Low-dose ipilimumab plus nivolumab combined with IL-2 and hyperthermia in cancer patients with advanced disease: exploratory findings of a case series of 131 stage IV cancers - a retrospective study of a single institution.

The 3-year overall survival (OS) rate of patients with previously treated or untreated stage III or IV melanoma has by now reached 63% using ipilimumab and nivolumab therapy. However, immune-related adverse events (irAEs) of grade 3 or 4 occurred in 59% of patients leading to discontinuation of therapy in 24.5% of patients and one death. Therapy with checkpoint inhibitors could be safer and more effective in combination with hyperthermia and fever inducing therapies. We conducted a retrospective analysis to test the safety and efficacy of a new combination immune therapy in 131 unselected stage IV solid cancer patients with 23 different histological types of cancer who exhausted all conventional treatments. Treatment consisted of locoregional- and whole-body hyperthermia, individually dose adapted interleukin 2 (IL-2) combined with low-dose ipilimumab (0.3 mg/kg) plus nivolumab (0.5 mg/kg). The objective response rate (ORR) was 31.3%, progression-free survival (PFS) was 10 months, survival probabilities at 6 months was 86.7% (95% CI, 81.0-92.8%), at 9 months was 73.5% (95% CI, 66.2-81.7%), at 12 months was 66.5% (95% CI, 58.6-75.4%), while at 24 months survival was 36.6% (95% CI:28.2%; 47.3%). irAEs of World Health Organization (WHO) Toxicity Scale grade 1, 2, 3, and 4 were observed in 23.66%, 16.03%, 6.11%, and 2.29% of patients, respectively. Our results suggest that the irAEs profile of the combined treatment is safer than that of the established protocols without compromising efficacy.

Some aspects of complementarity in the immune system. A bird's eye view.

The burden of this paper is the suggestion that the defence capacity of the immune system is rather limited. It cannot stand in readiness to deal with a practically endless diversity and abundance of microbes. In contrast to conventional thinking the current model proposes: (1) The core idea that cells of the immune system are basically and constantly interconnected with host cells (e.g., through TCR-MHC interactions) and that foreign antigens (peptides) may tend to obstruct such interactions. Peptides presented during a viral infection typically decrease complementarity between the structures that are the products of the major histocompatibility complex (MHC) genes (or other genes related to it) and T cells. The altered MHC profile exposes infected cells to a polyclonal immune attack from other T cells such that tissue destruction occurs in an allograft rejection-like fashion. This may explain why a substantial portion of T cell numbers is activated when only a small number of specific T cells is 'obstructed' from functioning by the presence of nonself peptides. (2) Phagocytes 'see' targets even in a non-immune host because complement distribution associated with polyreactive natural antibodies magnifies sensitization differences between pathogens and host cells. (3) There is only a probability that hypermutation will successfully change the genome in some B cell clones to produce high affinity antibodies that prevent the re-infection of the host by the same pathogen, but cannot conquer primary infections. (4) The history of the development of the immune responses suggests that during prolonged interaction between host and microbes in our natural habitat, carried on over many generations, the adaptive antibody population may facilitate the evolution of the natural antibody repertoire. The model predicts that microbes, which are not a part of the local environment, may invade the organism without significant resistance. The model is discussed in various interactions for survival in the context of infection and tumorigenicity.

Successful treatment of decompensated chronic viral hepatitis by bursal disease virus vaccine.

Three cases of women with chronic liver inflammation caused by hepatitis B (two) and C (one) viral infections, were followed up to twelve years after diagnosis. As conventional therapy was ineffective and the patients progressed into decompensated liver disease, they were superinfected with massive doses of an attenuated variant (MTH-68/B) of the apathogenic avian Bursal Disease virus (a double-stranded RNA virus from the Birnaviridae family). Clinical symptoms and biochemical abnormalities were resolved in two patients following few months of virus treatment. Cirrhosis was stabilized and significant clinical improvement was achieved in the third patient--who before the virus therapy was moribund with recurring, diuretic-resistant ascites, variceal bleedings, portal encephalopathy and renal failure. To our knowledge, these are the first recorded cases of decompensated chronic viral hepatitis which went to long-lasting remission or were stabilized by superinfection with an apathogenic virus.

Beneficial treatment of patients with advanced cancer using a Newcastle disease virus vaccine (MTH-68/H).

Newcastle Disease Virus Vaccine (MTH-68/H) was administered to patients suffering from advanced neoplastic diseases after non-efficient tumor-destructive treatment. Case reports of selected patients suggest promising effects of this treatment. A prospectively-randomized clinical study (phase III; in accordance with Good Clinical Practice, GCP) was proposed to confirm these results and is currently under consideration.

Preliminary report of a controlled trial of MTH-68/B virus vaccine treatment in acute B and C hepatitis: a phase II study.

Eighty four patients with viral hepatitis attributed to infection with hepatitis B virus (HBV) (n = 43) or hepatitis C virus (HCV) (n = 41) were included in this study employing the MTH-68/B vaccine, an attenuated variant of Bursal Disease Virus. Twenty of the 43 patients in the HBV group, and 22 of the 41 HCV patients were treated with MTH-68/B. The remaining patients received conventional therapy. Significantly more patients progressed into active chronic hepatitis on conventional therapy (13% of HBV and 26% of HCV cases respectively) than in the vaccine treated groups (0% and 9%). Relapses occurred less frequently in the vaccine treated groups (5% of HBV and 32% of HCV) than in the control groups (9% and 79%), while remissions within one month of treatment were observed more often in the vaccine treated groups (both 50% respectively) than in the control groups (26% of HBV and 21% of HCV patients respectively). The duration of hepatitis was also considerably shortened by MTH-68/B treatment in both HBV (from 7.5 +/- 3.7 to 5.9 +/- 3.0 weeks) and HCV patient groups (from 8.9 +/- 7.4 to 5.3 +/- 4.4 weeks). The data presented suggest that attenuated, non-pathogenic viruses may be of significant benefit for patients with viral hepatitis B and C infections.

Targeted adenovirus-mediated gene delivery to T cells via CD3.

T cells are primary targets in numerous gene therapy protocols. However, the use of subgroup C adenovirus serotype 2 or 5 (Ad2 or Ad5) as a vector to transduce T cells is limited by its poor transduction efficiency for these cells. In this report we show that poor T-cell transduction results from these cells lacking both the primary Ad2-Ad5 receptor, used in attachment, and the secondary Ad receptor, which mediates entry of most adenovirus serotypes. These deficiencies were overcome by using a bispecific antibody (bsAb) with specificities for human CD3 and for a FLAG epitope genetically introduced into Ad5 (Ad.FLAG) to redirect the virus to human T cells. The anti-FLAG x anti-CD3 bsAb increased Ad.FLAG binding 30-fold, induced the efficient uptake of Ad.FLAG into the cells, and led to a 100- to 500-fold increase in the transduction of resting T cells. Moreover, fluorescence-activated cell sorter analysis showed that 25 to 90% of the T cells were transduced by the bsAb-complexed Ad.FLAG at multiplicities of infection between 20 and 100 active particles per cell. These results demonstrate that bsAbs can target Ad to non-Ad receptors on cells that are normally resistant to Ad, resulting in their efficient and specific transduction.

A bispecific antibody prolongs survival in mice bearing lung metastases of syngeneic mammary adenocarcinoma.

In the present study we tested whether T cells retargeted with a bispecific antibody (bsAb) could block the growth of lung metastases of syngeneic mammary adenocarcinoma in immunocompetent mice. BALB/c mice were injected i.v. with tumor and i.p. with a genetically engineered bispecific F(ab')2 [bs(Fab')2] having specificity for murine CD3 epsilon chain and for the gp52 mouse mammary tumor viral glycoprotein, which is expressed on the tumor cells. The bs(Fab')2 was physically stable in blood and serum, was removed from the body with a half-time of 12-15 h, and accumulated in lymphoid tissue where it bound to T cells. We show that treatment of tumor bearing mice with the bs(Fab')2 significantly prolonged their survival relative to untreated controls. Two other genetically engineered bs(Fab')2s having specificity for murine CD3 epsilon chain and irrelevant antigens did not inhibit tumor growth. In addition, survival was not affected by bsAb therapy using a variant tumor cell line that expressed low levels of the gp52 target antigen. Inhibition of tumor growth was even more evident by histologic analysis. Treatment with the relevant bs(Fab')2 resulted in a marked reduction of tumor burden in lung sections taken on days 7, 9 and 11. This is the first report demonstrating that a bsAb can inhibit the growth of syngeneic solid tumor metastases in mice without addition of T cell activators.

Bispecific antibodies retarget murine T cell cytotoxicity against syngeneic breast cancer in vitro and in vivo.

Bispecific antibodies with specificity for CD3 and a tumor antigen can redirect cytolytic T cells to kill tumor targets, regardless of their natural specificity. To assess the clinical potential of bispecific antibodies for treatment of human cancers we have, in the present study, adapted a totally synergeic mouse model to the targeting of mouse T cells against mouse tumors in immunocompetent mice. We show that gp52 of the mouse mammary tumor virus (MTV) can serve as a tumor-specific antigen for redirected cellular cytotoxicity. Chemically crosslinked and genetically engineered bispecific antibodies with specificities for gp52 and murine CD3 epsilon-chain induced activated mouse T cells to specifically lyse mouse mammary tumor cells from cultured lines and primary tumors from C3H-MTV+ mice. Retargeted T cells also blocked the growth of mammary tumors in vitro as well as their growth in syngeneic mice. These findings identify murine MTV-induced mammary adenocarcinomas as a solid-tumor, animal model for retargeting T cells with bispecific antibodies against syngeneic breast cancer.

Comparison of the ability of red cells sensitized with a bispecific anti-D x anti-Fc gamma RIII Fab fragment to activate human K cells and peritoneal macrophages through Fc gamma RIII.

The functional activity of Fc gamma RIII on human K cells from peripheral blood was compared with that of Fc gamma RIII on peritoneal macrophages (PM) separated from the waste material of patients undergoing peritoneal dialysis. Fc gamma R function was assessed in vitro using human monoclonal IgG1 anti-D (AB5) or a bispecific antibody comprising Fab fragments of AB5 chemically linked to Fab fragments of monoclonal anti-Fc gamma RIII, 3G8 (AB5 x 3G8). In antibody-dependent cell-mediated cytotoxicity (ADCC) assays, K cells mediated the lysis of papainized red cells sensitized with the AB5 x 3G8 bispecific antibody but not with AB5. In contrast, red cell lysis by PM was not promoted by AB5 x 3G8 although AB5 was active. However, this lysis, being inhibited by monomeric IgG, was presumably mediated via Fc gamma RI. AB5 x 3G8 also failed to promote the binding and phagocytosis of both papainized and native red cells by PM although 99% of red cells and over 90% of peritoneal cells bound the bispecific antibody. In marked contrast to K cells therefore, Fc gamma RIII on PM was unable to mediate functional interactions with red cells sensitized with anti-D x anti-Fc gamma RIII bispecific antibody.

An indirect effect of an antibody on complement deposition and lysis of differently sensitized surrounding cells.

Lysis of papain-treated group A and B erythrocytes by human complement was studied by an anti-A (BRIC. 131) and an anti-B (BRIC. 30) IgM monoclonal antibody in 51Cr release assays. The indirect effect of membrane-bound antibody, i.e. its influence on complement binding to sensitized surrounding cells, was examined in a cold target competition test in which sensitized, non-labelled cells are present along with sensitized labelled cells and complement. The mode by which anti-A antibodies indirectly suppressed lysis of sensitized B cells up to 20-fold was studied by following C1q and C3b binding. C1q binding to both types of erythrocytes was not altered in mixed populations of erythrocytes in the presence of both antibodies. Binding of C3b to a mixture of both cell types was, however, suppressed, when both antibodies were present. C3b deposition in mixed cell populations did not reach a significantly higher extent than deposited to one type of erythrocyte alone. This was consistent with the results from competitive lysis and suggests that the anti-A captured most C3b at high anti-A concentrations and deprived the similarly sensitized B erythrocytes of complement. We think that this phenomenon is not due to an uneven removal of complement regulatory proteins from A and B erythrocytes by papain. Instead, the phenomenon might be due to an inherent property of anti-A mAb to better produce nucleation sites for C3 convertases which, upon binding factor B, better compete for the limiting factor D. A mathematical analysis of cold target competition experiment (containing 2430 individual measurements) also shows that the distribution of complement between the competing A and B erythrocyte population is uneven, since it predicts that in any given antibody combination the majority of complement is bound to A erythrocytes. This is consistent with the measured average percentage of lysis.

Red-cell bound anti-A is more efficient than anti-B in competition for fluid phase complement.

Lysis of group A and B erythrocytes by human complement was studied by an anti-A (BRIC.131) and an anti-B (BRIC.30) IgM monoclonal antibody in a 51Cr-release assay. The relative concentration of membrane-bound immunoglobulins was detected by flow cytometric analysis, and the amount of C1q and C3 bound to the sensitized red cells was measured by using purified, 125I-labelled molecules. The direct haemolysis was identical with both reagents in the presence of excess and suboptimal complement over a wide range of antibody concentration (between 50 and 7000 ng/ml). The indirect effect of membrane-bound antibody, i.e. its influence on complement binding by sensitized bystander cells, was examined in a cold target competition assay in which sensitized, non-labelled cells are present when complement is incubated with sensitized labelled cells. We have found that the competitive capacity of sensitized erythrocytes correlated with the amount of membrane-bound immunoglobulins. In accordance with our earlier findings, an equal level of target and competitor cell lysis was obtained only if the fluid phase anti-B antibody concentration was 2 to 4 times higher than that of the anti-A antibodies. We demonstrate in this paper that the different competitive activity of IgM anti-A and anti-B monoclonal antibodies might be accounted for by differences in their C1q and C3 binding capacities.

Interactions in the complement-mediated lysis of blood group AB erythrocytes sensitized simultaneously with anti-A and anti-B monoclonal antibodies.

The lysis of group AB erythrocytes by human complement was studied by different anti-A and anti-B IgM monoclonal antibodies (mabs) in a 51Cr-release assay. The concentration of membrane-bound immunoglobulin was detected by ELISA, and the amount of C1q and C3 bound to sensitized red cells was measured by using purified, 125I-labelled molecules. We have demonstrated that there is an exponential relationship between the concentration of the sensitizing IgM mabs and C1q binding to the sensitized AB cell. The efficiency of binding was related to the number of antibodies bound; thus, anti-A sensitized cells bound 3-6 times more C1q than anti-B sensitized cells did. AB cells, on the other hand, bound similar amounts of C3 whether anti-A or anti-B was present. The lytic efficiencies of the various IgM mabs during short incubation times were different, suggesting that the complement activation rates vary widely with different antibodies on the AB cell membrane. The binding of C1q to an antibody-sensitized target activates a cascade, whose components may migrate away from the sensitizing antibody; interactions between the activation processes generated by the anti-A and anti-B antibodies may thus occur. Choosing appropriate pairs of anti-A and anti-B mabs for the simultaneous sensitization of AB cells has indeed resulted in stimulation in some and inhibition in other combinations of mabs. It is suggested that stimulation is observed when the activated intermediates are produced in excess, whereas inhibition occurs when a shortage of activated intermediates prevents mutual utilization.

Cold target competition analysis of the classical activation pathway of complement-mediated cytotoxicity: a non-interaction model for competing lysis.

A mathematical analysis of cold target competition experiments of complement-mediated lysis is presented, aimed at developing a minimal model of lysis where no interaction between the competing populations of sensitized blood group A and B erythrocytes is presumed. The model is able to predict the extent of lysis from the input values with remarkable accuracy suggesting that under the conditions used no stimulation and/or inhibition of the lysis of the sensitized erythrocytes occurs. The distribution of complement between the competing A and B erythrocyte populations is approximated by the model and found to be proportional to the 5th and 4th power of the ratios of the antibody and target cell concentrations, respectively. In accordance with earlier observations, suggesting that the interaction between the antibody and the C1q molecules is based on polar electrostatic charges, we propose that the sensitizing antibody provides an electrostatic field around the erythrocytes which attracts C1q molecules towards their membranes.

Inhibition of the classical activation pathway of complement-mediated lysis by monoclonal antibodies to complement components C3c and C3d.

Eight epitope-mapped monoclonal antibodies (MoAbs) to complement component C3d and five to complement component C3c were investigated to determine whether they could inhibit the classical activation pathway of complement-mediated lysis (CML) by using blood group AB red cells sensitized by A or B MoAbs. Three IgM C3d MoAbs and one IgG1 C3c MoAb were able to inhibit CML in a dose-dependent manner. In the presence of excess complement, no inhibition was observed. The greatest inhibition was observed with two high-affinity IgM antibodies that were specific for epitope 1 on the C3d component. Some inhibition was observed with a high-affinity IgM antibody specific for epitope 3 of the C3d component and also with a lower-affinity IgG antibody specific for epitope 1 of the C3c component. The results indicate that some complement MoAbs have the capacity to distinguish between conformationally and/or functionally different forms of red cell-bound C3.

Haemolysis mediated by anti-D monoclonal antibodies in direct and cold target competition ADCC assays.

Thirteen IgG anti-D human monoclonal antibodies (mAbs) were compared for their ability to mediate lysis of D-positive erythrocytes by PBMC in direct and cold target competition antibody-dependent cell-mediated cytotoxicity (ADCC) assays. In the latter assay, lysis of fluid-phase anti-D-sensitised O Rh D-positive papainised erythrocytes (E-IgG) was inhibited by A (or B) Rh D-negative papainised erythrocytes sensitised by fluid-phase anti-A (or anti-B) mAbs. The competitive and lytic activities of the anti-D mAbs were characterised by the equilibrium dilution (ED) values, which were the reciprocal of the dilution of anti-A (or anti-B) at which lysis of target E-IgG and competitor E-IgG were identical. There was a poor correlation between the number of erythrocyte-bound anti-D molecules and either the sensitivity of E-IgG anti-D to haemolysis in the direct ADCC assay, or to the ED values of the mAbs obtained in the cold target competition ADCC. The discriminatory power of the cold target competition ADCC was better than than of the direct ADCC to detect differences in the lytic potential of the anti-D mAbs.

[K-cell activity in patients with germ cell testicular tumors. Effect of cytostatic therapy]. / Csírasejt eredetü heredaganatban szenvedök K-sejt aktivitásának vizsgálata. A citosztatikus kezelés hatása.

The antibody-dependent cell mediated cytotoxicity of peripheral blood mononuclear cells from 92 patients with germinal cell tumours and 60 healthy male controls was measured against 0, Rh(D) positive human red blood cells sensitized with anti-D antibody. To determine the maximal K-cell activity the enzym-like kinetic model of citotoxicity was employed in which maximal activity was measured in presence of target-cell excess. To avoid variation due to the individual sensitivity of target erythrocytes red blood cells were obtained from a single donor. It was demonstrated that compared to the control group the K-cell activity of patients with germinal cell tumours was significantly enhanced. Cytotoxic activity of patients with clinically detectable tumours was significantly higher than that of patients with no detectable tumour. The K-cell activity of patients with detectable tumours was significantly increased after chemotherapy.

Comparison of lysis of bromelain and papain treated red cells in ADCC assays.

Red cells were pretreated with the proteolytic enzymes bromelain or papain prior to use in antibody-dependent cell-mediated cytotoxicity (ADCC) assays with lymphocytes or peripheral blood mononuclear cells (PBMC) as effector cells. At low concentrations of anti-D or anti-A, lysis of papain-treated cells by lymphocytes was greater than that of bromelain-treated cells. Papain digestion resulted in both greater sensitivity to haemolysis by lymphocytes or PBMC and higher agglutination titres of anti-D-sensitised red cells than bromelain. With anti-A, however, although papain also promoted greater haemolysis, it was slightly less effective at red cell agglutination than bromelain.

Differences in the competitive capacity of monoclonal antibody sensitized human A and B erythrocytes in complement mediated cytotoxicity.

Lysis of group A and B erythrocytes by human complement was studied using 9 anti-A (IgM) and 10 anti-B (IgM) monoclonal antibodies in direct and cold target competition 51Cr-release assays. The relative concentration of membrane-bound immunoglobulins was detected by indirect immunofluorescence in flow cytometric analysis. The number of binding sites/cell and affinity of the monoclonal antibodies was measured in ELISA assays. Complete haemolysis was obtained at 2 micrograms.ml-1 antibody concentrations with the majority of reagents when only a small proportion of the antibody binding sites were occupied. The influence of membrane-bound antibody on complement binding was examined by the complement consumption capacity of sensitized erythrocytes at supra-haemolytic antibody concentration. We found that complement consumption correlated with the amount of membrane-bound immunoglobulins. To obtain equal level of target and competitor cell lysis group B erythrocytes had to be equipped with higher amounts of antibodies than group A erythrocytes. The different lytic activity of IgM anti-A and anti-B monoclonal antibodies could not be accounted for merely by differences in their surface density.