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. Author manuscript; available in PMC: 2010 Mar 1.
Published in final edited form as: Cancer Res. 2009 Feb 24;69(5):1933–1940. doi: 10.1158/0008-5472.CAN-08-2707

Design and Activity of a Murine and Humanized anti-CEACAM6 scFv in the Treatment of Pancreatic Cancer

Christopher J Riley 1, Kevin P Engelhardt 1, Jose W Saldanha 4, Wenqing Qi 1, Laurence S Cooke 1, Yingting Zhu 1, Satya T Narayan 1, Kishore Shakalya 1, Kimiko Della Croce 1, Ivan G Georgiev 1, Raymond B Nagle 1, Harinder Garewal 2, Daniel D Von Hoff 3, Daruka Mahadevan 1
PMCID: PMC2672909  NIHMSID: NIHMS100540  PMID: 19244123

Abstract

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with surgery the only curative modality for localized disease; gemcitabine with or without erlotinib remains standard therapy for unresectable or metastatic disease. CEACAM6 is over-expressed in human PDA independent of stage or grade, and causes anoikis resistance when dysregulated. Because murine Mab 13-1 possesses target-specific cytotoxicity in human PDA cell lines, we designed humanized anti-CEACAM6 single chain variable fragments (scFv’s) based on Mab 13-1. PEGylation of the glycine-serine linker was used to enhance plasma half-life. These scFv’s bound CEACAM6 with high affinity, exhibited cytotoxic activity and induced dose-dependent PARP-cleavage. Murine PDA xenograft models treated with humanized scFv alone elicited tumor growth inhibition (TGI), which was enhanced in combination with gemcitabine. Immunohistochemistry (IHC) showed significant apoptosis, with inhibition of angiogenesis and proliferation, with preservation of the target. Collectively, our results have important implications for development of novel antibody-based therapies against CEACAM6 in PDA.

Keywords: Pancreatic carcinoma, Carcinoembryonic antigen cell adhesion molecule 6, Anoikis resistance, Monoclonal antibody, Humanization

Introduction

In 2007, pancreatic ductal adenocarcinoma (PDA) accounted for ~37,170 cancer cases, of which there were ~33,370 deaths, providing a dismal prognosis (1). This is attributable to a lack of early diagnosis and effective treatments. Most patients present with unresectable and/or metastatic PDA, with a median overall survival of 12 and 6 months respectively (2). Therefore, the molecular basis of PDA is being intensely investigated in the hope of identifying disease mechanisms and associated therapeutic targets (3, 4). Genetic lesions linked to PDA have identified germline mutations in KRAS, CDKN2A (also known as INK4a or ARF), BRCA2, MLH1, STK11 (also known as LKB1), TP53 and SMAD4 (also known as DPC4) (5) and a progression model similar to colon cancer has been postulated with the identification of a precursor lesion, pancreatic intra-epithelial neoplasia (PanIN) (6–8). These lesions appear to acquire mutations in the above genes in a temporal sequence at progressive stages of PanIN (9). Genetically engineered mice (GEM) have been established with the implicated mutated genes but do not completely recapitulate human PDA (10). A hallmark in PDA is the presence of a ‘desmoplastic reaction’ (DR) due to proliferation of fibrotic tissue with an altered extracellular matrix (ECM) conducive to tumor growth and metastasis. Novel therapies targeting the DR are required (3, 4) and CEACAM6 is one such target, expressed only in higher vertebrates (dogs, monkeys) and humans (11).

CEACAM6 (CD66c) is an integral member of the CEA family (CEACAM1, 5, 7, 8) and is an important tumor-associated antigen (12). It is a cell surface glycoprotein composed of an extracellular region containing 3 immunoglobulin-like (Ig-like) domains (344 residues; MW ~35,200 Daltons) and is linked to the plasma membrane via a glycophosphoinositol-anchor (GPI-A) (13). CEACAM6 is capable of homophilic and/or heterophilic adhesion to other CEACAM family members (14). CEACAM6 is expressed on normal human epithelial and myeloid cells but the level of expression is 1–2 log lower compared to expression in malignant tissue (15). Several gene expression profiling studies on PDA cell lines (16, 17) and human PDA biopsy samples (18) have shown CEACAM6 to be 20 to 25-fold over-expressed compared to normal pancreatic ductal epithelial cells (16, 18). CEACAM6 is also over-expressed in several other epithelial carcinomas (colon, breast, ovarian, non-small cell lung cancer) (15, 19).

De-regulated over-expression of CEACAM6 inhibits differentiation and apoptosis of cells when deprived of their anchorage to the ECM, a process known as anoikis (20). A small interfering RNA (siRNA) targeting CEACAM6 reversed anoikis resistance and inhibited the in vivo metastatic potential in a mouse xenograft model of PDA by enhancing caspase-3 mediate apoptosis (21). In BxPC-3 PDA cells, gene silencing of CEACAM6 markedly increased sensitivity to gemcitabine mediated cytotoxicity (22). In a similar model, a maytansinoid (tubulin interactive agent) conjugated murine Mab but not unconjugated Mab against CEACAM6 led to TGI in a dose-dependent manner (Strickland, L., et al. Evaluation of efficacy and toxicity of CEACAM6 targeted immunotherapy in pancreatic ductal adenocarcinoma: April 2005 AACR Annual Meeting Abstract #4195 CA). Cynomolgus monkeys, when administered the unconjugated or immuno-conjugated Mab, demonstrated a decrease in absolute neutrophil count 7 days after dosing, but only with the immuno-conjugated group; no other toxicities were detected, and these effects were absent in the group treated with the unconjugated Mab. These results demonstrate that CEACAM6 is a potential therapeutic target for Mab therapy with a safe therapeutic index. We have designed and developed novel humanized scFv antibody fragments based on murine Mab 13-1 (Saldanha, J.W., et al. A Humanized anti-CEACAM6 monoclonal antibody targeting pancreatic adenocarcinoma demonstrates potent in vitro and in vivo activity. March 2004 AACR Annual Meeting, Abstract 2180, Florida), utilizing in-silico modeling methods. The novelty and uniqueness of this scFv-based therapeutic is that it promotes apoptosis without either cellular or humoral immune assistance. Furthermore, the PEGylated scFv enhances TGI alone and sensitizes with gemcitabine in mice xenograft models of PDA. These results have important implications for development of novel pancreas cancer therapies.

Materials and Methods

Histopathology

Thirty human PDA biopsy samples were deparaffinized and microwaved for antigen retrieval, or if fixed frozen above step was omitted. Both types of sections were acetone fixed and stained with α-NCA monoclonal antibody (13-1, Kamiya, CA) and processed using a mixture of anti-Ms and anti-Rb immunoglobulins. After rinsing, slides were incubated with Avidin-HRP reagent, rinsed, and incubated in DAB (3,3′-diaminobenzidine). The slides were counter-stained in hematoxylin. Mouse xenograft tumors (both control and treated) were divided in half and either snap frozen or processed for paraffin embedding. Paraffin block sections were analyzed by IHC for proliferation (MKI67, also known as Ki-67) angiogenesis (PECAM1, also known as CD31) and target (CEACAM6). The extent of apoptosis was estimated by hematoxylin-eosin staining. Counting was performed by summing the number of positively stained cells in 5 random fields under the microscope under 20X magnification.

PDA Cell Lines

Ten human pancreatic cancer cell lines (CAPAN-2, CFPAC-1, Panc-1, AsPC-1, MiaPaCa-2, CAPAN-1, BxPC-3, Hs766T, Su.86.86 and HPAF-2) were grown in RPMI 1640 supplemented with 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO) in a CO2 incubator. For Western blotting applications, cells were grown to approximately 75% confluence, pelletted via centrifugation, lysed with native lysis buffer, run on SDS-PAGE, transferred to nitrocellulose, and probed with the appropriate antibody. Native lysis buffer composition: 50mM HEPES, 150mM NaCl, 1% (v/v) Triton-100, 5mM EDTA, 20mM NaF, 20mM MgCl2, and 20mM Na4P2O7, with Na3VO4 (1mM), phenylmethanesulphonylfluoride (1mM), and protease inhibitor cocktail (10μL/107 cells; Sigma Aldrich, St. Louis, MO) added immediately before use.

Mouse Xenograft Models

BxPC-3, HPAF-2 or Panc-1 cells (~10×106) were injected into the flank region of SCID mice to form small nodules (~60–100mm3). Treatment with unconjugated murine anti-CEACAM6 scFv or PEGylated humanized anti-CEACAM6 scFv (version 8) was initiated when tumors reached ≥60mm3. Unconjugated scFv was administered daily, intra-peritoneally (IP), for 4 weeks. The PEGylated scFv was administered twice a week for 4 weeks via the same method. The primary endpoint was TGI compared to control utilizing the formula [(width)2 × length]/2. The secondary endpoint was effect of treatment on apoptosis, proliferation, angiogenesis and continued expression of CEACAM6, evaluated by IHC. Supernatant (media) from BxPC-3 cells in culture as well as serum from BxPC-3 xenograft mice were collected and analyzed for shedding of CEACAM6 by Western blotting utilizing Mab 13-1. The positive control was recombinant GST-CEACAM6 or BxPC-3 cell lysate.

Cell Viability and Apoptosis Assays

BxPC-3, HPAF-2 and CAPAN-2 PDA cells were plated onto 96 well plates, such that cell confluency would reach ~80% by the end of assay. After overnight incubation to allow for adhesion, cells were exposed to anti-CEACAM6 Mab 13-1 or scFv for four days. All studies were performed in quadruplicate and were repeated 3 different times. After incubation, 20μL of 2mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) and 0.92mg/mL phenazine methosulfate (PMS) were added to each well and incubated an additional four hours; absorbance was read at 490nm on a plate reader (Wallac Vector, PerkinElmer). Data are expressed as percent survival, compared to controls, calculated from the absorbance and corrected for background. For apoptosis assays, PDA cells were treated with scFv at 10 and 20μg/mL for 24 and 48 hr, lysed and Western blotting was performed to compare PARP cleavage in treated cells and untreated controls (PARP and cleaved-PARP antibodies from Cell Signaling, Danvers, MS).

Humanization by Design

Mab 13-1 VL and VH region sequence was characterized by mass spectroscopic peptide mapping, and compared to that in the NCBI protein sequence database (#JC5810 for VL; #PC4436 for VH). For homology modeling and design (23) of scFv fragments, a PSI-BLAST search (NCBI) was conducted to identify the crystal structure(s) of a mouse Mab with the highest sequence similarity to the VL and VH of Mab13-1. Sequence alignments of VL and VH domains of 1MCP (24) was performed with the VL and VH of 13-1 in Clustal W. Molecular modeling of 13-1 VL and VH was performed using 1MCP (Octane Silicon Graphics workstation) using ICM (Internal Coordinate Mechanism) and refined in Sybyl 6.9 (Tripos). A glycine-serine linker was constructed linking the VL C-terminus to VH N-terminus (~33° A) with a cysteine residue in the following order (GGGGSGGGGS (cys) GGGGS). The linker length of 16 residues was chosen to avoid oligomer formation. The cysteine residue was introduced into the linker as it is a potential site for PEGylation. The mouse scFv homology model and sequence was used to identify the best human acceptor for the VL and VH domains. The design was based on searching through the Kabat database (25) using the program FASTA (26) with analysis of the modeled structure utilizing the program QUANTA (Accelrys, 2000). Care was taken to conserve the canonical forms and residues at the interface between the light and heavy chains. For the VH the NCBI non-redundant database was searched with FASTA (26) and the modeled structure analyzed as above.

Gene synthesis, Expression, Purification and PEGylation

We utilized a gene synthesis approach to construct the murine and nine humanized scFv versions. All ten constructs were cloned into a pET25 expression vector with a C-terminally located hexa-histidine tag. DNA sequencing confirmed the authenticity of these constructs. Five scFv constructs (the murine version and humanized versions 1, 2, 7, 8) were expressed in BL21 (DE3) PlysS competent bacteria; inclusion bodies were isolated, denatured in 8M urea buffer and refolded by drop-wise introduction to tris-HCl refolding buffer, pH 7.4. Refolded scFv’s were concentrated to 0.3mg/ml and buffer exchanged using a PD-10 desalting column into cysteine reducing buffer (100mM NaPO4, 100mM imidazole, 2mM DTT, 2mM EDTA, pH 7.0). Samples were rocked gently for 2 hours at room temperature and buffer exchanged into PEGylation buffer (100mM NaPO4, 100mM imidazole, 2mM EDTA, pH 6.0), then placed in a reaction apparatus under Argon gas. PEG-maleimide 20kDa (Nektar, CA) was added to the mixture and the reaction continued for 2 hours under a constant Argon gas stream. Once the reaction was complete the samples were stored at −4°C (32). Western blotting confirms protein PEGylation due to a 20 or 40kDa shift in molecular weight, to a molecular weight of 40 or 60kDa depending on 1- or 2-site PEGylation (one site is located in the linker, and the other in the C-terminus of the VH domain).

Homology Models, Protein-protein docking and Binding studies

The 3 Ig-like ECDs of human CEACAM6 (NCBI #P40199) were homology modeled based on the crystal structure of rat NCAM (residues 1–291) (pdb:1QZ1) (27) utilizing “Modeller”1. Rat NCAM was identified and chosen for use by structure-based sequence analysis (3D-PSSM)2 and using sequence alignment in ClustalW3. The model was energy minimized (Powell method), and Procheck v.3.5.44 evaluated the correctness of this refined/energy minimized model. Protein-protein docking of human CEACAM6 ECD (1–291) with mouse or humanized anti-CEACAM6 scFv (versions 1 to 9) was performed in ClusPro5. Electrostatic energy (kcal/mol), desolvation energy (kcal/mol) and theoretical affinity was calculated using FastContact6.

Immunoblotting

For in vitro studies for humanized scFv (V1, 2, 7 and 8), Western blotting and immunoprecipitation (IP) were utilized with the PDA cell lines (BxPC-3, HPAF-2, CAPAN-2). For IP, scFv was added to cell lysates (1μg/μL total protein content, calculated via BCA assay; proteins lysed with native lysis buffer as discussed previously) and incubated, rocking, at 4°C overnight, then precipitated with 20μL Ni-NTA Superflow beads (Qiagen, Valencia, CA) under the same conditions. Beads were pelletted via centrifugation, washed 3 times with cold PBS, and protein was removed by addition of Laemmli loading buffer and heating to 95°C for two minutes followed by centrifugation; supernatant was removed and stored at −20°C. For Western blotting, cell lysates were prepared after treatment with scFv for 6 hours. SDS-PAGE and Western blotting were performed with anti-CEACAM6 antibody (Abcam, Cambridge, MA). Also used for immunoblotting were the murine monoclonal antibody to CEACAM6 (13-1) (Kamiya, CA) and an anti-β-actin control.

Statistical Analysis

Statistical analysis was computed using STATA software (StataCorp LP, College Station, TX, USA). P-values were calculated using ANOVA with the Bonferroni correction, calculating a lower critical α level to allow for multiple testing.

Results

CEACAM6 is over-expressed in human PDA

Relative to normal pancreatic tissue, ~50% PDA cell lines (Figure 1A) and >90% patient biopsies over-express CEACAM6 irrespective of stage or grade of disease (Figure 1B). Of the 10 human PDA cell lines (CAPAN-2, CFPAC-1, Panc-1, AsPC-1, MiaPaCa-2, CAPAN-1, BxPC-3, Hs766T, Su.86.86 and HPAF-2) evaluated by Western blotting with the murine Mab13.1, five are over-expressers (CFPAC-1, AsPC-1, CAPAN-1, BxPC-3 and HPAF-2), two are low expressers (Hs766T, Su.86.86) and three are non-expressers (CAPAN-2, Panc-1, MiaPaCa-2). The protein migrates at a molecular weight 60–90 kDa due to variable glycosylation patterns. Of the 30 patient biopsies, 26 (>90%) showed intense cell surface staining of neoplastic pancreatic ductal cells while surrounding normal tissues are not stained, clearly delineating tumor cells and normal pancreatic tissue. Cell culture medium and serum from mouse BxPC-3 xenograft tumors showed that CEACAM6 is not shed from the cell surface (data not shown). Hence, CEACAM6 is a feasible target for development of a therapeutic Mab, and may have additional utility in identifying micrometastatic sites via imaging during initial workup for potential surgical intervention and to follow disease status during therapy.

Figure 1.

Figure 1

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a. Western blotting demonstrating CEACAM6 expression in 10 human pancreatic cancer cell lines (M-Marker, 1: CAPAN-2, 2: CFPAC-1, 3: Panc-1, 4: AsPC-1, 5: MiaPaCa-2, 6: CAPAN-1, 7: BxPC-3, 8: Hs766T, 9: Su.86.86, 10: HPAF-2). CEACAM6 migrates at 90kDa due to variable glycosylation and the band at 45kDa is the β-actin control. b. Immunohistochemical staining of 4 representative PDA patient biopsy samples with the murine anti-CEACAM6 Mab 13-1. The intense dark brown staining of the malignant pancreatic ductal surface epithelium is evident (apical and basal staining).

Humanization by design

The VL design was based on the human sequence 163.15 (kabat database id 047292) (28). The first three residues of this sequence were changed to the most common residues found in human sequences (namely Asp-Ile-Val). Humanized version 1 (V1) is a straight graft of the CEACAM6 complementarity determining regions (CDRs) into the human frameworks. Humanized V2 changes the human Tyr at position 49 to the Ser found in the CEACAM6 VL chain. From the model, Ser49 is the most prominent framework residue in the binding site and binding affinity may be evaluated by mutation. The model shows that it might interact with Met100a in the heavy chain of CDR-H3. It can also interact with nearby CDR residues in the VL chain. The N-terminus is occasionally important in binding antigen (29), hence version 3 changes the human Asp at position 1 to the Asn found in the CEACAM6 light chain. From the model, this Asn interacts with Pro95 in CDR-L3 and Lys27 in CDR-L1 and may also interact with Glu61 in CDR-H2. Version 3 only has two backmutations (Figure 2A).

Figure 2.

Figure 2

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a. The structure based sequence alignment of the murine anti-CEACAM6 VH and VL chains with the VH and VL of 1MCP. b. Amino acid differences between the 9 humanized versions of the anti-CEACAM6 scFv. c. Coomassie stained fractions collected from purification on a nickel affinity column (FPLC-AKTA) of humanized scFv anti-CEACAM6-V8. d. Coomassie stained PEGylated anti-CEACAM6-V8; “CC8” lanes are un-PEGylated protein controls; molecular weight shift is approximately 40kDa in the PEGylated version.

The VH design was based on the human sequence AAC51024 (NCBI accession 1791061) (30). It is notable that all three CDRs are the same length as 13-1. Humanized V1 is a straight graft of the CEACAM6 CDRs into these human frameworks. Version 2 changes the human Ala24 to Thr found in the VH CEACAM6 and similarly Val48 to Ile. Thr24 is a canonical residue for CDR-H1 and possibly interacts with Phe27 and 29. Ile48 is a common back mutation in humanization experiments and from the model appears to support the conformation of CDR-H2. Version 3 mutates the unusual Cys93 to Ala and Val37 to Leu. Leu37 is a residue at the light/heavy interface and was kept murine. Cys93 in the model appears to be interacting with Tyr99. There is a possibility that Cys93 may be forming a disulphide bridge with Cys102 in CDR-H3 (31), although this is unlikely from the model or is contributing to a metal binding site (Figure 2A). Since, the CDRs in both the VL and VH regions are identical in the chosen human acceptors the only residues that require mutations are in the framework regions at the base of the CDR loops (VL-1, 53; VH- 24, 37, 48, 99). Humanized V4 to 9 were designed based on these mutational changes (Figure 2B).

Purification and PEGylation

Recombinant scFv (VL-ggggsggggscggggs-VH) versions (murine and humanized 1, 2, 7 and 8) yielded 5–10 mg per liter of bacterial culture, with purity >95% (AKTA FPLC) and refolded into PBS (Figure 2C). All four humanized versions were PEGylated successfully and ran as a 70 kDa band on SDS-PAGE due to a shift in the molecular weight by 40kDa, as there are 2 PEGylation sites for version 8: one in the linker and the other in the C-terminus of the VH domain (Figure 2D). The PEGylated scFv is stable in PBS at 4°C.

Anti-CEACAM6 scFv disrupts Ig-like D1-D1 homophilic dimer formation

Computational protein-protein docking of the 3 Ig-like ECDs demonstrated that domain 1 (D1) formed the optimal homophilic dimer based on electrostatic and desolvation free energies. These data correlate well with structure-function and mutational data conducted on CEACAM6 (32, 33). Moreover, docking studies of scFv Vs CEACAM6 ECD indicated that the best binder based on engagement of all CDRs from both the VL and VH domains was scFv version 7 (V.7), followed by V. 8, 1 and 2 respectively (Figures 3A and 3B). The scFv versions bind to Ig-like D1–2 and thereby disrupt Ig-like D1-D1 homophilic interactions. These results correlate with the estimates of direct electrostatic and desolvation interaction free energy (Figure 3B) for the 4 humanized scFv versions. Specificity of the scFv (V.1, 2, 7 and 8) for CEACAM6 was shown by immunoprecipitation and Western blotting analysis of two CEACAM6 expressing PDA cell lines (BxPC-3, HPAF-2) compared to non-CEACAM6 expressing CAPAN-2 cells (Figure 3C).

Figure 3.

Figure 3

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a. Protein-protein docking shows that the anti-CEACAM6 scFv binds to CEACAM6 Ig-like D1–2 and disrupts Ig-like D1-D1 homophilic dimer formation. b. Theoretical binding energy and affinity for 4 scFv versions with CEACAM6. c. Western blotting with CEACAM6 [9A6] of immunoprecipitation products using anti-CEACAM6-v7 (1, 5, or 10μg) with 3 PDA cell line lysates.

Anti-CEACAM6 scFv decreases tumor cell viability and increases TGI in a PDA mouse model

Mab 13-1 promoted a target specific decrease in tumor cell viability in CAPAN-1 and HPAF-2 with an IC50 = 1–10μg/ml while no change in cell viability was observed for Panc-1 and MiaPaCa-2 cells (data not shown). Murine scFv also promoted a decrease in PDA cell viability with an IC50 of 0.01–0.5μg/ml (data not shown). Humanized scFv V1, 2, 7 and 8 (un-PEGylated) similarly decreased cell viability in a target specific manner with an IC50 in the range 5–10μg/ml (Figure 4A) similar to that observed for Mab 13-1, and promoted PARP-cleavage in a dose dependent manner (Figure 4B).

Figure 4.

Figure 4

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a. MTS assay showing that anti-CEACAM6-V7 antibody fragment is efficient at killing 2 different CEACAM6+ pancreatic cancer cell lines; error bars indicate standard error of the mean (SEM). b. Western blot showing PARP-cleavage following treatment of HPAF-2 cells with anti-CEACAM6-V8. c. Mouse scFv given intraperitoneally is effective in delaying tumor growth in a dose-dependent manner (n=8); error bars indicate SEM. * Statistically significant: control versus 10mg/kg anti-CEACAM6 mouse scFv p=0.015. d. Mean tumor burden in a mouse model treated twice weekly with Gemcitabine with or without PEGylated anti-CEACAM6-V8 (n=8); error bars indicate SEM. *Statistically significant: control versus gemcitabine alone p=0.031; control versus gemcitabine plus CEACAM6 V8 p=0.001.

In order to evaluate efficacy, several mouse PDA xenograft models were conducted. The first study evaluated the murine scFv (5, 10mg/kg) in SCID mice bearing BxPC-3 xenografts, which showed a significant dose-dependent TGI of >50% (Figure 4C). Since scFv’s have a t1/2 of ~30 min (32), the persistent TGI observed is remarkable. The second study evaluated humanized PEGylated scFv (V8) at 3mg/kg and 6mg/kg with and without gemcitabine. The TGI was modest at both dose levels at ~25% but sensitizes with gemcitabine with a TGI >50% (Figure 4D; 6mg/kg data not shown). The tumors harvested at the end of the study showed persistent CEACAM6 expression in all treated arms (scFv plus gemcitabine, scFv alone, gemcitabine alone) by IHC suggesting the possibility of continuous treatment with the scFv or combination with gemcitabine was feasible. The third study evaluated treatment with PEGylated humanized scFv (V8) at 3mg/kg and 6mg/kg given twice a week for 3 weeks, tumors harvested and analyzed by IHC for CEACAM6 (Figure 5A), apoptosis and angiogenesis (Figure 5B) and proliferation (Figure 5C). There were four tumors per arm which showed a significant dose-dependent increase in apoptosis (3-fold by morphology), decrease in angiogenesis (20–60%) and proliferation (Ki-67) (25–48%), but persistence of the target CEACAM6 (~60%).

Figure 5.

Figure 5

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a. Immunohistochemical staining of mouse tumor tissue treated with anti-CEACAM6-V8 and stained for CEACAM6 [9A6]. b. Numbers of apoptotic bodies and PECAM+ cells counted on slides from anti-CEACAM6-V8 treated mice. c. Percent positivity for Ki67 from anti-CEACAM6-V8 treated mice.

Discussion

De-regulated cell surface over-expression of CEACAM6 is observed in >50% human hematological and solid tumors. CEACAM6 is a well validated antibody target in gastrointestinal malignancies such as colorectal cancer (15, 34) and PDA (3, 22). Over-expression of CEACAM6 inhibits differentiation and apoptosis of cells when deprived of their anchorage to the ECM, a process known as anoikis (Greek for ‘homelessness’), which accompanies malignant transformation (20). Cross-linking CEACAM6 in a BxPC3 mouse xenograft model with an anti-CEACAM6 Mab (By114) induces cytoplasmic accumulation but with no associated TGI. However, a secondary saporin-conjugated IgG does induce cytotoxicity, via caspase-3 mediated apoptosis with associated TGI (22).

Mouse Mab 13-1 directed against CEACAM6 markedly reduced PDA cell viability in a target specific manner with an IC50 of 1–10μg/mL, independent of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Further, CEACAM6 was not detected in either the culture medium or in serum samples from BxPC-3 xenograft mice, indicating a lack shedding from the cell surface. Therefore, we designed a murine and nine humanized scFv versions utilizing a structure-based in-silico method to evaluate activity without additional activity (ADCC and CDC). Recombinant murine and humanized versions (1, 2, 7, 8) bound CEACAM6 specifically, markedly reduced PDA cell viability (IC50 5–50μg/mL), and exhibited associated dose-dependent PARP-cleavage (10 and 20μg/mL). The non-PEGylated mouse scFv showed 50–70% TGI in a BxPC-3 murine xenograft model in a dose-dependent manner (5mg/kg, 10mg/kg). Subsequently, humanized PEGylated (35) scFv version 8 alone at 3mg/kg showed TGI of ~25%, but with the addition of gemcitabine demonstrated TGI of >50%.

The novel and unique characteristic of these humanized anti-CEACAM6 scFv antibody fragments lies in their ability to specifically induce targeted tumor cell apoptosis without dependence on ADCC, CDC, or conjugation with a cytotoxic agent. We postulate that this ability originates from their mode and high affinity of binding to CEACAM6 which enhances the disruption of D1-D1 homophilic dimer with consequent functional inhibition. Moreover, deletion of the Fc-fragment from Mab 13-1 in the design prevents additional toxicity mediated by ADCC and CDC to normal tissue without compromising efficacy. Finally, the PEGylated anti-CEACAM6 scFv is soluble, has a longer half-life and likely penetrates tumors more effectively than a full antibody molecule.

A pharmacodynamic study utilizing IHC of BxPC-3 xenograft mice tumors treated with humanized PEGylated scFv version 8 at two different doses (3mg/kg, 6mg/kg) twice a week for 3 weeks demonstrated that CEACAM6 continued to be expressed on tumor cells despite an increase in apoptosis (apoptotic bodies as well as cleaved caspase-3), and a decrease in proliferation (Ki-67) and angiogenesis (PECAM). Taken together, the data implicate that CEACAM6 continues to provide a survival benefit to PDA cells and therefore increasing the dose and duration of scFv therapy is likely to further enhance TGI. Furthermore, there is an anti-CEACAM6 scFv dose-dependent promotion of apoptosis with an associated decrease in proliferation and angiogenesis. Therefore, in conclusion this study demonstrates for the first time that a mouse and/or a humanized scFv alone directed against CEACAM6 promotes targeted killing of PDA cells independent of ADCC and CDC. The fact that an anti-CEACAM6 scFv sensitized with gemcitabine provides a rationale for combination therapy for patients with PDA.

Acknowledgments

This manuscript is dedicated to Mr. Stephen R. Smith whose fight against pancreatic cancer was heroic and dedication to this project inspirational. We thank Dr David Bearss and Dr. Li for their contributions to target discovery and mass spectroscopic analysis. Immunohistochemical data was generated by the TACMASS Core (Tissue Acquisition and Cellular/Molecular Analysis Shared Service) and Experimental Mouse Shared Service (EMSS) at the Arizona Cancer Center, supported by NIH grant CA23074. We also wish to thank the GI-SPORE for funding (CA95060-03).

Footnotes

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