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Purpose To evaluate a new immunoassay for identification and quantitation of MUC1 in the sera of patients with pancreatic cancer or pancreatitis. The sensitivity and specificity of the assay are examined and compared to results from a CA19-9 immunoassay. Methods An in vitro enzyme immunoassay was established with monoclonal antibody PAM4 as the capture reagent, and a polyclonal anti-MUC1 antibody as the probe. Patient sera were obtained from healthy, adult patients with acute and chronic pancreatitis, and those with pancreatic and other forms of cancer, and were measured for PAM4-reactive MUC1. Results At a cutoff of 10.2 units/mL, 41 (77%) of 53 pancreatic cancer patients, none of the healthy individuals (n = 43), and only four (5%) of 87 patients with pancreatitis were positive above this value. Among nonpancreatic cancers investigated, colorectal cancers gave the highest percentage of positives (14%; five of 36). Overall, the sensitivity and specificity of the immunoassay for pancreatic cancer were 77% and 95%, respectively. Receiver operator characteristic analyses for discrimination of pancreatic cancer from pancreatitis provided an area under the curve of 0.89 (95% CI, 0.82 to 0.93), with a specificity of 95.4% and a positive likelihood ratio of 16.8. A direct pair-wise comparison of PAM4 and CA19-9 immunoassays for discrimination of pancreatic cancer and pancreatitis resulted in a significant difference (P < .003), with the PAM4 immunoassay demonstrating superior sensitivity and specificity. Conclusion The high sensitivity and specificity observed suggest that the PAM4-based immunoassay of circulating MUC1 may be useful in the diagnosis of pancreatic cancer.

98 Background: Bispecific antibodies (bsAbs) for redirecting T cells to cancers have shown promise in both preclinical and clinical studies. However, clinical results have been disappointing in solid cancers. We have applied the DOCK-AND-LOCK method to generate a novel class of trivalent bsAbs, each comprising an anti-CD3 scFv covalently conjugated to a stabilized dimer of different anti-tumor Fabs. Herein we report the characterization of two such constructs, (E1)-3s and (14)-3s, which activate T cells and target Trop-2- and CEACAM5-expressing cancer cells, respectively. Methods: Human breast and colonic cancer cell lines were grown in monolayer cultures or as 3D spheroids for in vitro evaluation. NOD/SCID mice carrying xenografts of MDA-MB-231 (a TNBC line constitutively expressing Trop-2 and PD-L1) were used for in vivo studies. A human PD-1 antagonistic murine hybridoma antibody was subsequently converted to its chimeric form (IMMU-cPD-1). Human PBMCs, or T cells isolated from buffy coats by negative selection, were used as effector cells in cytotoxicity assays. The effect of IMMU-cPD-1 on cancer cells pretreated with IFN-γ to induce the expression of PD-L1 was compared with those not pretreated. Results: (E1)-3s and (14)-3s, in the presence of human T cells, killed target cells grown as monolayers at low picomolar concentrations, with similar potency observed for drug-resistant cells. The antitumor efficacy was demonstrated for (E1)-3s plus human PBMCs in NOD/SCID mice bearing MDA-MB-231, and for human PBMCs combined with (E1)-3s or (14)-3s in 3D spheroids generated from target cell lines to mimic the in vivo behavior and microenvironment of these tumors. Moreover, with the addition of IMMU-cPD-1, the benefit of PD-1 blockade was indicated by increased cell death in 3D spheroids and longer survival of MDA-MB-231-bearing mice. Conclusions: These results highlight the potency of (E1)-3s and (14)-3s as T-cell redirecting bsAbs, emphasize the potential of combining such bsAbs with immune checkpoint inhibitors to improve the therapeutic activity in the immunotherapy of solid cancers, and provide a basis for using 3D spheroids as an alternative to in vivo models for evaluating T-cell functions.

625 Background: IMMU-130 is a CEACAM5-targeted ADC, labetuzumab-SN-38, with the drug being the active form of the topoisomerase I inhibitor, CPT-11, and substituted at 7-8 moles/mole of IgG. This agent is in phase I/II clinical trials in patients with relapsed mCRC (NCT01270698; NCT01605318). Methods: Therapy experiments were conducted in female athymic nude mice (n=5-10 per group), 4-6 weeks of age, bearing s.c. LS174T human colon carcinoma xenografts of ~0.2 cm3 size, or 2 weeks after lung metastases were generated by i.v. injection of GW-39 human colon carcinoma cells. Untreated controls, including a non-targeting ADC, were included. Biodistribution was examined in the s.c model using a single 12.5-mg/kg dose of the ADC or unconjugated labetuzumab, each spiked with 111In-labeled substrate. Tolerability studies were conducted in white New Zealand rabbits. Results: In the metastatic model, fractionated dosing of a total of 50 mg/kg of ADC showed that 2 × the schedules of twice-weekly × 2 weeks with 1 week off and once-weekly × 2 weeks with 1 week off doubled the median survival vs. untreated mice, and were better than a 25-mg/kg dose given on days 1 and 15 (p < 0.0474; log-rank). Pre-dosing with as much as twice the dose of labetuzumab as the ADC dose in the metastatic model did not affect median survival (p > 0.15). Therapy experiments in the s.c. model revealed that IMMU-130, with 50% of drug released in ~20 h, was superior to the conjugate with an ultrastable linker, that the ADC was better than an MTD of 5FU/leucovorin chemotherapy (p < 0.0001), and that the ADC could be combined with bevacizumab for improved efficacy (p < 0.031). Significantly better efficacy for the specific ADC vs. nonspecific ADC was observed. Pharmacokinetics in mice indicated ~25% longer half-life for the unconjugated MAb vs. ADC, but with minimal impact on tumor uptake. A tolerability study in rabbits showed the NOAEL to be the human equivalent dose of 40-60 mg/kg, given as two doses. Conclusions: Preclinical data show an excellent therapeutic window for IMMU-130, which appears to be translated into the clinical experience thus far. The potential for combination therapy is also indicated.

262 Background: Trop-2 [also called tumor-associated calcium signal transducer 2 (TACSTD2), EGP-1 (epithelial glycoprotein-1), GA733-1, or M1S1]is a 35 kDa transmembrane glycoprotein that is overexpressed relative to normal tissues in a variety of human cancers, including pancreatic and gastric carcinomas, where increased expression correlates with poor prognosis. Trop-2 appears to be more tumor-specific than the related molecule, EpCAM (Trop-1). MT110, the EpCAM antibody x CD3 bispecific T-cell engager (BiTE), is currently undergoing a Phase I study in various solid tumors, including lung, gastric, colorectal, breast, prostate, and ovarian cancers. We produced a similar T-cell redirecting bispecific tandem scFv, E1-3, using the variable domains of hRS7 (humanized anti-Trop-2 mAb) and Okt-3 (anti-CD3 mAb). Methods: T-cell activation, cytokine induction and cytotoxicity were evaluated ex vivo using PBMCs or purified T cells with human pancreatic (Capan-1 and BxPC3) and gastric (NCI-N87) cancer cell lines as target cells. In vivo activity was assayed with NCI-N87 xenografts that were inoculated s.c. in a mixture with twice the number of human PBMCs and matrigel. Results: In the presence of target cells and PBMCs, E1-3 potently induced T-cell activation, proliferation, and dose-dependent cytokine production of IL-2 (>2 ng/mL), IL-6 (>1 ng/mL), IL-10 (>7 ng/mL), TNF-α (>1 ng/mL) and IFN-γ (>50 ng/mL). In vitro, E1-3 mediated a highly potent T-cell lysis of BxPC3 [IC50=0.09(±0.04) pM], Capan-1 [IC50=1.2(±1.1) pM] and NCI-N87 [IC50=1.2(±1.2) pM] target cells. In vivo, two 50-µg doses of E1-3 given three days apart cured all of the mice (N=8) bearing NCI-N87 xenografts (P=0.0005; Log-Rank). Tumors in the control group (PBMCs only) reached the endpoint (TV>1 cm3) with a median of 39.5 days. All mice remained tumor-free in the E1-3 group at 78 days. Conclusions: Trop-2 is an attractive target for T-cell-mediated killing of pancreatic, gastric and other epithelial cancers.

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...

3091 Background: CD74 is commonly considered to be an antigen present in hematopoietic cancers, but it is also expressed in a number of solid tumors. The humanized anti-CD74 antibody, milatuzumab (hLL1), was previously shown to have exquisite internalization properties, making it an attractive carrier for drug delivery. In this study, we evaluated hLL1 conjugates of a potent topo I inhibitor, SN-38, for treating solid tumors. Methods: Two hLL1-SN-38 conjugates, possessing either a pH-sensitive linker (‘CL2A’ form) or a cathepsin-B-cleavable linker ("CL2E" form), were examined. In vitro analyses were carried out in the A-375 human melanoma cell line, and therapy experiments were performed in female athymic nude mice bearing established s.c. A-375 or s.c. human pancreatic adenocarcinoma (Capan-1) xenografts using specified doses (i.p.) at a twice weekly × 4 wks schedule; animals were sacrificed when tumor volumes (TVs) reached 1 cm3. Results: Both A-375 and Capan-1 cell lines tested positive for CD74 expression by IHC. In vitro, both SN-38 derivatives liberated free drug at the same rate when exposed to cathepsin-B at pH 5, while serum stabilities for CL2A and CL2E forms of the conjugates were ~ 1 d and >10 d, respectively. In the A-375 melanoma cell line, IC50 for the hLL1-SN-38 conjugates of CL2A and CL2E forms were 5 nM and 34 nM, respectively. In the aggressive A-375 s.c. model of melanoma, in nude mice (n =8; TV: 0.23 ± 0.06 cm3) treated with 12.5 mg/kg protein dose (total 1.7 mg/kg SN-38 eq.) of specific or non-specific SN-38 conjugates of CL2A and CL2E forms, only hLL1-CL2A-SN-38 conjugate was efficacious (Log-rank: P3), two low doses of hLL1-CL2A-SN-38 (12.5 or 5 mg/kg) significantly improved survival in comparison to saline control mice (p

3043 Background: Veltuzumab (hA20) is a humanized 2nd generation anti-CD20 monoclonal antibody (mAb) under evaluation for B- cell malignancies and autoimmune diseases that differs from chimeric rit...

4102 Background: At the present time there are no reliable methods for the early detection and diagnosis of pancreatic cancer. Since the PAM4 MAb identifies an epitope that is more restricted to pa...

6535 Background: CD74, a cell-surface-expressed epitope of the HLA class II-associated invariant chain [Ii], is a unique target for antibody-drug immunoconjugate therapy, being a transmembrane chaperone molecule that actively directs transport from the cell surface to an endosomal compartment. IMMU-110 is a drug immunoconjugate comprised of doxorubicin (dox) conjugated to the humanized anti-CD74 monoclonal antibody (MAb), hLL1, at 6–8 drug molecules per IgG molecule. The dox is linked to partially-reduced antibody inter-chain disulfide groups using 4-[N-maleimidomethyl] cyclohexane-1 carboxylhydrazide. We recently reported that IMMU-110 has potent therapeutic effects in a non-Hodgkin's lymphoma-SCID mouse model. Methods: Here we examine the efficacy of IMMU-110 for therapy of multiple myeloma (MM), based on the high prevalence of CD74 expression on MM specimens, using in vitro assays on MM cell lines and in vivo studies in a SCID mouse model of disseminated MM. Results: In vitro cytotoxicity assays demons...