1/2 defective (HCC1806)and wild-type (MDA-MB-468) TNBC tumor xenografts. Procedures: In vitro, human TNBC cell lines were incubated with IMMU-132 and olaparib to determine a combination index number and whether the interaction was synergistic, as well as incubating with SN-38 or IMMU-132 ± olaparib with analysis by western blot or flow cytometry (FACS) for double-stranded DNA breaks, as evidenced by increases in phosphorylated histone H2AX (p-H2AX). In vivo, mice bearing MDA-MB-468 or HCC1806 tumors were treated with either paclitaxel (qwklyx5wks) or eribulin mesylate (wks 1, 2, 4, & 5) alone or in combination with IMMU-132 (wks 1, 2, 4, & 5). Additionally, mice bearing TNBC tumors were treated with olaparib (qdx5d) plus IMMU-132 (qwkly) for 4 wks. Study survival endpoint was tumor progression to >1.0 cm3. Results: Treatment with IMMU-132 plus paclitaxel in HCC1806 or MDA-MB-468 tumor-bearing mice significantly inhibited tumor growth compared to monotherapy (P Conclusions: Combining IMMU-132 with a PARP inhibitor achieves synergistic growth inhibition in TNBC, regardless of BRCA1/2 status. The combination of IMMU-132 therapy with either microtubule or PARP inhibitors results in significant anti-tumor effects in TNBC disease models with no observable toxicity. These data provide the rationale for the clinical evaluation of IMMU-132 in combination with these chemotherapeutics in TNBC patients. Citation Format: Thomas M. Cardillo, Serengulam V. Govindan, Maria Zalath, Ali Mostafa, Roberto Arrojo, Robert M. Sharkey, David M. Goldenberg. Significant enhancement of efficacy of an anti-Trop-2 antibody-drug conjugate, sacituzumab govitecan (IMMU-132), in experimental triple-negative breast cancer (TNBC) when combined with microtubule or PARP inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 584.

Purpose: Determine whether combining sacituzumab govitecan (IMMU-132), an anti-Trop-2/SN-38 antibody-drug conjugate, with microtubule inhibitors (paclitaxel or eribulin mesylate) or a poly(adenosine diphosphoribose) polymerase (PARP) inhibitor (olaparib) in mice bearing human TNBC xenografts improves anti-tumor effects. Experimental Procedures: Mice bearing human TNBC xenografts (MDA-MB-468 or HCC1806; ∼0.3 cm3) were treated with the maximum tolerated dose of paclitaxel (15 mg/kg weekly x 5 wks) and IMMU-132 at either 10 mg/kg or 12.5 mg/kg on days 1, 8, 22, and 29. Mice bearing HCC1806 tumors (∼0.28 cm3) were treated for 2 cycles with IMMU-132 (12.5 mg/kg) and 0.5 mg/kg of eribulin mesylate (equivalent to human dose of 1.4 mg/m2) weekly for 2 weeks on a 21-day cycle. Studies examining PARP inhibition used mice bearing MDA-MB-468 tumors (∼0.32 cm3) treated with olaparib (50 mg/kg, qdx5d, x 4 wks; 33% of human dose equaling 800 mg daily) and IMMU-132 (10 mg/kg, twice weekly x 4 wks). The primary endpoint was the median survival time (MST), defined as the time for tumors to progress to 1.0 cm3. Results: Mice with MDA-MB-468 tumors given the combination of IMMU-132 and paclitaxel exhibited superior anti-tumor effects, with >11-fold tumor shrinkage, in comparison to 1.4-fold shrinkage in the IMMU-132 group alone (P = 0.0003; area under the curve, AUC) or 11.4-fold increase in tumor size in mice treated with paclitaxel alone (P Conclusions: IMMU-132 is a humanized anti-Trop-2 antibody conjugated to SN-38, the active metabolite of irinotecan, a topoisomerase I inhibitor. Clinically, IMMU-132 has shown manageable toxicity and encouraging responses in patients with relapsed/refractory TNBC (ClinicalTrials.gov, NCT01631552). Since preclinical studies indicate IMMU-132 can be combined with two different microtubule-inhibitors or a PARP-inhibitor with significantly enhanced anti-tumor activity, these data provide a rationale for future clinical evaluation of IMMU-132 in combination with these and other chemotherapeutics that likewise target cell division through microtubule inhibition or DNA-repair mechanisms in patients with TNBC. Citation Format: Thomas M. Cardillo, Serengulam V. Govindan, Maria Zalath, Roberto Arrojo, Robert M. Sharkey, David M. Goldenberg. Combining an anti-Trop-2 antibody-SN-38 conjugate (sacituzumab govitecan) with microtubule inhibitors (paclitaxel and eribulin mesylate) or PARP inhibitor (olaparib) significantly improves therapeutic outcome in experimental triple-negative breast cancer (TNBC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C166.

Background: Trop-2 is a type I transmembrane protein that is highly expressed in diverse epithelial cancers, including lung, gastric, colorectal, pancreatic, bladder, mammary, ovarian, uterine, and prostate carcinomas, with limited presence on normal human tissues. Whereas various formats of bispecific antibodies (bsAbs) for redirecting T cells to cancers have shown promise in both pre-clinical and clinical studies, efforts to prolong the circulating half-life of the scFv-based platforms and to prevent the commonly observed cytokine release syndrome are continuing. Herein, we describe a novel T-cell redirecting trivalent bsAb, designated (E1)-3s, which comprises an anti-CD3 scFv covalently conjugated to a stabilized dimer of a Trop-2-targeting Fab. Potential advantages of (E1)-3s include high-level surface expression of Trop-2 on various solid cancers, bivalent binding to tumor cells, a larger size (∼130 kDa) to preclude rapid renal clearance, and potent T-cell mediated cytotoxicity. Methods: DOCK-AND-LOCKTM was employed to site-specifically link an anti-CD3 (Okt3) scFv to a stabilized Fab dimer of the humanized anti-Trop-2 mAb, hRS7. LC-MS confirmed the mass of the conjugate, which resolved as >90% of the desired monomeric peak by size-exclusion HPLC. Purity was demonstrated by reducing SDS-PAGE, which resolved only the three constituent polypeptide bands comprising (E1)-3s. Results: Fluorescence microscopy showed that (E1)-3s, but not a control conjugate [(19)-3s, anti-CD19/CD3], induced synapse formation between Jurkat (T) and Capan-1 (human pancreatic cancer) cells. Using a 3-fold excess of stimulated T cells in vitro, (E1)-3s induced a potent and specific T-cell-mediated lysis in Capan-1 (115,000 Trop-2 copies/cell) pancreatic cancer (IC50 = 29 pM, Lysismax = 60%), and NCI-N87 (383,000 Trop-2 copies/cell) human gastric cancer cells (IC50 = 0.85 pM, Lysismax> 90%). In vivo efficacy was demonstrated with Capan-1 and NCI-N87 xenografts in NOD-SCID mice by co-injection of purified human T cells and tumor cells at an effector-to-target ratio of 2, followed by five daily injections of (E1)-3s. With Capan-1, the control group reached the endpoint of disease progression (tumors exceeding 1 cm3) on day 24, at which time, the (E1)-3s group had significantly smaller tumors (0.257 ± 0.102 cm3 versus 1.011 ± 0.532 cm3; P=0.0092). For NCI-N87, the (E1)-3s group had significantly smaller tumors than the control group (0.147 ± 0.062 cm3 versus 0.824 ± 0.342 cm3, respectively; P=0.0017) when the latter reached the disease-progression endpoint on day 31. Conclusions: (E1)-3s effectively induced T-cell-mediated killing of Trop-2-expressing pancreatic and gastric cancer cells in vitro and in vivo. Additional cell lines of different solid cancer types, and ones with higher and lower Trop-2 antigen density, are under evaluation. Citation Format: Diane L. Rossi, Thomas M. Cardillo, Edmund A. Rossi, Maria Zalath, David M. Goldenberg, Chien-Hsing Chang. A novel Trop-2/CD3 trivalent bispecific antibody effectively redirects T cells to kill target human pancreatic and gastric cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2655. doi:10.1158/1538-7445.AM2014-2655

3056 Background: Trop-2 is highly expressed in diverse epithelial cancers with limited presence on normal tissues. (E1)-3s is a T-cell redirecting trivalent bsAb, which comprises an anti-CD3 scFv c...

Among kidney cancer types, approximately 90% are renal cell carcinomas (RCC). Advanced or metastatic RCC, which presents in about one third of the patients, has a poor prognosis as it is resistant to conventional chemotherapy or radiotherapy. Treatments with human interferon-α2b (IFN-γ2b) alone or in combination with mammalian target of rapamycin (mTOR) inhibitors have led to only modest improvements in clinical outcomes. One observation made with mTOR inhibitors is that cancer cells can overcome the effects of the inhibitor by activating the insulin-like growth factor-I (IGF-I) signaling pathways. Clinically, there is an association of IGF-I receptor (IGF-IR) expression in RCC and poor long-term patient survival, particularly among patients with high-grade tumors. We have developed a humanized anti-IGF-IR monoclonal antibody, hR1, which binds to multiple tumor types, including RCC, resulting in effective down-regulation of IGF-IR and moderate inhibition of cell proliferation in vitro. To enhance the anti-tumor activity of hR1, we applied the Dock-and-Lock (DNL) platform technology to generate 1R-2b, comprising a conjugate of hR1 IgG with two dimers of interferon-α2b, and Hex-hR1, comprising 6 Fab fragments of hR1 tethered onto a common Fc. There was no loss in cell binding for both 1R-2b and Hex-hR1 when compared to parental hR1 as determined by flow cytometry. Units of activity for 1R-2b as measured by a luciferase reporter gene fused to a promoter containing the interferon-stimulated response element (iLite kit), yielded a specific activity of 3750 U/pmole versus 180 U/pmole and 3255 U/pmole for two different forms of peginterferon alpha-2a (60 and 31 kDa), respectively. An in vitro cytotoxicity assay with1R-2b demonstrated growth inhibition of two different RCC cell lines, 786-0 and ACHN with EC50-values of 0.049 and 0.062 pmole/mL, respectively. In terms of receptor down-regulation, Hex-hR1 could effectively down-regulate IGF-IR at concentrations 10-fold lower than parental hR1 IgG. In soft-agar growth assays, all three agents (hR1, Hex-hR1 and 1R-2b) could significantly inhibit colony formation of 786-0 and ACHN (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4402. doi:1538-7445.AM2012-4402

Introduction: IFNα2 has been used in therapy of a variety of hematopoietic neoplasias, including myeloma (MM), NHL, hairy cell leukemia (HCL), and CML. However, its therapeutic potential is limited by its short circulating half-life and systemic toxicity. Fusion of IFNα2 to a monoclonal antibody (mAb) improves solubility and stability, and markedly increases circulating half-life. In addition to allowing less frequent and lower doses via extended Pk, targeting of IFNα2 to tumor sites using a tumor-directed mAb can reduce systemic concentrations and increase local concentration and tumor retention of IFNα2, thereby improving the therapeutic index. Increased tumor concentrations of IFNα2 can augment its direct anti-proliferative, apoptotic and anti-angiogenic activity, as well as prime an antitumor immune response. Methods: We previously used the DNL method to generate stably-tethered immunocytokines (Rossi et al., Blood 2009;114:3864-71; Rossi et al., Cancer Res 2010;70:7600-9), which include 20-2b-2b (CD20-targeted mAb-IFNα comprising tetrameric IFNα2b and veltuzumab) and 20-C2-2b (bispecific CD20/HLA-DR-targeting dimeric IFNα2b). Here we describe the potent in vitro and in vivo anti-tumor activities of a third immunocytokine, C2-2b-2b, comprising tetrameric IFNα2b and the anti-HLA-DR mAb, hL243, against myeloma, lymphoma and leukemia cell lines. Results: C2-2b-2b bound target cells with similar avidity to hL243 and exhibited high IFNα specific activity. In vitro, C2-2b-2b inhibited a panel of 20 cell lines comprising NHL (Burkitt, mantle cell & follicular), leukemia (HCL, AML, ALL & CLL), and MM, and in most cases was more effective than CD20-targeted mAb-IFNα or a mixture comprising hL243 and IFNα. Our findings indicate that a given cell's responsiveness depends on HLA-DR expression/density and its sensitivity to IFNα and hL243. C2-2b-2b was remarkably potent in vivo, where a single 1 μg dose significantly improved survival in advanced Daudi (NHL) and CAG (MM) xenograft models, and doses of ≥10 μg resulted in 70-100% long-term survivors (cures). C2-2b-2b showed superior anti-tumor efficacy compared to hL243 IgG, hL243 + rIFNα2b, Peginterferonalfa-2a or non-targeting mAb-IFNα. Ex-vivo pharmacodynamics studies using whole blood spiked with either NHL or MM cells showed that C2-2b-2b depleted either tumor cell type more efficiently than normal B cells and monocytes, did not deplete T cells, and was cytotoxic to dendritic cells, with myeloid DCs more susceptible than plasmacytoid DCs. Conclusions: The DNL method provides a modular approach to efficiently tether multimeric cytokines onto a targeting antibody, resulting in higher in vivo potency than the original cytokines due to improved pharmacokinetics, targeting and reduced systemic toxicity. These results suggest that C2-2b-2b might be useful in the treatment of various hematopoietic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1803. doi:10.1158/1538-7445.AM2011-1803

Antibody (MAb) conjugates of the potent topoisomerase I inhibitor, SN-38, have been previously shown by our groups to be efficacious in the targeted therapies of human cancer xenografts in nude mice. The conjugates have since been redesigned with a new bifunctional SN-38, ‘CL2A-SN-38’ (maleimido-[x]-Lys-PABOCO-20-O-SN-38, where PAB is p-aminobenzyl and ‘x’ contains a short PEG), without a cleavable dipeptide, to simplify process and improve quality. CL2A-SN-38 was conjugated to humanized MAbs, hRS7 (anti-EGP-1), hPAM4 (anti-mucin), hMN-14 (anti-CEACAM5), hLL2 (anti-CD22), and hA20 (anti-CD20). The conjugates, with a mean SN-38/MAb substitution of ∼ 6, were evaluated in the Capan-1 and BxPC-3 pancreatic human tumor xenografts and the Ramos human NHL tumor xenograft grown s.c. in female athymic nude mice. When the starting tumor sizes in animals reached 0.2 to 0.3 cm3, specific and non-targeting control conjugates were administered i.p. in a twice-weekly × 4 weeks schedule using 0.39 mg/kg (protocol-1) or 0.20 mg/kg (protocol-2) or 0.08 mg/kg (protocol 3) of SN-38 equivalent. Saline controls were included. In the therapy of Capan-1 xenograft (protocol 2; n = 10), specific targeting by SN-38 conjugates of hRS7, hPAM4, and hMN-14 produced significant tumor growth control versus control hA20-SN-38 conjugate on day 35 (AUC, P < 0.034), but there was no difference among the specific conjugates. Median survival time (MST) for untreated was 35 days, while MST was not reached on day 56 for treatments with all the three specific conjugates (protocol 2). In therapy in the BxPC-3 model (protocol 1; n = 10), MST for treatment with specific hRS7-SN-38 was significantly better than that for untreated or treatment with control hA20-SN-38 (P < 0.0006) or treatment with hMN-14-SN-38 (P < 0.0046). In the Ramos lymphoma model (n = 5), MST in untreated animals was only 7 days owing to rapid tumor growth, while MST was not reached on day 35 with the specific hLL2-SN-38 treatment (protocol 2). Moreover, combination of hA20 immunotherapy and hLL2-SN-38 immunoconjugate therapy (protocol 3) extended MST significantly versus conjugate treatment alone (P < 0.015). A dose of up to 47 mg/kg of SN-38 equivalent [240 mg/kg human equivalent protein dose of the conjugate], examined thus far, was very well tolerated in normal mice, as determined by blood counts and serum chemistries, which indicated an excellent therapeutic window. The redesigned conjugates, without the cathepsin B cleavable peptide, retained the efficacies of the CL2-SN-38-based conjugates, were monomeric, and were amenable to scale-up. These features, together with very favorable safety profiles, portend well for the clinical development of these immunoconjugates for different cancer indications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2438.

Antibody (MAb) conjugates of the potent topoisomerase I inhibitor, SN-38, prepared using the bifunctional derivative, ‘CL2-SN-38’ (maleimido-[x]-Phe-Lys-PABOCO-20-O-SN-38), were previously shown to produce significant and selective therapeutic efficacies of human tumor xenografts in nude mice. With a view to examining alternative linkers for possible improvement, we prepared a variant, namely maleimido-[x]-Lys-PABOCO-20-O-SN-38 (‘CL2A-SN-38’), and two other derivatives with cross-linker attachment at the 10-position of SN-38 instead of the 20-position: maleimido-[x]-Phe-Lys-N(Me)-(CH2)2-N(Me)-CO-10-O-SN-38 (‘CL2D-SN-38’) and maleimido-[x]-Phe-Lys-PABOCO-N(Me)-(CH2)2-N(Me)-CO-10-O-SN-38 (‘CL2E-SN-38’). In these, PAB is p-aminobenzyl moiety and ‘x’ contains a short PEG group to increase solubility. Conjugates of the humanized anti-CEACAM5 MAb, hMN-14 (labetuzumab), were prepared by reacting the maleimide-appended SN-38 derivatives with mildly reduced hMN-14. The SN-38/MAb molar substitutions were in the range of 5.9 to 6.3. In vitro studies showed a similarity in the stabilities, in mouse serum, of CL2 and CL2A-based linkers, but an order of magnitude greater stability for conjugates with linker at the 10-position; however, CL2D-SN-38 linker was inert to dipeptide cleavage by cathepsin B at the lysosomal pH of ∼ 5. The latter finding was also reflected in the loss of cytotoxicity due to hMN-14-CL2D-SN-38 conjugate in the LS174T colon cancer cell line in vitro. All conjugates were also evaluated in an exploratory preclinical therapy experiment in nude mice with aggressive s.c. LS174T human colon cancer xenografts. Conjugates were administered i.p. in groups of tumor-bearing animals (n = 10) at a schedule of 0.39 mg/kg of SN-38 equivalent twice weekly × 2 weeks. Untreated animals received saline. Mean tumor volumes (cm3) of animals in various treatment groups, on day 18, were: 0.073 ± 0.035 for the hMN-14-CL2-SN-38 positive control group; 0.055 ± 0.032 for the hMN-14-CL2A-SN-38 group; 0.518 ± 0.239 for the hMN-14-CL2D-SN-38 group; 0.490 ± 0.224 for the hMN-14-CL2E-SN-38 group; and 1.09 ± 0.88 for the untreated group. Median survival times (days) were 54.5, 55, 30, 25, and 26.5 for these groups, respectively. These results showed that a cleavable peptide was not necessary in the SN-38 conjugate design, as hMN-14-CL2A-SN-38 was equivalent to hMN-14-CL2-SN-38 in efficacy, and that hMN-14-CL2E-SN-38 was surprisingly much less efficacious than the CL2 and CL2A-based conjugates despite its higher relative stability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2439.