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The gut microbiome, composed of the microflora that inhabit the gastrointestinal tract and their genomes, make up a complex ecosystem that can be disrupted by antibiotic use. The ensuing dysbiosis is conducive to the emergence of opportunistic pathogens such as Clostridium difficile. A novel approach to protect the microbiome from antibiotic-mediated dysbiosis is the use of beta-lactamase enzymes to degrade residual antibiotics in the gastrointestinal tract before the microflora are harmed. Here we present the preclinical development and early clinical studies of the beta-lactamase enzymes, P3A, currently referred to as SYN-004, and its precursor, P1A. Both P1A and SYN-004 were designed as orally-delivered, non-systemically available therapeutics for use with intravenous beta-lactam antibiotics. SYN-004 was engineered from P1A, a beta-lactamase isolated from Bacillus licheniformis, to broaden its antibiotic degradation profile. SYN-004 efficiently hydrolyses penicillins and cephalosporins, the most widely used IV beta-lactam antibiotics. In animal studies, SYN-004 degraded ceftriaxone in the GI tract of dogs and protected the microbiome of pigs from ceftriaxone-induced changes. Phase I clinical studies demonstrated SYN-004 safety and tolerability. Phase 2 studies are in progress to assess the utility of SYN-004 for the prevention of antibiotic-associated diarrhea and Clostridium difficile disease.

SYN-004 is an orally administered β-lactamase enzyme, designed to be given concurrently with certain intravenous β-lactam antibiotics like cephalosporins. SYN-004 is intended to degrade residual antibiotics excreted into the intestine as a result of hepatobiliary excretion and to prevent the disruption of the gut microbiome by these excess antibiotics. Preserving the gut microbiome is expected to prevent secondary infections by pathogens like Clostridium difficile and protect against other antibiotic-associated diarrheas. Two, randomized, double blind, placebo-controlled Phase 1 clinical studies were conducted in normal healthy adult volunteers to assess the tolerability and systemic absorption of single and multiple doses of SYN-004. A single-ascending dose study investigated single oral doses of 75–750 mg SYN-004 and was conducted in 40 subjects (five cohorts of six active and two placebo subjects). A multiple-ascending dose study investigated doses of 75–300 mg SYN-004, administered every 6 h for 7 days and was conducted in 24 subjects (three cohorts of six active and two placebo subjects). The safety and tolerability of SYN-004 was assessed and serial plasma and serum samples were collected to assess the pharmacokinetics and potential immunogenicity of SYN-004. Minimal and mild adverse events were reported in ~30 % of the subjects who received active drug and placebo and no antidrug antibodies were detected in any subject. Analysis of serial plasma samples demonstrated negligible systemic bioavailability of SYN-004 with most plasma concentrations being below the lower limit of quantitation (0.8 ng/mL) for the assay. SYN-004 was well tolerated in the 48 subjects who received active drug, and adverse events in those subjects were comparable to the 16 subjects who received placebo, up to the maximum doses administered in each study. SYN-004 was well tolerated up to a single oral dose of 750 mg and multiple doses of 300 mg every 6 h for 7 days. The pharmacokinetic results support that SYN-004 remained localized in the intestine.

SYN-004 (ribaxamase) delayed release drug product is a multi-particulate, hard capsule for oral delivery of a recombinant β-lactamase enzyme designed to degrade β-lactam antibiotics administered intravenously, and thus prevent colon dysbiosis. Here we describe the development of the SYN-004 enteric coated pellet formulation, which has been tested in multiple clinical trials. Since the SYN-004 drug substance is a buffered liquid, several binder excipients in different ratios were tested to facilitate binding of SYN-004 to sugar spheres. The binding systems were evaluated by droplet pre-evaluation and film casting tests. The most promising formulations were produced in small scale fluidized bed application runs and analyzed by dissolution tests and complementary analytical assays. Hydroxypropyl cellulose was selected as the preferred SYN-004 binding excipient. The formulation included a second, outer coat containing the enteric EUDRAGIT® L 30 D-55 polymer-based formulation to achieve gastric protection, and rapid SYN-004 release in the intestinal tract, when the pH rises above 5.5. Additional formulation improvements resulted in an increase in the SYN-004 load compared to a predecessor oral enzyme formulation (Ipsat P1A). Thus, a novel formulation and process for an orally administered enzyme was developed and used to manufacture drug product for clinical trials.

There is growing evidence that methane production, predominantly by Methanobrevibacter smithii , in the intestines is a cause of constipation, pain, and bloating in irritable bowel syndrome with constipation (IBS-C). M smithii resides primarily in the large intestine but can also colonize the small intestine. In vitro studies found that the prodrug lactone form of lovastatin, found in cholesterol-lowering drugs, inhibited methane production in stool samples from patients with IBS-C. However, the cholesterol-lowering lovastatin β-hydroxyacid was ineffective at inhibiting methane production in this system. A considerable amount of lovastatin is converted to hydroxyacid in the stomach and is absorbed. It was hypothesized that galenic innovations could protect lovastatin from the stomach and allow release in 2 strategic locations, the duodenum and the ileocecal region, to reach M smithii . The desired release profile was achieved by developing an oral dosage form containing lovastatin and coated with 2 different enteric polymers that enabled a pH-dependent "dual pulse" drug release. Combinations of the 2 coated tablets were encapsulated together to deliver the desired amount of lovastatin to the targeted intestinal locations. The capsules have been tested in vitro and in vivo and show promise in treating IBS-C.

NPC-1C is a chimeric immunoglobulin IgG1 developed from antigen tested in the Hollinshead tumor vaccine trials that recognizes an immunogenic MUC5AC-related tumor-associated antigen. In this article, we describe the pre-clinical characterization of this antibody that is currently being tested in human clinical trials. The specificity of NPC-1C for pancreatic and colorectal cancer cell lines was tested by flow cytometry assays and immunohistochemical staining. Antibody-dependent cell cytotoxicity was measured using a tumor cell line lysis assay. Anti-tumor efficacy and biodistribution were assessed in nude mice bearing human pancreatic tumor xenografts. Human tumor cell binding measured by flow cytometry ranged from 52 to 94 % of cells stained positive with NPC-1C in three colorectal and one pancreatic cell lines, while IHC demonstrated staining of 43 % of colon cancers and 48 % of pancreatic cancer tissues, with little or no cross-reactivity of NPC-1C with normal colon or pancreas tissues. In vitro NPC-1C-mediated tumor cell killing occurred in a median of 44.5 % of four colorectal and three pancreatic tumor cell lines. In vivo anti-tumor efficacy in a human pancreatic CFPAC-1 tumor xenograft model was demonstrated with a twofold to threefold reduction in tumor growth in the NPC-1C-treated mice compared to saline and human IgG controls. Pharmacodynamic studies indicate NPC-1C localizes in antigen-positive tumors and has minimal uptake in normal mouse tissues. NPC-1C, a chimeric monoclonal antibody that reacts with a MUC5AC-related antigen expressed by pancreatic and colorectal tumor tissues, has promising preclinical activity in pancreatic and colorectal adenocarcinoma.

A serum ELISA using a monoclonal antibody that detects a MUC5AC-related antigen (NPC-1C antigen) expressed by pancreatic and colorectal cancer was developed. The NPC-1C antibody reacts with specific epitopes expressed by tumor-associated MUC5AC that does not appear on MUC5AC from normal tissues. Based on observations of a highly specific antibody, we tested the ELISA to differentiate serum from healthy blood donors compared to serum from patients with colorectal or pancreatic cancer. Additionally, patient tumor tissue was stained to examine the expression pattern of MUC5AC-related antigen in pancreatic and colorectal cancers. The results indicate the NPC-1C antibody ELISA distinguished serum of cancer patients from normal donors with very good sensitivity and specificity. Most patient's tumor biopsy exhibited NPC-1C antibody reactivity, indicating that tumor-associated MUC5AC antigen from tumor is shed into blood, where it can be detected by the NPC-1C antibody ELISA. This serum test provides a new tool to aid in the diagnosis of these cancers and immune monitoring of cancer treatment regimens.

SYN-004 is a first in class, recombinant β-lactamase that degrades β-lactam antibiotics and has been formulated to be administered orally to patients receiving intravenous β-lactam antibiotics including cephalosporins. SYN-004 is intended to degrade unmetabolized antibiotics excreted into the intestines and thus has the potential to protect the gut microbiome from disruption by these antibiotics. Protection of the gut microbiome is expected to protect against opportunistic enteric infections such as Clostridium difficile infection as well as antibiotic-associated diarrhea. In order to demonstrate that oral SYN-004 is safe for human clinical trials, 2 Good Laboratory Practice-compliant toxicity studies were conducted in Beagle dogs. In both studies, SYN-004 was administered orally 3 times per day up to the maximum tolerated dose of the formulation. In the first study, doses of SYN-004 administered over 28 days were safe and well tolerated in dogs with the no-observed-adverse-effect level at the high dose of 57 mg/kg/day. Systemic absorption of SYN-004 was minimal and sporadic and showed no accumulation during the study. In the second study, doses up to 57 mg/kg/day were administered to dogs in combination with an intravenous dose of ceftriaxone (300 mg/kg) given once per day for 14 days. Coadministration of oral SYN-004 with intravenous ceftriaxone was safe and well tolerated, with SYN-004 having no noticeable effect on the plasma pharmacokinetics of ceftriaxone. These preclinical studies demonstrate that SYN-004 is well tolerated and, when coadministered with ceftriaxone, does not interfere with its systemic pharmacokinetics. These data supported advancing SYN-004 into human clinical trials.

Out of 51 human adenoviral serotypes recognized to date, 32 of them belong to species D. Members of species D adenoviruses are commonly isolated from immune suppressed patients (organ transplant) and patients suffering from AIDS. The role of species D adenoviruses in pathogenesis is currently unclear. To derive new insights into the genetic content and evolution of species D adenoviruses and as a first step towards development of human adenovirus serotype 46 (Ad46) as vector, the complete nucleotide sequence of the virus was determined. The size of the genome is 35,178 bp in length with a G+C content of 56.9%. All the early and late region genes are present in the expected locations of the genome. The deduced amino acid sequences of all late region genes, with the exception of fiber, exhibited high degree of homology with the corresponding proteins of other adenoviruses. The deduced amino acid sequences of early regions E1, E3 and E4 showed a high degree of homology with the corresponding proteins of adenoviruses belonging to species D and less homology with the corresponding proteins of adenoviruses of other species. The homologues of Ad5 E3 region genes encoding 12.5K, gp19K, 10.4K, 14.5K and 14.7K are conserved in the genome of Ad46. However, the E3 region of Ad46 lacks genes encoding 6.7K and adenovirus death protein (ADP) but contains two additional open reading frames with a coding capacity of 433 and 281 amino acids. The fiber protein of Ad46 is 200 amino acids smaller than the fiber protein of Ad5 and contains only 10 pseudo-repeats in the shaft region. To facilitate the manipulation of the genome, the complete genome of Ad46 was cloned into a single bacterial plasmid. Following transfection into E1 complementing cell lines, the virus was recovered demonstrating the feasibility of viral genome manipulation for generation of recombinant viruses.

Historically, the adenoviral E3 region was found to be nonessential for viral replication in vitro. In addition, adenoviruses whose genome was more than approximately 105% the size of the native genome were inefficiently packaged. These profound observations were used experimentally to insert transgenes into the adenoviral backbone. More recently, however, the reintroduction of the E3 region into oncolytic adenoviruses has been found to positively influence antitumor efficacy in preclinical models and clinical trials. In the studies reported here, the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA sequence has been substituted for the E3-gp19 gene in oncolytic adenoviruses that otherwise retained the E3 region. Five viruses that differed slightly in the method of transgene insertion were generated and compared to Ar6pAE2fGmF (E2F/GM/ΔE3), a previously described E3-deleted oncolytic adenovirus encoding GM-CSF. In all of the viruses, the human E2F-1 promoter regulated E1A expression and GM-CSF expression was under the control of the adenoviral E3 promoter and the packaging signal was relocated immediately upstream from the right terminal repeat. The E3-gp19-deleted viruses had similar cytolytic properties, as measured in vitro by cytotoxicity assays, but differed markedly in their capacity to express and secrete GM-CSF. Ar15pAE2fGmF (E2F/GM/E3b), the virus that produced the highest levels of GM-CSF and retained the native GM-CSF leader sequence, was selected for further analysis. The E2F/GM/E3b and E2F/GM/ΔE3 viruses exhibited similar cytotoxic activity and GM-CSF production in several tumor cell lines in vitro. However, when compared in vivo in nude mouse xenograft tumor models, E2F/GM/E3b spread through tumors to a greater extent, resulted in higher peak GM-CSF and total exposure levels in both tumor and serum, and was more efficacious than the E3-deleted virus. Using the matched WI-38 (parental) and WI-38-VA13 (simian virus 40 large T antigen transformed) cell pair, GM-CSF was shown to be selectively produced in cells expressing high levels of E2F, indicating that the tumor-selective E2F promoter controlled E1A and GM-CSF expression.

Oncolytic adenoviral vectors selectively replicate in and lyse human tumor cells, providing a promising means for targeted tumor destruction. However, oncolytic vectors have limited capacity for incorporation of additional genetic material that could encode therapeutic transgenes and/or transcriptional regulatory control elements to augment the efficacy and/or safety of the vector. Therefore, we hypothesized that coadministration of an oncolytic vector with a replication-defective, gutless adenoviral vector encoding a therapeutic transgene would result in replication of both vectors within a tumor and potentiate antitumor efficacy relative to the use of either vector alone. We constructed gutless vectors encoding the murine granulocyte-macrophage colony-stimulating factor (AGVmGMF) or human tumor necrosis factor alpha-related apoptosis-inducing ligand (AGVhTRAIL) gene and tested the ability of these vectors to augment the efficacy of an oncolytic vector (Ar6pAE2fE3F) in a potentiating vector strategy. In Hep3B cells in vitro, cotreatment with Ar6pAE2fE3F increased transgene expression from AGVhTRAIL and permitted replication of AGVhTRAIL, suggesting that an oncolytic vector can propagate gutless vector spread in vivo. In pre-established Hep3B xenograft tumors, neither gutless vector alone inhibited tumor growth; however, coadministration of AGVmGMF or AGVhTRAIL with Ar6pAE2fE3F significantly reduced tumor growth relative to Ar6pAE2fE3F alone. Additionally, use of AGVhTRAIL with Ar6pAE2fE3F increased the number of complete or partial tumor regressions observed at study end. These data provide evidence that coadministration of an oncolytic vector with a gutless vector holds promise for potentiating tumor ablation efficacy.

In this study, we developed a mouse model of adoptive immunotherapy reflecting immune recognition of syngeneic tumor cells naturally expressing an endogenous rejection Ag. Specifically, in a pulmonary metastases model, we examined the potency and maintenance of an antitumor CD8+ CTL response in vivo, as well as its effectiveness against an “extensive” tumor burden. The approach taken was to first generate tumor-specific CTL from mice challenged with the CMS4 sarcoma coadministered with anti-CTLA4 mAb, which has been shown to facilitate the induction of Ag-specific T cell responses in vivo. An H-2Ld-restricted nonamer peptide, derived from an endogenous murine leukemia provirus was identified as a CMS4-reactive CTL epitope based upon the following: CTL cross-recognition of another syngeneic tumor cell line (CT26 colon carcinoma) previously characterized to express that gene product; sensitization of Ag-negative lymphoblasts or P815 targets with the peptide; and by cold target inhibition assays. In vivo, the adoptive transfer of CMS4-reactive CTL (≥1 × 106) resulted in nearly the complete regression of 3-day established lung metastases. Furthermore, mice that rejected CMS4 following a single adoptive transfer of CTL displayed antitumor activity to a rechallenge 45 days later, not only in the lung, but also at a s.c. distal site. Lastly, the adoptive transfer of CTL to mice harboring extensive pulmonary metastases (>150 nodules) led to a substantial reduction in tumor burden. Overall, these data suggest that the adoptive transfer of tumor-specific CTL may have therapeutic potential for malignancies that proliferate in or metastasize to the lung.

While much is known about adenovirus biology from its development as a therapeutic gene delivery vehicle, an important question remains regarding the appropriate in vivo vector dose. We describe here an in vivo dose threshold effect with an adenoviral vector expressing human Factor VIII (FVIII) in hemophiliac mice. Upon administration of vector doses between 6 × 10 10 and 2 × 10 10 vector particles per mouse, FVIII was expressed linearly, whereas a dose of 1 × 10 10 vector particles per mouse did not result in detectable levels of FVIII activity. In contrast, in vitro transduction studies demonstrated linear transgene expression over 2 to 3 log units. To further define this dose threshold effect, a vector-mixing study was performed. Mice were injected with a total vector dose of 6 × 10 10 particles containing admixtures of FVIII vector plus a control vector lacking a transgene (null vector). With the admixture, FVIII activity was detected in mice that received 1 × 10 10 particles of the FVIII vector, indicating that maintenance of the total viral input at 6 × 10 10 particles per mouse circumvented the threshold dose effect. This threshold dose effect could not be attributed to dose-dependent differences in liver toxicity nor to dose-dependent induction of cellular and humoral immune responses. Southern blot analysis of livers revealed that mice receiving the vector admixture contained FVIII DNA, accounting for the observed FVIII expression, whereas mice receiving 1 × 10 10 particles of FVIII vector had barely detectable FVIII DNA. These results suggest that the threshold effect is an in vivo phenomenon that will have important implications in defining the therapeutic window of adenoviral vectors for clinical applications.

We recently identified a murine mutant Ras p21 CD8+ CTL epitope reflecting residues 4 to 12, containing the mutation of Gly to Val at codon 12, that bound weakly to H-2Kd in vitro and generated a weak primary CTL response in immunized BALB/c mice. Here, we explored the hypothesis that specific modifications to the Ras4–12 peptide sequence can improve MHC binding, leading to enhanced immunogenicity without altering immune specificity. We synthesized Ras4–12 peptides in which Val at residue 12 was replaced with the more dominant H-2Kd C-terminus anchor residue Leu or Ile. In functional H-2Kd binding assays, Ras4–12(L12 or I12) peptide variants competed more effectively than the Ras4–12(V12) peptide. Ras4–12(L12 or I12) peptide variants enhanced both in vitro cytotoxicity and proliferation responses of anti-Ras4–12 CTL compared with the mutant Ras4–12(V12) peptide. Additionally, the Ras4–12(L12) peptide variant induced a quantitatively greater T cell response in vivo compared with that produced by Ras4–12(V12) as determined by IFN-γ production. Mice immunized with Ras4–12(L12) peptide elicited CD8+ CTL activity specific for target cells presenting the Ras4–12(V12) epitope exogenously and endogenously. Moreover, both anti-Ras4–12(V12)-derived and anti-Ras4–12(L12)-derived CTL lines were similar insofar as their TCR usage and amino acid contact residues in the Ras4–12(V12) peptide. These experiments demonstrate that modifications can be introduced in tumor-specific peptide epitopes to enhance both in vitro and in vivo immunogenicity. The design of oncogene-specific peptide epitope variants as immunogens may accelerate the generation of anti-tumor T cell responses for cancer immunotherapy.

Oncolytic adenoviruses are being tested as biological cancer therapeutics. Ar6pAE2fF (E2F vector) contains the E2F-1 promoter to regulate the expression of the E1a gene in cells with a disregulated retinoblastoma pathway. Ar6pAE2fmGmF (E2F-GM vector) includes the murine granulocyte–macrophage colony-stimulating factor (GM-CSF) transgene to enhance anti-tumor activity. Both vectors selectively killed human tumor cells in vitro. The E2F-GM vector expressed biologically active murine GM-CSF in vitro and GM-CSF was detected for several days in serum and tumor extracts following injections of established human xenograft tumors. In vivo, both vectors showed significant dose-dependent anti-tumor responses. The E2F-GM vector elicited greater efficacy compared to the E2F vector, demonstrating that GM-CSF enhanced the anti-tumor activity, even in immunodeficient nude mice. Histological analysis showed that both vectors induced necrosis and mononuclear cell infiltration, but only the E2F-GM vector resulted in eosinophil infiltration. Vector replication in vivo was demonstrated. The data showed that intratumoral injection of a GM-CSF-armed oncolytic vector induced potent anti-tumor responses in xenograft tumor models, likely as the result of both oncolytic vector activity and the induction of GM-CSF-mediated inflammation and innate immunity.

Phospholipase C (PLC) isozymes are known to be regulated, in part, by heterotrimeric GTP-binding protein (G-protein) subunits, including Galpha subunits of the G(q) family and Gbetagamma subunits. New data show that PLC can also be regulated by a high molecular weight G-protein that doubles as a cellular transglutaminase. Furthermore, a soluble phosphatidylinositol transfer protein (PITP) has been implicated in sustaining the activity of PLC by delivering substrate to the plasma membrane. Such diverse regulatory mechanisms imply that the PLC isozymes are precisely controlled and have specific roles in generating second messengers in response to various external stimuli.

Background: Monoclonal antibodies (mAbs) were raised against human colon tumor tissue extracts. Antibodies that specifically recognized tumor tissues, but did not cross-react with normal tissues, were selected for further characterization and development as diagnostic reagents and therapeutic candidates. Three mAbs that react with variant forms of MUC5AC, CEACAM molecules, and A33 were identified. The immunohistochemical staining profiles of these antibodies (NEO-101, NEO-201, and NEO-301) will be discussed. Methods: The three antibodies (NEO-101, NEO-201, and NEO-301) were each biotin-labeled, and used to stain a variety of tumor and normal human tissue types, typically at 10 ug/mL. Binding of the primary antibody was detected using streptavidin-HRP conjugate. Slides were reviewed and scored by light microscopy. Results: The NEO-101 antibody recognizes a variant form of MUC5AC expressed by colorectal (79%) and pancreatic (48%) cancer tissues. NEO-101 did not significantly cross-react with normal colon or pancreas tissues. Staining appeared to be cytoplasmic, membrane-associated, and elaborated into the lumenal spaces of positively stained tissues. The NEO-201 antibody recognizes variant forms of both CEACAM-5 and CEACAM-6. NEO-201 reacts with a wider variety of tumor tissue types including colorectal (98%), pancreatic (80%), lung (71%), stomach (100%), esophagus (61%), breast (31%), and ovarian (31%). Cross-reactivity to normal tissues was weak and sporadic. The NEO-301 antibody recognizes a variant form of A33, that is expressed by colorectal (46%), pancreatic (52%), lung (65%), and breast (45%) tumor tissues. NEO-301 also does not cross-react significantly with normal tissues. Antibodies NEO-201 and NEO-301 stained both cytoplasm and membranes of tissues examined. Conclusions: NEO-101, NEO-201, and NEO-301 antibodies react specifically with tumor tissues without significant cross-reactivity to normal tissues, an attractive characteristic for developing effective therapeutic and diagnostic products for oncology indications. We suggest that the antigens they react with are novel variants of commonly known wild type proteins that can be used as biomarker since they do not cross-react with healthy tissues. Interestingly, although these 3 antibodies were generated from extracts of pooled human colon tumor tissues, the variant antigens they cross-react with are not restricted to expression by colon tumor tissues, supporting the idea that variant protein expression may be a general property of tumor cells compared to normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C14.

Introduction: NPC-1C is a chimeric monoclonal antibody being developed as a novel biological treatment for pancreatic and colorectal cancers. NPC-1C appears to recognize a variant form of MUC5AC expressed specifically by human pancreatic and colorectal tumor tissues and cell lines. Here we show pre-clinical studies in preparation for testing this therapeutic for a Phase I clinical trial. Methods: The NPC-1C antibody was selected from a panel of hybridomas generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. In vitro assays and in vivo studies were performed to characterize the GMP-grade antibody. Results: Here we show that NPC-1C reacts with colorectal and pancreatic tumor tissues, but does not cross-react with normal human tissues, except for occasional, weak binding to certain GI tract tissues, which may indicate a pre-malignant state. The NPC-1C antibody exhibits cell-specific binding and ADCC activity against human colorectal and pancreatic tumor cells, but not against control tumor cell lines. In vivo, the anti-tumor efficacy of NPC-1C was tested using pre-established subcutaneous human tumor xenograft models. The data showed significant, and reproducible, anti-tumor action, including some complete tumor regressions. The pre-clinical toxicity profile of NPC-1C in normal BALB/c mice demonstrated little, if any, toxicity following single or multiple intravenous injections. The bio-distribution of radiolabeled NPC-1C in mice with pre-established human tumors revealed specific accumulation of NPC-1C at the tumor site, with little or no binding or accumulation in several major organ systems. Finally, the cytokine and antibody responses to NPC-1C were studied in the pre-clinical mouse model. Although anti-NPC-1C antibodies were detected (as expected) in a time-dependent fashion, there were no changes detect in Type I and Type II cytokines either 3 days or 14 days after intravenous administration. Conclusions: The pre-clinical development of NPC-1C indicates that it is safe and efficacious, and suggests that it may have clinical activity in patients whose tumor expresses the variant MUC5AC epitope. The potential clinical application for this antibody was bolstered by showing approximately 50-70% of human pancreatic and colon tumor tissues stained positively with NPC-1C. The therapeutic antibody is now being tested in a Phase I clinical trial in patients with advanced pancreatic and colorectal cancer. 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 4584. doi:10.1158/1538-7445.AM2011-4584

Introduction: h16C3 is a humanized monoclonal antibody being developed as a novel biological treatment for several types of solid tumor. By mass spectroscopy identification and target validation research, we determined that h16C3 appears to recognize a variant form of CEACAM-5 and CEACAM-6, molecules with a well-known association to cancer. Here we show the pre-clinical activities of this new mAb which shows great potential as a therapeutic agent to treat solid tumors. Methods: The 16C3 antibody was selected from hundreds of hybridoma clones generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. In vitro assays and in vivo studies were performed to characterize the potential of h16C3 as a therapeutic modality for solid tumors that express the variant CEACAM antigens. Results: Here we show that unlike most modalities targeting the CEACAM molecules, h16C3 reacts with variant CEACAM-5/6 expressed specifically by human tumor tissues as follows: 98% of human colorectal tumor tissues, 80% of pancreatic tumor tissues, 76% of lung tumor tissues, 43% of uterus tumor tissues, and 29% of breast tumor tissues stained positively with h16C3. Importantly, h16C3 did not cross-react significantly with normal human tissues, except for occasional, weak binding to certain GI tract tissues, which may indicate a pre-malignant state. We showed further that the h16C3 antibody exhibits cell-specific binding and ADCC activity against human colorectal and pancreatic tumor cells, but not against antigen-negative control tumor cell lines. In vivo, the anti-tumor efficacy of h16C3 was tested using pre-established subcutaneous human tumor xenograft models. The data showed significant anti-tumor action compared to control treated mice, including some complete tumor regressions. The bio-distribution of radiolabeled h16C3 in mice with pre-established human tumors exhibited specific accumulation of h16C3 at the tumor site, with little or no binding or accumulation in several major organ systems. Conclusions: The pre-clinical studies of h16C3 to date indicate that it has significant, specific anti-tumor potency, and suggest that it may have clinical activity in cancer patients whose tumor expresses the variant CEACAM-5/6 epitope. The therapeutic humanized 16C3 antibody is being developed for testing in a Phase I clinical trial. 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 4582. doi:10.1158/1538-7445.AM2011-4582

NPC-1C is a chimeric monoclonal antibody being developed as a novel biological treatment for pancreatic and colorectal cancers. This antibody was selected from a panel of hybridomas generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. NPC-1C appears to recognize an aberrantly expressed form of MUC5AC expressed specifically by human colon and pancreatic tumor tissues and cell lines. NPC-1C does not cross-react with normal human tissues (and with MUC5AC from other type of cancers), however, except for occasional, weak binding to certain GI tract tissues, which may indicate a pre-malignant state. In vitro, the NPC-1C antibody exhibits specific ADCC activity against human colon and pancreatic tumor cells, but not against control tumor cell lines. In vivo, the anti-tumor efficacy of NPC-1C was tested using preestablished subcutaneous human pancreatic tumor xenograft models. The data showed significant, and reproducible, anti-tumor action, including some complete tumor regressions. The clinical application for this antibody was bolstered by several examples of human tumor tissues were stained with biotin-conjugated NPC-1C which show a strong correlation of NPC-1C staining against pancreatic and colon tumors, with approximately 45% of tumors staining strongly positive. The staining pattern was typical of mucin expression, but also showed cytoplasmic and cell membrane staining. The pre-clinical toxicity profile of NPC-1C in normal BALB/c mice demonstrated little, if any, toxicity following single or multiple intravenous injections of 10 mg/kg, 50 mg/kg, or 100 mg/kg of clinical-grade NPC-1C antibody. Toxicity parameters measured include body weight, food consumption, complete blood analysis, serum chemistries, gross pathology, and microscopic histopathology. The bio-distribution of radio-labeled NPC-1C in mice with pre-established subcutaneous human tumors revealed specific accumulation of NPC-1C at the tumor site, with little or no binding or accumulation in several major organ systems. Finally, we studied the cytokine and antibody responses to NPC-1C in the pre-clinical mouse model. Although anti-NPC-1C antibodies were detected (as expected) in a time-dependent fashion, we did not detect changes in Type I (IL-2 or g-IFN) and Type II (IL-4 or IL-5) cytokines in response to up to 100 mg/kg of NPC-1C either 3 days or 14 days after intravenous administration. The pre-clinical development of NPC-1C indicates that it is safe and efficacious, and suggest that it should be well-tolerated and may have clinical activity in patients whose tumor expresses the altered MUC5AC epitope. The FDA has approved the Investigational New Drug (IND) application to initiate a Phase I clinical trial with NPC-1C for patients with advanced pancreatic and colorectal cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B124.