T lymphocyte killing by a xanthine-oxidase-containing immunotoxin

Summary We report on the preparation of an immunotoxin consisting of xanthine oxidase, a free-radicalproducing enzyme, covalently linked to an anti-CD3 monoclonal antibody. The immunotoxin retained both enzymic and immunological properties and its toxicity to target cells (a) was greater than that of the free enzyme, (b) was proportional to the enzyme concentration, and (c) was reduced either in the absence of hypoxanthine or by an excess of free anti-CD3 monoclonal antibody. The cytotoxicity and selectivity of the hypoxanthine/conjugated xanthine oxidase system were potentiated by the addition of chelated iron and by washing away the unbound immunotoxin prior to the addition of substrate. The same system was not toxic to bone marrow progenitor cells. A possible use of this immunotoxin for the ex vivo purging of organs to be transplanted from T lymphocytes, to avoid the graft-versus-host reaction, is suggested.     

Colon adenocarcinoma cells inhibit anti-CD3-activated killer cell induction

Adoptive immunotherapy with lymphokine-activated killer (LAK) cells has shown some promise in the treatment of certain cancers that are unresponsive to conventional treatment approaches. However, colon adenocarcinomas tend to respond poorly to LAK therapy, possibly as a result of tumor-induced immunosupprression. Recently, in vivo administration of anti-CD3 antibody has been shown to induce mouse T lymphocytes to mediate major-histocompatibility-complex(MHC)-unrestricted tumoricidal activity which is distinet from natural-killer-cell-derived LAK activity. It has therfore been suggested that anti-CD3 therapy may find application in tumor immunotherapy in humans. However, the effectiveness of anti-CD3-activated killer cell induction within the environment found in the vicinity of colon adenocarcinoma cells has not been evaluated. The present report demonstrates that colon cancer cells of human (HT-29) and mouse (MCA-38) origin markedly inhibit the generation of activated killer cells in murine spleen cell cultures. DNA synthesis and interleukin-2 production by spleen cells following stimulation with anti-CD3 antibody are also profoundly depressed in the presence of MCA-38 and HT-29 adenocarcinoma cells. MCA-38- and HT-29-mediated inhibition of activated killer cell development is exerted through the production of a tumor-associated soluble factor that is distinct from transforming growth factor or prostaglandins. Local immunosupression associated with sites of tumor growth may therefore represent a major obstacle to successful anti-CD3 immunotherapy of certain colon adenocarcinomas.     

The antitumor effect of tumor-draining lymph node cells activated by both anti-CD3 monoclonal antibody and activated B cells as costimulatory-signal-providing cells

To establish an efficient cell-culture system for adoptive immunotherapy, we attempted to use lipopolysacharide (LPS)-activated B cells (LPS blasts) as costimulatory-signal-providing cells in the in vitro induction of antitumor effector cells. Both normal and tumor-draining lymph node cells were efficiently activated by both anti-CD3 monoclonal antibody (mAb) and LPS blasts, and subsequently expanded by a low dose of interleukin-2 (IL-2; anti-CD3 mAb and LPS blasts/IL-2). The expanded cells were predominantly CD8⁺ T cells and showed a low level of tumor-specific cytotoxic T lymphocyte (CTL) activity. The adoptive transfer of B16-melanoma-draining lymph node cells expanded by anti-CD3 mAb and LPS blasts/IL-2 showed significant antitumor effect against the established metastases of B16 in combination with intraperitoneal injections of IL-2. This treatment cured all B16-bearing mice. In addition, these mice also showed tumorspecific protective immunity against B16 at the rechallenge. Considering that activated B cells express several kinds of costimulatory molecules, these findings thus indicate an efficacy of costimulation that is derived from activated B cells for the in vitro induction of tumor-specific CTL, in co-operation with anti-CD3 mAb. The culture system presented here may thus be therapeutically useful, providing potent effectors for adoptive immunotherapy against various types of cancer.     

Down-Regulation of T-Cell Proliferation in Response to Soluble Anti-CD3 Antibodies through Development of Redirected Cytolytic Activity Eliminating Costimulatory Cells

CD4⁺ T-depleted spleen cells (CD8⁺ T cells) activated by anti-CD3 antibodies (aCD3) suppressed proliferation of CD8⁺ T-depleted spleen cells (CD4⁺ T cells) and fresh normal T cells in response to aCD3. Antigen-nonspecific cytolytic activity was induced in splenic CD8⁺ T cells by stimulation with aCD3 and showed the peak level on day 3, whereas cytolytic activity induced in CD4⁺ T cells was weak. Intact Ig but not F(ab')₂ of aCD3 induced and mediated cytolytic activity. Correspondingly, the cytolytic activity induced by aCD3 was directed against target cells bearing Ig-binding Fc-receptor activity and cytolysis was inhibited by the addition of free Ig into the assay system. We showed that aCD3-activated T cells carried a high level of aCD3 on their surface at the time after the peak proliferation when they attained high cytolytic activity. This raised the possibility that the anti-CD3-induced aCD3-redirected cytolytic activity eliminated Fc-receptor-bearing costimulatory cells in the culture for down-regulation of the T-cell proliferation. This view was supported by partial restoration of anti-CD3-induced low responsiveness of CD8⁺ T cells by the addition of fresh costimulatory cells. These results suggested a new pathway of down-regulation of T-cell proliferation by aCD3-activated cytolytic CD8⁺ T cells.     

Attenuated signaling associated with immune activation in HIV-1-infected individuals

Chronic immune activation is associated with impaired signal transduction. Since such activation is commonly found during HIV-1 infection, we studied cellular responses to non-specific T-cell receptor stimulation of PBMC obtained from 20 HIV-1 non-infected individuals and 23 highly or partially immune activated HIV-1 infected individuals. PBMC proliferation and ERK-1/2 phosphorylation following anti-CD3 stimulation, and constitutive levels of Cbl-b, were determined. Increased levels of Cbl-b, decreased proliferation, and lower ERK-1/2 phosphorylation were found in PBMC of highly immune activated HIV-1 infected individuals. The elevated expression of Cbl-b and impaired phosphorylation of ERK-1/2 associated with immune activation probably contribute to the attenuated proliferative and cellular responses characteristic of HIV-1 infection. Therefore, targeting immune negative modulators, such as Cbl-b, may serve as a novel approach for controlling HIV-1 disease progression.     

In vivo cytotoxic efficacy of immunotoxins prepared from anti-CD5 antibody linked to ricin A-chain

Summary The antitumoral efficacy of various anti-CD5 immunotoxins, prepared with whole monoclonal antibody (mAb), F(ab)₂ or Fab fragment linked to native ricin A-chain (RTA) or partially deglycosylated ricin A-chain (dRTA), was examined in vivo in ascitic nude mice bearing a large burden of Ichikawa human tumour cells. We first demonstrated that after systemic administration of IgG-RTA or F(ab)₂-dRTA, the cytotoxic activity of immunotoxin molecules specifically bound to tumour cells was preserved. Secondly we showed, by using different immunotoxins with various targeting capacities, that their cytotoxic effect in vivo was related to the number of immunotoxin molecules bound per cell. However, even when antigen saturation was achieved after i.p. injection, the cytotoxic effect did not exceed 53% of the tumour burden. By contrast, when the immunotoxin was administered i.p. or i.v. with the enhancer monensin conjugated to human serum albumin and injected i.p., 90% of the tumour cells were killed. This potentiating effect was demonstrated even when the tumour localisation was as low as 5% of the saturation level. Such an effect could be completely prevented by addition of unconjugated monoclonal antibody, demonstrating the specificity of the immunotoxin-induced cytotoxicity in the presence of the enhancer. However this enhancement was demonstrated whatever the route of immunotoxin administration, i.p. or i.v., but was only observed when the enhancer was injected i.p. and not i.v.. These results emphasize the importance of optimizing the therapeutic course to improve the antitumoral efficacy of immunotoxins.     

Treatment of low-grade non-Hodgkin's lymphoma with continuous infusion of low-dose recombinant interleukin-2 in combination with the B-cell-specific monoclonal antibody CLB-CD19

Seven patients with low-grade non-Hodgkin's lymphoma were treated with a combination of a murine monoclonal antibody directed against the B-cell-specific antigen CD19 (CLB-CD10), given twice weekly, and continuous infusion of low-dose recombinant interleukin-2 (rIL-2). We demonstrated stable serum CLB-CD19 levels throughout the 12 weeks of treatment, and homing of the antibody into the tumour sites. A variable degree of antigenic modulation was noted. Prolonged treatment resulted in a sustained increase in the number of natural killer cells in the circulation with enhanced cytotoxic capacity, including antibody-dependent cellular cytotoxicity. During the first weeks of treatment, T cell activation occurred in the majority of patients. Toxicity was related to the rIL-2 treatment and consisted of transient constitutional symptoms and a flu-like syndrome without organ dysfunction. A partial remission occurred in one patient, and in another patient who was primarily leukaemic a greater than 50% reduction of circulating B cells was noted. An antitumour effect occurred early during treatment and could not be related to rIL-2-induced modulation of natural killer cell or T lymphocyte activation.     

Preclinical investigation of the antitumour effects of anti-CD19-idarubicin immunoconjugates

Idarubicin (Ida), an analogue of daunomycin, was linked to a mouse monoclonal antibody against the B cell differentiation antigen CD19. Determination of the activity of both the antibody and drug moieties was carried out in vitro using a pre-B cell human acute lymphoblastic leukaemia cell line (NALM-6). A 23% loss in antibody binding and a 20-fold loss in drug activity were observed upon conjugation. Selective cytotoxicity for CD19⁺ cells, however, was obtained. Measurement of the cytotoxicity, antibody activity and release of the breakdown product, 14-OH-Ida, showed that the conjugates remained stable for more than 100 days after lyophilization and storage at –20° C. In vivo studies were performed in irradiatednu/nu mice bearing NALM-6 tumours. Initial dose response studies with free idarubicin demonstrated that three i.p. doses (every other day) of 10 g resulted in little antitumour activity, but the death of all the animals by day 23. The same treatment regime using 15 g Ida-anti-CD19 conjugate caused the disappearance of four out of five tumours with three complete cures and no evidence of toxicity as assessed by weight loss. Administration of a conjugate of idarubicin with an irrelevant antibody at this dose led to no significant antitumour response. The administration of free drug at a dose of 6 g resulted in a minor antitumour response but high toxicity, whereas injection of Ida-anti-CD19 conjugate at this dose caused no toxicity and a substantial antitumour effect with eradication of two out of five tumours. These results clearly demonstrate that the administration of Ida-anti-CD19 conjugates can result in complete tumour regression in an experimental model. Idarubicin-containing immunoconjugates should be useful for the treatment of patients with non-Hodgkin's lymphoma.     

巨型引物PCR法对抗CD3改型单链抗体

亲和力是影响改型单链抗体应用于临床的重要因素之一.利用巨型引物PCR定点诱变方法,设计并化学合成出两组含多个突变位点的简并引物,在第一轮PCR中使用简并引物分别扩增出含突变碱基的两条特异性的DNA片段,即巨型引物,将其经琼脂糖凝胶电泳分离纯化后,作为3′和5′的两端引物应用于第二轮PCR反应中.通过改变标准PCR反应条件,调整引物与模板的浓度,扩增出特异性较强的目的DNA条带.PCR产物经回收后,进行DNA测序.测序结果表明利用该方法扩增得到特异的抗CD3改型单链抗体的突变体库.     

Optimisation of ITK inhibitors through successive iterative design cycles

Based on a hit cluster of compounds inhibiting interleukin-2 inducible T-cell kinase (ITK) in the submicromolar range a series of ITK inhibitor libraries were synthesized. Through iterative design cycles including kinase crystal structure information, indolylindazole libraries were identified which showed low nanomolar activity in enzymatic and cellular assays. The potential of these novel lead series was confirmed through in vivo tests in an anti-CD3-IL2 mouse model. The intravenous administration of highly potent ITK inhibitor 11o resulted in dose-dependent, efficient suppression of IL-2.     

Pharmacokinetics and biodistribution of radioimmunoconjugates of anti-CD19 antibody and single-chain Fv for treatment of human B-cell Malignancy

The comparative advantages and disadvantages of intact antibodies and single-chain Fv as immunotoxins and radioimmunoconjugates have been widely discussed but not directly compared. In this study, the in vivo properties of anti-CD19 B43 monoclonal antibody and its derived single-chain Fv (FVS191) were studied in athymic nude mice bearing CD19-positive human lymphomas. B43 mab and FVS191 were labeled with iodine-125 using iodine-beads, and immunoreactivities were determined to be 57% and 72%, respectively. Scatchard analysis showed a similar high affinity for both. The results of pharmacokinetic studies revealed that FVS191 had a rapid biphasic clearance from the circulation (T1/2α = 2.5 min, T1/2β = 3.7 h); The T1/2α and T1/2β phases of B43 mab were determined to be 0.72 h and 57 h respectively. Biodistribution studies compared the uptake of labeled antibodies by CD19-positive and by CD19-negative tumors. The peak percentages of injected dose were 5.7% at 12 h for B43 and 2.45% at 1 h for FVS191. Radiolocalization indices (RI) demonstrated tumor-specific uptake for both, but higher uptake for B43. The optimal RI was seen at 15 min for FVS191 and 6 h for B43. FVS191 was unstable in vivo, approximately 50% of the injected dose being degraded in blood in 100 min. Radioactivity detected in the urine was present mainly as the deiodinized form of FVS191. The results suggest that B43 mab is favored over FVS191 in biodistribution properties and in vivo stability. Because B43 Mab showed early tumor-specific uptake, high RI values, and favorable tissue-to-blood ratios, it is a potential candidate for radioimmunotherapy and immunotoxin therapy of B-cell leukemia and lymphoma. Received: 17 June 1997 / Accepted: 17 June 1998     

High level expression of a functional human/mouse chimeric anti-CD20 monoclonal antibody in milk of transgenic mice

Rituximab, a chimeric anti-CD20 monoclonal antibody, is one of the most successful biomedicines and has been used to treat at least 370,000 patients with indolent, aggressive non-Hodgkin's lymphoma and other malignant diseases. However, the global demand for rituximab and other therapeutic monoclonal antibodies is exponentially increasing and barely able to be met by current manufacturing capacities of mammalian cell culture. The mammary gland bioreactor has been regarded as an ideal substitute for mammalian cell culture to mass-produce recombinant monoclonal antibodies at the lowest possible cost. Here, we show a feasible model to produce recombinant anti-CD20 antibodies in the mammary glands of transgenic animals. Six lines of transgenic mice were generated by co-microinjection of the two expression cassettes that can specially express the chimeric light and heavy chain of anti-CD20 mAbs in the milk of transgenic animals. The recombinant antibodies were detected in the milk of transgenic mice with the highest expression level up to 17 microg/mul and could specifically bind the CD20 surface antigens on human B-lymphoma cells.     

CD40 ligation in vivo can induce T cell independent antitumor effects even against immunogenic tumors

Antitumor effects of CD40 ligation appear to involve distinct antitumor effector cells in different experimental models. In this study, we tested whether T cells were required for antitumor effects of agonistic anti-CD40 mAb (alphaCD40) against immunogenic versus poorly immunogenic tumors. Treatment of mice bearing poorly immunogenic B16 melanoma and its more immunogenic variant, B16-hsp72.1, with alphaCD40 resulted in a similar level of tumor growth suppression. Depletion of T cells did not reduce the antitumor effects in these 2 tumor models. To generate antitumor T cell responses, C57BL/6 mice were immunized with irradiated B16-hsp72.1. Treatment of these vaccinated mice challenged with a high dose of B16-hsp72.1 tumor cells with alphaCD40 induced tumor growth suppression, which was reduced by T-cell depletion, demonstrating that T cells were involved in the antitumor effect of alphaCD40. However, immunized mice depleted of T cells and treated with alphaCD40 were still able to suppress tumor growth as compared to tumor growth in immunized, T cell-depleted mice not treated with alphaCD40, suggesting that T cells were not required for the antitumor effect of alphaCD40. To confirm a lack of correlation between tumor immunogenicity and T-cell requirement in antitumor effects of CD40 ligation, we found that alphaCD40 induced tumor growth suppression in nude and SCID/beige mice bearing highly immunogenic tumors such as Meth A sarcoma, suggesting that macrophages may play a role. Indeed, both poorly immunogenic and highly immunogenic tumors were sensitive to in vitro growth inhibition by macrophages from alphaCD40-treated mice. Taken together, our results indicate that antitumor effects induced by alphaCD40, even against immunogenic tumors, can be observed in the absence of T cells and may involve macrophages.     

Expression and purification of two anti-CD19 single chain Fv fragments for targeting of liposomes to CD19-expressing cells

Antibody-targeted liposomal anticancer drugs combine the specificity of antibodies with large payloads of entrapped drugs. We previously showed that liposomal doxorubicin (DXR) targeted via anti-CD19 monoclonal antibodies (mAb) or their Fab' fragments against the B-cell antigen CD19 led to improved therapeutic effects in murine B-cell lymphoma models relative to non-targeted liposomal DXR. We now are examining the use of anti-CD19 single chain fragments of the antibody variable region (scFv) as a targeting moiety, to test the hypothesis that scFv have advantages over full-sized mAb or Fab' fragments. We expressed two different anti-CD19 scFv constructs, HD37-C and HD37-CCH in E. coli, and purified the scFvs using two different methods. The HD37-CCH construct was selected for coupling studies due to its relative stability and activity in comparison to HD37-C. When coupled to liposomes, the HD37-CCH scFv showed increased binding in vitro to CD19-positive Raji cells, compared to non-targeted liposomes. Cytotoxicity data showed that HD37-CCH scFv-targeted liposomes loaded with DXR were more cytotoxic than non-targeted liposomal DXR. Our results suggest that anti-CD19 scFv constructs should be explored further for their potential in treating B-lymphoid leukemias and lymphomas.     

Enhancement of antitumor effect by peptide vaccine therapy in combination with anti-CD4 antibody: Study in a murine model

PurposeThe clinical efficacy of cancer peptide vaccine therapy is insufficient. To enhance the anti-tumor effect of peptide vaccine therapy, we combined this therapy with an anti-CD4 mAb (GK1.5), which is known to deplete CD4⁺ cells, including regulatory T cells (Tregs).MethodsTo determine the treatment schedule, the number of lymphocyte subsets in the peripheral blood of mice was traced by flow cytometry after administration of anti-CD4 mAb. The ovalbumin (OVA)_257–264 peptide vaccine was injected intradermally and anti-CD4 mAb was administered intraperitoneally into C57BL/6 mice at different schedules. We evaluated the enhancement of OVA peptide-specific cytotoxic T lymphocyte (CTL) induction in the combination therapy using the ELISPOT assay, CD107a assay, and cytokine assay. We then examined the in vivo metastasis inhibitory effect by OVA peptide vaccine therapy in combination with anti-CD4 mAb against OVA-expressing thymoma (EG7) in a murine liver metastatic model.ResultsWe showed that peptide-specific CTL induction was enhanced by the peptide vaccine in combination with anti-CD4 mAb and that the optimized treatment schedule had the strongest induction effect of peptide-specific CTLs using an IFN-γ ELISPOT assay. We also confirmed that the CD107a⁺ cells secreted perforin and granzyme B and the amount of IL-2 and TNF produced by these CTLs increased when the peptide vaccine was combined with anti-CD4 mAb. Furthermore, metastasis was inhibited by peptide vaccines in combination with anti-CD4 mAb compared to peptide vaccine alone in a murine liver metastatic model.ConclusionThe use of anti-CD4 mAb in combination with the OVA peptide vaccine therapy increased the number of peptide-specific CTLs and showed a higher therapeutic effect against OVA-expressing tumors. The combination with anti-CD4 mAb may provide a new cancer vaccine strategy.     

DNA repair after DNA fragmentation in mouse small intestinal epithelial cells

In our earlier work, we found that, in mice, i.p. injection of anti-CD3 monoclonal antibody activated intraepithelial lymphocytes (iIEL), leading to DNA fragmentation in villous epithelial cells of the duodenum and jejunum within 30 min. By 2 h after injection, nearly half of the enterocytes had detached from the villi, and DNA fragmentation could barely be detected in the remaining villous epithelium. We hypothesized that DNA had been repaired in enterocytes in which DNA fragmentation had previously been induced. In this study, enterocytes became negative for TUNEL staining at 60 min after anti-CD3 treatment, prior to detachment. The remaining villous epithelial cells, after DNA fragmentation and detachment, were found to be positive for 5-bromo-2-deoxyuridine labeling. To confirm whether fragmented DNA had been repaired in situ, we investigated the appearance and/or mobilization of DNA-repair-related proteins. Focus formation, a typical staining pattern of repair-related proteins including phosphorylated H2AX, phospo-ATM substrate, and Nbs1, was observed 30 min after anti-CD3 injection, with the kinetics virtually identical to that of DNA fragmentation. The co-localization of γ-H2AX and phospo-ATM substrate was also confirmed. The disappearance of a positive reaction for TUNEL staining in previously fragmented DNA, the appearance of representative DNA-repair-related proteins, the coincidence of the kinetics of DNA fragmentation and this appearance of DNA-repair-related proteins, and the co-localization of two of the repair-related proteins strongly indicated that enterocyte DNA could be repaired after it had been fragmented in vivo. Thus, DNA fragmentation per se may not necessarily be an immediate sign of cell death. This work was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (16590132 to T.M., 16390045 to T.I., and 20590181 to M.O.).     

Further characterization of cytotoxic T cells generated by short-term culture of human peripheral blood lymphocytes with interleukin-2 and anti-CD3 mAb

 In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3⁺ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fcγ receptor (FcγRI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab′)₂, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18⁺ ovarian tumor cell line), was also compared in the presence of a bispecific antibody (OC/TR, anti-CD3 × MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 ×antitumoral bispecific antibodies. Received: 6 December 1995 / Accepted: 4 June 1996