Adenovirus-mediated intratumoral expression of immunostimulatory proteins in combination with systemic Treg inactivation induces tumor-destructive immune responses in mouse models.

Tumor-associated antigens (TAAs) include overexpressed self-antigens (for example, Her2/neu) and tumor virus antigens (for example, HPV-16 E6/E7). Although in cancer patients, TAA-specific CD4+ and CD8+ cells are often present, they are not able to control tumor growth. In recent studies, it became apparent that tumor site-located immune evasion mechanisms contribute to this phenomenon and that regulatory T cells have a major role. We tested in Her2/neu+ breast cancer and HPV-16 E6/E7+ cervical cancer mouse models, whether intratumoral expression of immunostimulatory proteins (ISPs), for example, recombinant antibodies (αCTLA-4, αCD137, αCD3), cyto/chemokines (IL-15, LIGHT, mda-7) and costimulatory ligands (CD80), through adenovirus(Ad)-mediated gene transfer would overcome resistance. In both the breast and cervical cancer model, none of the Ad.ISP vectors displayed a significant therapeutic effect when compared with an Ad vector that lacked a transgene (Ad.zero). However, the combination of Ad.ISP vectors with systemic T regulatory (Treg) depletion, using anti-CD25 mAb (breast cancer model) or low-dose cyclophosphamide (cervical cancer model) resulted in a significant delay of tumor growth in mice treated with Ad.αCTLA4. In the cervical cancer model, we also demonstrated the induction of a systemic antitumor immune response that was able to delay the growth of distant tumors. Ad.αCTLA4-mediated tumor-destructive immune responses involved NKT and CD8+ T cells. In both models no autoimmune reactions were observed. This study shows that Ad.αCTLA4 in combination with systemic Treg depletion has potentials in the immunotherapy of cancer.

Combining CA 125 and SMR serum markers for diagnosis and early detection of ovarian carcinoma.

OBJECTIVES: The serum tumor marker CA 125 is elevated in most clinically advanced ovarian carcinomas. Because these elevations may precede clinical detection by a year or more, CA 125 is potentially useful for early detection as part of an ovarian cancer screening program. However, CA 125 is often not elevated in clinically detected cancer and is frequently elevated in women with benign ovarian tumors. CA 125 may be more useful in conjunction with one or more other tumor biomarkers. Additional markers could play a role if, when used with CA 125, they identify some carcinomas missed by CA 125 (i.e., they improve sensitivity), rule out false positives (i.e., improve specificity), or are able to detect the same cancers earlier. METHODS: We have evaluated a composite marker (CM) that combines CA 125 and a previously described soluble mesothelin related (SMR) marker in sera from 52 ovarian cancer cases, 43 controls with benign ovarian tumors, and 220 normal risk controls who participated in a screening program, including 25 healthy women having two serum samples collected 1 year apart. CA 125, SMR, and CM were evaluated for their ability to identify clinical disease and for their temporal stability, which assesses their ability to obtain even greater sensitivity when used in a longitudinal screening program. RESULTS: CM has the best sensitivity, with specificity equal to CA 125. Importantly, CM has temporal stability at least as high as CA 125. CONCLUSION: The CM may outperform CA 125 alone in a longitudinal screening program as well as in a diagnostic setting.

CD3-mediated activation of tumor-reactive lymphocytes from patients with advanced cancer.

Lymphocytes from blood or tumors of patients with advanced cancer did not proliferate and produced very low levels of tumor necrosis factor and IFN-gamma when cultured with autologous tumor cells. Proliferation and lymphokine production dramatically increased in the presence of beads conjugated with mAbs to CD3 plus mAbs to CD28 and/or CD40, and the lymphocytes destroyed the tumor cells. Expression density of CD3 concomitantly increased from low to normal levels. Furthermore, beads providing a CD3 signal (in combination with CD28 or CD28 plus CD40) gave partial protection against the inhibitory effect of transforming growth factor type beta1 on lymphocyte proliferation and production of tumor necrosis factor and IFN-gamma. MHC class I-restricted cytolytic T cells lysing autologous tumor cells in a 4-h Cr(51) release assay were generated when peripheral blood leukocytes were activated in the presence of autologous tumor cells and anti-CD3/CD28 or anti-CD3/CD28/CD40 beads. Experiments performed in a model system using anti-V-beta1 or anti-V-beta2 mAbs to activate subsets of T cells expressing restricted T cell receptor showed that lymphocytes previously activated by anti-V-beta can respond to CD3 stimulation with vigorous proliferation and lymphokine production while retaining their specificity, also in the presence of transforming growth factor type beta1. Our results suggest that T lymphocytes from cancer patients can proliferate and form Th1 type lymphokines in the presence of autologous tumor cell when properly activated, and that antigen released from killed tumor cells and presented by antigen-presenting cells in the cultures facilitates the selective expansion of tumor-directed, CD8(+) cytolytic T cells.

Overexpression of HER-2 in ovarian carcinomas.

The transmembrane receptor encoded by the HER-2 cellular oncogene is amplified in several types of human carcinomas and provides an attractive therapeutic target. Shown by immunohistology, <25% of newly diagnosed ovarian carcinomas express the HER-2 protein. However, now we report that this protein was expressed in all 20 tumor cell lines derived from stage III and IV ovarian cancers as well as in tumor cells harvested from patients with malignant ascites and in tumor samples taken at a second surgery, suggesting that cells with excess expression may have a selective growth advantage. HER-2-positive ovarian carcinoma cells were shown to be sensitive to antibody-dependent cellular cytotoxicity, and their in vitro proliferation was inhibited by anti-HER-2 MAb Herceptin. We postulate, therefore, that therapy which targets HER-2 may be more efficacious in patients with ovarian carcinoma than indicated by the commonly low expression of HER-2 in tumors removed at the time of primary surgery.

CD83 is an I-type lectin adhesion receptor that binds monocytes and a subset of activated CD8+ T cells [corrected].

To help determine CD83 function, a cDNA encoding a soluble protein containing the CD83 extracellular domain was fused with a mutated human IgG1 constant region (CD83Ig) and expressed by stable transfection of Chinese hamster ovary cells. Purified CD83Ig bound to peripheral blood monocytes and a subset of activated CD3(+)CD8(+) lymphocytes but did not bind to FcR. Monocytes that had adhered to plastic lost their ability to bind to CD83Ig after 90 min of in vitro incubation. CD83Ig bound to two of five T cell lines tested, HPB-ALL and Jurkat. The binding to HPB-ALL cells significantly increased when they were grown at a low pH (pH 6.5), whereas binding to Jurkat cells increased after apoptosis was induced with anti-Fas mAb. B cell and monocytic lines did not bind CD83Ig and neither did CD56(+) NK cells or granulocytes. Full-length CD83 expressed by a transfected carcinoma line mediated CD83-dependent adhesion to HPB-ALL cells. CD83Ig immunoprecipitated and immunoblotted a 72-kDa protein from HPB-ALL cells. Binding of CD83Ig to HPB-ALL cells was eliminated by neuraminidase treatment of the cells. We conclude that CD83 is an adhesion receptor with a counterreceptor expressed on monocytes and a subset of activated or stressed T lymphocytes, and that interaction between CD83 and its counterreceptor is dependent upon the state of glycosylation of a 72-kDa counterreceptor by sialic acid residues. In view of the selectivity of the expression of CD83 and its ligand, we postulate that the interaction between the two plays an important role in the induction and regulation of immune responses.

Enhanced delivery improves the efficacy of a tumor-specific doxorubicin immunoconjugate in a human brain tumor xenograft model.

OBJECTIVE: To evaluate dose intensification with osmotic blood-brain barrier disruption (BBBD) and the potential use of drug targeting with monoclonal antibody (MAb) BR96 conjugated to doxorubicin (BR96-DOX, now called SGN15) for treatment of intracerebral and subcutaneous human LX-1 small cell lung carcinoma xenografts in rats. METHODS: LX-1 tumors with high, low, or heterogeneous levels of the Lewis(y) antigen for BR96 were evaluated. Rats were treated with intracarotid or intravenous BR96-DOX, with or without osmotic BBBD. RESULTS: Both BR96-DOX and MAb BR96 treatment resulted in significant regression of subcutaneous tumors, in contrast to control groups including doxorubicin alone, saline, or nonbinding doxorubicin immunoconjugate. BR96-DOX delivered with BBBD to brain tumors with low antigen expression resulted in significantly (P < 0.001) increased rat survival time compared with animals that received intravenous or intra-arterial BR96-DOX. CONCLUSION: The combination of an effective drug such as doxorubicin with a MAb to facilitate tumor-selective localization and osmotic BBBD to increase tumor delivery may have practical application in the clinic, because an increased delivery of drug to tumor can be obtained without increasing the dose of systemic drug.

Enhanced antitumor activity of paclitaxel in combination with the anticarcinoma immunoconjugate BR96-doxorubicin.

The efficacy of chemotherapy has been improved by regimens that combine several cytotoxic drugs with different mechanisms of action and/or different dose-limiting toxicities. Here we demonstrate clearly, and for the first time, that combined therapy using an anticarcinoma immunoconjugate, BR96-doxorubicin, and the cytotoxic drug paclitaxel results in a significant increase in antitumor activity over that of either agent alone. Synergistic activity was seen at doses of BR96-doxorubicin that were minimally active as single agents. A dramatic increase in regression rates was seen when a regimen that combined BR96-doxorubicin and paclitaxel was used to treat both paclitaxel-sensitive and paclitaxel-insensitive carcinomas. Importantly, combined therapy resulted in increased antitumor activity against lung, colon, and breast tumors xenografted in athymic mice and large, paclitaxel-insensitive colon tumors xenografted in athymic rats that also express the Lewis(y) target antigen in normal tissues.

Soluble member(s) of the mesothelin/megakaryocyte potentiating factor family are detectable in sera from patients with ovarian carcinoma.

mAb OV569 was made by immunizing mice with ovarian carcinoma cells. It binds to cells from ovarian carcinomas and, to a lesser extent, to cells from certain other carcinomas whereas the binding to normal tissues is low to nondetectable. It also binds to soluble molecule(s) in culture supernatants from antigen-positive carcinomas. OV569 recognizes a protein(s) of 42-45 kDa with the same N-terminal amino acid sequence as the membrane-bound portion of mesothelin and megakaryocyte potentiating factor (MPF). Binding assays with fusion proteins comprising either the N-terminal part of mesothelin/MPF (D1Ig), reported to be easily cleaved off, or a noncleavable, membrane-associated part (D2Ig) showed that OV569 only binds to D2hIg. A new member of the mesothelin/MPF family was discovered, which has an 82-bp insert in the membrane-associated part, leading to a frameshift of 212 bp, and whose predicted molecular structure indicates that it is soluble. To test patient sera for soluble tumor antigen, antigen was isolated from cell-free tumor culture supernatants via immunoadsorption with OV569 and used to generate murine mAbs to an epitope different from the one to which OV569 binds, after which mAbs to two different epitopes were used to develop a "sandwich ELISA." Using this assay, the level of circulating antigen was elevated significantly in 23 of 30 sera from patients with ovarian carcinoma, as compared with 0 of 68 sera from healthy controls, 0 of 3 sera from patients with nonneoplastic diseases, and 25 of 75 sera from patients with other tumors. Soluble molecules of the mesothelin/MPF family may provide useful new marker(s) for diagnosis of ovarian carcinoma and/or monitoring its response to therapy.

Monoclonal antibody conjugates of doxorubicin prepared with branched linkers: A novel method for increasing the potency of doxorubicin immunoconjugates.

Immunoconjugates of monoclonal antibody BR96 and Doxorubicin have been prepared using a novel series of branched hydrazone linkers. Since each linker bound to the mAb carries two DOX molecules, the DOX/mAb molar ratios of these conjugates were approximately 16, twice that of those previously prepared with single-chain hydrazone linkers. The conjugates were stable at a physiological pH of 7, but released DOX rapidly at lysosomal pH 5. The branched series of BR96 conjugates demonstrated antigen-specific cytotoxicity, and were more potent in vitro than the single-chain conjugate on both a DOX (4-14-fold) and mAb (7-23-fold) basis. The results suggest that, by using the branched linker methodology, it is possible to significantly reduce the amount of mAb required to achieve antigen-specific cytotoxic activity. In this paper, the synthesis and in vitro biology of branched chain immunoconjugates are described.

Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway.

We have explored the role of an activation-induced T cell molecule, 4-1BB (CDw137), in the amplification of tumor immunity by retrovirus-mediated transduction of the 4-1BB ligand (4-1BBL) into tumor cells. Mice inoculated with P815 tumor cells expressing 4-1BBL developed a strong cytotoxic T lymphocyte (CTL) response and long-term immunity against wild-type tumor. The optimal effect of 4-1BBL in CTL stimulation required B7-CD28 interaction since blockade of this interaction by antibodies down-regulated the expression of 4-1BB on T cells and decreased CTL activity. Furthermore, co-expression of 4-1BBL and B7-1 in the poorly immunogenic AG104A sarcoma enhanced the induction of effector CTL and the rejection of the wild-type tumor while neither 4-1BBL nor B7-1 single transfectants were effective, suggesting a synergistic effect between the 4-1BB and the CD28 co-stimulatory pathways. Our results underscore the importance of the 4-1BB T cell stimulation pathway in the amplification of an antitumor immune response.

Role of epidermal growth factor receptor family members in growth and differentiation of breast carcinoma.

Members of the epidermal growth factor (EGF) family of tyrosine kinase receptors are involved in the regulation of cell growth and differentiation, and are found to be expressed in many types of cancers. Activation of these receptors can be elicited by multiple ligands, resulting in the formation of a spectrum of heterodimer complexes and a number of biological outcomes. A clear demonstration of biological activation by a single complex has been difficult to address because of the endogenous expression of HERs (human EGF-like receptors) in many cell lines. We have generated a collection of cell lines expressing all HERs alone or in all pairwise combinations in a clone of NIH 3T3 cells (3T3-7d) devoid of detectable EGF receptor family members. Transformation, as measured by growth in soft agar, only occurred in cells expressing two different HER family members. Transformation with activated Neu and the rate of in vivo tumour formation were also correlated with the expression of multiple HERs in the same cell. To further our understanding of the role of heterodimer signalling, we demonstrated that, within a breast carcinoma cell line, activation of HER-3 results in cellular differentiation, prolonged activation of extracellular-signal-related kinase 1 (ERK1) activity and an increase in p21CIP1/WAF1 nuclear staining. In contrast, activation of HER-4 is mitogenic, induces transient activation of ERK1 activity and decreases the nuclear staining of p21CIP1/WAF1. These differences in biochemical and biological responses are correlated with the contrasting abilities of HER-3 and HER-4 to be down-regulated from the cell surface. The cell-surface localization of HER-3 does not change in response to ligand, whereas activation of HER-4 results in a loss of cell-surface staining followed by accumulation into a perinuclear compartment.

Antitumor activity of carcinoma-reactive BR96-doxorubicin conjugate against human carcinomas in athymic mice and rats and syngeneic rat carcinomas in immunocompetent rats.

The internalizing monoclonal antibody BR96 was conjugated to the anticancer drug doxorubicin (DOX) using an acid-labile hydrazone bond to DOX and a thioether bond to the monoclonal antibody. The resulting conjugate, termed BR96-DOX, binds to a tumor-associated Lewis(y) antigen that is abundantly expressed on the surface of human carcinoma cells. BR96-DOX binds to RCA, a human colon carcinoma cell line, and BN7005, a transplantable colon carcinoma induced in a Brown Norway (BN) rat by 1,2-dimethyl-hydrazine. BR96-DOX produces cures of established s.c. RCA human colon carcinomas in athymic mice and rats. BR96-DOX also cured both s.c. and intrahepatic BN7005 tumors in immunocompetent BN rats. Unconjugated DOX, given at its maximum tolerated dose, and matching doses of nonbinding IgG-DOX conjugate were not active against RCA or BN7005 carcinomas. An anticonjugate antibody response was produced in BN rats treated with BR96-DOX. However, this could be largely prevented by administering the immunosuppressive drug deoxyspergualin. These results confirm the concept of antibody-directed therapy in models in which the targeted antigen is expressed both in normal tissues and tumors. The findings in BN7005 further demonstrate efficacy of BR96-DOX therapy in a model in which the tumor is syngeneic and the host is immunocompetent.

Cancer vaccines: challenges and potential solutions.

Almost a century has passed since immunotherapy of cancer was first attempted using cancer immunogens (vaccines); however, its clinical impact remains modest. Although initial concerns about a lack of human tumor antigens have decreased, prevailing issues include inefficient procedures for immunization and downregulated expression of major histocompatibility complex (MHC) class I molecules in tumor cells. While immunization can be improved, deficient MHC class I expression remains a problem, because it hampers the ability of tumor cells to present antigens for killing by CD8+ T cells. These are the major mediators of tumor destruction, and they have little or no activity against antigen-negative bystander cells. However, there are reasons to be optimistic that therapeutic vaccination against cancer antigens might become a reality at last.

Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment.

Previously, we have reported on successful imaging of colon, rectal, and pancreatic carcinomas in patients by using a radiolabeled all-human monoclonal antibody, COU-1, directed against modified cytokeratin. To further develop this antibody for use as an immunoconjugate, COU-1 was cloned by phage display selection and the human Fab fragment was expressed in bacteria. Analysis by confocal laser scanning microscopy demonstrated that COU-1 bound in a uniform punctate pattern to the surface of viable carcinoma cells stained at 4 degrees C, and binding increased significantly when cells were cultured on fibronectin, laminin, or collagen IV. In the case of fibronectin, COU-1 staining was particularly enhanced at intercellular junctions. When carcinoma cells were cultured with COU-1 at 37 degrees C for 6 hr, the antibody was found in large perinuclear vesicles and the punctate surface staining was significantly reduced. Similar results were obtained using intact IgM COU-1 and the recombinant Fab fragment. Immunohistological studies indicated that COU-1, in contrast to murine monoclonal antibodies against normal cytokeratin 8 and 18, could differentiate between malignant and normal colon epithelia, and between colon cancer metastasis in the liver and surrounding normal hepatocytes. Within biopsies of malignant tissue, COU-1 exhibited membrane-associated staining of proliferating cells, while resting cells had a filamentous pattern. Thus, modified cytokeratin at the surface of carcinoma cells may represent a new target for immunoconjugates and may explain the promising results of the phase I/II clinical study.

Construction, expression, and activities of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation.

The L49 (IgG1) monoclonal antibody binds to p97 (melanotransferrin), a tumor-selective antigen that is expressed on human melanomas and carcinomas. A recombinant fusion protein, L49-sFv-bL, that contains the antibody binding regions of L49 fused to the Enterobacter cloacae r2-1 beta-lactamase (bL) was constructed, expressed, and purified to homogeneity in an Escherichia coli soluble expression system. The variable regions of L49 were cloned by reverse transcription-polymerase chain reaction from L49 hybridoma mRNA using signal sequence and constant region primers. Construction of the gene encoding L49-sFv-bL was accomplished by hybridization insertion of VH, VL, and sFv linker sequences onto a pET phagemid template containing the bL gene fused to the pelB leader sequence. Optimal soluble expression of L49-sFv-bL in E. coli was found to take place at 23 degrees C with 50 microM isopropyl beta-D-thiogalactopyranoside induction and the use of the nonionic detergent Nonidet P-40 for isolation from the bacteria. Construction and expression of a soluble form of the p97 antigen in Chinese hamster ovary cells allowed affinity-based methods for analysis and purification of the fusion protein. Surface plasmon resonance, fluorescent activated cell sorting, and Michaelis-Menten kinetic analyses showed that L49-sFv-bL retained the antigen binding capability of monovalent L49 as well as the enzymatic activity of bL. In vitro experiments demonstrated that L49-sFv-bL bound to 3677 melanoma cells expressing the p97 antigen and effected the activation of 7-(4-carboxybutanamido)cephalosporin mustard (CCM), a cephalosporin nitrogen mustard prodrug. On the basis of these results, L49-sFv-bL was injected into nude mice with subcutaneous 3677 tumors, and localization was determined by measuring bL activity. Tumor to blood conjugate ratios of 13 and 150 were obtained 4 and 48 h post conjugate administration, respectively, and the tumor to liver, spleen, and kidney ratios were even higher. A chemically produced L49-Fab'-bL conjugate yielded a much lower tumor to blood ratio (5.6 at 72 h post administration) than L49-sFv-bL. Therapy experiments established that well-tolerated doses of L49-sFv-bL/CCM combinations resulted in cures of 3677 tumors in nude mice. The favorable pharmacokinetic properties of L49-sFv-bL allowed prodrug treatment to be initiated 12 h after the conjugate was administered. Thus, L49-sFv-bL appears to have promising characteristics for site-selective anticancer prodrug activation.

Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors.

The 4-1BB glycoprotein is a member of the tumor necrosis factor receptor superfamily and binds to a high-affinity ligand (4-1BBL) expressed on several antigen-presenting cells such as macrophages and activated B cells. Expression of 4-1BB is restricted to primed CD4+ and CD8+ T cells, and 4-1BB signaling either by binding to 4-1BBL or by antibody ligation delivers a dual mitogenic signal for T-cell activation and growth. These observations suggest an important role for 4-1BB in the amplification of T cell-mediated immune responses. We now show that administration of anti-4-1BB monoclonal antibodies can eradicate established large tumors in mice, including the poorly immunogenic Ag104A sarcoma and the highly tumorigenic P815 masto cytoma. The immune response induced by anti-4- 1BB monoclonal antibodies is mediated by both CD8+ and CD4+ T cells and is accompanied by a marked augmentation of tumor-selective cytolytic T-cell activity. Our data suggest that a similar approach may be efficacious for immunotherapy of human cancer.

The role of B7-2 (CD86) in tumour immunity.

Tumour cells engineered to express co-stimulatory molecules on their surface provide researchers with powerful new tools to manipulate antitumour responses. It has been demonstrated that B7-1+ and B7-2+ tumour cells can elicit effective responses against their wild-type counterparts. This response is primarily mediated by CD8+ cytolytic T-lymphocytes. The co-stimulatory ability of B7-2+ tumour cells is comparable to that of B7-1+ tumour cells, though with some exceptions. However, on host antigen-presenting cells (APC), B7-2 plays a dominant role in inducing T-cell-mediated immune responses. Up-regulation of B7-2 on host APC may, therefore, present an effective means of generating potent antitumour immunity.