The promise and pitfalls of monoclonal antibody therapeutics

Recent experience has helped to clarify the best ways to use monoclonal antibodies to solve clinical problems. For example, imaging based on tumor antigens, rather than tumor size, will permit early detection of cancer and accurate staging. Blocking receptor—ligand interactions may permit therapeutic intervention in cell growth or function but activity may depend on the choice of an antiligand or antireceptor strategy. Humanized antibodies will achieve greater intensity and duration of therapy, while allowing repeat administration in chronic diseases.     

Therapeutic Use of Antimelanoma Monoclonal Antibodies

Monoclonal antibodies (MAb) to tumor-associated antigens are attracting much attention for tumor therapy. Melanomas belong to the tumors most studied in this respect, and several melanoma-associated antigens have been studied in great detail. These include the melanoma-associated glycoprotein p97, the melanoma-associated proteoglycan, and glycolipid antigens. Although none of the antigens is absolutely specific for tumor, the degree of relative specificity appears to be sufficient to use several of the melanoma antigens as therapeutic “targets”. Antimelanoma MAb can be applied therapeutically in several ways. The most straightforward approach is use of MAb without further modification. MAb which kill melanoma cells in the presence of human serum as the source of complement or mediate antibody-dependent cellular cytotoxicity with human natural killer (NK) cells or macrophages as effectors are logical choices for this. Some cases of partial or even complete regression of metastatic melanoma have been observed in patients treated with such MAb. Combinations of such MAb with interleukin 2 (IL-2) or other immunological response modifiers are of great interest. Alternatively, one may use antimelanoma MAb (or fragments prepared from MAb) as carriers of antitumor agents, including radioactive isotopes, toxins, or chemotherapeutic drugs. Although it is premature to make any conclusions about the efficacy of such conjugates, we are optimistic that it will be feasible by using the right combination of MAb and antitumor agent to achieve therapeutic benefit. Another approach is to develop therapeutic “vaccines” for active immunization, once an antigen characterized by using a MAb has proven to have a relatively high level of tumor selectively. Anti-idiotypic antibodies and live recombinant viruses inducing tumor antigen expression in infected cells provide alternative strategies to this approach.     

Methods to label monoclonal antibodies for use in tumor imaging

The use of radiolabeled monoclonal antibodies as a diagnostic tool in nuclear medicine has grown rapidly over the past several years. Early studies utilized antibodies labeled with radionuclides of iodine (i.e. ¹²⁵I and ¹³¹I) although these radionuclides are not ideal for use in tumor imaging. Recent advances and the development of new chemical methods has made it possible to label antibodies with other radionuclides (i.e. ⁷⁷Br, ¹¹¹In and ^99mTc). The advantages and disadvantages associated with all of the different radionuclides and labeling methods will be discussed.     

Serotherapy of ovarian cancer

The development of monoclonal antibodies has permitted the identification of several ovarian-tumor-associated antigens which might serve as targets for serotherapy in vivo. With the exception of antibodies directed against growth factor receptors, unmodified monoclonal reagents must activate complement (C') components or bind effector cells to destroy tumor targets. Antibody-dependent cell-mediated cytotoxicity (ADCC) may be particularly important for eliminating tumor cells in vivo. A shortage of functionally active effector cells can limit the efficacy of serotherapy with heteroantisera or monoclonal reagents. The use of immunostimulants such as Corynebacterium parvum has increased the number and activity of effector cells for ADCC within the peritoneal compartment of mice and of patients with ovarian cancer. Intraperitoneal serotherapy can achieve direct contact between antibody and microscopic deposits of ovarian tumor cells which persist following cytoreductive operations and cytotoxic chemotherapy. Conjugation of monoclonal antibodies with radionuclides, drugs or toxins might increase the potency of serotherapy and circumvent the effector shortage. Clinical studies to date have evaluated radionuclide conjugates for imaging and for therapy. Patients with a small volume of disease have responded to treatment. Preclinical models suggest that drug and toxin conjugates might also prove active. Recent studies have demonstrated a synergistic interaction between different immunotoxins. Ovarian carcinoma is likely to be a valuable clinical model for evaluating immunoconjugates which react with epithelial tumor cells.     

Development of humanized antibodies as cancer therapeutics

Recent success in the development of monoclonal antibody-based anti-cancer drugs has largely benefitted from the advancements made in recombinant technologies and cell culture production. These reagents, derived from the antibodies of mouse origin, while maintaining the exquisite specificity and affinity to the tumor antigens, have low immunogenicity and toxicity in human. High-level expressing cell clones are generated and used to produce large quantities of the recombinant antibodies in bioreactors in order to meet the clinical demand for therapeutic applications. In this report, the systems and general methodologies developed by us to construct and produce humanized antibodies from the parent mouse antibodies are described. Once the humanized antibodies are available, they can be applied in three principal forms for cancer therapy: (1) naked antibodies, (2) drug- or toxin conjugates, and (3) radioconjugates. Using the humanized anti-CD22 (epratuzumab) and anti-carcinoembryonic antigen (ant-CEA; labetuzumab) antibody prototypes, clinical applications of naked and radiolabeled humanized monoclonal antibodies are described.     

Tumor-directed targeting of liposomes

Tumor-specific targeting is a critical goal in the research area of liposomal drug delivery. Identification of the specific interactions between ligands and target tumor cells is a principle prerequisite in achieving this goal. Generally, tumor cells aberrantly express tumor-associated antigens that can be utilized as appropriate target molecules. Monoclonal antibodies against tumor-associated antigens have been successfully adopted for targeting to various types of cancer cells. The incorporation of humanized monoclonal antibodies or single chain human antibodies, instead of rodent antibodies into immunoliposomes has resulted in better clinical applicability. Tumor-specific ligands other than monoclonal antibodies have also been investigated as in vivo tumor-directing molecules. However, the number of pre-clinical studies of anticancer treatments using tumor-specific liposomal drugs reporting successful targeting and enhanced therapeutic efficacy has been limited. Further refinement of tumor-specific interactions and liposomal formulations will be necessary for the application of the tumor-specific liposomal drug strategy for anticancer chemotherapy or gene therapy.     

The combination of a chemokine,cytokine and TCR-based T cell stimulus for effective gene therapy of cancer

Cytotoxic T cells can recognize and kill tumor cells that present peptides derived from tumor-associated antigens (TAA) on their surface when associated with major histocompatibility complex (MHC) class I molecules. However, immune responses to tumor-associated antigens are often suppressed by a tumor-induced state of immune anergy. Previous work has attempted to overcome tumor-induced T cell anergy by the direct injection of vectors carrying genes encoding one of a variety of cytokines. Polyclonal stimulation of T cells, preferably via the TCR complex, results in a cascade of cytokines associated with T cell activation and thus may be better able to overcome T cell anergy. We have previously reported the use of the highly attenuated MVA poxvirus to express on tumor cells, in vitro and in vivo, antibodies specific for the CD3epsilon chain (KT3). When injected into growing tumors, these constructs induce the activation of immune effector cells and result in rejection of the tumor. A variety of recombinant adenovirus (Ad) vectors expressing immunostimulatory and/or immunoattractant molecules have now been produced. With this collection of viruses, we have carried out in vivo analyses of combinations of vectors in tumor therapy experiments. For example, we have tested, in murine tumor models, the combination of MVA-KT3 with Ad expressing recently identified cytokines [for example interleukin-12 (IL-12), IL-18] as well as chemokines (e.g. RANTES, MIP1beta). One combination, MVA-KT3/Ad-IL-12/Ad-MIP1beta causes rejection of 100% of growing RENCA tumors. Much attention has been focused on cancer gene therapy using gene transfer of single agents. These data show that antigenic stimulation via the MHCI/TCR-CD3+cytokine+chemokine combination may provide a new and promising approach to cancer gene therapy which is more likely to bypass tumor immunosuppression mechanisms.     

The reverse proteomics for identification of tumor antigens

The identification of tumor antigens is essential for the development of anticancer therapeutic vaccines and clinical diagnosis of cancer. SEREX (serological analysis of recombinant cDNA expression libraries) has been used to identify such tumor antigens by screening sera of patients with cDNA expression libraries. SEREX-defined antigens provide markers for the diagnosis of cancers. Potential diagnostic values of these SEREX-defined antigens have been evaluated. SEREX is also a powerful method for the development of anticancer therapeutics. The development of anticancer vaccines requires that tumor antigens can elicit antigen-specific antibodies or T lymphocytes. More than 2000 antigens have been discovered by SEREX. Peptides derived from some of these antigens have been evaluated in clinical trials. This review provides information on the application of SEREX for identification of tumor-associated antigens (TAA) for the development of cancer diagnostics and anticancer therapeutics.     

Role of tumor-associated gangliosides in cancer progression

Neuroectodermic tumors can mostly be characterized by the presence of tumor-associated glycosphingolipid antigens, such as gangliosides, defined by monoclonal antibodies. Recently, cumulative evidence indicates that gangliosides modify the biological effects of several trophic factors, in vitro and in vivo, as well as the mitogenic signaling cascade that these factors generate. The functional roles of gangliosides in tumor progression can be revisited: (i) ganglioside antigens on the cell surface, or shed from the cells, act as immunosuppressors, as typically observed for the suppression of cytotoxic T cells and dendritic cells, (ii) certain gangliosides, such as GD3 or GM2, promote tumor-associated angiogenesis, (iii) gangliosides strongly regulate cell adhesion/motility and thus initiate tumor metastasis, (iv) ganglioside antigens are directly connected with transducer molecules in microdomains to initiate adhesion coupled with signaling, and (v) ganglioside antigens and their catabolites are modulators of signal transduction through interaction with tyrosine kinases associated with growth factor receptors or other protein kinases. Given the potential importance of these sialylated gangliosides and their modulating biological behavior in vivo, further studies on the role of gangliosides are warranted.     

A human IgE bispecific antibody shows potent cytotoxic capacity mediated by monocytes

The generation of bispecific antibodies (bsAbs) targeting two different antigens opens a new level of specificity and, compared to mAbs, improved clinical efficacy in cancer therapy. Currently, the different strategies for development of bsAbs primarily focus on IgG isotypes. Nevertheless, in comparison to IgG isotypes, IgE has been shown to offer superior tumor control in preclinical models. Therefore, in order to combine the promising potential of IgE molecules with increased target selectivity of bsAbs, we developed dual tumor-associated antigen-targeting bispecific human IgE antibodies. As proof of principle, we used two different pairing approaches - knobs-into-holes and leucine zipper–mediated pairing. Our data show that both strategies were highly efficient in driving bispecific IgE formation, with no undesired pairings observed. Bispecific IgE antibodies also showed a dose-dependent binding to their target antigens, and cell bridging experiments demonstrated simultaneous binding of two different antigens. As antibodies mediate a major part of their effector functions through interaction with Fc receptors (FcRs) expressed on immune cells, we confirmed FcεR binding by inducing in vitro mast cell degranulation and demonstrating in vitro and in vivo monocyte-mediated cytotoxicity against target antigen-expressing Chinese hamster ovary cells. Moreover, we demonstrated that the IgE bsAb construct was significantly more efficient in mediating antibody-dependent cell toxicity than its IgG1 counterpart. In conclusion, we describe the successful development of first bispecific IgE antibodies with superior antibody-dependent cell toxicity–mediated cell killing in comparison to IgG bispecific antibodies. These findings highlight the relevance of IgE-based bispecific antibodies for clinical application.     

Monoclonal antibody drug immunoconjugates for targeted treatment of cancer

Monoclonal antibodies (mAb) directed to tumor-associated antigens (TAA) or antigens differentially expressed on the tumor vasculature have been covalently linked to drugs that have different mechanisms of action and various levels of potency. The use of these mAb immunoconjugates to selectively deliver drugs to tumors has the potential to both improve antitumor efficacy and reduce the systemic toxicity of therapy. Several immunoconjugates, particularly those that incorporate internalizing antibodies and tumor-selective linkers, have demonstrated impressive activity in preclinical models. Immunoconjugates that deliver doxorubicin, maytansine and calicheamicin are currently being evaluated in clinical trials. The feasibility of using immunoconjugates as cancer therapeutics has been clearly demonstrated. Gemtuzumab ozogamicin, a calicheamicin conjugate that targets CD33, has recently been approved by the Food and Drug Administration (FDA) for treatment of acute myelogenous leukemia (AML). This review concentrates on the properties of the tumor and the characteristics of the mAb, linker, and drugs that influence the efficacy, potency, and selectivity of immunconjugates selected for cancer treatment.     

Liposome encapsulated tumor-associated antigens elicited humoral and cellular immune responses in mice bearing tumor

Chemically induced tumors in mice provide a system to investigate tumor-associated antigens (TAA). The cell surface glycoprotein antigens on such tumor cells have been identified as suitable targets for immune attack. The induction of immune responses against (TAA) in N-nitrosodiethylamine (DEN) exposed mice has been examined. In order to present antigens to the immune system, the liposome was used as vehicle to deliver the TAA. Liposomal-TAA formulation, elicited both humoral and the cellular immune responses, when administered intramuscularly in DEN-exposed mice. Presence of circulatory antibodies against TAA and the induction of cellular responses in immunized mice were monitored using ELISA and in vitro cell proliferation assay of lymphocytes respectively. Specificity of antibody against TAA in immune sera was analysed using immunoblotting technique. Based on these results, it is proposed that the liposome encapsulated TAA may successfully be used to induce humoral and cellular immune responses against tumor.     

Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors

Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, and a number of such reagents are in clinical use. However, responses could not be achieved in all patients with tumors expressing high levels of the respective target antigens, suggesting that other factors such as limited recruitment of endogenous immune effector mechanisms can also influence treatment outcome. This justifies the search for alternative, potentially more effective reagents. Antibody-toxins and cytolytic effector cells genetically modified to carry antibody-based receptors on the surface, represent such tailor-made targeting vehicles with the potential of improved tumor localization and enhanced efficacy. In this way, advances in recombinant antibody technology have made it possible to circumvent problems inherent in chemical coupling of antibodies and toxins, and have allowed construction via gene fusion of recombinant molecules which combine antibody-mediated recognition of tumor cells with specific delivery of potent protein toxins of bacterial or plant origin. Likewise, recombinant antibody fragments provide the basis for the construction of chimeric antigen receptors that, upon expression in cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells, link antibody-mediated recognition of tumor antigens with these effector cells potent cytolytic activities, thereby making them promising cellular therapeutics for adoptive cancer therapy. Here, general principles for the derivation of cytotoxic proteins and effector cells with antibody-dependent tumor specificity are summarized, and current strategies to employ these molecules and cells for directed cancer therapy are discussed, focusing mainly on the tumor-associated antigens epidermal growth factor receptor (EGFR) and the closely related ErbB2 (HER2) as targets.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

A versatile bifunctional chelate for radiolabeling humanized anti-CEA antibody with In-111 and Cu-64 at either thiol or amino groups: PET imaging of CEA-positive tumors with whole antibodies

Radiolabeled anti-carcinoembryonic antigen (CEA) antibodies have the potential to give excellent images of a wide variety of human tumors, including tumors of the colon, breast, lung, and medullar thyroid. In order to realize the goals of routine and repetitive clinical imaging with anti-CEA antibodies, it is necessary that the antibodies have a high affinity for CEA, low cross reactivity and uptake in normal tissues, and low immunogenicity. The humanized anti-CEA antibody hT84.66-M5A (M5A) fulfills these criteria with an affinity constant of >10 (10) M (-1), no reactivity with CEA cross-reacting antigens found in normal tissues, and >90% human protein sequence. A further requirement for routine clinical use of radiolabeled antibodies is a versatile method of radiolabeling that allows the use of multiple radionuclides that differ in their radioemissions and half-lives. We describe a versatile bifunctional chelator, DO3A-VS (1,4,7-tris(carboxymethyl)-10-(vinylsulfone)-1,4,7,10-tetraazacyclododecane) that binds a range of radiometals including 111 In for gamma-ray imaging and 64Cu for positron emission tomography (PET), and which can be conjugated with negligible loss of immunoreactivity either to sulfhydryls (SH) in the hinge region of lightly reduced immunoglobulins or surface lysines (NH) of immunoglobulins. Based on our correlative studies comparing the kinetics of radiolabeled anti-CEA antibodies in murine models with those in man, we predict that 64Cu-labeled intact, humanized antibodies can be used to image CEA positive tumors in the clinic.     

Vaccine therapy for renal cell carcinoma

Several potential vaccines have been evaluated for the treatment of patients with renal cell carcinoma (RCC). They include dendritic cells pulsed with tumor lysate, a dendritic cell-tumor cell hybrid, irradiated tumor cells admixed with adjuvants, and a heat shock protein-peptide complex. Promising results have been obtained in several early clinical trials, but issues of tumor immunosuppression and lack of identified tumor-associated antigens must be addressed before vaccine therapy can be applied successfully in advanced RCC. In this patient population, vaccine therapy will likely be required in combination with other forms of immunotherapy, such as interleukin-2 and thalidomide. In contrast, vaccine therapy alone may be sufficient for high-risk patients in the adjuvant setting.     

Activation of prodrugs by antibody-enzyme conjugates: a new approach to cancer therapy

A new strategy for the delivery of cytotoxic agents to solid tumors is described in which monoclonal antibodies are used as carriers for enzymes to tumor cell surfaces. The enzymes are chosen for their abilities to convert relatively noncytotoxic drug precursors (pro-drugs) into active anticancer drugs. The drugs thus formed can then penetrate into nearby tumor cells, resulting in cell death. A number of prodrugs have been developed that can be transformed into active anti-cancer drugs by enzymes of both mammalian and non-mammalian origin. The enzymes have been shown to localize into tumors when linked to monoclonal antibodies that bind to tumor-associated antigens. In vivo studies indicate that MAb-enzyme/prodrug combinations can result in antitumor activities significantly greater than those of the prodrugs or drugs given alone. This is most likely due to the generation of large amounts of active drug at the tumor site.     

Malignant Glial Neoplasms: Definition of a Humoral Host Response to Tumor-Associated Antigen(s)

There is increasing evidence that human tumors possess tumor-associated neo-antigens. The host mounts an immunological response to these antigens, as evidenced by the detection of circulating humoral antibodies in a variety of human neoplasia.An indirect immunofluorescent antibody technique was employed to detect antibodies to tumor-associated antigens in the sera of patients with malignant gliomas. Viable single cell suspensions were used to demonstrate antibodies to surface contents of tumor cells and cell preparations were snap-frozen at −160° C to demonstrate antibodies to cytoplasmic components of tumor cells. After incubation with serum, the preparations were treated with polyvalent sheep antihuman globulin conjugated to isomer-1-fluorescein isothiocyanate, washed, and examined with a Leitz incident fluorescent microscope.Of the 17 sera from histologically proven malignant glial neoplasm patients, 2 (11%) were positive for an autologous surface antibody reaction. Five (23%) of 21 were positive for an autologus cytoplasmic antibody, however, 10 (47%) of 21 of the sera gave a positive reaction for cross-reacting cytoplasmic antibodies when tested with a battery of tumor cells obtained from different patients with malignant glial tumors.No reaction was observed with normal brain tissue. Absorption studies indicated the presence of a tumor-associated antigen.This study demonstrated that certain patients with malignant gliomas possess circulating antibodies to cytoplasmic components of their own tumor cells. The fact that a number of sera cross-reacted with tumor cells obtained from different patients suggests that antigenic cross-reactivity exists between malignant glioma cells from different patients. It is suggested that with further refinement, immunofluorescent detection of antibodies could evolve as a useful diagnostic adjunct in malignant glioma.     

Biological response modifiers in cancer

We have seen a surge in the use of immunotherapy for the treatment of cancer. Biological response modifiers can act passively by enhancing the immunologic response to tumor cells or actively by altering the differentiation/growth of tumor cells. Active immunotherapy with cytokines such as interferons (IFNs) and interleukins (IL-2) is a form of nonspecific active immune stimulation. The use of IL-2 has recently been approved by the United States Food and Drug Administration (FDA) for the treatment of renal cell carcinoma and metastatic colorectal cancer. Considerable success has been achieved with the use of immunotherapy, especially in the area of passive immunotherapy using monoclonal antibodies--in particular, radiolabeled monoclonal antibodies. In addition to the various monoclonal antibodies that have been used in clinical trials, other strategies such as the use of antiangiogenic agents and matrix metalloprotease inhibitors (MMPIs) have also met with some success. Recently, the FDA approved bevacizumab, an anti-vascular endothelial growth factor (VEGF) agent, for the treatment of metastatic melanoma. This review also sheds light on the various angiogenesis inhibitors in clinical trials, the increasing use of thalidomide in cancer, and the upcoming potential cancer vaccines designed to activate cell-mediated immune responses against tumor antigens.     

Antigen forks: bispecific reagents that inhibit cell growth by binding selected pairs of tumor antigens

Bispecific antibodies of a new category, termed antigen forks, were constructed by crosslinking antibodies that recognized pairs of distinct tumor cell surface antigens. At concentrations of 1–100 nM, several such forks inhibited the growth of human tumor cell lines bearing both relevant antigens. The same cells were not inhibited by unconjugated component antibodies, and the active conjugates did not inhibit the growth of human cell lines that expressed lower levels of relevant antigens. The three most active antigen forks all contained monoclonal antibody 454A12, which recognizes human transferrin receptor. This antibody was conjugated respectively to antibodies 113F1 (against a tumor-associated glycoprotein complex), 317G5 (against a 42-kDa tumor-associated glycoprotein), or 520C9 (against the c-erbB-2 protooncogene product). The 317G5-454A12 fork strongly inhibited the HT-29 and SW948 human colorectal cancer cell lines, while the 113F1-454A12 fork was also effective against SW948. By designing forks against antigens of incompatible function that are co-expressed at high levels on tumor cells but not on normal tissues, it may be possible to generate reagents that inhibit tumor growth with enhanced selectivity.     

Interleukin 15 as a promising candidate for tumor immunotherapy

Interleukin 15 participates in the development of important immune antitumor mechanisms. It activates CD8(+) T cells, natural killer (NK) cells, NK T cells, and can promote the formation of antitumor antibodies. IL-15 can also protect T effector cells from the action of T regulatory cells and reverse tolerance to tumor-associated antigens. In pre-clinical studies IL-15 has been found to demonstrate potentiated antitumor effects following pre-association with IL-15Rα, or when used in combination with chemotherapy, adoptive therapy, monoclonal antibodies, and tumor vaccines. Although a clinical trial based on application of IL-15 in tumor patients has already begun, it is important to be aware of its potential side effects, including induction of autoimmunity and promotion of proliferation, survival, and dissemination of some tumor cells.