Specific immunotherapy of cancer in elderly patients.

The concept of immunotherapy of cancer is more than a century old, but only recently have molecularly defined therapeutic approaches been developed. In this review, we focus on the most promising approach, active therapeutic vaccination. The identification of tumour antigens can now be accelerated by methods allowing the amplification of gene products selectively or preferentially transcribed in the tumour. However, determining the potential immunogenicity of such gene products remains a demanding task, since major histocompatibility complex (MHC) restriction of T cells implies that for any newly defined antigen, immunogenicity will have to be defined for any individual MHC haplotype. Tumour-derived peptides eluted from MHC molecules of tumour tissue are also a promising source of antigen. Tumour antigens are mostly of weak immunogenicity, because the vast majority are tumour-associated differentiation antigens already 'seen' by the patient's immune system. Effective therapeutic vaccination will thus require adjuvant support, possibly by new approaches to immunomodulation such as bispecific antibodies or antibody-cytokine fusion proteins. Tumour-specific antigens, which could be a more potent target for immunotherapy, mostly arise by point mutations and have the disadvantage of being not only tumour-specific, but also individual-specific. Therapeutic vaccination will probably focus on defined antigens offered as protein, peptide or nucleic acid. Irrespective of the form in which the antigen is applied, emphasis will be given to the activation of dendritic cells as professional antigen presenters. Dendritic cells may be loaded in vitro with antigen, or, alternatively, initiation of an immune response may be approached in vivo by vaccination with RNA or DNA, given as such or packed into attenuated bacteria. The importance of activation of T helper cells has only recently been taken into account in cancer vaccination. Activation of cytotoxic T cells is facilitated by the provision of T helper cell-derived cytokines. T helper cell-dependent recruitment of elements of non-adaptive defence, such as leucocytes, natural killer cells and monocytes, is of particular importance when the tumour has lost MHC class I expression. Barriers to successful therapeutic vaccination include: (i) the escape mechanisms developed by tumour cells in response to immune attack; (ii) tolerance or anergy of the evoked immune response; (iii) the theoretical possibility of provoking an autoimmune reaction by vaccination against tumour-associated antigens; and (iv) the advanced age of many patients, implying reduced responsiveness of the senescent immune system.

Interleukin 2-antibody and tumor necrosis factor-antibody fusion proteins induce different antitumor immune responses in vivo.

Two fusion proteins, composed of interleukin 2 (IL-2) or tumor necrosis factor (TNF) coupled to an antibody [fusion protein (FuP); IL-2-FuP or TNF-FuP], were capable of retarding growth of a human malignant melanoma in the severe combined immunodeficient mouse depending on the concomitant application of human peripheral blood leukocytes. Here we have analyzed the mechanisms that determine the therapeutic effect. Tumor-bearing severe combined immunodeficient mice received once per week an i.v. injection of HLA-matched peripheral blood leukocytes and twice per week i.v. or intratumoral injections of FUPS: Leukocyte recovery and their activation state were monitored. The number of draining lymph node cells (LNCs) and tumor-infiltrating leukocytes increased continuously, and the yield of draining LNCs was improved significantly when the FuPs were applied locally. In IL-2-FuP-treated mice, the majority of draining LNCs and tumor-infiltrating lymphocytes expressed T-cell activation markers and IL-2, thus being classified as T helper type 1 cells. These cells displayed strong proliferative activity and initiated activation of lymphokine-activated killer cells and CTLS: TNF-FuP supported activation of CTLs and of monocytes as revealed by TNF expression and cytostatic activity. Neither the antibody, nor IL-2, nor TNF, nor the mixture of antibody and cytokines exhibited the full-fledged activational potency of the FUPS: Notably, activation of immune effector mechanisms was much stronger when the FuPs were applied intratumorally. This is the first report to show that FuPs are efficient immunostimulants in vivo for native leukocytes. Although IL-2-FuP induced a T helper type 1 response with recruitment of LAK and CTL, TNF-FuP efficiently recruited and activated monocytes and, in a less pronounced manner, CTLS:

Efficacy of local versus systemic application of antibody-cytokine fusion proteins in tumor therapy.

Application of immunocytokines [fusion proteins (FuPs)] where the cytokine has been coupled to an antibody may not produce the severe side effects frequently observed during systemic application of cytokines in cancer therapy. However, it has not been explored whether FuPs are sufficient for intratumoral activation of leukocytes or whether intratumoral versus systemic application may be of greater efficacy. Interleukin 2 (IL2) or tumor necrosis factor (TNF) coupled to an anti-epidermal growth factor receptor monoclonal antibody (IL2-FuP or TNF-FuP) were tested in SCID mice bearing a human epidermal growth factor receptor-positive melanoma transplant and being reconstituted with human HLA-matched peripheral blood leukocytes. Whole-body autoradiography revealed larger accumulation and prolonged retention of i.v. or intratumorally applied IL2-FuP or TNF-FuP compared with the antibody. Even with low doses of FuP, tumor growth was significantly retarded, with the survival time being further prolonged by the intratumoral application. Furthermore, outgrowth of the tumor was prevented in approximately 50% of mice as long as they received weekly injections of peripheral blood leukocytes concomitantly with the FuPs, which confirmed that it was the donor leukocytes activated in vivo that retarded tumor growth. An in vitro analysis revealed that the IL2-FuP supported mainly proliferation and lymphokine-activated killer cell activity, whereas TNF-FuP stimulated cytokine production and cytotoxic activity of monocytes and, to a low degree, of T cells. Both TNF-FuP and IL2-FuP significantly accumulated in the tumor, which led to retardation of tumor growth. The therapeutic effect was improved by intratumoral application. Importantly, the efficacy of both IL2-FuP and TNF-FuP depended on the induction of an immune response in vivo.

Cloning of the human homologue of the metastasis-associated rat C4.4A.

We have previously described a rat metastasis-associated molecule, C4.4A, which has some common features with the uPAR. Because of its restricted expression in non-transformed tissues a search for the human homologue became of interest. Human C4.4A was cloned from a placental cDNA library. As in the rat, the human uPAR and the human C4.4A genes appear to belong to the same family. Both genes are located on chromosome 19q13.1-q13.2 and both molecules have a glycolipid anchor site and are composed of three extracellular domains. Only domains one and two of the human C4.4A and the uPAR protein show a significant degree of identity. Expression of the human C4.4A was observed by RT-PCR and Northern blotting in placental tissue, skin, esophagus and peripheral blood leukocytes, but not in brain, lung, liver, kidney, stomach, colon and lymphoid organs. Yet, tumors derived from the latter tissues frequently contained C4.4A mRNA. As demonstrated for malignant melanoma, C4.4A mRNA expression correlated with tumor progression. While nevi were negative and only a minority of primary malignant melanoma expressed C4.4A, all metastases were C4.4A-positive. Taking into account the high degree of homology between rat and human C4.4A, the conformity of the expression profiles and the association of rat C4.4A with tumor progression, human C4.4A might well become a prognostic marker and possibly a target of therapy.

Neovascular targeting with cyclic RGD peptide (cRGDf-ACHA) to enhance delivery of radioimmunotherapy.

Radioimmunotherapy (RIT) has been hampered by delivery of only a small fraction of the administered dose of radiolabeled MAb to tumor. A strategy for creating and controlling tumor vascular permeability would enable more effective RIT. The alpha v beta 3 integrin receptor is an appealing target for strategies designed to enhance permeability of tumor vessels because it is highly and preferentially expressed in most tumors. In human tumor mouse models, apoptosis of neovascular endothelial cells has been demonstrated after treatment with alpha v beta 3 antagonists. Since this apoptotic effect could transiently increase permeability of tumor blood vessels, radiolabeled antibodies (MAb) circulating during this period would have increased access to extravascular tumor. To determine if this hypothesis was correct, a pharmacokinetic study of an immunospecific MAb given after an alpha v beta 3 antagonist was performed in nude mice bearing human breast cancer xenografts. The alpha v beta 3 antagonist, cyclic RGD pentapeptide (c-RGDf-ACHA; cyclo arginine glycine aspartic acid D-phenylalanine -1 amino cyclohexane carboxylic acid), inhibits alpha v beta 3 binding to its vitronectin ligand at nanomolar levels. Cyclic RGD peptide (250 micrograms i.p.) given 1 hour before 111In-ChL6 MAb resulted in a 40-50% increase in tumor uptake (concentration), when compared to the control tumor uptake, of MAb 24 hours after administration. When cyclic RGD peptide was given as a continuous infusion (17.5 micrograms/hr) for 1 or 24 hours before 111In-ChL6, tumor uptake of 111In-ChL6 was increased less, and, these data were not statistically different from the control data. There were no differences for any of the groups in the groups in the concentrations of 111In-ChL6 in normal organs or blood when compared to the control group. The results suggest that cyclic RGD peptide provided a temporary, selective increase in tumor vascular permeability, that allowed a larger fraction of the 111In-ChL6 to accumulate in the tumor.

Vaccination: a future modality of cancer therapy.

With an expanding list of known tumor antigens and increasing knowledge of the requirements for activation of immune effector functions, immunotherapy of cancer is about to become the fourth weapon in the fight against cancer, together with surgery, radiation and chemotherapy. Although our knowledge has come mostly from experimental models, the mechanistic premises observed in these models will provide a reasonable basis for immunotherapeutic protocols in cancer patients.

Cancer therapy: new concepts on active immunization.

There is increasing evidence that tumors express putative target molecules for a therapeutic immune reaction. Yet, tumor cells lack the prerequisites for appropriate antigen presentation and--hence--the immune system does not respond. This difficulty can probably be circumvented when tumor antigens are processed by conventional antigen presenting cells. Thus, the identification of immunogenic tumor-associated antigens may allow new modes of vaccination with the hope of adding a fourth and hopefully powerful weapon to surgery, radiation and chemotherapy in the fight against cancer.

Tumor therapy with bispecific antibody: the targeting and triggering steps can be separated employing a CD2-based strategy.

For tumor therapy with unprimed effector cells, we developed a novel combination of a CD2 x tumor Ag bispecific targeting Ab and an anti-CD2 triggering Ab. These Ab constructs were derived from two novel CD2 mAbs, termed M1 and M2 that, together, but not individually activate T cells. Unlike many other CD2 Abs, M1 and M2 do not interfere with TCR/CD3 triggering nor do they inhibit binding of CD2 to its ligand CD58, thus preserving the physiological functions of these important effector cell molecules. M2 was chemically conjugated with an Ab recognizing the epidermal growth factor-receptor (EGF-R). Incubation of unprimed peripheral blood mononuclear cells with the bispecific F(ab')2 construct (M2xEGF-R) in the presence of trigger Ab M1 led to efficient selective lysis of EGF-R-positive targets by CTL and NK cells. Importantly, the need for trigger Ab M1 for effector cell stimulation allowed to separate targeting from triggering steps in vitro and should thus enable to focus immune responses to sites of target Ag expression in vivo.

High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions.

Functional bivalent miniantibodies, directed against the epidermal growth factor receptor, accumulated to more than 3 gl-1 in high-cell-density cultures of Escherichia coli RV308(pHKK) on a pilot scale. The miniantibodies consist of scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif, which homodimerizes in vivo. The improved expression vector pHKK is characterized by the hoklsok suicide system, improving plasmid maintenance, and the inducible lac pl o promoter system with the very strong T7g10 Shine-Dalgarno sequence. The expression unit is flanked by terminators. The prototrophic RV308 cells were cultivated in glucose mineral salt medium and reached a cell density of 145 g dry biomass l-1 after 33 h. After induction, growth continued almost unchanged for a further 4 h with concomitant miniantibody formation. In the fedbatch phase, the concentration of glucose was kept almost constant at the physiological level of approximately 1.5 gl-1, using on-line flow injection analysis for control. Surprisingly, E. coli RV308(pHKK) did not accumulate significant amounts of the metabolic by-product acetate under these unlimited aerobic growth conditions.

Development of a bispecific F(ab')2 conjugate against the complement receptor CR3 of macrophages and a variant CD44 antigen of rat pancreatic adenocarcinoma for redirecting macrophage-mediated tumor cytotoxicity.

A bispecific F(ab')2 antibody conjugate (BAC) was constructed against the complement receptor CR3 of macrophages and variant CD44 (CD44v6) antigen of rat pancreatic adenocarcinoma cells to redirect macrophage-mediated tumor cytotoxicity. The Fab' fragments of monoclonal antibodies (mAb) 1.1ASML and OX42, recognizing the CD44v6 and the CR3 antigens respectively, were chemically coupled at the hinge region using 5,5'-dithiobis(2-nitrobenzoate). The BAC was characterized in vitro for its specific, dual binding capacity to CD44v6 and CR3 antigens. Although the monovalence of the BAC resulted in lower avidities to both the antigens as expected, it was still able to form stable cross-linkages between tumor cells and macrophages in culture leading to the formation of "clump-like" cell aggregates. The in vitro and in vivo tumor-targeting capacity of the BAC was compared with that of the parental antitumor mAb 1.1ASML, which mediates tumor killing by antibody-dependent cell cytotoxicity. These results showed that, even though the bivalent mAb 1.1ASML did not mediate stable cross-linking of target and effector cells, its Fc-receptor-mediated killing of tumor cells was more effective when compared to the BAC. Thus, this study strongly supports the hypothesis that firm persistent binding between effector and target cells per se is not as important as the choice of trigger molecule used for macrophage activation to redirect their tumor cytotoxic potential effectively.

Developmentally regulated expression of metastasis-associated antigens in the rat.

The specificity of monoclonal antibodies (mABs) obtained after immunization with a metastasizing rat tumor line was evaluated by screening expression in a variety of nonmetastasizing and metastasizing rat tumor lines. mABs, which by immunohistology and Western blotting recognized metastasizing lines, were used to define the physiological expression of the corresponding antigens during ontogeny as well as in adult rats. From a panel of 12 mABs, 2 recognized structures on metastasizing and nonmetastasizing tumor lines, while 10 stained exclusively metastasizing lines. Five of the latter bound to tumor lines metastasizing either hematogenously or via the lymphatic system. All five recognized an epitope on CD44 variant exon v6. The five remaining mABs, recognizing four independent antigenic entities, only stained tumor lines metastasizing via the lymphatics. Surprisingly, these antigens were also detected in normal tissues: three on epithelial cells either widespread or of the upper gastrointestinal tract or the urogenital system, the fourth preferentially on epithelial cells, but also on nerves and hematopoietic precursor cells, and the fifth on many tissues and cells with a predominance of mesenchyme-derived structures. Notably, during ontogeny, expression on these five antigens was induced in different compartments of the developing fetal and/or maternal part of the placenta. The five newly described metastasis-associated antigens share with CD44v the absence of expression on nonmetastasizing tumor lines as well as expression on distinct, nontransformed cells and induction of expression during ontogeny. Thus, tumor progression may rather be initiated by inappropriate expression or up-regulation of genes, which do not display transforming features, than by de novo appearance of "metastasis genes." Accordingly, metastasizing tumor lines may be a valuable tool to identify developmentally regulated gene products.

A fusion protein of IL-8 and a Fab antibody fragments binds to IL-8 receptors and induces neutrophil activation.

A fusion protein was generated by genetic engineering which combined a Fab fragment of a monoclonal antibody directed to the human epidermal growth factor receptor with the biologically active N-terminally truncated 2-72 amino acid form of the human chemokine IL-8. The Fab IL-8 fusion protein was expressed in E. coli and antibody binding and IL-8 activity were determined. Our data indicate that the N-terminus of IL-8 remains functional for receptor interaction. The fusion protein showed specific binding to IL-8 receptors, induced IL-8 mediated chemotactic activity, and the release of MPO activity. However, N-terminal fusion of IL-8 to the carboxyl terminus of the Fab fragment resulted in reduced binding to IL-8 receptors and consequently to reduced biologic activity of IL-8. The affinity of the antibody arm for EGF-R was improved when compared to a monovalent Fab. Fusion proteins as described herein may represent improved therapeutics for cancer therapy based on their potential to selectively increase and prolong cytokine concentration in the tumour. Since chemokines such as IL-8 recruit effector cells and stimulate effector cell function in situ, a lymphocyte-independent anti-tumour activity followed by tumour-specific immunity could be proposed.

Systemic radiotherapy with monoclonal antibodies. An experimental study with human neuroblastoma xenografts in nude mice.

In this experimental study, feasibility and efficiency of systemic radiotherapy with the I-131 labelled monoclonal antibody BW575/9 (radioimmunotherapy) are investigated using human SK-N-SH neuroblastoma transplanted into nude mice. Series of six nude mice were treated with intravenous application of 400 microCi (group 1), 700 microCi (group 2) of the I-131 labelled and of the unlabelled MAb (group 3). An untreated group (group 4) served as control. Tumors of group (3) and (4) showed an identical growth. In group (1), tumor growth was arrested for seven days. In group (2), the tumor showed complete regression after eight days which lasted for 55 days. Thereafter, the tumor started to regrow. This growth characteristics are correlated with the doses achieved in the tumor using a medical internal radiation dose (MIRD) formulation. The biodistribution data necessary for MIRD calculation were obtained by previously performed experiments with the I-125 labelled MAb. The doses assessed in the tumor turned out to be five to ten times greater than those in normal tissues (liver, bone, etc.) These results confirm feasibility, selectivity and efficiency of radioimmunotherapy in the above described model. Moreover, this in vivo model seems suitable for further investigations concerning fundamental issues of radioimmunotherapy.

Development of a bispecific monoclonal antibody against a gallium-67 chelate and the human melanoma-associated antigen p97 for potential use in pretargeted immunoscintigraphy.

A bispecific monoclonal antibody (bsmAb) has been developed against the human melanoma-associated antigen p97 and an octahedral gallium chelate (Ga-HBED) using the hybrid hybridoma technology. As tetradomas were expected to produce a maximum of ten different molecular species of immunoglobulins, the bispecific antibody was purified from this mixture by consecutive protein A affinity and cation-exchange chromatographic techniques. Although it was established by sodium dodecyl sulphate/polyacrylamide gel electrophoresis that the heavy (H) and light (L) chains of the two parental immunoglobulins were mismatched in the bispecific antibody, results from cell enzyme-linked immunosorbent assay indicated significant dual specific binding to both the melanoma cells and 67Ga-HBED. Other in vitro techniques further confirmed that the bsmAb Bi 5-56-II-17 still retained about 30%-40% simultaneous binding capacity to both the antigens, as would have been expected in a bsmAb that has ideally matched H and L chains. Preliminary in vivo experiments using nude mice bearing the human melanoma xenografts showed that the bsmAb Bi 5-56-II-17 was able to target the radioactive gallium chelate to the tumours twice as efficiently compared to the monospecific, bivalent gallium chelate antibody.

Prevention of tumor metastasis formation by anti-variant CD44.

A splice variant of CD44 (CD44v) originally discovered on metastases of a rat pancreatic adenocarcinoma (BSp73ASML) has been shown by transfection to confer metastatic behavior to nonmetastatic tumor cells (Günthert U., M. Hofmann, W. Rudy, S. Reber, M. Zöller, I. Haussmann, S. Matzku, A. Wenzel, H. Ponta, and P. Herrlich. 1991. Cell. 65:13). A monoclonal antibody (mAb), 1.1ASML, to the metastasis-specific domain of the CD44v molecule retards growth of lymph node and lung metastases of the metastatic tumor line BSp73ASML, and can efficiently prevent formation of metastases by the transfected line. The antibody is only effective when given before lymph node colonization. Anti-CD44v does not downregulate the expression of CD44v, and prevention of metastatic growth by anti-CD44v is not due to activation of any kind of immune defense. We suggest that the mAb interferes with proliferation of metastasizing tumor cells in the draining lymph node, most probably by blocking a ligand interaction. The interference with metastatic spread will greatly facilitate the exploration of the function of CD44v and, in particular, may also open new strategies for the therapy of human metastases.

Expression of CD44 isoforms carrying metastasis-associated sequences in newborn and adult rats.

Expression of a splice variant of CD44, recognised by the monoclonal antibody (Mab) 1.1ASML, confers metastatic potential to non-metastasising tumour cells (Cell 1991, 65, 13-24). To explore whether the metastasis-associated variant of CD44 (CD44v) is expressed under physiological conditions, tissues of newborn and adult rats were stained with the Mab 1.1ASML. The 1.1ASML epitope is, indeed, expressed on the basal layer of the epidermis and the hair follicles as well as on cryptic epithelia in the gut. In addition, ductal epithelia of the pancreatic gland of newborn rats express CD44v. This pattern of expression differs from that of standard lymphocyte CD44 (CD44s). The anti-CD44s mAB Ox50 predominantly stains connective tissue. Although different variants of CD44 may express the epitope recognised by 1.1ASML, cells expressing CD44v share properties with metastasising tumour cells: the stage of proliferation and a restricted degree of mobility. Thus, during metastatic progression tumour cells may reactivate the expression of gene segments which serve highly specialised functions in embryonic and adult tissues.

Monoclonal antibody uptake in B-cell lymphomas: experimental studies in nude mouse xenografts.

Accumulation of radiolabelled monoclonal antibodies (mAb) in human B-lymphoma xenografts was found to result in two distinct patterns. The basic elements leading to these patterns were elucidated by autoradiographic and immunohistological analysis applied to the nude mouse xenografts BJAB and OCI.LY1. With BJAB, accumulation occurred exclusively in peripheral cell layers of the lymphoma nodule, while central areas were not accessible irrespective of mAb dose. This feature was the consequence of an inefficient transport across intratumoral vessels together with peripheral mAb supply through a subcapsular pseudosinus. With OCI.LY1, intratumoral vessels showed generalized leakiness. Furthermore, interstitial transport was operative to a fair extent, such that in early images multiple sites of mAb extravasation were obvious, which coalesced during the course of prolonged uptake. The pattern of peripheral mAb uptake resulted in a low overall tumour uptake, while multifocal uptake yielded substantial accumulation values.

Monoclonal antibody accumulation in experimental and spontaneous lung metastases of human malignant melanoma in rodents.

Monoclonal antibody (mAb) uptake in metastatic lung lesions was depicted and evaluated by digital autoradiography. The models examined were experimental metastases of a human melanoma in nude mice and spontaneous metastases of human melanoma in immunocompromised young rats. By comparing uptake patterns in local (s.c.) tumours and in lung processes of various sizes it was found that patterns were essentially similar in both types of malignant tissue. From the point of view of visualization, however, the high blood content of lung tissue resulted in high background and low contrast. This could be overcome by the use of rapidly cleared antibody fragments.

Preferential antibody targeting to small lymphoma metastases in the absence of the primary tumour.

Targeting of spontaneous liver metastases of the ESb.MP murine lymphoma was achieved with anti-CD2 monoclonal antibody (MAb) 12-15A, which does not react with normal liver tissue. Using quantitative autoradiography on whole body sections of animals that had received a standard dose of 1.1 MBq of 125I-labelled monoclonal antibody, metastases accumulated up to > 90% of the injected dose per gram (id/g). The average uptake of primary tumour lesions was at a low level of 24 Bq/mg (corresponding to 2.2% id/g) because of highly non-uniform accumulation, while metastatic lesions were all above 50 Bq/mg. Uptake was particularly pronounced in animals tested after resection of the primary tumour: 85% of metastases showed levels above 300 Bq/mg, which was the upper limit of uptake in metastases of non-resected animals. These findings demonstrate the potential of the antibody approach with regard to attacking residual metastatic lesions after debulking.