anti-CEA mAb Research Articles

Bright and Specific Targeting of Metastatic Lymph Nodes in Orthotopic Mouse Models of Gastric Cancer with a Fluorescent Anti-CEA Antibody.

Gastric cancer poses a major diagnostic and therapeutic challenge. Improved visualization of tumor margins and lymph node metastaseswith tumor-specific fluorescent markers could improve outcomes. To establish orthotopic modelsof gastric cancer, one million cells of the humangastric cancer cell line, MKN45, were suspended in 50 μl of equal parts PBS and Matrigel and injected into the nude mouse stomach with a 29-gauge needle. Tumors were allowed to grow for 8-12 weeks before performing imaging studies. For tumor labeling, M5A (humanized anti-CEA mAb) and IgG as a control, were conjugated with the near-infrared dye IRDye800CW. Mice were randomized to receive 50 μg of M5A-IR800 (n = 14) or 50 μg of IgG-IR800 (n = 14) intravenously and were imaged 72 hours later. Fluorescence imaging was performed using the LI-COR Pearl Imaging System. Forty-two lymph nodes were collected from 28 mice, of which 59.5% were found to contain metastatic gastric cancer cells on pathologic examination. In mice that received M5A-IR800, there was a statistically significant difference in the mean fluorescence signal for cancer-positive lymphnodes at 0.431 (SE ± 0.224) compared with 0.105 (SE ± 0.009) for cancer-negative nodes (p: 0.002). For IgG-IR800, there was no significant difference in the mean fluorescence signal for cancer-positive nodes (0.057) compared with cancer-negative nodes (0.064), p-value 0.677. Humanized anti-CEA (M5A) antibodies conjugated to fluorescent dyes provide bright labeling of lymph nodes containing metastatic gastric cancer. This tumor-specific fluorescent antibody is a promising clinical tool for identifying lymph nodes containing metastatic gastric cancer.

Pretargeted radioimmunotherapy and SPECT imaging of peritoneal carcinomatosis using bioorthogonal click chemistry: probe selection and first proof-of-concept.

Rationale: Pretargeted radioimmunotherapy (PRIT) based upon bioorthogonal click chemistry has been investigated for the first time in the context of peritoneal carcinomatosis using a CEA-targeting 35A7 mAb bearing trans-cyclooctene (TCO) moieties and several 177Lu-labeled tetrazine (Tz) radioligands. Starting from three Tz probes containing PEG linkers of varying lengths between the DOTA and Tz groups (i.e. PEGn = 3, 7, or 11, respectively, for Tz-1, Tz-2, and Tz-3), we selected [177Lu]Lu-Tz-2 as the most appropriate for pretargeted SPECT imaging and demonstrated its efficacy in tumor growth control. Methods: An orthotopic model of peritoneal carcinomatosis (PC) was obtained following the intraperitoneal (i.p.) injection of A431-CEA-Luc cells in nude mice. Tumor growth was assessed using bioluminescence imaging. Anti-CEA 35A7 mAb was grafted with 2-3 TCO per immunoglobulin. Pretargeted SPECT imaging and biodistribution experiments were performed to quantify the activity concentrations of [177Lu]Lu-Tz-1-3 in tumors and non-target organs to determine the optimal Tz probe for the PRIT of PC. Results: The pharmacokinetic profiles of [177Lu]Lu-Tz-1-3 alone were determined using both SPECT imaging and biodistribution experiments. These data revealed that [177Lu]Lu-Tz-1 was cleared via both the renal and hepatic systems, while [177Lu]Lu-Tz-2 and [177Lu]Lu-Tz-3 were predominantly excreted via the renal system. In addition, these results illuminated that the longer the PEG linker, the more rapidly the Tz radioligand was cleared from the peritoneal cavity. The absorbed radiation dose corresponding to pretargeting with 35A7-TCO followed 24 h later by [177Lu]Lu-Tz-1-4 was higher for tumors following the administration of [177Lu]Lu-Tz-2 (i.e. 0.59 Gy/MBq) compared to either [177Lu]Lu-Tz-1 (i.e. 0.25 Gy/MBq) and [177Lu]Lu-Tz-3 (i.e. 0.18 Gy/MBq). In a longitudinal PRIT study, we showed that the i.p. injection of 40 MBq of [177Lu]Lu-Tz-2 24 hours after the systemic administration of 35A7-TCO significantly slowed tumor growth compared to control mice receiving only saline or 40 MBq of [177Lu]Lu-Tz-2 alone. Ex vivo measurement of the peritoneal carcinomatosis index (PCI) confirmed that PRIT significantly reduced tumor growth (PCI = 15.5 ± 2.3 after PRIT vs 30.0 ± 2.3 and 30.8 ± 1.4 for the NaCl and [177Lu]Lu-Tz-2 alone groups, respectively). Conclusion: Our results clearly demonstrate the impact of the length of PEG linkers upon the biodistribution profiles of 177Lu-labeled Tz radioligands. Furthermore, we demonstrated for the first time the possibility of using bioorthogonal chemistry for both the pretargeted SPECT and PRIT of peritoneal carcinomatosis.

Physiologically Based Modeling of the Pharmacokinetics of “Catch-and-Release” Anti-Carcinoembryonic Antigen Monoclonal Antibodies in Colorectal Cancer Xenograft Mouse Models

Engineered monoclonal antibodies (mAbs) with pH-sensitive target release, or “catch-and-release” (CAR) binding, have shown promise in decreasing the extent of target-mediated mAb elimination, increasing mAb exposure, and increasing dose potency. This study developed a mechanistic physiologically based pharmacokinetic (PBPK) model to evaluate the effects of pH-sensitive CAR target binding on the disposition of anti-carcinoembryonic antigen (CEA) mAbs in mouse models of colorectal cancer. The PBPK model was qualified by comparing model-predicted plasma concentration-time data with data observed in tumor-bearing mice following the administration of T84.66, a “standard” anti-CEA mAb that demonstrates strong binding at pH 7.4 and 5.5. Further simulations evaluated the effects CAR pH-dependent binding, with decreasing CEA affinity with decreasing pH, on anti-CEA mAb plasma pharmacokinetics. Simulated data were compared with data observed for a novel CAR mAb, 10H6. The PBPK model provided precise parameter estimates, and excellent data characterization (median prediction error 18.4%) following fitting to T84.66 data. Simulations well predicted 10H6 data (median prediction error 21.4%). Sensitivity analyses demonstrated that key determinants of the disposition of CAR mAbs include the following: antigen binding affinity, the rate constant of mAb-CEA dissociation in acidified endosomes, antigen concentration, and the tumor vasculature reflection coefficient.

Improving biosensing activity to carcinoembryonic antigen with orientated single domain antibodies

Carcinoembryonic antigen (CEA), also referred as CEACAM5, is integral to the adhesion process during cancer invasion and metastasis and is one of the most widely used tumor markers for assisting the diagnosis of cancer recurrence and cancer metastasis. Antibodies against CEA molecules have been developed for detection and diagnostic applications following tumor removal. Single domain antibodies (sdAbs) against CEA isolated from dromedary and llama exhibited high specificity in binding to tumor cells. However, because these CEA sdAbs were not designed to be orientated when conjugated to surface sensors, there is potential for significant improvements in their activity and limit of detection. Herein we modified the CEA sdAbs with two different C-terminal fusions designed to aid with orientation by way of the tail’s charge and biotin binding. A fusion which incorporated the C-terminus addition of a positively charged tail (B5-GS3K) improved biosensor sensitivity to CEA while also retaining the sub-nanomolar binding affinity and thermal stability of the unmodified sdAb. Using our fabricated surfaces on bare gold chips and a multiplexed surface plasmon resonance imager (SPRi), we quantified the specific binding activities, defined as the percentage of bound epitopes to the total immobilized, of the sdAb fusions and anti-CEA mAb. Our results demonstrate that monovalent B5-GS3K exhibited significantly improved binding activity, approximately 3-fold higher than bivalent mAb.

Engineering a high-affinity peptide binding site into the anti-CEA mAb M5A.

We have previously identified a cyclic peptide called meditope which binds to the central cavity of the Fab portion of cetuximab and shown that this peptide binding site can be grafted, or 'meditope-enabled', onto trastuzumab. This peptide has been shown to act as a hitch for the non-covalent attachment of imaging agents to meditope-enabled antibodies. Herein, we explore the process of grafting this peptide binding site onto M5A, an anti-CEA antibody in clinical trials for cancer diagnostics. In order to explore the contributions of the amino acids, we sequentially introduced pairs of amino acid substitutions into the Fab and then we reverse-substituted key residues in the presence of the other substitutions. We demonstrate that Pro40Thr, Gly41Asn, Phe83Ile and Thr85Asp in the light chain are sufficient to recreate the meditope binding site in M5A with single-digit micromolar affinity. We show that Pro40 abrogates peptide binding in the presence of the other 12 residue substitutions, and that the presence of all 13 substitutions does not interfere with antibody:antigen recognition. Collectively, these studies provide detailed insight for defining and fine-tuning the binding affinity of the meditope binding site within an antibody.

Sorafenib Decreases Tumor Exposure to an Anti-carcinoembryonic Antigen Monoclonal Antibody in a Mouse Model of Colorectal Cancer.

In this investigation, we test the hypothesis that treatment with sorafenib, an anti-angiogenic agent, decreases tumor vascularization and, consequently, hinders the delivery of monoclonal antibodies (mAb) to xenograft tumors. Severe combined immunodeficiency mice bearing carcinoembryonic antigen (CEA) expressing tumor xenografts were divided into control and sorafenib-treated groups. Sorafenib was administered to the latter group at 50mg/kg IP every 48h, starting 4days post-tumor implantation. When tumors attained a size of 200-300mm(3), mice were evaluated for (a) tumor microvessel density (using immunohistochemical analysis), (b) tumor macromolecular extravasation (using Evans Blue Dye (EBD)), (c) pharmacokinetics of an anti-CEA mAb, T84.66, following an intravenous dose of 10mg/kg, and (d) intra-tumoral spatial distribution of T84.66 (using autoradiography). Sorafenib treatment resulted in a substantial reduction in tumor growth rate, a visible reduction in tumor microvessel density, and in a 46.4% decrease in EBD extravasation in tumor tissue (p < 0.0455). For control and treated mice, no significant difference was found for the area under the mAb plasma concentration-time curve (AUC(0-7d): 1.67 × 10(3) ± 1.28 × 10(2) vs. 1.76 × 10(3) ± 1.75 × 10(2) nM × day, p = 0.51). However, tumor AUC(0-7d) was reduced by 40.8% in sorafenib-treated mice relative to that observed in control mice (5.61 × 10(2) ± 4.27 × 10(1) vs. 9.48 × 10(2) ± 5.61 × 10(1) nM × day, p < 0.001). Sorafenib therapy was also found to markedly alter mAb tumor spatial distribution. The results collectively suggest that sorafenib treatment causes a significant reduction in mAb delivery to, and distribution within, solid tumors.

Combination of antibody targeting and PTD-mediated intracellular toxin delivery for colorectal cancer therapy.

The bottlenecks of current chemotherapy in the treatment of colorectal cancer lie in the ineffectiveness of the existing anti-cancer small molecule drugs as well as the dose-limiting toxicity caused by the nonselective action on normal tissues by such drugs. To address these problems, we introduce a novel therapeutic strategy based on tumor targeting using a non-internalizing anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) and intracellular delivery of the extremely potent yet cell-impermeable protein toxin gelonin via the aid of a cell-penetrating peptide (also termed as protein transduction domain; PTD). A chimeric TAT-gelonin fusion protein was genetically engineered, and it displayed remarkably enhanced anti-cancer activity against human colorectal cancer cells, with IC50 values being several orders of magnitude lower than the unmodified gelonin. On the other hand, a chemically synthesized conjugate of heparin and a murine anti-CEA mAb, T84.66 (termed T84.66-Hep) was found able to bind highly specifically to CEA over-expressing LS174T colorectal cancer cells. When mixing together, TAT-gelonin and T84.66-Hep could associate tightly and automatically through an electrostatic interaction between the cationic TAT and anionic heparin. In preliminary in vivo studies using LS174T s.c. xenograft tumor bearing mouse, selective and significantly augmented (58-fold) delivery of TAT-gelonin to the tumor target was observed, when compared with administration of TAT-gelonin alone. More importantly, efficacy studies also revealed that only the TAT-gelonin/T84.66-Hep complex yielded a significant inhibition of tumor growth (46%) without causing gelonin-induced systemic toxicity. Overall, this study suggested a generic strategy to effectively yet safely deliver potent PTD-modified protein toxins to the tumor.

A series of anti-CEA/anti-DOTA bispecific antibody formats evaluated for pre-targeting: comparison of tumor uptake and blood clearance

A series of anti-tumor/anti-chelate bispecific antibody formats were developed for pre-targeted radioimmunotherapy. Based on the anti-carcinoembryonic antigen humanized hT84.66-M5A monoclonal antibody and the anti-DOTA C8.2.5 scFv antibody fragment, this cognate series of bispecific antibodies were radioiodinated to determine their tumor targeting, biodistribution and pharmacokinetic properties in a mouse xenograft tumor model. The in vivo biodistribution studies showed that all the bispecific antibodies exhibited specific high tumor uptake but the tumor targeting was approximately one-half of the parental anti-CEA mAb due to faster blood clearance. Serum stability and FcRn studies showed no apparent reason for the faster blood clearance. A dual radiolabel biodistribution study revealed that the (111)In-DOTA bispecific antibody had increased liver and spleen uptake, not seen for the (125)I-version due to metabolism and release of the radioiodine from the cells. These data suggest increased clearance of the antibody fusion formats by the mononuclear phagocyte system. Importantly, a pre-targeted study showed specific tumor uptake of (177)Lu-DOTA and a tumor : blood ratio of 199 : 1. This pre-targeted radiotherapeutic and substantial reduction in the radioactive exposure to the bone marrow should enhance the therapeutic potential of RIT.

Pharmacokinetic mAb–mAb Interaction: Anti-VEGF mAb Decreases the Distribution of Anti-CEA mAb into Colorectal Tumor Xenografts

To date, there has been little investigation of the risk for drug-drug interactions involving monoclonal antibodies. The present work examined the effects of an anti-vascular endothelial growth factor (anti-VEGF) antibody on the plasma, tissue, and tumor disposition of T84.66, an anti-carcinoembryonic antigen (CEA) antibody, in mice. SCID mice bearing CEA-expressing human colorectal cancer (LS174T) xenografts were divided into control and anti-VEGF-treated groups. When tumors reached 200-300 mm(3) in size, (125)I-T84.66 was administered intravenously at 10 mg/kg (400 μCi/kg). Radioactivity in plasma and tissue samples was counted, and T84.66 concentrations were determined. Areas under the concentration vs. time curves (AUC) were calculated. In separate groups of mice, Evans Blue Dye was administered to evaluate the effect of anti-VEGF on tumor vascular permeability. The investigations did not demonstrate significant effects of anti-VEGF therapy on T84.66 pharmacokinetics in plasma or in non-tumor tissues. T84.66 plasma AUC((0-10 days)) values were 2.37 × 10(3) ± 1.54 × 10(2) and 2.56 × 10(3) ± 1.01 × 10(2) nM × day, for the control and treated groups (p = 0.148). Conversely, anti-VEGF treatment significantly reduced tumor vascular permeability to Evans Blue Dye by 45.0 % (p = 0.0012), and anti-VEGF therapy decreased T84.66 tumor AUC((0-10 days)) by more than 60 % (7.27 × 10(2) ± 51.4 vs. 1.98 × 10(3) ± 90.1 nM × day, p < 0.0001). Our findings suggest that anti-VEGF therapies may lead to a substantial reduction in the delivery of monoclonal antibodies to tumor tissues. It is interesting and important to note that this pharmacokinetic interaction occurs at the target site, and that no alterations in T84.66 disposition were visible based on assessment of plasma pharmacokinetics alone.

Quantitative serial imaging of an 124I anti-CEA monoclonal antibody in tumor-bearing mice.

The 4.2-day half-life of (124)I favors its use for positron emission tomography (PET) of monoclonal antibodies (mAbs). However, high positron energy and beta(+) -associated cascade gamma rays pose image resolution and background noise problems for (124)I. This study evaluated quantitative PET of an (124)I mAb in tumor-bearing mice. An R4 microPETtrade mark (Siemens/CTIMI, Knoxville, TN) was used with standard energy and coincidence timing windows (350-750 keV and 6 ns, respectively), delayed random coincidence subtraction, iterative image reconstruction, and no attenuation or scatter correction. Image resolution, contrast, and response linearity were compared for (124)I and (18)F, using phantoms. Nude mice bearing human colon tumors (LS-174T) were injected intravenously with a chimeric (124)I anti-CEA mAb (cT84.66) and imaged serially 1 hour to 7 days postinjection. Venous blood was sampled to validate image-derived blood curves. Mice were sacrificed after the final scan, and the biodistribution of (124)I was measured by direct tissue assay. Images were converted to units of kBq/g for each tissue of interest by comparing the final scans with the direct assays. Measured resolution (FWHM) 0-16 mm from the scanner axis was 2.3-2.7 mm for (124)I versus 1.9-2.0 mm for (18)F. Due to true coincidence events between annihilation photons and cascade gamma rays, background was greater for (124)I than (18)F, but the signal-to-background ratio was still more than 20, and (124)I image intensities varied linearly with activity concentration. Tissue-based calibration worked well (i.e., PET blood curves agreed with direct measurements within 12% at all time points), while calibration, based on a cylindrical phantom approximating the mouse body, yielded tumor quantitation that was 46%-66% low, compared with direct assay. Images of quantitative accuracy sufficient for biodistribution measurements can be obtained from tumor-bearing mice by using (124)I anti-CEA mAbs with standard microPET acquisition and processing techniques, provided the calibration is based on the direct assay of excised tissue samples.

Use of radiolabeled monoclonal antibody to enhance vaccine-mediated antitumor effects.

Radiolabeled monoclonal antibodies (mAb) have demonstrated measurable antitumor effects in hematologic malignancies. This outcome has been more difficult to achieve for solid tumors due, for the most part, to difficulties in delivering sufficient quantities of mAb to the tumor mass. Previous studies have shown that nonlytic levels of external beam radiation can render tumor cells more susceptible to T cell-mediated killing. The goal of these studies was to determine if the selective delivery of a radiolabeled mAb to tumors would modulate tumor cell phenotype so as to enhance vaccine-mediated T-cell killing. Here, mice transgenic for human carcinoembryonic antigen (CEA) were transplanted with a CEA expressing murine carcinoma cell line. Radioimmunotherapy consisted of yttrium-90 (Y-90)-labeled anti-CEA mAb, used either alone or in combination with vaccine therapy. A single dose of Y-90-labeled anti-CEA mAb, in combination with vaccine therapy, resulted in a statistically significant increase in survival in tumor-bearing mice over vaccine or mAb alone; this was shown to be mediated by engagement of the Fas/Fas ligand pathway. Mice receiving the combination therapy also showed a significant increase in the percentage of viable tumor-infiltrating CEA-specific CD8(+) T cells compared to vaccine alone. Mice cured of tumors demonstrated an antigen cascade resulting in CD4(+) and CD8(+) T-cell responses not only for CEA, but for p53 and gp70. These results show that systemic radiotherapy in the form of radiolabeled mAb, in combination with vaccine, promotes effective antitumor response, which may have implications in the design of future clinical trials.

300. Gene Therapy for Gastric Cancer Cells Via CEA-Targeted Adv-FZ33

IntroductionAt present, adenoviral vectors have been widely used for gene transfer into tumor cells primarily because they can be produced in high titers and can infect many different cell types. However, a major problem when utilizing the adenoviral vectors for gene therapy applications is thought to be related to low transduction efficiency due to weak expression of the adenovirus receptor, coxsackie– adenovirus receptor (CAR) in cancer cells, or side effects due to expression of CAR in normal cells. There is an urgent requirement to improve the specificity of gene delivery via adenoviral vectors in the context of cancer gene therapy.Experimental designWe constructed a fiber modified Ad5 vector (Adv-FZ33) in which a IgG-binding domain (Z33) derived from staphylococcal protein A was inserted into the HI loop of knob protein. We then evaluated on both in vitro and in vivo levels the extent of retargeting towards and therapeutic effectiveness against CEA-positive gastric cancers when using the fully human CEA antibody (C2-45) complex with this modified vector.ResultsIn vitro LacZ or EGFP reporter gene expression after infection with Adv-FZ33 vectors conjugated to anti-CEA mAb was approximately 20 times higher than that obtained with vector conjugated to the control mAb (human IgG4). We generated Ax3CAUP-FZ33, which is an Adv-FZ33 derivative vector expressing a therapeutic gene, namely E. coli uracil phosphoribosyltransferase (UPRT) which converts 5FU directly to 5-fluorouridine monophosphate. When conjugated with the anti-CEA mAb, C2-45, Ax3CAUP-FZ33 enhanced the cytotoxicity of 5FU (19.7-fold in terms of IC50 values) against gastric cancer cells. In a nude mouse peritoneal dissemination model, use of Ax3CAUP- FZ33 conjugated with anti-CEA mAb C2-45, in combination with 5FU administration, resulted in greatly enhanced anti-tumor effects against CEA-positive cancer cells. Tumor growth in mice treated with Ax3UP-FZ33/C2-45/5FU was significantly suppressed, and tumor volumes were less than a quarter of those of the control IgG4 group (total weight, 0.12 ± 0.11 g; P < 0.05). Furthermore, we examined the survival effects of Ax3UP-FZ33 conjugated with anti- CEA mAb plus 5FU in mice with peritoneal disseminated gastric cancer. The median survival time of the Ax3CAUP-FZ33/C2-45/5FU group was significantly longer than those of the PBS and 5FU only treatment groups (p<0.01, versus PBS and 5FU only groups). On the other hand, no significant difference was present between the Ax3CAUP-FZ33/5FU or Ax3CAUP-FZ33/IgG4/5FU groups and the PBS or 5FU only groups.ConclusionsThese data suggest that CEA-targeted FZ33-mutant adenovirus- mediated gene delivery offers a strong and selective therapeutic modality against CEA-producing cancers. This novel strategy may be a promising tool for cancer gene therapy.

A Predictive Model of Therapeutic Monoclonal Antibody Dynamics and Regulation by the Neonatal Fc Receptor (FcRn)

We constructed a novel physiologically-based pharmacokinetic (PBPK) model for predicting interactions between the neonatal Fc receptor (FcRn) and anti-carcinoembryonic antigen (CEA) monoclonal antibodies (mAbs) with varying affinity for FcRn. Our new model, an integration and extension of several previously published models, includes aspects of mAb-FcRn dynamics within intracellular compartments not represented in previous PBPK models. We added mechanistic structure that details internalization of class G immunoglobulins by endothelial cells, subsequent FcRn binding, recycling into plasma of FcRn-bound IgG and degradation of free endosomal IgG. Degradation in liver is explicitly represented along with the FcRn submodel in skin and muscle. A variable tumor mass submodel is also included, used to estimate the growth of an avascular, necrotic tumor core, providing a more realistic picture of mAb uptake by tumor. We fitted the new multiscale model to published anti-CEA mAb biodistribution data, i.e. concentration-time profiles in tumor and various healthy tissues in mice, providing new estimates of mAb-FcRn related kinetic parameters. The model was further validated by successful prediction of F(ab')2 mAb fragment biodistribution, providing additional evidence of its potential value in optimizing intact mAb and mAb fragment dosing for clinical imaging and immunotherapy applications.

Advantage of a Residualizing Iodine Radiolabel in the Therapy of a Colon Cancer Xenograft Targeted with an Anticarcinoembryonic Antigen Monoclonal Antibody

A disadvantage of conventionally radioiodinated monoclonal antibodies (mAb) for cancer therapy is the short retention time of the radionuclide within target cells. To address this issue, we recently developed a method in which radioiodine is introduced onto antibodies using an adduct consisting of a nonmetabolizable peptide attached to the aminopolycarboxylate diethylenetriaminepentaacetic acid, designated IMP-R4. This adduct causes the radioiodine to become trapped in lysosomes following antibody catabolism. Clinical-scale production of 131I-IMP-R4-labeled antibodies is possible using a recently developed facile method. The properties of 131I-IMP-R4-labeled anticarcinoembryonic antigen (CEA) humanized mAb hMN-14 were compared with the directly radioiodinated hMN-14 (131I-hMN-14) in CEA-expressing human colon cancer cell lines, LoVo and LS174T, and in nude mice bearing established LoVo tumor xenografts. 125I-IMP-R4-hMN-14 retention in the cell lines was significantly increased (61.5% after 3 days) compared with 125I-hMN-14. In vivo, a significant improvement in tumor accretion of radiolabel was obtained using 131I-IMP-R4-hMN-14, which led to a marked improvement in therapeutic efficacy. Eight weeks post-treatment, mean tumor volumes were 0.16 +/- 0.19 and 1.99 +/- 1.35 cm3 in mice treated with 131I-IMP-R4-hMN-14 and 131I-hMN-14, respectively, with complete remissions observed in 27% of mice treated with 131I-IMP-R4-hMN-14 and none using 131I-hMN-14. 131I-IMP-R4-hMN-14 provides a significant therapeutic advantage in comparison to the conventionally 131I-labeled antibody. The ability of this labeling method to lend itself to clinical-scale labeling, the broad applicability of a humanized anti-CEA mAb for CEA-expressing cancers, and the clinical benefits of radioimmunotherapy with anti-CEA mAb shown recently for small-volume and minimal residual disease combine to make 131I-IMP-R4-hMN-14 a promising new agent for radioimmunotherapy.

Fully human IgG and IgM antibodies directed against the carcinoembryonic antigen (CEA) Gold 4 epitope and designed for radioimmunotherapy (RIT) of colorectal cancers

BackgroundHuman monoclonal antibodies (MAbs) are needed for colon cancer radioimmunotherapy (RIT) to allow for repeated injections. Carcinoembryonic antigen (CEA) being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs.MethodsXenoMouse®-G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2κ and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice.ResultsFlow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2κ and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2κ and VG-IgM were determined to be 0.19 ± 0.06 × 108 M-1 and 1.30 ± 0.06 × 108 M-1, respectively, as compared with 0.61 ± 0.05 × 108 M-1 for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2κ and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10-7 M was needed to precipitate approximatively 1 ng 125I-rhCEA as compared with 10-9 M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo, 24 h post-injection, 125I-VG-IgG2κ demonstrated a high tumor uptake (25.4 ± 7.3%ID/g), close to that of 131I-X4 (21.7 ± 7.2%ID/g). At 72 h post-injection, 125I-VG-IgG2κ was still concentrated in the tumor (28.4 ± 11.0%ID/g) whereas the tumor concentration of 131I-X4 was significantly reduced (12.5 ± 4.8%ID/g). At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver.ConclusionOur human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab')2 fragments, or as a bispecific antibody.

Regulatory role of the carcinoembryonic antigen (CEA)-family molecules in the innate eosinophil response to staphylococcus aureus

Rationale The CEA family belongs to the immunoglobulin superfamily and is expressed mainly on mucosal tissue cells and granulocytes. While CEA molecules are highly expressed on eosinophils from patients with helminth infection, the significance of these molecules is unknown. Hence, we investigated in vitro the expression and functions of the CEA family of molecules in eosinophils. Methods We examined the expression of two CEA family molecules, CGM6 (CD66b) and CEA (CD66e), before and after incubation with IL-5 or heat-killed Staphylococcus aureus (Staph) by FACS and laser scanning microscopy. The functions of these molecules in the eosinophils' responses to Staph, such as binding and superoxide production, were examined by ligating them with specific monoclonal antibodies (mAbs). Results Both CGM6 and CEA were constitutively expressed on eosinophils; their expression was upregulated when cells were incubated with IL-5 or Staph. By laser scanning microscopy, CGM6 and CEA were stained on the peripheral surface and on pseudopods of adherent eosinophils, suggesting their roles in adhesion. Incubation of eosinophils with anti-CGM6 or anti-CEA mAb significantly enhanced eosinophil binding to Staph. Furthermore, anti-CEA, but not anti-CGM6 mAb, significantly enhanced superoxide production by eosinophils in response to Staph. In contrast, IL-5-induced superoxide production by eosinophils was not affected by anti-CEA or anti-CGM6 mAb. Conclusions The CEA family of molecules is upregulated in activated eosinophils and is involved in regulating the eosinophil response to Staph. Further studies will address the roles of these molecules in the innate immune response of eosinophils.

Antibody targeting studies in a transgenic murine model of spontaneous colorectal tumors.

Monoclonal antibodies (mAbs) have been used to treat malignancies in humans with varying degrees of success. Progress has been hindered by the lack of suitable animal models, which would ideally consist of immunocompetent animals that are tolerant to tumor-associated antigens. Suitable models would allow the study and optimization of anti-tumor immunotherapy. We describe a murine model for the study of immunotherapy in colorectal cancers. Carcinoembryonic antigen (CEA) is a cell-surface glycoprotein that is expressed on normal human intestinal epithelium and that is overexpressed in intestinal tumors. Mice that are transgenic for the human CEA gene (CEA.Tg) were crossed with multiple intestinal neoplasia (MIN) mice. MIN mice carry a germline APC mutation and are prone to the development of intestinal adenomas. The offspring from the MIN x CEA.Tg cross developed intestinal adenomas that were shown by immunohistochemistry to overexpress CEA. Pharmacokinetic studies by using (125)I-labeled anti-CEA mAb PR1A3 showed rapid localization of antibody to tissues expressing CEA, especially the gastrointestinal tract. Macroscopic and microscopic radioautographic analysis of the gastrointestinal tracts from MIN/CEA.Tg mice indicated that PR1A3 targeted and was retained in tumors at levels higher than in areas of normal gut. These results demonstrate the utility of the MIN/CEA.Tg mouse as a model for the study of anti-CEA immunotherapy and, furthermore, demonstrate the efficiency of tumor localization by PR1A3.

Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin

Phage display is a powerful method of isolating of antibody fragments from highly diverse naive human antibody repertoires. However, the affinity of the selected antibodies is usually low and current methods of affinity maturation are complex and time-consuming. In this paper, we describe an easy way to increase the functional affinity (avidity) of single chain variable fragments (scFvs) by tetramerization on streptavidin, following their site-specific biotinylation by the enzyme BirA. Expression vectors have been constructed that enable addition of the 15 amino acid biotin acceptor domain (BAD) on selected scFvs. Different domains were cloned at the C-terminus of scFv in the following order: a semi-rigid hinge region (of 16 residues), the BAD, and a histidine tail. Two such recombinant scFvs directed against the carcinoembryonic antigen (CEA) were previously selected from human non-immune and murine immune phage display libraries. The scFvs were first synthesized in Escherichia coli carrying the plasmid encoding the BirA enzyme, and then purified from the cytoplasmic extracts by Ni–NTA affinity chromatography. Purified biotinylated scFvs were tetramerized on the streptavidin molecule to create a streptabody (StAb). The avidity of various forms of anti-CEA StAbs, tested on purified CEA by competitive assays and surface plasmon resonance showed an increase of more than one log, as compared with the scFv monomer counterparts. Furthermore, the percentage of direct binding of 125I-labeled StAb or monomeric scFv on CEA–Sepharose beads and on CEA-expressing cells showed a dramatic increase for the tetramerized scFv (>80%), as compared with the monomeric scFv (<20%). Interestingly, the percentage binding of 125I-labeled anti-CEA StAbs to CEA-expressing colon carcinoma cells was definitely higher (>80%) than that obtained with a reference high affinity murine anti-CEA mAb (30%). Another advantage of using scFvs in a StAb format was demonstrated by Western blot analysis, where tetramerized anti-CEA scFv could detect a small quantity of CEA at a concentration 100-fold lower than the monomeric scFv.

Radioimmunotherapy of colorectal cancer liver metastases: combination with radiotherapy.

The expected therapeutic gain of a combined radioimmunotherapy (RIT) with conventional radiotherapy (RT) would be a synergy of tumor irradiation, provided that toxic, dose-limiting side effects concern different organs. We have shown in a model of subcutaneous human colon cancer transplants in nude mice that RIT with 131I-labeled anti-CEA antibody fragments combined with fractionated RT give an additive therapeutic effect without increase of side effects. A second study of different timing schedules of RIT and RT has shown that close association of both therapies without delay is more efficient than a therapy with a treatment-free interval of two weeks. In a new model of human colon cancer liver metastases in nude mice, early treatment with RIT and with RT has been curative, whereas therapies initiated later were less efficient, suggesting that the combined therapy is likely to be more efficient in an adjuvant situation after surgery. At the clinical level, six patients with limited liver metastatic disease from colorectal cancer were treated with RIT using 200 mCi 131I-labeled anti-CEA MAb F(ab')2 fragments combined with fractionated external beam RT of 20 Gy to the entire liver. As expected, spontaneously reversible bone marrow toxicity grade 3 to 4 and reversible liver toxicity grade 1 to 3 have been observed. By computerized tomography, three patients showed stable disease and one patient partial remission, whereas two patients had progressive disease. In conclusion, animal experiments have shown a clear advantage of combined RT and RIT, and the clinical study shows the feasibility of such a therapy in patients with colorectal cancer liver metastases.

A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA.

Chimeric T cell receptors with specificity for tumor-associated antigens are successfully used to target T cells to tumor cells. The efficacy of this approach, however, is reduced by soluble antigen that is frequently present in high serum concentrations. To overcome this situation, we constructed an anti-CEA chimeric receptor whose extracellular moiety is composed of a humanized single chain antibody fragment (scFv) derived from the anti-CEA mAb BW431/26 and the CH2/CH3 constant domains of human IgG. The intracellular moiety consists of the gamma-signaling chain of the human Fc epsilon RI receptor constituting a completely humanized chimeric receptor. After transfection, the humBW431/26 scFv-CH2CH3-gamma receptor is expressed as a homodimer on the surface of MD45 T cells. Co-incubation with CEA+ tumor cells specifically activates grafted MD45 T cells indicated by IL-2 secretion and cytolytic activity against CEA+ tumor cells. Notably, the efficacy of receptor-mediated activation is not affected by soluble CEA up to 25 micrograms/ml demonstrating the usefulness of this chimeric receptor for specific cellular activation by membrane-bound CEA even in the presence of high concentrations of CEA, as found in patients during progression of the disease.