Role of M-CSF-dependent macrophages in colitis is driven by the nature of the inflammatory stimulus

Although macrophages are considered a critical factor in determining the severity of acute inflammatory responses in the gut, recent evidence has indicated that macrophages may also play a counterinflammatory role. In this study, we examined the role of a macrophage subset in two models of colitis. Macrophage colony-stimulating factor (M-CSF)-deficient osteopetrotic mice (op/op) and M-CSF-expressing heterozygote (+/?) mice were studied following the induction of colitis by either dinitrobenzene sulfonic acid (DNBS) or dextran sulfate sodium (DSS). DNBS induced a severe colitis in M-CSF-deficient op/op mice compared with +/? mice. This was associated with increased mortality and more severe macroscopic and microscopic injury. Colonic tissue myeloperoxidase (MPO) activity as well as concentrations of TNF-alpha, IL-1beta, and IL-6 were higher and IL-10 lower in op/op mice with DNBS colitis. The severity of inflammation and mortality was attenuated in op/op mice that had received human recombinant M-CSF prior to the induction of colitis. In contrast, op/op mice appeared less vulnerable to colitis induced by DSS. Macroscopic damage, microscopic injury, MPO activity, and tissue concentrations of TNF-alpha, IL-1beta, and IL-6 were all lower in op/op mice compared with +/? mice with DSS colitis, and no changes were seen in IL-10. Macrophage inflammatory protein-1alpha concentrations were increased in op/op but not +/? mice following colitis induced by DNBS but not DSS. These results indicate that M-CSF-dependent macrophages may play either a pro- or counterinflammatory role in acute experimental colitis, depending on the stimulus used to induce colitis.     

Recognition by human Vγ9/Vδ2 T cells of melanoma cells upon fusion with Daudi cells

 Daudi Burkitt’s lymphoma cells, unlike other tumor cell lines, stimulate human T cells coexpressing the variable (V) region genes TCRG-V9 and V TCRD-V2 to proliferate and secrete lymphokines. Hybrids, derived by the fusion of Daudi cells with the human melanoma cell line MZ2-MEL 2.2, retain the morphology of melanoma cells. Unlike the parental melanoma cell line, these Daudi × MZ2-MEL 2.2 hybrids stimulate secretion of tumor necrosis factor (TNF) and granulocyte/macrophage colony stimulating factor (GM-CSF) by CD4-positive Vγ9/Vδ2 T-cell clones. Whereas the stimulator phenotype of Daudi cells behaves as a dominant trait in Daudi × melanoma hybrids, the expression of B-cell differentiation markers is suppressed. Thus, the γ/δ T-cell ligand expressed by Daudi cells behaves as a dominant tumor antigen in Daudi × melanoma hybrids and is unrelated to the differentiated B-cell phenotype. Dominant expression of the Daudi ligand for human Vγ9/Vδ2 T cells in these hybrids may provide a basis for defining the stimulatory principle at the molecular level. Received: 2 May 1996 / Revised: 15 July 1996     

Tumor associated osteoclast-like giant cells promote tumor growth and lymphangiogenesis by secreting vascular endothelial growth factor-C

Tumors with osteoclast-like giant cells (OGCs) have been reported in a variety of organs and exert an invasive and prometastatic phenotype, but the functional role of OGCs in the tumor environment has not been fully clarified. We established tumors containing OGCs to clarify the role of OGCs in tumor phenotype. A mixture of HeLa cells expressing macrophage colony-stimulating factor (M-CSF, HeLa-M) and receptor activator of nuclear factor-κB ligand (RANKL, HeLa-R) effectively supported the differentiation of osteoclast-like cells from bone marrow macrophages in vitro. Moreover, a xenograft study showed OGC formation in a tumor composed of HeLa-M and HeLa-R. Surprisingly, the tumors containing OGCs were significantly larger than the tumors without OGCs, although the growth rates were not different in vitro. Histological analysis showed that lymphangiogenesis and macrophage infiltration in the tumor containing OGCs, but not in other tumors were accelerated. According to quantitative PCR analysis, vascular endothelial growth factor (VEGF)-C mRNA expression increased with differentiation of osteoclast-like cells. To investigate whether VEGF-C expression is responsible for tumor growth and macrophage infiltration, HeLa cells overexpressing VEGF-C (HeLa-VC) were established and transplanted into mice. Tumors composed of HeLa-VC mimicked the phenotype of the tumors containing OGCs. Furthermore, the vascular permeability of tumor microvessels also increased in tumors containing OGCs and to some extent in VEGF-C-expressing tumors. These results suggest that macrophage infiltration and vascular permeability are possible mediators in these tumors. These findings revealed that OGCs in the tumor environment promoted tumor growth and lymphangiogenesis, at least in part, by secreting VEGF-C.     

Diverse manifestations of tumorigenicity and immunogenicity displayed by the poorly immunogenic B16-BL6 melanoma transduced with cytokine genes

 We evaluated the in vivo response to the poorly immunogenic B16-BL6 (BL6) murine melanoma genetically altered to secrete interleukin-2 (IL-2), IL-4, interferon γ (IFNγ) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). Three parameters were evaluated: (1) tumorigenicity, (2) vaccination of naive animals, and (3) assessment of antitumor reactivity of T cells derived from tumor-draining lymph nodes (TDLN). Secretion of IL-2 abrogated the tumorigenicity of BL6, while IFNγ and IL-4 partially reduced tumorigenicity, and GM-CSF had no effect. Protective immunity to wild-type tumor challenge could not be achieved by vaccination with irradiated cytokine-secreting tumors, although IL-2 and IL-4 secretion appeared to retard the growth of the challenge inoculum significantly. An alternative method to evaluate the immunogenicity of the cytokine-secreting tumors was to measure the ability of T cells obtained from TDLN to mediate regression of wild-type tumor in adoptive immunotherapy. Neither IL-2 nor IFNγ secretion resulted in the induction of immune T cells. By contrast, GM-CSF and IL-4 secretion were found to induce immune T cells in the TDLN with GM-CSF being superior to IL-4. The combined secretion of GM-CSF and IL-4 did not lead to enhanced induction of immune T cells. GM-CSF secretion was found to up-regulate B7-1 expression in TDLN, consistent with an increase in the population of antigen-presenting cells. These studies demonstrated that reduced tumorigenicity by cytokine secretion did not correlate with increased immunogenicity. With the cytokines examined, there was limited capability of developing protective immunity against the BL6 tumor. Nevertheless, GM-CSF and IL-4 secretion significantly enhanced T cell immune reactivity to the poorly immunogenic BL6 tumor. Received: 30 January 1996 / Accepted: 22 March 1996     

The neutrophil as an information transmitter in tumor inhibition by a streptococcal biological response modifier,OK-432

 Effective treatment of a rat transplanted ascites tumor by i. p. injection of a streptococcal biological response modifier, OK-432, was abrogated by selective in vivo depletion of neutrophils by a monoclonal antibody, RP-3. The mechanisms by which neutrophils participate in the therapeutic action of OK-432 were studied with Winn’s assay using peritoneal exudate cells periodically obtained from rats i. p. injected with this biological response modifier. Intraperitoneal resident macrophages were first activated with OK-432, and within 3 h, tumor-inhibitory activity had moved to the early exuded neutrophils. However, 6 h after injection, exuded macrophages were the only cells involved in tumor inhibition. Considered together with other findings, it is likely that, in this system, neutrophils may transmit information from resident macrophages to exuded inflammatory macrophages in a series of responses induced by i. p. injection of OK-432. Received: 29 April 1996 / Accepted: 27 July 1996     

Macrophage colony-stimulating factor is indispensable for repopulation and differentiation of Kupffer cells but not for splenic red pulp macrophages in osteopetrotic (<Emphasis Type="Italic">op</Emphasis>/<Emphasis Type="Italic">op</Emphasis>) mice after macrophage depletion

We previously reported that macrophage colony-stimulating factor (M-CSF, CSF-1) played important roles in the process of the repopulation of Kupffer cells after their elimination by administration of liposome-entrapped dichloromethylene diphosphonate (lipo-MDP). In this study, we examined the repopulation of Kupffer cells and splenic red pulp macrophages in osteopetrotic (op/op) mice defective in the production of functional M-CSF and their littermate mice by using the lipo-MDP model. In untreated op/op mice, numbers of F4/80-positive Kupffer cells in the liver and F4/80-positive splenic red pulp macrophages were reduced. Repopulation of Kupffer cells and splenic macrophages was observed in littermate (op/+) mice liver by 14 days after depletion. However, in op/op mice, repopulation of Kupffer cells was not observed in Kupffer-cell-depleted op/op mice until 56 days after depletion, whereas splenic red pulp macrophages repopulated and recovered to the level of control op/op mice by 10 days after depletion. Single injection of M-CSF was effective for the induction of the repopulation of Kupffer cells, and daily administration of M-CSF induced remarkable repopulation and maturation of Kupffer cells and proliferation of macrophage precursor cells in the liver of Kupffer-cell-depleted op/op mice. These results suggest that Kupffer cells are completely M-CSF-dependent tissue macrophages, whereas splenic red pulp macrophages are composed of M-CSF-dependent macrophages and M-CSF-independent macrophages. This mouse model provides a useful tool for the study of effects of growth factor on Kupffer cell differentiation in vivo. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, NIH grant CA20408, and a Tsukada Memorial Grant (2000).     

Type I interferons inhibit the generation of tumor-associated macrophages

Tumor-associated macrophages (TAM) are very abundant in tumors and are thought to play a major role in promoting tumor growth. The generation of TAM is positively regulated by several cytokines, including colony stimulating factor-1 (CSF-1) and monocyte chemoattractant protein-1 (CCL2). However, endogenous factors that suppress the generation of TAM within tumors have not been previously identified. An earlier study showed that endogenously produced type I interferons (IFN) suppressed tumor growth via their effects on hematopoietic cells rather than through direct effects on tumor cells. Therefore, we used mouse tumor models to investigate the effects of endogenously produced type I IFNs on the generation of TAM. We found using immunohistochemistry and flow cytometry that TAM density was significantly increased in tumors of mice lacking the type I IFN receptor (IFN-α/βR^−/− mice) compared to wild type mice. Moreover, the increase in TAM density was associated with a significant increase in tumor growth rate and angiogenesis. The phenotype of TAM was similar in IFN-α/βR^−/− mice and wild type mice and tumors in both mice produced similar amounts of CSF-1 and CCL2. However, in vitro assays indicated that low concentrations of type I IFNs significantly inhibited the generation of bone marrow macrophages in response to CSF-1. These findings indicate that endogenously produced type I IFNs suppress the generation of TAM, which may in turn account for inhibition of tumor growth and angiogenesis.     

Macrophage colony stimulating factor (M-CSF) is essential for osteoclast formation in vitro

The op/op mouse, in which the M-CSF gene is mutated, has greatly reduced numbers of macrophages and osteoclasts. We assessed the ability of M-CSF to induce osteoclast and macrophage formation in op/op hemopoietic cells in vitro. Osteoclast production was undetectable in op/op cell cultures, but was restored by M-CSF at concentrations approximately an order of magnitude higher than those that induced macrophages. In normal hemopoietic tissue M-CSF similarly increased macrophage numbers, but inhibited osteoclast formation. Despite cure of the macrophage defect, neither interleukin 3 nor granulocyte-macrophage CSF were able to induce osteoclastic differentiation in op/op cells. The results suggest that M-CSF induces osteoclastic differentiation but that macrophages, which are also induced by M-CSF, suppress osteoclast differentiation. Macrophages induced by other cytokines seem unable to contribute to osteoclast-formation.     

Targeting macrophages rescues age‐related immune deficiencies in C57BL/6J geriatric mice

Changes to innate cells, such as macrophages and myeloid‐derived suppressor cells (MDSCs), during aging in healthy or tumor‐bearing hosts are not well understood. We compared macrophage subpopulations and MDSCs from healthy young (6–8 weeks) C57BL/6J mice to those from healthy geriatric (24–28 months) mice. Spleens, lymph nodes, and bone marrow of geriatric hosts contained significantly more M2 macrophages and MDSCs than their younger counterparts. Peritoneal macrophages from geriatric, but not young, mice co‐expressed CD40 and CX3CR1 that are usually mutually exclusively expressed by M1 or M2 macrophages. Nonetheless, macrophages from geriatric mice responded to M1 or M2 stimuli similarly to macrophages from young mice, although they secreted higher levels of TGF‐β in response to IL‐4. We mimicked conditions that may occur within tumors by exposing macrophages from young vs. geriatric mice to mesothelioma or lung carcinoma tumor cell–derived supernatants. While both supernatants skewed macrophages toward the M2‐phenotype regardless of age, only geriatric‐derived macrophages produced IL‐4, suggesting a more immunosuppressive tumor microenvironment will be established in the elderly. Both geriatric‐ and young‐derived macrophages induced allogeneic T‐cell proliferation, regardless of the stimuli used, including tumor supernatant. However, only macrophages from young mice induced T‐cell IFN‐γ production. We examined the potential of an IL‐2/agonist anti‐CD40 antibody immunotherapy that eradicates large tumors in young hosts to activate macrophages from geriatric mice. IL‐2‐/CD40‐activated macrophages rescued T‐cell production of IFN‐γ in geriatric mice. Therefore, targeting macrophages with IL‐2/anti‐CD40 antibody may improve innate and T‐cell immunity in aging hosts.     

Characterization of a high-affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas

 Variant isoforms of CD44, a family of cell-surface glycoproteins generated by alternative splicing and post-translational modifications, are expressed in a variety of human tumors and play important roles in tumor progression and metastasis formation. The murine monoclonal IgG1 antibody VFF18, specific for an epitope encoded by human CD44 variant exon 6, binds with high affinity to the recombinant protein (K _d = 1.7×10^–10 M) as well as to tumor cell lines in vitro, and is suitable for immunohistochemical analysis of human tumors. Screening of more than 500 tumor samples of different histogenesis showed that VFF18 most strongly and uniformly reacts with squamous cell carcinomas (SCC). Detailed analysis of 185 SCC (head and neck, lung, skin) confirmed reactivity of the antibody with 99% of the samples, with intense and homogeneous staining of the tumor cells in the majority of cases, whereas reactivity of VFF18 with normal tissues is limited to certain epithelia and activated lymphocytes. When radiolabelled VFF18 was administered to nude mice bearing human epidermoid carcinoma (A-431) xenograft, fast and selective tumor uptake of the radioimmunoconjugate with a maximum of 18% of the injected dose per gram of tissue was observed. Taken together, these data suggest that mAb VFF18 is a promising targeting vehicle for radioimmunotherapy of squamous cell carcinomas in humans. Received: 9 September 1996 / Accepted: 25 September 1996     

Immune modulation by interleukin-12 in tumor-bearing mice receiving vitamin D3 treatments to block induction of immunosuppressive granulocyte/macrophage progenitor cells

 Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and increase the presence of granulocyte/macrophage (GM) progenitor cells having natural suppressor activity. Treatment of these tumor-bearing mice with interleukin-12 (IL-12) resulted in minimal immune modulation. The objective of this study was to determine whether eliminating natural suppressor activity would allow for immune stimulation by IL-12. Treatment of LLC-LN7 tumor-bearing mice with vitamin D₃ eliminated natural suppressor activity. In mice that were first treated with vitamin D₃ and then also with IL-12, there was stimulation of splenic T cell proliferation in response to immobilized anti-CD3 plus IL-2. In addition, spleen and lymph node cells from vitamin-D₃/IL-12-treated tumor-bearing mice became stimulated in response to autologous tumor to produce interferon γ (IFNγ), although IL-2 production was not stimulated. A prominent effect of the combined vitamin-D₃/IL-12 treatment regimen was the synergistic augmentation of autologous tumor-specific cytolytic activity within the regional lymph nodes. The generation of these tumor-specific effector cells required the presence of the tumor mass since such activity was not elicited in the lymph nodes of mice from which the tumors had been surgically excised. The results of this study show that, after treatment of tumor bearers with vitamin D₃ to eliminate GM-suppressor cells, IL-12 can induce select regional antitumor immune responses, particularly IFNγ production and cytolysis by regional lymph node cells of autologous tumor. Received: 15 December 1995 / Accepted: 22 March 1996     

Bcl-2 protects cells from cytokine-induced nitric-oxide-dependent apoptosis

 Cytokine-mediated cell death in tumor cells can be achieved through endogenous nitric oxide (NO) from within tumor cells or exogenous NO from either activated macrophages or endothelial cells. The purpose of this study was to determine the role of Bcl-2 in NO-mediated apoptosis. The incubation of murine L929 and NIH3T3 cells with interleukin-1α (IL-1α) and interferon γ (IFNγ) induced high endogenous NO production only in the L929 cells that also underwent apoptosis. NIH3T3 cells were not resistant to NO-mediated apoptosis. In fact, the incubation of L929 and NIH3T3 cells with exogenous NO derived from NO donors, sodium nitroprusside, or S-nitroso-N-acetyl-DL-penicillamine (SNAP) induced death, characterized by typical apoptotic morphology and DNA fragmentation, in both cell types, but to a higher degree in NIH3T3 cells than in the L929 cells. We then measured the effect of Bcl-2 expression on exogenous NO-induced apoptosis. At both the mRNA and protein levels, L929 fibroblasts expressed higher levels of endogenous mouse Bcl-2 than did NIH3T3 cells. At the same time, L929 cells were much more resistant to exogenous NO-induced cell death than were NIH3T3 cells. The inverse correlation between mouse Bcl-2 expression and sensitivity to exogenous NO-mediated cell death was also found in the murine K-1735 melanoma C-23 and X-21 clonal populations. Transfection of both NIH3T3 cells and L929 cells with the human bcl-2 gene led to resistance to both exogenous and endogenous NO-mediated apoptosis. These data demonstrate that NO-mediated apoptosis can be suppressed by expression of Bcl-2, suggesting that abnormal expression of Bcl-2 may influence the efficacy of tumor immunotherapy. Received: 28 June 1998 / Accepted: 23 August 1996     

Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF)

Recently, we have shown that macrophage uptake of low density lipoprotein (LDL) and cholesterol accumulation can occur by nonreceptor mediated fluid-phase macropinocytosis when macrophages are differentiated from human monocytes in human serum and the macrophages are activated by stimulation of protein kinase C (Kruth, H. S., Jones, N. L., Huang, W., Zhao, B., Ishii, I., Chang, J., Combs, C. A., Malide, D., and Zhang, W. Y. (2005) J. Biol. Chem. 280, 2352-2360). Differentiation of human monocytes in human serum produces a distinct macrophage phenotype. In this study, we examined the effect on LDL uptake of an alternative macrophage differentiation phenotype. Differentiation of macrophages from human monocytes in fetal bovine serum with macrophage-colony-stimulating factor (M-CSF) produced a macrophage phenotype demonstrating constitutive fluid-phase uptake of native LDL leading to macrophage cholesterol accumulation. Fluid-phase endocytosis of LDL by M-CSF human macrophages showed non-saturable uptake of LDL that did not down-regulate over 48 h. LDL uptake was mediated by continuous actin-dependent macropinocytosis of LDL by these M-CSF-differentiated macrophages. M-CSF is a cytokine present within atherosclerotic lesions. Thus, macropinocytosis of LDL by macrophages differentiated from monocytes under the influence of M-CSF is a plausible mechanism to account for macrophage foam cell formation in atherosclerotic lesions. This mechanism of macrophage foam cell formation does not depend on LDL modification or macrophage receptors.     

Prognostic markers for survival in patients with metastatic renal cell carcinoma treated with interleukin-2

 Interleukin-2 (IL-2)-based immunotherapy can induce antitumor responses in about 25% of patients with metastatic renal cell carcinoma (RCC). The limited effect and the severe side-effects of IL-2 have led us to perform a prognostic factor analysis. Twenty-four patients with metastatic RCC were treated with IL-2. Flow cytometry and immunohistology were used to determine DNA ploidy, HLA-II expression on tumor cells, and the presence of macrophages in the primary tumor. These variables were examined in relation to survival. The 4-year overall survival rate was 38%. Forty-six percent of the primary tumors were aneuploid. All tumors, except one, showed HLA-II expression and macrophage presence. A statistically significant correlation (r = 0.66, P = 0.002) was found between HLA-II expression and macrophage presence. Patients with high HLA-II expression had a lower 4-year survival (22% compared to 50%), as had patients with high macrophage presence (20% compared to 42%). Of note, patients characterized by both high HLA-II and high macrophage expression had the worst survival (13% compared to 50%). We concluded that DNA ploidy was not predictive for survival, whereas HLA-II expression and macrophage presence may represent valuable prognostic factors related to survival. The present data suggest that more of the patients with no or moderate HLA-II expression and/or no or moderate macrophage presence in the primary tumor could survive with persistance of their malignant disease after having received IL-2 immunotherapy, as compared to patients with both high HLA-II and high macrophage expression. Received: 2 April 1996 / Accepted: 15 October 1996     

Contrasting effects of T cell growth factors on T cell responses to melanoma in vitro

 Determinants of T cell responses to tumor cells remain largely unknown. In the present study we have used long-term cultures of human melanoma cells and autologous peripheral blood lymphocytes to examine the influence of cytokines with T cell growth activity on the phenotype and cytotoxic and proliferative response of T cells to melanoma. It was found that addition of interleukin-4 (IL-4) inhibited the response of CD8⁺ T cells and promoted the response of the CD4 subset. IL-2 or IL-7 was effective in increasing melanoma-specific cytotoxic T lymphocyte (CTL) activity in cultures where CD8 T cells were predominant, whereas IL-4 followed by IL-2 was most effective in cultures where CD4 T cells predominated. IL-10 or IL-12 inhibited proliferation and CTL activity against melanoma in long-term cultures. The effects of IL-12 were reproduced in long-term cultures of T cells stimulated with mAb against CD3 and were shown to depend on prior exposure of T cells to IL-12 before IL-2. As yet unidentified factors, such as co-factor expression on melanoma, appear to be as important as exogenous cytokines in determining the nature of T cell responses to melanoma. These results suggest that analysis of responses in long-term culture may assist in defining the role of key cytokines and other determinants of immune responses to melanoma. Received: 4 June 1996 / Accepted: 12 November 1996     

Effect of intraperitoneal administration of granulocyte/macrophage-colony-stimulating factor in rats on omental milky-spot composition and tumoricidal activity in vivo and in vitro

 Milky spots in the greater omentum are small accumulations of leucocytes that consist mainly of macrophages and have recently shown to be a selective dissemination site of intraperitoneal (i. p.) inoculated tumour cells. However, milky-spot macrophages show tumoricidal activity and may, therefore, be an excellent source of effector cells suited for local immunotherapy. In the present study we first examined whether granulocyte/macrophage- colony-stimulating factor (GM-CSF) treatment of isolated milky-spot macrophages affects the cytotoxicity against syngeneic colon carcinoma cells (CC531) in vitro. Secondly, we studied the influence of intraperitoneal GM-CSF administration on the number and antitumour activity of milky-spot and peritoneal macrophages. All studies were performed in Wag/Rij rats in which a syngeneic colon carcinoma cell line (CC531) is available. The results of the in vitro study showed that GM-CSF treatment of the omental macrophages led to an increased cytotoxicity against the tumour cell line. Intraperitoneal administration of 1000 U GM-CSF daily for 7 consecutive days demonstrated both an enhanced antitumour activity of the milky-spot macrophages and an increase in the milky-spot macrophage population. An increase in the proliferative capacity, according to bromodeoxyuridine incorporation, was shown in the milky-spot macrophages. Taking into account both the enhanced macrophage number and their enhanced activity upon i. p. GM-CSF treatment, the milky-spot macrophages may provide a rationale for local intraperitoneal immunotherapy in the prevention of intra-abdominal tumour growth. Received: 11 April 1996 / Accepted: 21 May 1996     

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 a 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. Received: 2 May 1996 / Accepted: 21 May 1996     

Immunogenicity increase of autologous tumor cell vaccines by virus infection and attachment of bispecific antibodies

 A new type of cancer vaccine for therapeutic application in cancer patients is described. It consists of three components. (1) autologous tumor cells, (2) Newcastle Disease Virus (NDV), to be used for infection and (3) bispecific antibodies (bsAb) which attach to the viral hemagglutinin neuraminidase (HN) molecule on the infected tumor cells. A standardized procedure has been developed for generating virus infected human autologous tumor cell vaccines (ATV-NDV) which includes cell dissociation, removal of leukocytes and cell debris, gamma-irradiation and cryopreservation. Infection with the non-virulent strain NDV Ulster is performed within 30 min of co-incubation. While virus infection already increased immunogenicity of the tumor vaccine, further augmentation of T cell stimulatory capacity is achieved by attachment of specially designed bi-specific antibodies (bs HN × CD28 or bs HN × CD3). Received: 6 August 1996 / Accepted: 20 September 1996