CEA-expressing Tumor Cells Research Articles

Biodistribution of a Bispecific Single-chain Diabody and Its Half-life Extended Derivatives

Small recombinant antibody molecules such as bispecific single-chain diabodies (scDb) possessing a molecular mass of approximately 55 kDa are rapidly cleared from circulation. We have recently extended the plasma half-life of scDb applying various strategies including PEGylation, N-glycosylation and fusion to an albumin-binding domain (ABD) from streptococcal protein G. Here, we further analyzed the influence of these modifications on the biodistribution of a scDb directed against carcinoembryonic antigen (CEA) and CD3 capable of retargeting T cells to CEA-expressing tumor cells. We show that a prolonged circulation time results in an increased accumulation in CEA+ tumors, which was most pronounced for scDb-ABD and PEGylated scDb. Interestingly, tumor accumulation of the scDb-ABD fusion protein was approximately 2-fold higher compared with PEGylated scDb, although both molecules exhibit similar plasma half-lives and similar affinities for CEA. Comparing half-lives in neonatal Fc receptor (FcRn) wild-type and FcRn heavy chain knock-out mice the contribution of the FcRn to the long plasma half-life of scDb-ABD was confirmed. The half-life of scDb-ABD was approximately 2-fold lower in the knock-out mice, while no differences were observed for PEGylated scDb. Binding of the scDb derivatives to target and effector cells was not or only marginally affected by the modifications, although, compared with scDb, a reduced cytotoxic activity was observed for scDb-ABD, which was further reduced in the presence of albumin. In summary, these findings demonstrate that the extended half-life of a bispecific scDb translates into improved accumulation in antigen-positive tumors but that modifications might also affect scDb-mediated cytotoxicity.

Potent Control of Tumor Growth by CEA/CD3-bispecific Single-chain Antibody Constructs That Are Not Competitively Inhibited by Soluble CEA

Carcinoembryonic antigen (CEA, CD66e) is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors. Phospholipases release CEA from tumor cells resulting in high circulating serum levels of soluble CEA (sCEA) that has been validated as marker for progression of colorectal, breast, and lung cancers. sCEA also acts as a competitive inhibitor for anticancer strategies targeting membrane-bound CEA. As a novel therapeutic approach for treatment of tumors expressing CEA on their cell surface, we constructed a series of bispecific single-chain antibodies (bscAb) combining various single-chain variable fragments recognizing human CEA with a deimmunized single-chain variable fragments recognizing human CD3. CEA/CD3-bscAbs redirected human T cells to lyse CEA-expressing tumor cells in vitro and in vivo. Efficient tumor cell lysis was achieved in vitro at bscAb concentrations from 1 pg/mL (19 fM) to 8.9 pg/mL with preactivated CD8 T cells, and 200 to 500 pg/mL with unstimulated peripheral blood mononuclear cell. The cytotoxic activity of a subset of CEA/CD3-bscAbs was not competitively inhibited by sCEA at concentrations that exceeded levels found in the serum of most cancer patients. Treatment with CEA/CD3-bscAbs prevented the growth of human colorectal cancer lines in a severe combined immunodeficiency mouse model modified to show human T cell killing of tumors. A murine surrogate CEA/CD3-bscAb capable of recruiting murine T cells for redirected tumor lysis in immunocompetent mice prevented the growth of lung tumors expressing human CEA. Together, our results reveal a unique opportunity for targeting cytotoxic T cells toward CEA-expressing tumors without being competitively inhibited by sCEA and establish CEA/CD3-bscAb as a promising and potent therapeutic approach.

An in vitro characterization study of new near infrared dyes for molecular imaging

The spectroscopic properties, stability, and cytotoxicity of series of cyanine labels, the dyes DY-681, DY-731, DY-751, and DY-776, were studied to identify new tools for in vivo fluorescence imaging and to find substitutes for DY-676 recently used by us as fluorescent label in a target-specific probe directed against carcinoembryonic antigen (CEA). This probe enables the selective monitoring of CEA-expressing tumor cells in mice, yet displays only a low fluorescence quantum yield and thus, a non-optimum sensitivity. All the DY dyes revealed enhanced fluorescence quantum yields, a superior stability, and a lower cytotoxicity in comparison to clinically approved indocyanine green (ICG). With DY-681 and far-red excitable DY-731 and DY-751, we identified three dyes with improved properties compared to DY-676 and ICG.

N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies

The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because of their small size. Here, we have investigated the effects of N-glycosylation on the activity and pharmacokinetics of a small bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. We could show that the introduction of N-glycosylation sequons into the flanking linker and a C-terminal extension results in the production of N-glycosylated molecules after expression in transfected HEK293 cells. N-Glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites, respectively, retained antigen binding activity and bispecificity for target and effector cells as shown in a target cell-dependent IL-2 release assay, although activity was reduced approximately 3-5-fold compared with the unmodified scDb. All N-glycosylated scDb variants exhibited a prolonged circulation time compared with scDb, leading to a 2-3-fold increase of the area under curve (AUC). In comparison, conjugation of a branched 40-kDa PEG chain increased AUC by a factor of 10.6, while a chimeric anti-CEA IgG1 molecule had the longest circulation time with a 17-fold increase in AUC. Thus, N-glycosylation complements the repertoire of strategies to modulate pharmacokinetics of small recombinant antibody molecules by an approach that moderately prolongs circulation time.

Immunization with Mimotopes Prevents Growth of Carcinoembryonic Antigen–Positive Tumors in BALB/c Mice

The carcinoembryonic antigen (CEA) is a glycoprotein that is overexpressed in nearly 50% of all human and veterinarian tumors. At present, anti-CEA antibodies are being tested in clinical studies as passive immunotherapeutics. This study aims to establish an active immunotherapy for the poorly immunogenic CEA glycoprotein by generating antigen surrogates. We used the monoclonal anti-CEA antibody Col-1 and the biopanning method to generate peptide mimics (mimotopes) of the Col-1 epitope. The peptide showing the highest specificity and mimicry was synthesized as an octameric multiple antigenic mimotope (MAM). Subsequently, immunogenicity of the selected mimotope was examined in BALB/c mice. We assessed antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity mediated by the induced antibodies on CEA-expressing HT29 tumor cells. Furthermore, after immunization, the BALB/c mice were transplanted s.c. with Meth-A/CEA tumor cells. When BALB/c mice were immunized with this MAM, they generated a specific humoral immune response against CEA. The mimotope-induced polyclonal and poly-isotypic antibodies induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro. Furthermore, when MAM-immunized mice were transplanted s.c. with Meth-A/CEA cells expressing human CEA, a suppressed tumor growth was observed. From our results, we can conclude that the Col-1 epitope of the glycoprotein CEA can be translated into an immunogenic peptide mimic. The mimotope-induced antibodies recognize CEA and do effectively inhibit growth of CEA-positive tumors. Based on these finding, we suggest that the generated mimotopes are candidates for active immunotherapy of CEA-expressing tumors.

A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G

The therapeutic application of small recombinant antibody molecules is often limited by a short serum half-life. In order to improve the pharmacokinetic properties, we have investigated a strategy utilizing fusion with an albumin-binding domain (ABD) from streptococcal protein G. This strategy was applied to a bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. This novel tri-functional fusion protein (scDb-ABD) was expressed in mammalian cells and recognized both antigens as well as human and mouse serum albumin. scDb-ABD was capable to retarget T cells to CEA-expressing target cells in vitro and to activate the effector cells as measured by stimulation of IL-2 release. Although activity was reduced 3-fold compared with scDb and further reduced 4-fold in the presences of human serum albumin, this assay demonstrated that scDb-ABD is active when exposed to all three antigens. Compared with scDb, the circulation time of scDb-ABD in mice was prolonged 5- to 6-fold similar to a previously described scDb-HSA fusion protein. This strategy, which adds only a small protein domain (46 amino acids) and which utilizes high-affinity, non-covalent albumin interaction, should be broadly applicable to improve serum half-lives of small recombinant antibody molecules.

Impact of Regulatory T Cells on Antigen Specific T Cell Response Using Recombinant Chimeric T Cell Receptors In Vivo.

Recombinant T cell receptors with defined specificity against tumor cells are a promising experimental approach in the elimination of residual leukemia and lymphoma cells. It is so far unresolved whether regulatory T cells with suppressor activities impair the efficiency of cytolytic T cells grafted with a recombinant immunoreceptor. The frequency of regulatory T cells is highly increased in tumor patients and their suppressive function seems to play a role in the fail of an autologous T cell response against the malignant cells.In this study we analyzed the antigen-triggered, specific activation of receptor grafted T cells in the presence or absence of regulatory CD4+CD25high T cells. CD3+ T cells were grafted with CEA-specific immunoreceptors containing the CD3-zeta signaling domain for T cell activation. Co-cultivation of receptor grafted effector T cells together with regulatory T cells repressed proliferation of the effector cells and decreased IL-2 secretion. Secretion of IFN-gamma and IL-10 was not impaired. Interestingly, the cytotoxicity of grafted effector T cells towards CEA-expressing tumor cells was not impaired by regulatory T cells in vitro.To evaluate the relevance in vivo, we used a Crl:CD1 Nu/Nu mouse model to assess growth of CEA+ tumor cells in the presence of receptor grafted effector T cells and of regulatory T cells. Mice inoculated with tumor cells together with CD3+ effector T cells without immunoreceptor and regulatory T cells developed earlier tumors with faster growth kinetics compared to mice that were inoculated with tumor cells, CD3+ T cells and CD4+CD25- control T cells. Using effector T cells that were equipped with a recombinant CEA-specific CD3-zeta immunoreceptor, 2 of 5 mice developed a tumor in the presence of regulatory T cells while none of the mice developed a tumor in the absence of regulatory T cells.Taken together, regulatory T cells obviously impair an antigen-specific, anti-tumor T cell attack in vivo. This seems to be due to repression of proliferation of the effector T cells and not to diminished cytotoxicity. These findings have major impact on the design of clinical studies involving adoptively transferred effector T cells.

Antitumor activity of chimeric immunoreceptor gene‐modified Tc1 and Th1 cells against autologous carcinoembryonic antigen‐expressing colon cancer cells

To generate tumor-specific and interferon (IFN)-gamma-producing Tc1 and Th1 cells applicable for many cancer patients, we previously developed a protocol for generating carcinoembryonic antigen (CEA)-specific Tc1 and Th1 cells from healthy human T cells by transduction with a lentivirus containing a chimeric immunoglobulin T-cell receptor (cIgTCR) gene composed of single-chain variable fragments from an anti-CEA-specific monoclonal antibody fused to an intracellular signaling domain of CD28 and CD3zeta. These cells, designated Tc1-T and Th1-T bodies, respectively, showed strong antitumor activity against CEA-expressing tumor cells in RAG2-/- mice when both of them were transferred. However, it remains unclear whether it is possible to generate Tc1-T and Th1-T bodies from cancer patients with defective T-cell function because of significant immunosuppression. Here, we prepared Tc1-T and Th1-T bodies from T cells of a colon cancer patient, and asked whether these T bodies can exert effective T-cell function against autologous tumor cells. These T bodies showed high cytotoxicity and produced IFN-gamma in response to CEA-expressing autologous tumor cells, even in the presence of soluble CEA. It was also demonstrated that Th1-T bodies supported the survival of Tc1-T bodies through cell-to-cell interactions. Furthermore, our protocol utilized retrovirus for cIgTCR transduction to achieve better induction efficiency compared to lentivirus-mediated transduction. Taken together, our findings here indicate that retrovirally transduced Tc1-T and Th1-T bodies will become a promising strategy for adoptive immunotherapy of human cancer.

Identification of Novel Subdominant Epitopes on the Carcinoembryonic Antigen Recognized by CD4+ T Cells of Lung Cancer Patients

The carcinoembryonic Ag (CEA) is an attractive target for immunotherapy because of its expression profile and role in tumor progression. To verify the existence of spontaneous anti-CEA CD4+ T cells in lung cancer patients, we first identified CEA sequences forming naturally processed epitopes, and then used the identified epitopes to test their recognition by CD4+ T cells from the patients. We had previously identified CEA(177-189/355-367) as an immunodominant epitope recognized by CD4+ T cells in association with several HLA-DR alleles. In this study, we identified four additional subdominant CEA sequences (CEA(99-111), CEA(425-437), CEA(568-582), and CEA(666-678)), recognized in association with one or more HLA-DR alleles. Peptide-specific CD4+ T cells produced proinflammatory cytokines when challenged with the native protein and CEA-expressing tumor cells, thus demonstrating that the identified CEA sequences contain naturally processed epitopes. However, CEA is expressed in the thymus and belongs to the CD66 family that comprises highly homologous molecules expressed on hemopoietic cells, raising concerns about tolerance interfering with the in vivo development of anti-CEA immunity. We thus tested the spontaneous reactivity to the identified epitopes of peripheral blood CD4+ T lymphocytes from eight early-stage lung cancer patients bearing CEA-positive tumors. We found GM-CSF- and IFN-gamma-producing CD4+ T cells in two patients. Our data indicate that CD4+ immune responses against CEA develop in neoplastic patients, suggesting that tolerance toward CEA or cross-reactive CD66 homologous molecules might be either not absolute or be overcome in the neoplastic disease.

Human monocytes expressing a CEA-specific chimeric CD64 receptor specifically target CEA-expressing tumour cells in vitro and in vivo

Antibody-dependent cellular cytotoxicity (ADCC) is one means by which macrophages (as well as natural killer cells and granulocytes) elicit a cytotoxic response. This is achieved via interaction of the Fc-gamma-receptor (CD64) with the Fc portion of antibody bound to target cells. We have created a chimeric CD64 molecule that incorporates a single chain Fv molecule, targeted against human carcinoembryonic antigen (CEA), fused to the membrane spanning and cytosolic domains of human CD64. Following adenoviral transfer to primary human monocytes, this chimeric CD64 receptor induced antigen-specific cytokine secretion during culture on immobilised CEA protein or on CEA-expressing tumour cells. Moreover, CEA targeted, but not control, monocytes effectively retarded CEA-positive tumour cell growth in vitro. Importantly, targeted monocyte cultures significantly reduced in vivo tumour growth rates in xenograft studies resulting in improved survival rates over that of control monocyte cultures. These data suggest that genetically directing monocytes against tumour antigens may be a useful means of achieving an immunotherapeutic response.

Observation of circulating tumour cells in patients with non-small cell lung cancer by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction in peroperative period

To assess whether surgical manoeuvre or resection of lung cancer could lead to haematogenous dissemination of malignant cells. In the mean time, the relationship between the sequence of vessel ligation and the haematogenous dissemination of cancer cells during operation was determined. Exploiting cytokeratin 19 (CK19)/carcinoembryonic antigen (CEA) mRNA as markers, 69 peripheral blood samples were collected from 23 consecutive patients with non-small cell lung cancer (NSCLC) who underwent surgical resection with curative intention in preoperative, intraoperative and postoperative period, respectively. Before the operation, all patients were randomly assigned to one of the two surgical procedure groups according to the order of vessel ligation, PV-first group and PA-first group. Additionally, the ten patients with benign lung disease served as control subjects undergoing surgical resection. The quantity and timing of the shedding of lung cancer cells into the circulation of patients were also monitored by fluorescent quantitative-reverse transcriptase-polymerase chain reaction before, during and after surgery. (1) The CK19 diagnostic test: the value of CK19 mRNA in operation was significantly higher than that of preoperation (5.246+/-0.196 vs. 4.472+/-0.164, P=0.000) and postoperation (5.246+/-0.196 vs. 4.694+/-0.177, P=0.013). The values between adenocarcinoma and squamous carcinoma were strikingly different (4.9110+/-1.0315 vs. 4.1891+/-0.4126, t=2.364, P=0.028). The values between PV-first group and PA-first group during perioperative period appear to be different (4.503 vs. 5.085, P=0.086). Before operation, of the 23 cases studied, 14 cases were positive (60.9%). Surprisingly, circulating epithelial cells were detected in two patients resected for benign lung disease. (2) The CEA diagnostic test: the level of CEA mRNA ascended continuously within this period. The postoperative values were significantly higher than those of preoperation (4.874 vs. 4.483, P=0.000) and those of operative day (4.874 vs. 4.537, P=0.000). The values between PV-first group and PA-first group appear to reach statistical significance (4.397 vs. 4.817, P=0.075). At the same time, there was a correlation between preoperative T-stage and perioperative CEA mRNA (4.267 vs. 4.760, P=0.025). Among the 23 cases, 10 cases were positive (43.5%). Both patients with benign lung disease served as control subjects undergoing surgical resection and the volunteers were negative. A considerable proportion of patients who appear to have resectable NSCLC might be regarded as having systemic disease, which is often undetectable by current tumour staging method. In terms of a marker used for the NSCLC patients who undergo operation, CEA is more suitable than CK19. The CK19-expressing epithelial cells are released intraoperatively into the circulation, meanwhile CEA-expressing tumour cells are disseminated mostly postoperatively. Surgical manipulation could promote the release of tumour cells into the bloodstream, but the ligation of pulmonary vein before the ligation of the pulmonary artery may partly prevent such release during surgery.

540. Re-Targeting of Adenoviral Vector-Mediated Interferon-γ Gene Transfer to Colon Cancer Cells That Express the Human Carcinoembryonic Antigen

We hypothesized that re-targeting of an Ad5 vector encoding the interferon-|[gamma]| (IFN-|[gamma]|) cDNA to tumor cells that express human carcinoembryonic antigen (CEA) can be accomplished. In order to test the feasibility of this schema, we conducted a proof-of-principle experiment. In this experiment, we utilized a recombinant adapter protein (sCAR-MFE), consisting of the extracellular portion of the native Ad receptor, the coxsackievirus-adenovirus receptor (CAR), fused to a single chain antibody (scFv) specific for the human carcinoembryonic antigen (CEA). We tested the ability of this adapter protein to re-target an adenovirus expressing Ad5-IFN-|[gamma]| to cells expressing CEA. We chose as a model, a mouse colon cancer cell line derived from the MC38 cell line that expresses the human CEA constitutively and forms tumors in syngeneic immunocompetent C57BL6 mice (MC38-CEA2). The Ad5-IFN-|[gamma]| vector (1|[times]|107 viral particles) was first incubated in the absence or presence of 100 ng of the recombinant adapter protein sCAR-MFE for 15 min at room temperature. As a negative control, the Ad5-IFN-|[gamma]| vector was also incubated in the absence or presence of a non-specific recombinant adapter protein, CFm40L. The untargeted or re-targeted Ad5-IFN-g vector was used to infect either the MC38 cell line or the MC38-CEA2 cell line at a low multiplicity of infection (0.1 infectious units/cell). The cells were infected for 1 hour, and then the infected cells were cultured for 48 hours. At the end of the culture period, the levels of Ad-IFN-|[gamma]| in the culture media were determined by ELISA analysis. The levels of IFN-|[gamma]| in the culture media from MC38 cells infected with Ad5-IFN-g was low in the culture media, ranging from 454 to 572 pg/ml, regardless of the presence or absence of recombinant adapter proteins. In contrast, in the culture media from MC38-CEA2 cells infected with Ad5-IFN-g the level of IFN-g was low, at 1159 pg/ml. Similarly, in the culture media from MC38-CEA2 cells infected with the Ad5-IFN-|[gamma]| vector pre-incubated with the irrelevant adapter protein CFm40L, the level of IFN-|[gamma]| was 1648 pg/ml. In contrast, in the culture media from MC38-CEA2 cells infected with the Ad5-IFN-|[gamma]| vector pre-incubated with the re-targeting adapter protein sCAR-MFE, the level of IFN-|[gamma]| was nearly three orders of magnitude higher at 732,000 pg/ml. Importantly, the observation that re-targeting was inefficient on CEA-negative MC38 cells demonstrates the CEA-targeted mechanism of the targeting complex uptake by MC38-CEA2 cells. Therefore, we have shown that the re-targeting is highly specific. These data clearly show that our approach can be exploited to re-target an Ad vector, and support our hypothesis that re-targeting of an Ad5-IFN-|[gamma]| vector to CEA-expressing tumor cells can be accomplished. We anticipate that the selective transduction of tumor cells with Ad5-IFN-|[gamma]| in vivo will allow this cytokine to act locally while minimizing any systemic toxicity.

Monoclonal Anti-idiotype Antibody 6G6.C4 Fused to GM-CSF Is Capable of Breaking Tolerance to Carcinoembryonic Antigen (CEA) in CEA–Transgenic Mice

Internal image anti-idiotypic antibodies are capable of mimicking tumor-associated antigens and thus may serve as surrogate for vaccination strategies in cancer patients. The monoclonal antibody (mAb) 6G6.C4 mimics an epitope specific for the human carcinoembryonic antigen (CEA) and generates a CEA-specific response (Ab3) in various experimental animals. In humans, however, 6G6.C4 only yields a very limited humoral anti-CEA reaction presumably due to tolerance against the CEA autoantigen. In this study, we investigated the CEA-specific Ab3 response in mice transgenic for the human CEA and tested whether the antigen tolerance could be overcome by fusing a recombinant single-chain variable fragment of 6G6.C4 (scFv6G6.C4) to the murine granulocyte macrophage colony-stimulating factor (GM-CSF). Like mAb 6G6.C4, the fusion protein (scFv6G6.C4/GM-CSF) retained binding to the CEA-specific idiotype mAb T84.66. Also, scFv6G6.C4/GM-CSF was biologically active as measured by proliferation of the GM-CSF-dependent murine FDC-P1 cells in vitro. After immunization with the scFv6G6.C4/GM-CSF fusion protein, CEA-transgenic animals showed significantly enhanced Ab3 antibody responses to scFv6G6.C4 (P=0.005) and to CEA (P=0.012) compared with the scFV6G6.C4 alone. Sera from mice immunized with the fusion protein specifically recognized CEA in Western blot analyses with no cross-reaction to CEA-related antigens. Finally, the Ab3 antisera detected single CEA-expressing tumor cells in suspension as shown by flow cytometry. Taken together, these data show in a model antigenically related to the human system that vaccination with scFv6G6.C4/GM-CSF improves vaccination against an endogenous tumor-associated antigen resulting in a highly specific humoral Ab3 response in vivo that is capable of bind single circulating CEA-positive tumor cells.

Integrating Bench With Bedside: The Role of Vaccine Therapy in the Treatment of Solid Tumors

The treatment of solid tumors continues to be one of the major challenges facing both patients and the oncology community. An improved understanding of how the immune system recognizes and eradicates tumor cells has led to an intense interest in therapeutic vaccine development, representing one of the most successful applications of bench-to-bedside translational research. Extensive “proof of principle” evidence from the bench has used murine models to demonstrate that active immunization can mediate effective tumor treatment. In 1991, a vaccine strategy employing recombinant vaccinia virus engineered to express carcinoembryonic antigen (CEA) was reported. Initial animal experiments documented the ability of vaccinia-CEA to induce humoral and cellular immunity by targeting a commonly expressed solid tumor antigen in a highly immunogenic vaccine—a result since confirmed in transgenic mice and in vitro experiments with human T cells. The first clinical trial with this vaccine was published in 1996, and numerous other studies, largely phase I in format, were conducted throughout the last 8 years, demonstrating the safety and feasibility of various poxvirus vaccines in patients with advanced solid tumors. While these studies were not designed or powered to determine clinical effectiveness, intriguing anecdotal evidence suggested that vaccination induced CEA-specific T cells, and even objective clinical responses, in some patients. The completion of these early-phase clinical trials provided important safety data and guided additional bench experiments aimed at improving the therapeutic effectiveness of CEA-based poxvirus vaccines. For example, careful analysis of in vitro T-cell responses from vaccinated patients led to the identification of HLA-restricted T-cell epitopes and later demonstrated that poxvirus vaccines could break tolerance against these epitopes in patients. This also allowed Schlom et al to identify a modified CEA agonist peptide that was more efficient in inducing T-cell responses against the native CEA peptide and CEAexpressing tumor cells. Another problem identified from early clinical trials was that repeated administration of vaccinia virus induced strong neutralizing antibodies that prohibited subsequent boosting of immune responses against the tumor antigen. Animal studies utilizing a prime-boost approach, wherein vaccinia virus is used once for priming followed by booster vaccinations with nonreplicating poxvirus vectors such as fowlpox virus, were shown to be superior to using either vector alone. This was later confirmed in a clinical trial. Additional vaccine improvements were provided by the coexpression of powerful costimulatory molecules in the viral genome, designed to augment the activation of T cells after engagement of the T-cell receptor. Preclinical experiments in mice and with human T cells in vitro resulted in selecting a triad of costimulatory molecules (B7-1, ICAM-1, and LFA-3) that significantly enhanced immune responses of the poxvirus vaccines, and augmented tumor regression in animals. Other studies evaluated the benefits of adjuvant cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) for promoting CEA-specific immunity in clinical trials after a benefit was seen in murine models. In this issue of the Journal of Clinical Oncology, Marshall et al, combine many of these methods for improving poxvirus vaccination into a single vaccine strategy for patients with advanced CEA-expressing solid tumors. They utilized a vaccinia virus expressing CEA encoding the agonist epitope, along with three costimulatory molecules (B7-1, ICAM-1, and LFA-3) for priming an immune response, followed by booster vaccinations with a fowlpox virus expressing the same transgenes. In addition, they treated some cohorts with GM-CSF administered near the vaccine site to promote local recruitment of dendritic cells. They also evaluated a split dose of the booster vaccine in an JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 23 NUMBER 4 FEBRUARY 1 2005

Generation, immunologic characterization and antitumor effects of human monoclonal antibodies for carcinoembryonic antigen

We generated fully human mAbs (HmAbs) to carcinoembryonic antigen (CEA) using the KM mouse, which carries a human chromosome 14 fragment containing the entire Ig H chain loci and human kappa L chain segments in the mouse genome. Forty-six hybridoma clones producing HmAbs to CEA were thus obtained by fusing the P3-U1 mouse myeloma cells with splenocytes of the KM mice immunized with CEA. Among them, 22 clones produced HmAbs that reacted with CEA but not with 3 other CEA-related cell adhesion molecule (CEACAM) family members, CEACAM1, CEACAM6 and CEACAM8. In 12 HmAbs examined, 8 were IgG4, 2 were IgG3, 1 was IgG2, and the other was IgG1. The affinity constants for CEA of these HmAbs were comparable to those of the previously prepared mouse anti-CEA mAbs (MmAbs). BIAcore analyses revealed that 1 and 2 of the 22 HmAbs react with 2 epitopes defined by MmAbs on the domain N and the domain A1 or B1 of CEA, respectively. In the presence of human complement in vitro, 2 HmAbs tested showed substantial cytotoxicity, namely, 50-65%, against CEA-expressing tumor cells. With human lymphokine-activated killer cells in vitro, 3 HmAbs tested exhibited 40-65% Ab-dependent cell-mediated cytotoxicity against the tumor cells. Moreover, one of the HmAbs induced a significant inhibition of tumor growth when administered to mice xenografted with the CEA-expressing cells. Considering their lack of immunogenicity to humans, these CEA-specific HmAbs may be useful for immunotherapeutic approaches as well as for immunodiagnosis.

T lymphocytes isolated from patients with advanced colorectal cancer are suitable for gene immunotherapy approaches.

Despite improvements in treatment, the 5-year survival for metastatic colorectal cancer remains poor. Novel approaches such as gene immunotherapy are being investigated to improve treatment. Retroviral gene transfer methods have been shown to transduce primary human T lymphocytes effectively resulting in the expression of therapeutic genes. However, a number of defects have been identified in T lymphocytes isolated from patients bearing tumour, which may have critical implications for the development of gene-targeted T cells as an anticancer therapy. To address this issue, primary T lymphocytes were isolated from patients with advanced colorectal cancer and tested for their ability to be transduced and to express subsequently a chimeric immune receptor consisting of a single-chain antibody fragment antigen-binding moiety specific for carcinoembryonic antigen (CEA) fused to the T cell receptor (TCR) CD3ζ chain. In 10 out of 10 patients, T lymphocytes were transduced, expanded in the absence of selection and tested for functional activity against CEA-expressing tumour cells. In each case, functional-specific cytotoxic activity was observed. Negligible activity was found in control cultures. This study highlights the feasibility of patient-derived T lymphocytes as a source of immune cells for autologous gene immunotherapy approaches.

The carcinoembryonic antigen and its prognostic impact on immunocytologically detected intraperitoneal colorectal cancer cells

Background: Carcinoembryonic antigen (CEA) has been suggested to promote colon cancer progression. In this study we analyzed the prognostic impact of CEA expression on intraperitoneally detected single colon cancer cells. Methods: Peritoneal lavage samples of 135 colorectal cancer patients were imunocytologically analyzed, including a staining of cellular CEA; serum CEA levels were measured; and 5-year survival rates were calculated according to immunocytological findings and CEA expression. Results: The worst survival rate of 20% was found in patients suffering from CEA-expressing intraperitoneal tumor cells ( P = 0.0006). The prognostic impact of an intraperitoneal tumor cell finding significantly increased when serum CEA levels were elevated: only 23% survived 5 years in contrast to a 85% 5-year survival rate of patients who neither had signs of dissemination nor showed elevated serum CEA values ( P = 0.0010). Conclusions: This study shows that the determination of CEA expression improves the prognostic impact of an intraperitoneal tumor cell finding.

Fusion proteins of B7.1 and a carcinoembryonic antigen (CEA)-specific antibody fragment opsonize CEA-expressing tumor cells and coactivate T-cell immunity.

Genetic engineering can be used to generate antigen-specific molecules for improved tumor immunotherapy. We have constructed genes coding for fusion proteins consisting of a high-affinity antibody single-chain antibody fragment (scFv) specific for the human carcinoembryonic antigen (CEA) and the costimulation domain of the murine B7.1 molecule (mB7.1) linked to the antibody moiety by an IgG3 peptide linker. The hybrid genes were constructed in 2 orientations, one with the scFv located N-terminal to mB7.1 and one vice versa. Soluble proteins were expressed by CHO cells, purified using anti-idiotype-affinity chromatography and characterized by tumor-cell binding and costimulation activity. When tumor cells expressing CEA on the cell membrane were opsonized with the CEA-specific costimulators, both fusion proteins specifically stimulated murine T-cell preparations to proliferate in a similar manner. Our data suggest that "costimulation coating" of tumor cells may be a suitable approach for activation of a sustained cellular antitumor response. It also provides the opportunity to increase tumor immunogenicity using easily generated soluble fusion proteins that advantageously link biological functions of both the humoral and the cellular arm of the specific immune system.

Strategies for cancer therapy using carcinoembryonic antigen vaccines.

Advances in molecular biology and immunology have renewed interest in the development of vaccines for the treatment or prevention of cancer. Research over the past 10 years has focused on the identification of suitable tumour antigens to use as targets for a variety of vaccine strategies. Carcinoembryonic antigen (CEA) was one of the first tumour antigens described, and is commonly expressed by a wide range of adenocarcinomas. Recent studies have identified several human-leukocyte-antigen-restricted epitopes (short peptides) within the CEA protein that can be recognised by human T lymphocytes (T cells). Although CEA-expressing tumour cells are generally weakly recognised by the immune system, several new strategies have been used to enhance immune responses against CEA. This includes using antibodies directed against CEA; inserting the CEA gene into recombinant viruses and bacteria as viral and bacterial vaccines; pulsing the CEA protein, peptides, DNA or RNA onto dendritic cells (specialised antigen-presenting cells); and combining CEA vaccines with cytokines or co-stimulatory molecules to increase vaccine effectiveness. Other factors that might be important in establishing systemic immunity against CEA are the dose, route, timing, and choice of vector and adjuvants for vaccine administration. Further research in understanding the fundamental processes involved in tumour-cell recognition by the immune system, better animal models, and improved clinical trial designs will help to define the full potential of CEA as a target for cancer vaccine development.

Phase I Study of Recombinant Carcinoembryonic Antigen (CEA) Vaccinia Virus Vaccine With Post Vaccination Carcinoembryonic Antigen Peptide (CAP-1) Boost

227 February 2000 Rationale Seventy percent of non–small-cell lung cancers express carcinoembryonic antigen (CEA).1 A direct immunologic approach to CEA-bearing tumors has been recently defined using inoculation with a recombinant vaccinia virus that expresses the human CEA gene.2 Since vaccinia elicits both humoraland cell-mediated immune responses, copresentation of tumor-associated antigen products with vaccinia could enhance cell-mediated immunity against the tumor.3 A preclinical murine model has been established using cDNA coding for CEA inserted into a strain of vaccinia virus (rV-CEA) (Figure 1). Animals inoculated with rVCEA showed inhibition of CEA-positive tumor growth and did not allow tumor growth when rechallenged.4 Safety testing of rV-CEA in the rhesus monkey model noted only local skin reactions, low-grade fever, and lymphadenopathy.5 Based on this preclinical data, an initial phase I study using an escalating dose administration of the rV-CEA vaccine was performed without grade 3 or dose-limiting toxicities.6 A therapeutic immune response was not realized; however, in vitro priming of postvaccination peripheral blood lymphocytes (PBL) with CEA peptide demonstrated specific lytic activity against CEA-expressing tumor cells. Immune recognition, therefore, of the CEA peptide can be induced by the rVCEA vaccine.6,7 The in vitro response of the post-vaccination PBL to peptide stimulation suggests that a CEA-specific T-lymphocyte precursor population is present in vivo as a result of the rV-CEA vaccination. Administration of relevant tumor-associated epitopes with adjuvant to stimulate and significantly expand the number of antigen-specific T-lymphocyte precursor cells present in vivo holds promise.8 A logical next step, therefore, would be post-rV-CEA vaccination administration of CEA peptide with an adjuvant reagent in vivo. An enhanced CEA-bearing tumor T-cell population could then potentially lead to a direct therapeutic antitumor immune response. Furthermore, additional interventions, such as the ex vivo expansion of CEA-specific Tlymphocytes for adoptive immunotherapy, would then be possible. Therefore, we hypothesize that a significant in vivo precursor tumor-specific T-cell population can be established using rV-CEA vaccination and follow-up CEA peptide challenge that could lead to an effective antitumor response. Our objectives are: (1) to determine tolerance/side effects of repeated vaccination with rV-CEA followed by repeated CEA peptide David J. Cole,1 Paul L. Baron,1 Paul O'Brien,1 Carolyn E. Reed,1 Jeffrey Schlom,2 Kwon Y. Tsang2